Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms.

Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. This antimicrobial response of Drosophila is specific and can discriminate between various classes of microorganisms. The genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. The level of induction of the diptericin gene in immune-challenged adults varies strikingly with the microorganism tested. Gram-negative bacteria are potent inducers. In contrast, Gram-positives do not induce expression above the level of a simple injury. The pattern of cecropin A, drosocin, defensin, and attacin induction roughly corresponds to the pattern of diptericin induction Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides (especially drosomycin) with antifungal activities. The expression of metchnikowin combines both patterns, as this gene is strongly induced by all microorganisms. These responses is mediated through the selective activation of the Toll pathway. Genes whose expression levels are most strongly affected by the immune deficiency, imd, mutation and that code for strictly antibacterial peptides are also those that are most strongly induced by challenge with Gram-negative as compared with Gram-positive bacteria. In contrast, the metchnikowin and drosomycin genes that are strongly induces by Gram-positive bacteria retain most of their inducibility in imd mutants (Lemaitre, 1997).

Sequential activation of signaling pathways during innate immune responses

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. These transcriptional patterns can be dissected into distinct groups. In addition to signaling through either the Toll/NFkappaB or Imd/Relish pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, the results may imply a close link between the control of tissue repair and antimicrobial processes (Boutros, 2002).

Lipopolysaccharides (LPS) are the principal cell wall components of gram-negative bacteria. In mammals, exposure to LPS causes septic shock through a Toll-like receptor TLR4-dependent signaling pathway. LPS treatment of Drosophila SL2 cells leads to rapid expression of antimicrobial peptides, such as Cecropins (Cec). SL2 cells resemble embryonic hemocytes and have also been used as a model system to study JNK and other signaling pathways. LPS-responsive induction of the antimicrobial peptides AttacinA (AttA), Diptericin (Dipt), and Cec relies on IKK and Relish. In order to obtain a broad overview on the transcriptional response to LPS in Drosophila, genome-wide expression profiles of SL2 cells were generated at different time points following LPS treatment. Altered expression of 238 genes was detected (Boutros, 2002).

In time-course experiments, a complex pattern of gene expression was observed that can be separated into different temporal clusters. A first group, with peak expression at 60 min after LPS, primarily consists of cytoskeletal regulators, signaling, and proapoptotic factors. This group includes cytoskeletal and cell adhesion modulators such as Matrix metalloprotease-1, WASp, Myosin, and Ninjurin, proapoptotic factors such as Reaper, and signaling proteins such as Puckered and VEGF-2. A second group, with peak expression at 120 min, includes many known defense and immunity genes, such as Cec, Mtk, and AttA, but not the gram-positive-induced peptide Drs. Interestingly, this cluster also includes PGRP-SA, which is a gram-positive pattern recognition receptor in vivo, suggesting possible crossregulation between gram-positive- and gram-negative-induced factors. A third group is transiently downregulated upon LPS stimulation. This cluster includes genes that play a role in cell cycle control, such as String and Rca1. Altogether, these results show that, in response to LPS, a defined gram-negative stimulus, cells elicit a complex transcriptional response (Boutros, 2002).

In adult Drosophila, gram-negative bacteria elicit an antimicrobial response mediated by a signaling pathway that involves the intracellular factors Imd, Tak1, IkappaB kinase Kenny (Key), and Rel. On the basis the expression profiling results, it was reasoned that the temporal waves of transcriptional activity in SL2 cells might reflect different signaling pathway contributions. It was therefore asked whether selectively removing signaling components by RNA interference (RNAi) would block induction of all, or only parts, of the transcriptional response to LPS (Boutros, 2002).

The effect of removing key or rel by RNAi was investigated. The expression profiles demonstrate that removing key or rel diminishes the induction of antimicrobial peptides. However, the induction of cytoskeletal and proapoptotic factors was not affected. In contrast, removing tak1 reduces the level of induction or repression for all identified genes, indicating that LPS-induced signaling is transmitted through Tak1 and that specific pathways branch downstream of Tak1 (Boutros, 2002).

In the Rel-independent group, several transcripts were identified that are indicative of other signaling events. For example, puc is transcriptionally regulated by JNK signaling during embryonic development. Therefore, the effect of removing SAPK/JNK activity was tested on LPS-induced transcripts. mkk4/hep dsRNA-treated cells lose the ability to induce the Rel-independent cluster, indicating that LPS signaling branches downstream of Tak1 into separate Rel- and JNK-dependent branches. To validate the results obtained from the microarray experiments, quantitative PCR (qPCR) was performed using puc and cec mRNA levels as indicators for Imd/Rel- or Mkk4/Hep-dependent pathways. Additionally, the effect of removing imd, which, in vivo, acts upstream of Tak1, was tested to clarify whether, in addition to Tak1, other known upstream components of a gram-negative signaling pathway are required for both Rel- and Mkk4/Hep-dependent pathways. These qPCR experiments confirm that cec is dependent for its expression on Imd, Tak1, Rel, and Key, whereas LPS-induced puc expression is dependent on Imd, Tak1, and Mkk4/Hep. Hence, the immunity signaling pathway in response to LPS bifurcates downstream of Imd and Tak1 into Rel- and SAPK/JNK-dependent branches. Both the Rel and SAPK/JNK pathways regulate different functional groups of downstream target genes (Boutros, 2002).

While both Rel and Mkk4/Hep pathways are downstream of Imd and Tak1 in response to LPS, the two downstream branches elicit different temporal expression patterns. It was then asked whether the first transcriptional response is controlled by downstream targets that might negatively feed back into the signaling circuit. puc was a candidate for such a transcriptionally induced negative regulator. Expression profiles of cells depleted for puc were tested before and after a 60 min LPS treatment. These experiments showed that transcripts dependent on the Mkk4/Hep branch of LPS signaling are upregulated, even without further LPS stimulus. In contrast, Rel branch targets are not influenced. puc dsRNA-treated cells show loss of the typical round cell shape. These cells appear flat and have a delocalized Actin staining, consistent with a deregulation of cytoskeletal modulators in puc-deficient cells (Boutros, 2002).

The analysis of expression profiles shows that, while SAPK/JNK and Rel signaling are controlled by the same Imd/Tak1 cascade, they appear to have different feedback loops. Whereas Rel signaling induces Rel expression and thereby generates a self-sustaining loop, possibly leading to the maintenance of target gene expression, the SAPK/JNK branch induces an inhibitor and thereby establishes a self-correcting feedback loop. These results may explain how a single upstream cascade can lead to different dynamic patterns (Boutros, 2002).

Septic injury of adult Drosophila is a widely used model system to study innate immune responses in vivo. To explore the signaling pathways that control induced genes in vivo, genome-wide expression profiles were generated of adult Drosophila infected by septic injury. Equal numbers of male and female adult Oregon R flies were infected with a mixture of E. coli (gram negative) and M. luteus (gram positive). Subsequently, flies were collected at 1, 3, 6, 24, 48, and 72 hr time points post-septic injury to measure temporal changes in gene expression levels. Computational analysis identified a list of 223 genes that were differentially regulated and matched the filtering criteria for at least two time points after microbial infection. This set includes 197 genes that are transiently upregulated and 26 that are transiently downregulated upon immune challenge. Different temporal profiles of gene expression can be detected in this analysis; clusters of genes differed significantly in the timing and persistence of induction. For example, whereas many genes are expressed transiently shortly after infection, others are induced late and are still upregulated at a 72 hr time point. A significant number of genes of both early and late clusters are differentially expressed at a 6 hr time point after infection, which was chosen for further analysis (Boutros, 2002).

The signaling requirements for these differentially expressed transcripts were examined in mutant alleles of known Toll and Imd/Rel pathway components, reasoning that additional pathways might be uncovered by analyzing patterns that cannot be reconciled with expected signaling patterns. Flies homozygous for loss-of-function mutations in tube, key, or rel were infected with gram-negative and gram-positive bacteria, and expression profiles were generated for a 6 hr time point after infection. In addition, noninfected Tl^10b, a gain-of-function allele of the receptor, and cact, a homolog of the inhibitory factor IkappaB, were used to monitor transcripts that are constitutively expressed in gain-of-function signaling mutants. The antimicrobial peptides dipt and drosomycin (drs) are representative targets for the Toll and Imd/Rel pathways, respectively. dipt induction is not detectable in the expression profiles in either a rel or key mutant background, whereas its expression is not affected in tube mutants. In contrast, drs relies on Tube to convey a Toll-dependent signal. Consistently, the expression profiles show that, in a tube mutant background, drs expression is diminished. These experiments showed that the analysis of mutant expression profiles can be used to deduce signaling requirements for distinct target groups (Boutros, 2002).

Toward a computational annotation of signaling pathways, a pattern-matching strategy was employed to rank transcripts by similarity to bona fide Toll or Imd/Rel pathway targets, such as dipt and drs. A set of 91 transcripts that matched the filtering criteria was analyzed for differential expression at a 6 hr time point after septic injury. To determine their dependence on known immunity signaling pathways, the correlation coefficients were calculated of the individual gene expression level in mutant backgrounds to binary Toll or Imd/Rel patterns. Genes were subsequently ordered according to their correlation coefficients for each pathway signature. Using this strategy, transcripts were separated that primarily belong to either the Toll or Imd/Rel pathway groups. For example, genes that show a high correlation coefficient for a Toll pathway pattern include drs, transferrin, a secreted iron binding protein, IM2, and a cluster of homologous secreted peptides at 55C9. These genes have a low correlation coefficient for an Imd/Rel pattern, indicating that they are primarily dependent on Toll pathway signaling in response to microbial infection. In contrast, a group of genes score low for a Toll pathway pattern but have high correlation coefficients for an Imd/Rel pattern. This group includes known gram-negative antimicrobial peptides, such as cec and dipt, peptidoglycan receptor-like genes (PGRP-SD, PGRP-SB1), other small transcripts (CG10332), and genes coding for putative transmembrane proteins, such as CG3615 (Boutros, 2002).

Interestingly, some genes do not fit either pattern, suggesting that they are regulated by other pathways. One group of genes, including cytoskeletal factors such as actin88F, flightin, and tpnC41C, is induced in Tl^10b, but not in cact, mutants. In contrast, totM and CG11501 are expressed at high levels in cact mutant flies but are not expressed in Tl^10b mutant flies. In addition, these transcripts are highly inducible in a tube genetic background, but they are not inducible in key or rel. This may suggest that Toll, Tube, and Cact do not act in a linear pathway under all circumstances. Moreover, rel shows an expression pattern suggesting that it is regulated by both the Imd/Rel and Toll pathways. Thus, these results indicate that, in addition to the canonical Toll and Imd pathways, other signaling events and possibly signaling pathway branching contribute to the complex expression patterns after septic injury. Finally, there is a strong correlation between pathway requirement and temporal expression pattern. Whereas Toll targets are exclusively found in the sustained cluster, Imd/Rel targets are expressed early and transiently after septic injury. The two additional clusters with noncanonical patterns show temporal patterns distinct from either Toll or Imd pathways (Boutros, 2002).

It was reasoned that the patterns observed in the mutant analysis might reflect the contributions of additional signaling pathways. Also, these noncanonical clusters show distinct temporal expression patterns, suggesting that they are separately controlled. One group of genes consists primarily of cytoskeletal regulators and structural proteins that are expressed early on, with peak expression at 3 hr. These include several muscle-specific proteins, thus possibly reflecting the organ that is injured during injection. For example, flightin (fln) encodes a cytoskeletal structural protein expressed in the indirect flight muscle (Boutros, 2002).

Since the expression of cytoskeletal genes after LPS stimulation is dependent on a JNK cascade, whether removing JNK activity in vivo affects the induction of fln was examined. In Drosophila, JNK signaling pathways have been previously implicated in epithelial sheet movements during embryonic and pupal development, a process that has been likened to wound-healing responses. hep¹ (JNKK) mutants, which are impaired in JNK signaling, the induction of fln is diminished, whereas the expression of the antimicrobial peptide dipt is not affected. A test was performed to see whether fln induction in Tl loss-of-function alleles is affected. These experiments show that fln expression is lost in Tl mutants, suggesting that Toll acts upstream of a JNK pathway to induce septic injury-induced target genes (Boutros, 2002).

The clustering revealed a second noncanonical group with small proteins that are expressed late and transiently with peak expression at 6 hr after septic injury. One of the clustered transcripts, CG11501, encodes a small Cys-rich protein that is 115 amino acids long and is strongly induced after septic injury. By RT-PCR, it was confirmed that CG11501 is upregulated after septic injury. In order to characterize how CG11501 is controlled after microbial challenge, a candidate pathway approach was undertaken. In an independent study, it was found that totM gene induction, which is part of the same cluster, is dependent on a JAK/STAT signaling pathway. Whether CG11501 induction requires JAK/STAT signaling was examined. Mutations in JAK/STAT pathways in Drosophila have been implicated in various processes during embryonic and larval development. In Anopheles, STAT is activated in response to bacterial infection. Similarly, gain-of-function STAT has been implicated in the transcriptional control of thiolester proteins. Mutant alleles of hopscotch (hop), the Drosophila homolog of JAK were examined. Quantitative PCR shows that CG11501 induction after septic injury is diminished in hop loss-of-function mutants, whereas the expression of Toll and Imd targets drs, and cec is not affected (Boutros, 2002).

This study shows that in addition to known innate immune cascades, JNK and JAK/STAT are required for the transcriptional response during microbial challenge. One transcriptional signature of small secreted peptides can be traced to JAK/STAT signaling. Additionally, JNK signaling controls cytoskeletal genes after an LPS stimulus and after septic injury in vivo. Both in cells and in vivo, JNK pathways are connected to the same upstream signaling cassette that induces NFkappaB targets. Altogether, these results suggest that innate immune signaling pathways closely link cytoskeletal remodeling, as required for tissue repair, and direct antimicrobial actions. The data also provide insights into the connection of temporal patterns and the activation of distinct signaling pathways (Boutros, 2002).

NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).

Strikingly, cytoskeletal gene expression during innate immune responses is controlled by JNK through the same upstream signaling cascade that activates NFkappaB pathways. JNK pathways act downstream of microbial stimuli, both in vivo and in cells, to induce cytoskeletal regulators. In SL2 cells, JNK signaling is required for the induction of a cluster of cytoskeletal, cell adhesion regulators and proapoptotic factors. Interestingly, both NFkappaB and JNK branches share the same upstream components, Tak1 and Imd, indicating that the activation of both processes are tightly linked. MMP-1, a matrix metalloproteinase that is one of the most markedly upregulated genes after LPS stimulation, has been implicated in wound-healing responses in mammals. Compared with experiments in cells, the situation in vivo after septic injury is likely more complex. Gene expression profiling in whole organisms likely has a lower sensitivity for transcriptional changes that occur in rather small numbers of cells. Also, tissue-specific differences in signaling pathway activity may not reflect the transcriptional changes observed in the cell culture model. Muscle-specific cytoskeletal factors, possibly because they were injected into the thoracic muscle, are not inducible in a JNK-deficient genetic background. However, since it was necessary to remove both Mkk4 and Hep (Mkk7) in cells to deplete JNK pathway activity, an experiment that cannot be performed in vivo because of the lack of an Mkk4 mutant, these experiments might not have uncovered all JNK-dependent transcripts. SAPK/JNK modules can also be linked to different upstream activating cascades. For example, a recent study reported the activation of p38a through a cascade involving Toll, TRAF6, and TAB. Similarly, during innate immune responses JNK pathways can be activated by both Toll and Imd pathways in vivo (Boutros, 2002).

The activation of JNK signaling is reminiscent of signaling during dorsal and thorax closure. In dorsal closure, SAPK/JNK signaling controls cytoskeletal rearrangements that lead to the epithelial sheet movements of the embryonic epidermis. SAGE analysis of embryos with activated SAPK/JNK signaling has shown an induction of cytoskeletal factors. Also, dorsal closure movements are proposed to be similar to the reepithelization that occurs during wound healing. In other developmental contexts, SAPK/JNK signaling has been implicated in cytoskeletal rearrangements and cell motility, such as the generation of planar polarity in Drosophila and convergent-extension movements in vertebrates. A common theme of SAPK/JNK pathways might be their control of cytoskeletal regulators for diverse biological processes. The finding that, in response to LPS, SAPK/JNK and NFkappaB targets are coregulated through the same intracellular pathway suggests a close linkage of directed antimicrobial activities and tissue repair processes (Boutros, 2002).

In conclusion, genome-wide expression profiling was employed to examine the contribution of different signaling pathways in complex tissues and to assign targets to candidate pathways. Both a cell culture model system and an in vivo analysis were used to show the temporal order of NFkappaB-dependent and -independent pathways after septic injury. An interesting question that remains is, how do the extracellular events leading to pathway activation reflect the nature of the pathogen? Clean injury experiments induce a largely overlapping set of induced genes, but to a lower extent than septic injury. This is consistent with experiments showing that septic injury with only gram-negative E. coli induces both anti-gram-negative and anti-gram-positive responses. These results can be interpreted to suggest that wounding, in itself, might be sufficient to induce a transient (and unspecific) innate immune response. However, further studies are needed to understand the nature of the inducing agent (Boutros, 2002).

Immunodeficient Drosophila mutants: Constitutive expression of a single antimicrobial peptide can restore wild-type resistence to infection

One of the characteristics of the host defense of insects is the rapid synthesis of a variety of potent antibacterial and antifungal peptides. To date, seven types of inducible antimicrobial peptides (AMPs) have been characterized in Drosophila. The importance of these peptides in host defense is supported by the observation that flies deficient for the Toll or Immune deficiency (Imd) pathway, which affects AMP gene expression, are extremely susceptible to microbial infection. A genetic approach has been developed to address the functional relevance of a defined antifungal or antibacterial peptide in the host defense of Drosophila adults. AMP genes have been expressed via the control of the UAS/GAL4 system in imd;spätzle double mutants that do not express any known endogenous AMP gene. These results clearly show that constitutive expression of a single peptide in some cases is sufficient to rescue imd;spätzle susceptibility to microbial infection, highlighting the important role of AMPs in Drosophila adult host defense (Tzou, 2002).

Antimicrobial peptides (AMPs) are a key component of innate immunity. Their distribution throughout the animal and plant kingdom is ubiquitous, reflecting the importance of these molecules in host defense. In insects, systemic infection induces the synthesis of combinations of AMPs that are secreted from the immune organs, mainly the fat body, an analog of the mammalian liver, into the hemolymph, where the AMPs reach high concentrations. In Drosophila, at least seven types of AMPs (plus isoforms) have been described. Their activities have been either determined in vitro by using peptides directly purified from flies or produced in heterologous systems, or deduced by comparison with homologous peptides isolated in other insect species: (1) Drosomycin and Metchnikowin show antifungal activity; (2) Cecropins have both antibacterial and antifungal activities; (3) Drosocin and Defensin are predominantly active against Gram-negative and -positive bacteria, respectively, and (4) Attacins and Diptericins are similar to peptides from other insects that show antibacterial activity (Tzou, 2002 and references therein).

Analysis of the in vivo roles of each AMP on microbial infection is complicated by the numerous AMP genes present in the fly, as well as the redundant defense mechanisms within the innate immune system. The importance of AMPs, however, is supported by the sensitive phenotype of mutants that do not express AMP-encoding genes. A clear correlation is observed between the lack of expression of antibacterial peptide genes in mutants of the Immune deficiency (Imd) pathway and their susceptibility to Gram-negative bacteria. Conversely, mutations in the Toll pathway block Drosomycin expression and result in susceptibility to fungal infection. Finally, mutants deficient in both the Imd and Toll pathways failed to express any known AMP genes after infection and are extremely susceptible to both fungal and bacterial infections. These evidences of the importance of AMPs in fighting infection, however, are still indirect, because it cannot be exclude that these mutations affect other defense reactions. The Toll pathway, for example, has also been reported to regulate hemocyte proliferation. To study unambiguously the in vivo role of each AMP in Drosophila host defense, imd;spätzle (spz) double mutant flies have been created that are deficient for both the Imd and Toll pathways but that constitutively express different AMPs under the control of a noninducible promoter. These flies express only one AMP on infection and, consequently, a simple survival experiment can be used to monitor the contribution of this peptide in resistance to infection by various microorganisms. This powerful assay allowed the analysis, in vivo, of the spectrum of activity of each peptide and, by combining two different transgenes, any potential synergy among them. These results clearly show that expression of a single peptide, in some cases, is sufficient to rescue the imd;spz susceptibility to microbial infection, highlighting the important role of AMPs in Drosophila adult host defense (Tzou, 2002).

In this assay, the AMP genes are expressed via the UAS/GAL4 system at a level similar to that observed in wild-type induction of the endogenous AMP genes (except Defensin and Diptericin). However, there are still some differences between this assay and the wild-type physiological condition. In the UAS-Pep flies, AMP genes are expressed ubiquitously and constitutively, contrasting to the wild-type flies in which peptides are made mainly by the fat body in an acute phase profile. The accumulation of AMP, therefore, through constitutive gene expression before infection may be critical to confer an effective protection (Tzou, 2002).

This study provides an alternative method for monitoring and comparing the antimicrobial activity of the various Drosophila AMPs. Defensin is the most potent peptide against Gram-positive bacteria, whereas Attacin A and Drosomycin are active against Gram-negative bacteria and fungi, respectively. One copy of UAS-Def is sufficient to protect flies to wild-type level against M. luteus, B. subtilis, and S. aureus. The efficiency of Defensin may explain why the endogenous Defensin gene is transcribed to lower levels than the other AMP genes after infection. One copy of UAS-Drs is sufficient to protect against N. crassa, whereas two copies are required to induce a complete and partial protection against F. oxysporum and A. fumigatus, respectively. These results are consistent with the Minimum Inhibitory Concentration assay of Drosomycin required in vitro to kill these three fungi: 0.3-0.6 µM for N. crassa, 1.2-2.5 µM for F. oxysporum, and 20-40 µM for A. fumigatus. In addition, Diptericin in Drosophila contributes to resistance against some Gram-negative bacteria, although its activity is probably underestimated because of the low levels of Diptericin expression generated by the constructs used in this study. Surprisingly, no clear protective effect of Cecropin A could be detected in this assay, whereas Cecropin A peptide shows strong in vitro activity. The possibility cannot be excluded that in the lines used, Cecropin A is not effectively produced or well processed to the active form. Alternatively, a higher level of Cecropin A expression may be required to generate a protective effect, considering that the Drosophila genome contains three other inducible Cecropin genes (Tzou, 2002).

These results also underline the differential activities of Drosophila AMPs: such is the case of Attacin A and Drosocin in resistance to some Gram-negative bacterial species. Thus the existence of numerous AMPs may help widen the protection against a large number of microorganisms. In the case of Gram-negative bacterial infection, none of the peptides are able to restore a wild-type resistance in imd;spz double mutants. These results and the observation that the Drosophila genome encodes a high number of AMP genes with activity directed against Gram-negative bacteria suggest that the elimination of this class of bacteria may require the global toxicity generated by multiple, rather than one or two, AMPs (Tzou, 2002).

This study does not reveal a striking synergistic activity among any pair of AMPs tested. In some cases, a rather cooperative effect is observed between two AMPs such as Attacin A when coexpressed with either Diptericin or Drosocin in resistance to some Gram-negative bacteria. These observations suggest that the multiple Drosophila AMPs may function in an additive way, rather than synergistically (Tzou, 2002).

Host-pathogen interactions are antagonistic relationships in which the success of each organism depends on its ability to overcome the other. The production of AMPs is a common strategy to eliminate the invading microbes and, consequently, pathogens have evolved strategies to prevail over these defenses. The assay used provides a powerful tool to compare the resistance of various bacteria to different AMPs, because in these experiments, microbes were injected in an environment previously enriched in peptides. The time race between pathogen and the host defense is clearly illustrated by the observation that a preexisting level of Defensin is sufficient to ensure a complete resistance against B. subtilis, a Gram-positive bacterium highly pathogenic for flies. This observation indicates that B. subtilis is sensitive to Drosophila AMP but nevertheless can overtake the Drosophila immune response by its rapid growth. The observation that 'immunizing' flies with nonpathogenic bacteria fully protects Drosophila from a subsequent infection by B. subtilis is consistent with this hypothesis. These results also show that the kinetics of infection by P. aeruginosa or B. bassiana, two highly entomopathogenic microbes, are not delayed in flies expressing AMP genes, suggesting that these microbes have developed some mechanisms to escape the AMP activity. The observation that Drosomycin expression does not confer any protection against B. bassiana is unexpected, because Toll-mediated defense against this pathogen has been reported. This observation suggests that other antifungal peptides (e.g., Metchnikowin) or a yet uncharacterized defense reaction may be required to resist this fungus. Finally, the human pathogen, S. aureus, is also highly pathogenic to Drosophila and shows a better resistance to a high level of Defensin compared with other Gram-positive bacteria. These results underline the correlation between pathogenicity and increased resistance to AMPs (Tzou, 2002).

The immune phenotype of three Drosophila leukemia models

Many leukemia patients suffer from dysregulation of their immune system, making them more susceptible to infections and leading to general weakening (cachexia). Both adaptive and innate immunity are affected. The fruitfly Drosophila melanogaster has an innate immune system including cells of the myeloid lineage (hemocytes). To study Drosophila immunity and physiology during leukemia, three models were established by driving expression of a dominant-active version of the Ras oncogene (RasV12 ) alone or combined with knockdowns of tumor suppressors in Drosophila hemocytes. The results show that phagocytosis, hemocytes migration to wound sites, wound sealing and survival upon bacterial infection of leukemic lines are similar to wild type. In all leukemic models the two major immune pathways (Toll and Imd) are dysregulated. Toll-dependent signaling is activated to comparable extents as after wounding wild type larvae, leading to a proinflammatory status. In contrast, Imd signaling is suppressed. Finally, adult tissue formation was blocked, and degradation was observed of cell masses during metamorphosis of leukemic lines, which is akin to the state of cancer-dependent cachexia. To further analyze the immune competence of leukemic linesa natural infection model was used that involves insect-pathogenic nematodes. Two leukemic lines, which were sensitive to nematode infections, were identified. Further characterization demonstrates that despite the absence of behavioral abnormalities at the larval stage, leukemic larvae show reduced locomotion in the presence of nematodes. Taken together this work establishes new Drosophila models to study the physiological- immune- and behavioral consequences of various forms of leukemia (Arefin, 2017).

A time course transcriptomic analysis of host and injected oncogenic cells reveals new aspects of Drosophila immune defenses

Oncogenic RasV12 cells injected into adult males proliferated massively after a lag period of several days, and led to the demise of the flies after 2 to 3 wk. The injection induced an early massive transcriptomic response that, unexpectedly, included more than 100 genes encoding chemoreceptors of various families. The kinetics of induction and the identities of the induced genes differed markedly from the responses generated by injections of microbes. Subsequently, hundreds of genes were up-regulated, attesting to intense catabolic activities in the flies, active tracheogenesis, and cuticulogenesis, as well as stress and inflammation-type responses. At 11 d after the injections, GFP-positive oncogenic cells isolated from the host flies exhibited a markedly different transcriptomic profile from that of the host and distinct from that at the time of their injection, including in particular up-regulated expression of genes typical for cells engaged in the classical antimicrobial response of Drosophila (Chen, 2021).

Tissue- and ligand-specific sensing of gram-negative infection in Drosophila by PGRP-LC isoforms and PGRP-LE

The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. This study used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC, and the tissue-specific roles were assessed of PGRP-LC isoforms and PGRP-LE in the antibacterial response. In vivo studies demonstrate the key role of PGRPLCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. The contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria was highlighted through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. The results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. The in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, this study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila (Neyen, 2012).

In animals, the innate immune system detects bacterial infection through the use of germline-encoded pattern recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs), such as LPS, peptidoglycan, or flagellin. After the identification of PRRs and their respective ligands, a challenge in the field is to understand how each of these various PRRs contributes to an effective and adapted immune response. The study of innate immune recognition is complicated by the existence of multiple PRRs with various expression patterns, variation in PAMP exposure, and modifications through the action of host and bacterial enzymes during the course of infection. In addition, PAMP signals intersect with a less well understood but equally complex network of endogenous danger signals, which allow the immune system to discriminate between pathogenic and non-pathogenic microorganisms. A better understanding of the mode of action of PRRs ideally requires an in vivo approach in whole organisms using natural routes of infection. The Drosophila antimicrobial response is one of the best characterized systems of PRR-mediated defense in metazoans and provides a good model to understand both the logic of pattern recognition and how PRRs shape the ensuing immune response. This study used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of peptidoglycan recognition protein (PGRP)-LC, a Drosophila PRR involved in sensing Gram negative bacteria (Neyen, 2012).

Pattern recognition upstream of the two Drosophila innate immune response branches, the Toll and Imd pathways, relies to a large extent on peptidoglycan sensing by PGRPs. Peptidoglycan, a cell wall component found in almost all bacteria, is a polymer of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked by short peptide bridges whose amino acid composition and organization differs among bacteria. As evidenced by Gram staining, peptidoglycan (PGN) forms an abundant external layer in Gram-positive bacteria but is less abundant in Gram-negative bacteria where it is hidden under an external layer of LPS. The structure of PGN from Bacillus and Gram-negative bacteria differs from that of most Gram-positive PGN in the third amino acid position of the peptide bridge. Gram-negative and Bacillus-type PGNs are cross-linked by a peptide containing a meso-diaminopimelic residue, whereas in other Gram-positive bacterial PGNs a lysine is found in this position. In addition, diaminopimelic acid (DAP)-type PGN from Gram-negative bacteria but not DAP-type bacilli contains anhydro-MurNAc residues at the end of each PGN strand, which are distinctive footprints of bacterial PGN synthetic enzymes. Monomers of GlcNAc-1,6-anhydro-MurNAc-L-Ala-γ-D-Glu-meso- DAP-D-Ala, also called tracheal cytotoxin (TCT), represent ~5% of GlcNAc-MurNAc residues and were previously shown to be the minimal PGN motif to elicit Imd responses in flies. As anhydro-muropeptides are released during bacterial cell wall synthesis, TCT has been put forward as a specific indicator of potentially dangerous Gram-negative bacterial proliferation (Neyen, 2012).

Use of highly purified products has demonstrated that in contrast to vertebrates, sensing of Gram-negative bacteria in Drosophila is not based on recognition of LPS. Rather, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of PGN by PGRPs. The Drosophila genome carries a total number of 13 PGRP genes, which give rise to 19 known different receptors. The family comprises both enzymatically active, generally secreted, amidase PGRPs that cleave PGN into non-immunogenic fragments and catalytically inactive receptors, generally membrane-bound, which mediate ligand-dependent downstream signaling. All family members contain at least one PGRP domain, which is structurally related to bacterial T7 lysozymes and recognizes different types of PGN. Whereas PGRP-SA upstream of Toll recognizes mainly lysine-containing PGN from Gram-positive bacteria, Imd-activating PGRP-LC and PGRP-LE exclusively sense DAP-type PGN from Gram-negative bacteria and Gram-positive bacilli. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their PGN binding specificities but share identical signaling capacities. PGRP-LF, a highly similar but signaling-deficient receptor encoded by the locus adjacent to PGRP-LC, contains two functional PGRP domains but lacks the intracellular signaling domain and acts as a negative regulator of Imd activation. Crystal structures of ligand-binding domains of PGRP-LC isoforms in the presence of monomeric PGN have defined the molecular basis for ligand binding. Only PGRP-LCx contains the characteristic L-shaped PGN binding groove described for mammalian PGRP-Ia (Guan, 2004) and Drosophila PGRP-LB and can accommodate polymeric and monomeric PGN. Protruding residues in the ligand binding pocket of PGRPLCa prevent direct binding of TCT, but PGRP-LCa dimerizes with PGRP-LCx-TCT complexes via its PGN binding groove. Notably, PGRP domain affinity studies have determined equivalent binding constants for PGRP-LCa and PGRPLF to PGRP-LCx-TCT complexes. Because activation of the Imd pathway relies on ligand-induced receptor homo- or hetero-multimerization, this implies that the stoichiometry of signaling-efficient PGRP-LC isoforms to the signaling-deficient PGRP-LF determines the strength of pathway activation (Neyen, 2012).

Studies in cell culture using RNA interference (RNAi) specific for each PGRP-LC isoform have shown that PGRP-LCx is required for recognition of polymeric PGN, whereas both PGRPLCa and PGRP-LCx are mandatory for detection of monomeric PGN. It has been proposed that signaling is achieved by association of at least two PGRP-LCx molecules in close proximity through binding of polymeric PGN. Such an interaction cannot occur with monomeric PGN, and in this case PGRP-LCa is expected to act as an adapter. This model is supported by the crystallization of TCT in complex with both PGRP-LCa and PGRP-LCx (Neyen, 2012).

Although loss-of-function mutants established the fundamental role of PGRP-LC in survival to Gram-negative infection (Choe, 2002; Gottar, 2002; Ramet, 2002), the residual antimicrobial peptide response in flies lacking PGRP-LC compared with Imd-deficient flies suggested a second receptor upstream of Imd. PGRP-LE encodes a PGRP with affinity to DAP-type PGN and is expressed both extracellularly and intracellularly. A secreted fragment of PGRP-LE corresponding to the PGRP domain alone functions extracellularly to enhance PGRP-LC- mediated PGN recognition on the cell surface, a role evocative of that of mammalian CD14 in binding of LPS to TLR4. The full-length form of PGRP-LE is cytoplasmic and acts as an intracellular receptor for monomeric PGN, effectively bypassing the requirement for PGRP-LC. Thus, both PGRP-LC and PGRP-LE account for sensing of Gram-negative bacteria upstream of the Imd pathway. Finally, detection of DAP-type PGN in Drosophila is modulated by amidase PGRPs, which enzymatically degrade PGN and reduce the amount of available immunostimulatory compounds. Among these, PGRP-LB has been best characterized as a negative regulator of the Imd pathway. Despite a wealth of studies, several questions remain to be addressed, including the respective contribution of each PGRP-LC isoform and PGRP-LE in response to bacteria as well as the differential requirement of these PRRs in specific tissues, especially in barrier epithelia such as the gut that are constantly exposed to bacterial stimuli. Overexpression studies using full-length and ectodomain-truncated receptors lead to ligand-independent activation of the immune response, probably due to increased receptor proximity in the membrane. It is therefore crucial to use an in vivo model with wild-type receptor levels to interpret correctly the mechanism of ligand-specific Imd activation downstream of various PGRP-LC isoforms. This study therefore used a genomic complementation approach to supplement PGRP-LC-deficient mutants with isoform-specific PGRP-LC loci elsewhere in the genome (Neyen, 2012).

This approach allowed generation of wild-type levels of defined PGRP-LC isoforms in vivo and to assess the tissue-specific roles of each isoform, alone or in various combinations. The results confirm previously described roles of PGRP-LCx and PGRP-LCa/x dimers in polymeric and monomeric PGN sensing, respectively, and uncover a new role for PGRP-LE in the activation of the Imd pathway in the gut. In addition, the in vivo model definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a case of rupture of tolerance induced by knockdown of amidase PGRP-LB (Neyen, 2012).

The initial aim of this study was to define the role of each PGRP-LC isoform in vivo. Using Phi31-mediated recombineering, loci of full and isoform-specific PGRP-LC constructs were successfully inserted into the fly genome, and they were proved capable of complementing PGRP-LC null mutations. The in vivo approach confirms and extends previous in vitro and RNAi experiments in proving that PGRP-LCx is indeed necessary and sufficient to respond to challenge with live or dead Gram-negative bacteria and to Gram-positive, DAP-type bacilli. Moreover, PGRP-LCx alone induces the in vivo immune response to polymeric PGN, whereas combined presence of PGRP-LCx and PGRP-LCa is necessary to sense the anhydro-monomer TCT. The differential requirement of PGRP-LC isoforms in response to Gram-positive DAP-type (PGRP-LCx alone) and Gram-negative bacteria (both PGRP-LCx and PGRP-LCa/x) indicates that flies are able to discriminate between the two types of DAP-type PGN-containing bacteria and to mount appropriate responses. Notably, injection of TCT in contrast to polymeric PGN leads to an increase in amplitude and duration of Imd pathway activation. Thus, TCT detection by PGRP-LCa/x allows flies to mount a strong response to Gram-negative bacteria despite the fact that DAP-type PGN is not exposed (masked by the LPS layer) and is less abundant compared with DAP-type PGN-containing Gram-positive bacteria (Neyen, 2012).

Consistent with previous reports that showed no effect of PGRPLCy RNAi on PGN sensing in cells, PGRP-LCy on its own did not show any induction of the Imd pathway. However, bacterial infection or injection of immunostimulatory compounds repeatedly produced a stronger response in flies carrying PGRPLCa/ x isoforms than in flies carrying the whole PGRP-LC locus. Although subtle differences in isoform expression from the intact, full locus compared with the engineered isoform loci cannot be excluded, this suggests that the full locus carries an additional regulatory element lacking in heterozygous PGRP-LCa/x flies. It is tempting to speculate that PGRP-LCy, present in the full locus but absent from PGRP-LCa/x flies, might help to regulate response levels. PGRP-LCy is structurally unlikely to bind PGN but, unlike PGRP-LF, retains a signaling-competent cytoplasmic tail. If any regulatory activity was associated with the PGRP-LCy isoform, it would therefore have to act extracellularly, possibly by competing with other isoforms for cell surface localization and thereby diluting receptor availability. Thus, the only function that can be attributed to PGRP-LCy from this study is a regulatory role in adjusting the amplitude of Imd pathway activation. The importance of wild-type receptor levels in any study of isoform function is crucial because overexpression of receptors is sufficient on its own to stimulate the Imd pathway. The PGRP-LC complemented system mimics wild-type receptor expression dynamics, and no elevated background levels of Imd activation was detected in complemented PGRP-LC mutant flie. However, alterations in the genomic ratio of PGRP-LC to PGRP-LF, achieved by combining [LC] or [LC,LF] vector-carrying lines with wild-type or different PGRP-LC-deficient backgrounds, showed a significant correlation between Dpt levels and PGRP-LC/LF ratios in infected flies, consistent with an inhibitory role of PGRP-LF. This indicates that the stoichiometry of activating and regulating receptors matters, as foreshadowed by affinity studies between signaling-competent PGRP-LCx-TCT-PGRP-LCa and signaling-deficient PGRPLCx-TCT-PGRP-LF complexes (Neyen, 2012).

Several overexpression studies in S2 cells already localized PGRP-LC to the plasma membrane. This study extend this finding to wild-type receptor expression levels in an immunocompetent tissue and provides evidence that PGRP-LC localizes to the apical and lateral plasma membrane in fat body cells, revealing a previously undescribed polarity in this immune-responsive tissue. Similar to a previous study that found no significant difference between Diptericin expression in PGRP-LC versus PGRP-LE;; PGRP-LC mutants after stimulation with B. subtilis and Escherichia coli, no additional decrease was seen in survival rates to Erwinia carotovora carotovora 15 when comparing single PGRP-LC and double PGRP-LE;;LC mutants, and no significant underlying reduction in Diptericin levels. This underlines the major role of PGRP-LC to survey a defined compartment -- the insect hemolymph -- and to preferentially activate immune responses in the fat body. This study confirmed a role of PGRP-LE in the systemic immune response to TCT, albeit depending on the route of administration. On one hand, we note a predominant role of PGRP-LCa/x over PGRP-LE in sensing injected TCT in the hemolymph. In this context, the contribution of PGRP-LE was discernible in the presence of any PGRP-LC isoform but was less marked in the absence of the full locus, consistent with the concept that hemolymph PGRP-LE cannot signal directly but depends on membrane-bound PGRP-LC to relay information. However, even though secreted PGRP-LE might contribute to Imd activation by delivering hemolymph TCT/ PGN to membrane-bound PGRP-LC, the effect of complete PGRP-LE loss on systemic immune activation after injection of TCT into the hemocele was not significant. This suggests that the cytosolic, autonomous PGRP-LE form does not contribute significantly to the activation of the Imd pathway by injected TCT and establishes PGRP-LC as the predominant receptor eliciting systemic responses in the hemocele (Neyen, 2012).

In contrast, when Imd activation in the fat body was triggered by oral ingestion of TCT in the PGRP-LB mutant background, a non-negligible contribution of PGRP-LE was observed to this systemic response in the absence of PGRP-LC. This indicates that when TCT reached the hemocele by active or passive transport from the intestine, the role of cytosolic PGRP-LE became more prominent. Although these is no explanation for this discrepancy, one might speculate that even though cytosolic PGRP-LE does not significantly contribute to TCT sensing when injected into the hemolymph, possibly because the fat body lacks transporters present in absorptive organs, this intracellular mode of recognition gains in importance when TCT transits through cells. Taken together, the subordinate role of secreted PGRP-LE compared with PGRP-LC might suggest that the main contribution of PGRP-LE is as an intracellular sensor, which will only spring into action when systemic levels of TCT have reached a critical threshold and permeated the cytosol (Neyen, 2012).

Determining the mechanisms by which barrier epithelia sense bacteria and differentiate between acceptable and non-acceptable intruders is a major issue in the field of innate immunity. Previous studies proposed PRR compartmentalization as an essential mechanism to discriminate between pathogenic versus beneficial bacterial colonization. Although this study observed a clear role of PGRP-LC sensing in the gut, consistent with previous studies, it is not possible to conclude whether this reflects direct sampling of the gut lumen by PGRP-LC. Unfortunately, the expression of the PGRPLC- GFP fusion construct was not strong enough to determine whether PGRP-LC is expressed at the apical or the basal side of enterocytes. Of note, recognition PGRPs involved in the sensing of Gram-negative bacteria show differential expression patterns along the gut, with enrichment of PGRP-LE in the endodermally derived midgut and a modest enrichment of PGRP-LC in ectodermally derived foregut and hindgut. Moreover, PGRPs in these sections are more or less accessible to gut contents. A relatively impermeable cuticle protects ectodermal epithelia in the foregut and hindgut, whereas the peritrophic matrix covering the PGRP-LE- rich section of the midgut is permeable to allow passage of digested nutrients. It is therefore more likely for bacterial compounds to reach midgut epithelia, and a reduction in surface receptors capable of mounting potentially detrimental immune responses to commensals in this compartment would make sense. Cytosolic receptors expressed in this compartment would be able specifically to detect absorbed or diffusible bacterial compounds such as TCT, which may be a hallmark of proliferation and/or harmful bacteria. Consistent with this, a major contribution of PGRP-LE (most probably of the cytosolic form as PGRP-LE signaling did not depend on PGRP-LC) and less of PGRP-LC when the midgut-specific response to Gram-negative bacteria was assessed. More strikingly, the midgut response to ingested TCT relied mostly on PGRP-LE, supporting a role of this receptor in danger detection in the gut. Thus, this study uncovers a key role of PGRP-LE in the Drosophila midgut and suggests that intracellular sensing of TCT is used in Drosophila as a mechanism to recognize infectious bacteria (Neyen, 2012).

Previously a model was put forward whereby long-range activation of the systemic immune response in Drosophila is mediated by the translocation of small PGN fragments from the gut lumen or other barrier epithelia to the hemolymph. This view was supported by the observation that ingestion of monomeric PGN can stimulate a strong systemic immune response in PGRP-LB knockdown flies with reduced amidase activity and that deposition of PGN or TCT on the genitalia is sufficient to induce a systemic immune response. Moreover, because TCT consistently elicited stronger responses than PGN, these models proposed an involvement of active or passive transport of the elicitor to the hemocele. On the basis of the current results, the mechanism of TCT delivery to the hemocele is still uncertain. However, the unique and well-characterized interaction of TCT-PGRP-LCa-PGRPLCx (Chang, 2006) and the primordial role of PGRP-LCa/x heterodimers in mediating TCT-specific systemic activation of the Imd pathway demonstrates that TCT is indeed a crucial element in the long-range activation of the immune response (Neyen, 2012).

In conclusion, this study shows that a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila. It is probable that the absence of LPS sensing in Drosophila has imposed some constraints on the system and that sensing of TCT through PGRP-LCa/x and PGRP-LE evolved as a surrogate way to distinguish Gram-negative bacteria from Gram-positive DAP-type PGN-containing bacteria. Because TCT is released during bacterial division, intracellular sensing through PGRP-LE provides an adequate mechanism of detection in the gut, reminiscent of the intracellular sensing of Gram-negative muropeptides by intracellular NOD1 in epithelia. To date, the existence of a mode of recognition of lysine-type bacteria in the midgut remains unexplored. A simple explanation could be that lysine-type bacteria do not represent a threat for flies as they rarely infect via the oral route and are therefore not detected. Indeed, DAP-type PGN-containing bacteria of either Gram-negative type (Serratia, Pseudomonas) or bacillus-type (Bacillus thuringiensis) are the only characterized naturally occurring insect pathogens to date (Neyen, 2012).

Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis

How multicellular organisms maintain immune homeostasis across various organs and cell types is an outstanding question in immune biology and cell signaling. In Drosophila, blood cells (hemocytes) respond to local and systemic cues to mount an immune response. While endosomal regulation of Drosophila hematopoiesis is reported, the role of endosomal proteins in cellular and humoral immunity is not well-studied. This study demonstrated a functional role for endosomal proteins in immune homeostasis. The ubiquitous trafficking protein ADP Ribosylation Factor 1 (ARF1) and the hemocyte-specific endosomal regulator Asrij differentially regulate humoral immunity. Asrij and ARF1 play an important role in regulating the cellular immune response by controlling the crystal cell melanization and phenoloxidase activity. ARF1 and Asrij mutants show reduced survival and lifespan upon infection, indicating perturbed immune homeostasis. The ARF1-Asrij axis suppresses the Toll pathway anti-microbial peptides (AMPs) by regulating ubiquitination of the inhibitor Cactus. The Imd pathway is inversely regulated- while ARF1 suppresses AMPs, Asrij is essential for AMP production. Several immune mutants have reduced Asrij expression, suggesting that Asrij co-ordinates with these pathways to regulate the immune response. This study highlights the role of endosomal proteins in modulating the immune response by maintaining the balance of AMP production. Similar mechanisms can now be tested in mammalian hematopoiesis and immunity (Khadilkar, 2017).

A balanced cellular and humoral immune response is essential to achieve and maintain immune homeostasis. In Drosophila, aberrant hematopoiesis and impaired hemocyte function can both affect the ability to fight infection and maintain immune homeostasis. Endosomal proteins are known to regulate Drosophila hematopoiesis. This study shows an essential function for endosomal proteins in regulating immunity (Khadilkar, 2017).

Altered hemocyte number and distribution as a result of defective hematopoiesis, can also lead to immune phenotypes like increased melanization or phagocytosis. This study shows that perturbation of normal levels of endocytic molecules ARF1 or Asrij leads to aberrant hematopoiesis, affecting the circulating hemocyte number. This in turn leads to an impaired cellular immune response. The aberrant hematopoietic phenotypes with pan-hemocyte tissue-specific depletion of ARF1 using e33cGal4 or HmlGal4 are comparable to the phenotypes observed in the case of asrij null mutant. Hence this study has compared Gal4-mediated ARF1 knockdown to asrij null mutant (Khadilkar, 2017).

In addition, it was also shown that ARF1 and Asrij have a direct role in humoral immunity by regulating AMP gene expression. This is likely to be a contribution from the hemocyte compartment which is primarily affected upon perturbation of Asrij or ARF1. It is well established that hemocytes, apart from acting as the cellular arm of the immune response, also act as sentinels and relay signals to the immune organs that mount the humoral immune response. Hemocytes have been shown to produce ligands like Spaetzle and upd3 that activate immune pathways and induce anti-microbial peptide secretion from the fat body or gut. Asrij or ARF1 could also be affecting the production of such ligand molecules thereby affecting the target immune-activation pathways (Khadilkar, 2017).

Considering the involvement of Asrij and ARF1 in both the arms of immune response, a model is proposed for the role of the ARF1-Asrij axis in maintaining immune homeostasis that can be used for testing additional players in the process (Khadilkar, 2017).

It is known that ARF1 is involved in clathrin coat assembly and endocytosis and has a critical role in membrane bending and scission. In this context it is also intriguing to note that ARF1, like Asrij, does not seem to have an essential role in phagocytosis. This suggests that hemocytes could be involved in additional mechanisms beyond phagocytosis in order to combat an infection (Khadilkar, 2017).

Both ARF1 and Asrij control hemocyte proliferation as their individual depletion leads to an increase in the total and differential hemocyte counts. Also, both mutants have higher crystal cell numbers due to over-activation of Notch as a result of endocytic entrapment. This suggests that increased melanization accompanied by increase in phenoloxidase activity upon ARF1 or Asrij depletion is a consequence of aberrant hematopoiesis and not likely due to a cellular requirement in regulating the melanization response. Constitutive activation of the Toll pathway or impaired Jak/Stat or Imd pathway signaling in various mutants also leads to the formation of melanotic masses. Thus the phenotypes seen on Asrij or ARF1 depletion could either be due to the defective hematopoiesis which directly affects the cellular immune response or leads to a mis-regulation of the immune regulatory pathways (Khadilkar, 2017).

Regulation of many signaling pathways, including the immune regulatory pathways takes place at the endosomes. For example, endocytic proteins Mop and Hrs co-localize with the Toll receptor at endosomes and function upstream of MyD88 and Pelle, thus indicating that Toll signalling is regulated by endocytosis. This study shows that loss of function of the ARF1-Asrij axis leads to an upregulation of some AMP targets of the Toll pathway. Upon depletion of ARF1-Asrij endosomal axis, increased ubiquitination of Cactus, a negative regulator of the Toll pathway, was found in both hemocytes and fat bodies. This suggests non-autonomous regulation of signals by the ARF1-Asrij axis, which is in agreement with an earlier model of signalling through this route. Thus the endosomal axis may systemically control the sorting and thereby degradation of Cactus, which in turn promotes the nuclear translocation of Toll effector, Dorsal. This could explain the significant increase in Toll pathway reporter expression such as Drosomycin-GFP. Interestingly the effect of ARF1 depletion on the Toll pathway is more pronounced than that of Asrij depletion. This is not surprising as ARF1 is a ubiquitous and essential trafficking molecule that regulates a variety of signals. This suggests that ARF1 is likely to be involved with additional steps of the Toll pathway and may also interact with multiple regulators of AMP expression (Khadilkar, 2017).

ARF1 and Asrij show complementary effects on IMD pathway target AMPs. While ARF1 suppresses the production of IMD pathway AMPs, Asrij has a discriminatory role. Asrij seems to promote transcription of AttacinA and Drosocin, whereas it represses Cecropin. However in terms of AMP production only Drosocin and Diptericin are affected, but not to the extent of ARF1. In addition, Relish shows marked nuclear localization in fat body cells of hemocyte-specific arf1 knockdown larvae whereas there is no significant difference in the localization in Asrij depleted larval fat bodies. This indicates that ARF1-Asrij axis exerts differential control over the Imd pathway. Thus ARF1 causes strong generic suppression of the Imd pathway while the role of Asrij could be to fine tune this effect. Mass spectrometric analysis of purified protein complexes indicates that ARF1 and Imd interact. Hence it is very likely that ARF1 regulates Imd pathway activation at the endosomes. Whether this interaction involves Asrij or not remains to be tested and will give insight into modes of differential activation of immune pathways (Khadilkar, 2017).

This analysis shows that Asrij is the tuner for endosomal regulation of the humoral immune response by ARF1 and provides specialized tissue- specific and finer control over AMP regulation. This is in agreement with earlier data showing that Asrij acts downstream of ARF127. Since ARF1 is expressed in the fat body it could communicate with the hemocyte- specific molecule, Asrij, to mediate immune cross talk (Khadilkar, 2017).

As reduced Asrij expression is seen in Toll and Jak/Stat pathway mutants such as Rel E20 and Hop Tum1, it is likely that these effectors also regulate Asrij, setting up a feedback mechanism to modulate the immune response. Earlier work has shown that ARF1-Asrij axis modulates different signalling outputs like Notch by endosomal regulation of NICD (Notch Intracellular Domain) transport and activity and JAK/STAT by endosomal activation of Stat92e. Further, ARF1 along with Asrij regulates Pvr signaling in order to maintain HSC's. ARF1 acts downstream of Pvr. Surprisingly, Asrij levels are downregulated in the Pvr mutant. Hence it is likely that the ARF1-Asrij axis regulates trafficking of the Pvr receptor, which then also regulates Asrij levels thus providing feedback regulation. While active modulation of signal activity and outcome at endosomes could be orchestrated by ARF1 and Asrij, their activities in turn need to be modulated. The data suggest that targets of Asrij endosomal regulation may in turn regulate Asrij expression at the transcript level. Further, upon Gram positive infection in wild type flies, asrij transcript levels decrease with a concomitant increase in suppressed AMPs such as Cecropin. This indicates additional regulatory loops such as that mediated by the IMD pathway effector NFκB may regulate asrij transcription. Using bioinformatics tools, presence of binding sites for NFκβ and Rel family of transcription factors are seen in the upstream regulatory sequence (1kb upstream) of asrij and arf1. Hence, feedback regulation is proposed of Asrij and ARF1 by the effectors of the Toll and Imd pathway respectively. This is reflected in the regulation of Asrij expression by these pathways. This also implies multiple modes of regulation of asrij and arf1, which are likely important in its role as a tuner of the generic immune response, thereby allowing it to discriminate between AMPs that were thought to be uniformly regulated, such as those downstream of IMD. Thus this analysis gives insight into additional complex regulation of the Drosophila immune response that can now be investigated further (Khadilkar, 2017).

Asrij and ARF1 being endocytic proteins are likely to interact with a number of molecules that regulate different cell signalling cascades. Due to endosomal localization, molecular interactions may be favored that further translate into signalling output. Hence, it is not surprising that Asrij and ARF1 genetically interact with multiple signalling pathways and can aid crosstalk to regulate important developmental and physiological processes like hematopoiesis or immune response. It is quite likely that Asrij and ARF1 are themselves also part of different feedback loops or feed-forward mechanisms as their levels need to be tightly regulated. Evidence for this is found with respect to the Toll, JAK/STAT and Pvr pathway as described earlier. Hence it is proposed that the Asrij-ARF1 endosomal signalling axis genetically interacts with various signalling components thereby regulating blood cell and immune homeostasis (Khadilkar, 2017).

AMP transcript level changes upon ARF1 or Asrij depletion also correspond to reporter-AMP levels seen after infection. This suggests that although ARF1 is known to have a role in secretion, mutants do not have an AMP secretion defect. Hence aberrant regulation of immune pathways on perturbation of the ARF1-Asrij axis is most likely due to perturbed endosomal regulation (Khadilkar, 2017).

ARF1 has a ubiquitous function in the endosomal machinery and is well-positioned to regulate the interface between metabolism, hematopoiesis and immunity in order to achieve homeostasis. Along with Asrij and other tissue-specific modulators, it can actively modulate the metabolic and immune status in Drosophila. In this context, it is interesting to note that Asrij is a target of MEF253, which is required for the immune-metabolic switch in vivo. Thus Asrij could bring tissue specificity to ARF1 action, for example, by modulating insulin signalling in the hematopoietic system (Khadilkar, 2017).

It is likely that in Asrij or ARF1 mutants, the differentiated hemocytes mount a cellular immune response and perish as in the case of wild type flies where immunosenescence sets in with age and the ability of hemocytes to combat infection declines. Since their hematopoietic stem cell pool is exhausted, they may fail to replenish the blood cell population, thus compromising the ability to combat infections. Alternatively, mechanisms that downregulate the inflammatory responses and prevent sustained activation may be inefficient when the trafficking machinery is perturbed. This could result in constitutive upregulation thus compromising immune homeostasis (Khadilkar, 2017).

In summary, this study shows that in addition to its requirement in hematopoiesis, the ARF1-Asrij axis can differentially regulate humoral immunity in Drosophila, most likely by virtue of its endosomal function. ARF1 and Asrij bring about differential endocytic modulation of immune pathways and their depletion leads to aberrant pathway activity and an immune imbalance. In humans, loss of function mutations in molecules involved in vesicular machinery like Amphyphysin I in which clathrin coated vesicle formation is affected leads to autoimmune disorders like Paraneoplastic stiff-person syndrome. Synaptotagmin, involved in vesicle docking and fusion to the plasma membrane acts as an antigenic protein and its mutation leads to an autoimmune disorder called Lambert-Eaton myasthenic syndrome. Mutations in endosomal molecules like Rab27A, β subunit of AP3, SNARE also lead to immune diseases like Griscelli and Hermansky-Pudlak syndrome. Mutants of both ARF1 and Asrij are likely to have drastic effects on the immune system. Asrij has been associated with inflammatory conditions such as arthritis, thyroiditis, endothelitis and tonsillitis, whereas the ARF family is associated with a wide variety of diseases. ARF1 has been shown to be involved in mast cell degranulation and IgE mediated anaphylaxis response. Generation and analysis of vertebrate models for these genes such as knockout and transgenic mice will provide tools to understand their function in human immunity (Khadilkar, 2017).

Effect of aminergic signaling on the humoral innate immunity response of Drosophila

Biogenic amines are crucial signaling molecules that modulate various physiological life functions both in vertebrates and invertebrates. In humans, these neurotransmitters influence the innate and adaptive immunity systems. This work analyzed whether the aminergic neurotransmission of dopamine, serotonin, and octopamine could have an impact on the humoral innate immune response of Drosophila melanogaster. This is a powerful model system widely used to uncover the insect innate immunity mechanisms which are also conserved in mammals. The neurotransmission of all these amines positively modulates the Toll-responsive antimicrobial peptide (AMP) drosomycin (drs) gene in adult flies infected with the Micrococcus luteus bacterium. Indeed, either blocking the neurotransmission in their specific aminergic neurons by expressing shibire (Shi(ts)) or silencing the vesicular monoamine transporter gene (dVMAT) by RNAi caused a significantly reduced expression of the Toll-responsive drs gene. However, upon M. luteus infection, the block of aminergic transmission did not alter the expression of AMP attacin genes responding to the immune deficiency (Imd) and Toll pathways. Overall, the results not only reveal a neuroimmune function for biogenic amines in humoral immunity but also further highlight the complexity of the network controlling AMP gene regulation (Cattabriga, 2023).

NF-κB immunity in the brain determines fly lifespan in healthy aging and age-related neurodegeneration

During aging, innate immunity progresses to a chronically active state. However, what distinguishes those that "age well" from those developing age-related neurological conditions is unclear. This study used Drosophila to explore the cost of immunity in the aging brain. Mutations in intracellular negative regulators of the IMD/NF-κB pathway were shown to predispose flies to toxic levels of antimicrobial peptides, resulting in early locomotor defects, extensive neurodegeneration, and reduced lifespan. These phenotypes are rescued when immunity is suppressed in glia. In healthy flies, suppressing immunity in glial cells results in increased adipokinetic hormonal signaling with high nutrient levels in later life and an extension of active lifespan. Thus, when levels of IMD/NF-κB deviate from normal, two mechanisms are at play: lower levels derepress an immune-endocrine axis, which mobilizes nutrients, leading to lifespan extension, whereas higher levels increase antimicrobial peptides, causing neurodegeneration. Immunity in the fly brain is therefore a key lifespan determinant (Kounatidis, 2017).

Sex-specific routes to immune senescence in Drosophila melanogaster

Animal immune systems change dramatically during the ageing process, often accompanied by major increases in pathogen susceptibility. However, the extent to which senescent elevations in infection mortality are causally driven by deteriorations in canonical systemic immune processes is unclear. This study examined Drosophila melanogaster and compared the relative contributions of impaired systemic immune defences and deteriorating barrier defences to increased pathogen susceptibility in aged flies. To assess senescent changes in systemic immune response efficacy, one and four-week old flies with the entomopathogenic fungus Beauveria bassiana and subsequent mortality was studied; whereas to include the role of barrier defences flies were injected by dusting the cuticle with fungal spores. The processes underlying pathogen defence senescence differ between males and females. Both sexes became more susceptible to infection as they aged. However, it is concluded that for males, this was principally due to deterioration in barrier defences, whereas for females systemic immune defence senescence was mainly responsible. The potential roles of sex-specific selection on the immune system and behavioural variation between males and females in driving these different senescent trends is discussed (Kubiak, 2017).

Antimicrobial peptides extend lifespan in Drosophila

Antimicrobial peptides (AMPs) are important defense molecules of the innate immune system. High levels of AMPs are induced in response to infections to fight pathogens, whereas moderate levels induced by metabolic stress are thought to shape commensal microbial communities at barrier tissues. Single AMPs were expressed in adult flies either ubiquitously or in the gut by using the inducible GeneSwitch system to tightly regulate AMP expression. Activation of single AMPs, including Drosocin, were found to result in a significant extension of Drosophila lifespan. These animals showed reduced activity of immune pathways over lifetime, less intestinal regenerative processes, reduced stress response and a delayed loss of gut barrier integrity. Furthermore, intestinal Drosocin induction protected the animals against infections with the natural Drosophila pathogen Pseudomonas entomophila, whereas a germ-reduced environment prevented the lifespan extending effect of Drosocin. This study provides new insights into the crosstalk of innate immunity, intestinal homeostasis and ageing (Loch, 2017).

Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress

This study used Drosophila to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca(2+) signaling pathway mediates innate immune responses to GBP, this study conducted a dsRNA library screen for genes that modulate Ca(2+) mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. The metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This was established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. It was further reported that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these homeostatic attributes of GBP: Elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan (Sung, 2017).

The most important development to emerge from this study is the deorphanization of Mthl10, through the placement of this GPCR at the epicenter of a molecular pathway that pits stress responses against lifespan. Various immunological and metabolic properties of a single cytokine, GBP, are integrated through its interactions with Mthl10. In particular, it was shown how the operation of the GBP/Mthl10 axis usefully matches nutrient supply to the degree of a metabolically expensive inflammatory response; this is an important topic in immunology. The model for GBP/Mthl10 functionality also shows how it has the potential to exacerbate metabolic inflammation; this may be one of the reasons that nutrient excess in Drosophila can model human metabolic syndrome. Furthermore, these homeostatic functions for Mthl10 were linked to its strong influence upon longevity. This provides a molecular foundation for a theory of aging, namely, that a shortened lifespan can be the ultimate price that a young organism pays to successfully combat short-term environmental stresses (Sung, 2017).

These findings were considered in relation to previous work that provides a detailed analysis of the expression pattern of Mthl10 in Drosophila embryos and larvae. For example, due to extensive expression of Mthl10 in imaginal discs, it has been proposed this gene may influence organogenesis. It is therefore relevant that cytokines-including the Mthl10 ligand, GBP-are well-known to regulate tissue remodeling and development. Additionally, the determination that Mthl10 regulates GBP-mediated innate immune responses seems pertinent to earlier observations that Mthl10 is expressed in hematopoietic tissue (which has immunological functions) and also crystal cells, which encapsulate foreign material. Nevertheless, the possibility cannot be excluded that other ligands for Mthl10 remain to be identified, perhaps as a consequence of the expression of alternate Mthl10 transcripts (Sung, 2017).

The significance of Mthl10 to longevity and metabolism is shared by Mth. In fact, it was the first gene duplication within the Mth superclade that is believed to have given rise to Mthl10, which did not then undergo any further expansion in Drosophila. In contrast, five further rounds of gene duplication apparently occurred before Mth emerged. Thus, it is concluded that the connection between lifespan and metabolic homeostasis that was observed for Mthl10 is an ancestral trait rather than adaptive specifically to Mth (Sung, 2017).

It is not unusual for gene regulatory networks to be widely conserved, even when certain components might undergo evolutionary turnover. Indeed, recent work has shown that although selection pressure has caused GPCR ectodomains and their ligands to codiversify, there has nevertheless been considerable conservation of the receptor's intracellular interactions with G proteins; as a result, flies and mammals share many of the same downstream signaling cascades. Indeed, GBP exhibits some sequence similarity with the human defensin BD2; both are small, cationic cytokines produced by protease action upon larger, precursor proteins. Furthermore, human BD2 acts through an uncharacterized GPCR to stimulate PLC/Ca2+ signaling to initiate inflammatory responses; the current study demonstrates that GBP is also a GPCR ligand that initiates PLC/Ca2+ signaling. Thus, it is proposed that there is general applicability to the concepts that emerge from our integration of immunological, metabolic, and lifespan functions for the GBP/Mthl10 axis (Sung, 2017).

Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library - Smarcd3/Bap60 is a mechanosensitive pro-inflammatory gene

Disturbed blood flow (d-flow) induces atherosclerosis by altering the expression of mechanosensitive genes in the arterial endothelium. Previous studies have identified >580 mechanosensitive genes in the mouse arterial endothelium, but their role in endothelial inflammation is incompletely understood. From this set, 84 Drosophila RNAi lines were obtained that silence the target gene under the control of upstream activation sequence (UAS) promoter. These lines were crossed with C564-GAL4 flies expressing GFP under the control of drosomycin promoter, an NF-κB target gene and a marker of pathogen-induced inflammation. Silencing of psmd12 or ERN1 decreased infection-induced drosomycin expression, while Bap60 silencing significantly increased the drosomycin expression. Interestingly, knockdown of Bap60 in adult flies using temperature-inducible Bap60 RNAi enhanced drosomycin expression upon Gram-positive bacterial challenge but the basal drosomycin expression remained unchanged compared to the control. In the mammalian system, smarcd3 (mammalian ortholog of Bap60) expression was reduced in the human- and mouse aortic endothelial cells exposed to oscillatory shear in vitro as well as in the d-flow regions of mouse arterial endothelium in vivo. Moreover, siRNA-mediated knockdown of smarcd3 induced endothelial inflammation. In summary, an in vivo Drosophila RNAi screening method identified flow-sensitive genes that regulate endothelial inflammation (Kumar, 2016).

Regulation of dual oxidase activity by the Galphaq-phospholipase Cbeta-Ca2+ pathway in Drosophila gut immunity

All metazoan guts are in constant contact with diverse food-borne microorganisms. The signaling mechanisms by which the host regulates gut-microbe interactions, however, are not yet clear. This study shows that phospholipase C-β (PLCβ) signaling modulates dual oxidase (DUOX) activity to produce microbicidal reactive oxygen species (ROS) essential for normal host survival. Gut-microbe contact rapidly activates PLCβ through Gαq, which in turn mobilizes intracellular Ca²⁺ through inositol 1,4,5-trisphosphate generation for DUOX-dependent ROS production. PLCβ mutant flies have a short life span due to the uncontrolled propagation of an essential nutritional microbe, Saccharomyces cerevisiae, in the gut. Gut-specific reintroduction of the PLCβ restores efficient DUOX-dependent microbe-eliminating capacity and normal host survival. These results demonstrate that the Gαq-PLCβ-Ca²⁺-DUOX-ROS signaling pathway acts as a bona fide first line of defense that enables gut epithelia to dynamically control yeast during the Drosophila life cycle (Ha, 2009).

All organisms are in constant contact with a large number of different types of microbes. This is especially true in the case of the gut epithelia, which control life-threatening pathogens as well as food-borne microbes. In addition to this microbe-eliminating capacity, gut epithelia also need to protect normal commensal microbes which are in a mutually beneficial relationship. Therefore, gut epithelia must be equipped to differentially operate innate immunity in order to efficiently eliminate life-threatening microbes while protecting beneficial microbes. Studies using Drosophila as a genetic model have greatly enhanced understanding of the microbe-controlling mucosal immune strategy in gut epithelia. Previous studies in a gut infection model using oral ingestion of pathogens revealed that the redox system has an essential role in host survival by generating microbicidal effectors such as reactive oxygen species (ROS) (Ha, 2005a; Ha, 2005b). In this redox system, dual oxidase (DUOX), a member of the nicotinamide adenine dinucleotide phosphate (NADP)H oxidase family, is responsible for the production of ROS in response to gut infection (Ha, 2005a). Following microbe-induced ROS generation, ROS elimination is assured by immune-regulated catalase (IRC), thereby protecting the host from excessive oxidative stress (Ha, 2005b). In addition to the redox system, the mucosal immune deficiency (IMD)/NF-κB signaling pathway, which leads to the de novo synthesis of microbicidal effector molecules such as antimicrobial peptides (AMPs), has an essential complementary role to the redox system when the host encounters ROS-resistant pathogenic microbes. These findings indicate that the different spectra of microbicidal activity encompassed by ROS and AMPs may provide the versatility necessary for Drosophila gut immunity to control microbial infections. Furthermore, in the absence of gut infection, a selective repression of IMD/NF-κB-dependent AMPs is mediated by the homeobox gene Caudal, which is required for protection of the resident commensal community and host health. Therefore, fine-tuning of different gut immune systems appears to be essential for both the elimination of pathogens and the preservation of commensal flora (Ha, 2009).

Most studies evaluating gut immunity have been performed in an oral infection model in which the pathogens are ingested. However, the gut epithelia constitute the interface between the host and the microbial environment; therefore, it is likely that animals in nature have already been subjected to continuous microbial contact, even in the absence of oral infection. Thus, it is essential to determine the mechanism by which this natural and continuous microbial interaction produces ROS at a tightly controlled, yet adequate level that allows for healthy gut-microbe interactions and gut homeostasis, because deregulated generation of ROS is believed to lead to a pathophysiologic condition in the gut epithelia. Although the DUOX system is of central importance in gut immunity, the signaling pathway(s) by which gut epithelia regulate DUOX-dependent microbicidal ROS generation are poorly understood (Ha, 2009).

Drosophila feed on microbes, and one of their most essential microbial food sources is baker's yeast, Saccharomyces cerevisiae. As early as 1930, yeast was discovered to be an essential nutrient source for Drosophila and is now used as a major ingredient in standard laboratory Drosophila food recipes. Further, Drosophila-Saccharomyces interaction occurs in wild-captured Drosophila, which suggests that this interaction is an evolutionarily ancient natural phenomenon. Although many studies have investigated the effect of yeast on Drosophila metabolism and aging, very few works have been reported on the effect of yeast in terms of the host immunity. Specifically, it has previously been shown that dietary yeast contributes to the cellular immune responsiveness of Drosophila against a larval parasitoid, Leptopilina boulardi. However, the relationship between yeast and Drosophila gut immunity during the normal life cycle has never been closely examined. Therefore, in this study, a Drosophila-yeast model was used to investigate the intracellular signaling pathway by which the host mounts mucosal antimicrobial immunity, as well as the in vivo value of this pathway in the host's natural life. Through biochemical and genetic analyses, this study revealed that the Gαq-mediated phospholipase C-β (PLCβ) pathway is involved in the routine control of dietary yeast in the Drosophila gut. PLCβ is dynamically activated in the presence of ingested yeast and subsequently mobilizes the intracellular Ca²⁺ to produce ROS in a DUOX-dependent manner. The presence of all of these signaling components of the Gαq-PLCβ-Ca²⁺-DUOX-ROS pathway in the gut is essential to ensure routine control of dietary yeast and host fitness, highlighting the importance of this immune signaling as a bona fide first line of defense in Drosophila (Ha, 2009).

This study demonstrates that the Gαq-PLCβ-Ca²⁺ signaling pathway controls the mucosal gut epithelial defense system through DUOX-dependent ROS generation, which is responsible for routine microbial interactions in the gut epithelia in the absence of infection. The PLCβ pathway impacts a wide variety of biological processes through the generation of a lipid-derived second messenger. In this process, the hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, by PLCβ generates two intracellular messengers, IP₃ and diacylglycerol. This process is one of the earliest events through which more than 100 extracellular signaling molecules regulate functions in their target cells. It has been shown that Gαq-PLCβ signaling is essential for the activation of the phototransduction cascade in Drosophila. This study revealed a physiological role of PLCβ wherein it is involved in the regulation of DUOX enzymatic activity, which leads to the generation of microbicidal ROS in the mucosal epithelia (Ha, 2009).

PLCβ signaling is very rapid, with only a few seconds necessary to activate Ca²⁺ release and ROS production. This rapid response may be advantageous for the host and may be the mechanism by which dynamic and routine control of microbes in the gut epithelia is achieved. Because the gut is in continuous contact with microbes such as dietary microorganisms, it is conceivable that under normal conditions routine microbial contact dynamically induces a certain level of basal Gαq-PLCβ activity that varies depending on the local microbe concentration. This basal Gαq-PLCβ-DUOX activity seems to be sufficient for host survival. In such conditions of low bacterial burden, NF-κB-dependent AMP expression is known to be largely repressed by Caudal repressor for the preservation of commensal microbiota (Ryu, 2008). However, in the case of high bacterial burden (e.g., gut infection condition), the DUOX-ROS system would be strongly activated for full microbicidal activity. Furthermore, all of the flies that contained impaired signaling potentials for the Gαq-PLCβ-Ca²⁺-DUOX pathway were totally intact following septic injury but short-lived under natural rearing conditions or under gut infection conditions, indicating that the mucosal immune pathway is distinct from the systemic immune pathway (Ha, 2009).

It is not clear how Gαq- and PLCβ-induced Ca²⁺ modulates DUOX enzymatic activity. Because the DUOX lacking Ca²⁺-binding EF hand domains is unable to rescue the DUOX-RNAi flies (Ha, 2005a), it is plausible that Ca²⁺ directly modulates the enzymatic activity of DUOX through binding to the EF hand domains (Ha, 2009).

It is also important to determine what pathogen-associated molecular patterns (PAMPs) are responsible for the activation of PLCβ signaling. In Drosophila, peptidoglycan and β-1,3-glucan are the only two PAMPs known to induce the NF-κB signaling pathway in the systemic immunity. The results showed that neither peptidoglycan nor β-1,3-glucan was able to induce ROS in S2 cells, which suggests that a previously uncharacterized type(s) of PAMP is involved in the mucosal immunity. Because the Gαq protein acts as an upstream signaling component of the PLCβ-Ca²⁺ pathway, a microbe-derived ligand capable of activating G protein coupled receptor(s) and/or Gαq protein may be the best candidate for the Gαq-PLCβ-Ca²⁺-DUOX signaling pathway. Given the broad spectrum of microbes that activate the response, it remains possible that the unknown upstream sensors resemble a stress response more than a PAMP response. Elucidation of the molecular nature of such agonists will greatly enhance understanding of bacteria-modulated redox signaling in the gut epithelia. In conclusion, this study demonstrates that mucosal epithelia have evolved an innate immune strategy, which is functionally distinct from the NF-κB-dependent systemic innate immune system. The rapid Gαq-PLCβ-Ca²⁺-DUOX signaling is adapted to the routine and dynamic control of gut-associated microbes and may impact the long-term physiology of the intestine and host fitness (Ha, 2009).

Interaction between familial transmission and a constitutively active immune system shapes gut microbiota in Drosophila melanogaster

Resident gut bacteria are constantly influencing the immune system. Yet the role of the immune system in shaping microbiota composition during an organism's lifespan has remained unclear. This study used Drosophila as a genetically tractable system with a simple gut bacterial population structure and streamlined genetic backgrounds to address this issue. Depending on their genetic background, young flies had microbiota of different diversities that converged with age to the same Acetobacteraceae-dominated pattern in healthy flies. This pattern was accelerated in immune-compromised flies with higher bacterial load and gut cell death. Nevertheless, immune compromised flies resembled their genetic background, indicating that familial transmission was the main force regulating gut microbiota. In contrast, flies with a constitutively active immune system had microbiota readily distinguishable from their genetic background with the introduction and establishment of previously undetectable bacterial families. This indicated the influence of immunity over familial transmission. Moreover, hyper active immunity and increased enterocyte death resulted in the highest bacterial load observed starting from early adulthood. Cohousing experiments showed that the microenvironment also played an important role in the structure of the microbiota where flies with constitutive immunity defined the gut microbiota of their co-habitants. These data show that in Drosophila, constitutively active immunity shapes the structure and density of gut microbiota (Mistry, 2017).

Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila

Oxidative stress induced by high levels of reactive oxygen species (ROS) is associated with the development of different pathological conditions, including cancers and autoimmune diseases. This study analysed whether oxidatively challenged tissue can have systemic effects on the development of cellular immune responses using Drosophila as a model system. Indeed, the haematopoietic niche that normally maintains blood progenitors can sense oxidative stress and regulate the cellular immune response. Pathogen infection induces ROS in the niche cells, resulting in the secretion of an epidermal growth factor-like cytokine signal that leads to the differentiation of specialized cells involved in innate immune responses (Sinenko, 2011).

Abnormal metabolism is often associated with oxidative stress that results in increased production of ROS by mitochondria. Different concentrations of ROS and their derivatives are required for proper maintenance, proliferation, differentiation and apoptosis of stem cells and their committed progenitors. In Drosophila, developmentally regulated levels of ROS are critical for maintenance of haematopoietic progenitors within the medullary zone (MZ) of the lymph gland. In contrast, under normal growth conditions, posterior signaling center (PSC) cells in wild-type larvae had very low levels of ROS expression compared with that in the progenitor population of cells within the MZ. To induce oxidative stress in the PSC ND75, a component of complex I of the electron transport chain (ETC), was inactivated with double-stranded RNA (dsRNA) using the Gal4/UAS misexpression system and the PSC-specific Antp-Gal4 driver. ND75 inactivation causes a readily detectable increase in ROS in the PSC cells, rising to levels similar to those seen in the progenitor cells of the MZ. The phenotypic consequence of inducing oxidative stress in the cells of the PSC was a remarkably robust increase in numbers of circulating lamellocytes. Such an elevated number of lamellocytes was usually observed in wild-type larvae only if they were infested by parasitic wasps. Although Antp-Gal4 is not expressed anywhere in the blood system, except the PSC, this driver is also expressed in other larval tissues. To exclude the possibility that the effect was due to a non-PSC expression of Antp-Gal4, the function of ND75 was also eliminated using the Dot-Gal4 driver normally expressed at high levels in the PSC, and this resulted in an identical lamellocyte response. In contrast, oxidative challenge to various other larval tissues, including the fat body (LSP2-GaI4), the epidermis (A58-GaI4), the neurons (C127-GaI4), the dorsal vessel (Hand-GaI4), the ring gland (5015-GaI4), the wing imaginal disc (ap-Gal4) or the trachea (btl-GaI4), did not have a significant effect on lamellocyte differentiation. Furthermore, high ROS levels generated within the progenitor cells (dome-GaI4) of the lymph gland, which causes autonomous differentiation of this population, also did not have any significant effect on the non-autonomous differentiation of lamellocytes in the circulation. In contrast, oxidative challenge of the PSC caused non-autonomous lamellocyte response in circulation as well as within the lymph gland. The PSC-mediated effect was due to mitochondrial dysfunction and not specifically linked to the product of the ND75 gene, because attenuation of PDSW (another complex I component), cytochrome-c oxidase, subunit Va (CoVa, a component of ETC complex IV) or Marf (mitochondrial assembly regulatory factor) function in the PSC, all induced increases in lamellocyte differentiation. The strength of the lamellocyte response to complex I inactivation depended on the strength of the dsRNA construct used in the experiment. Temporally, induction of the mutation in the second-larval instar caused the lamellocyte response to be seen in the third instar. This correlates well with the timescale of response to parasitic wasp infection. Finally, this oxidative stress elicited a cell-specific response; for example, no significant effect was seen on the differentiation of crystal cells and plasmatocytes in circulation. These results establish that the oxidative status of the PSC has a specific and non-autonomous role in lamellocyte differentiation as an immune response to parasitic invasion (Sinenko, 2011).

The status of the PSC cells on oxidative stress conditions was further analysed in some detail. ND75 dysfunction does not affect proliferation or maintenance of the PSC, because the number of PSC cells, which maintain expression of Antp, remains intact in this mutant background. In addition, no apoptosis is detected in ND75-deficient PSC cells, and also, apoptosis in the PSC alone, specifically induced by overexpression of Hid/Rpr, has no effect on lamellocyte differentiation (Sinenko, 2011).

Overexpression of superoxide dismutase-2 (SOD2) as a scavenger for ROS in ND75-deficient PSC is able to suppress the lamellocyte response significantly. Furthermore, activation of the Forkhead box O (FoxO) transcription factor that positively regulates expression of antioxidant enzymes, including SOD2, completely suppresses the dsND75-induced lamellocyte response. Inactivation of the Akt1 protein kinase in PSC also results in a near-complete suppression of the dsND75-induced lamellocyte response, suggesting a role for the PI3K/Akt pathway in the regulation of FoxO. This is an important issue because FoxO activity can also be controlled by the Jun N-terminal kinase (JNK) pathway, but in the PSC the AKT pathway mediates this effect. The JNK reporter (puc69-lacZ) is not expressed in the PSC, and inactivation of JNK (encoded by the basket gene) using the dominant-negative form (bsk^DN) does not suppress dsND75-induced lamellocyte response. The FoxO reporter (4E-BP-lacZ) is robustly activated in the ND75-deficient PSC; however, loss of translational inhibition mediated by 4E-BP does not mimic this effect. It is important to point out that under wild-type non-stressed conditions, the PSC has relatively low levels of ROS, and therefore inactivation of either Foxo or SOD2 has no phenotypic consequence. These data are interpreted to indicate that metabolic dysfunction induces an oxidatively stressed PSC that causes the activation of this pathway and the lamellocyte response (Sinenko, 2011).

Differentiation of lamellocytes has been associated with the JAK/STAT, JNK and Ras/Erk signalling pathways. These pathways were genetically altered in an ND75-deficient PSC background to identify which, if any, is involved in the lamellocyte response. Inactivation of the unpaired ligands (upd3, upd2 or upd) that activate the JAK/STAT pathway or of eiger (egr), which activates JNK signalling, did not suppress the lamellocyte phenotype. This strongly suggests that these pathways are not involved in the process downstream of ROS in the PSC and is consistent with previous studies showing that components of the JAK/STAT pathway (upd3, dome and Tep4) and JNK (puc69-lacZ reporter) are not involved in the functioning of the PSC. However, these pathways are likely to be involved in direct regulation of lamellocyte differentiation independently of the PSC function. In contrast, inactivation of spitz (spi), encoding the ligand for epidermal growth factor receptor (EGFR), in the context of ND75-deficient PSC significantly suppresses the lamellocyte response. Furthermore, overexpression of the secreted form of Spi (s.Spi), but not the alternative EGFR ligand, Vein (Vn) in the PSC, causes increased differentiation of circulating lamellocytes in an otherwise wild-type larva. EGFR mutant Egfr^TS/Egfr¹⁸ lymph glands develop normally, suggesting that EGFR signalling is not required for normal lymph gland development but rather is involved in the regulation of a cellular immune response as a signalling event from the PSC only when the latter is oxidatively stressed (Sinenko, 2011).

The PSC-dependent parasitic challenge induced by wasp egg infestation and the mechanism described above both give rise to the same cellular response. Therefore, whether parasitization causes oxidative stress to the PSC was examined. Immune challenge caused by wasp infestation was found to induce high levels of ROS in the PSC cells as seen 12 h after invasion. The most prominent effect is on superoxide radicals detected with dihydroethidium staining; a smaller but detectable elevation of peroxide radicals revealed by RedoxSensor staining is also apparent in PSC cells on this immune challenge. Scavenging these ROS types in the PSC by overexpressing SOD2 or catalase (Cat) but not glutathione peroxidase (GPx), which reduces thioredoxin-mediated effects, significantly suppresses the lamellocyte response caused by wasp infestation. These genetic results are consistent with a model in which parasitic infection by wasp eggs raises ROS levels in the PSC, which then causes lamellocyte induction by expressing Spitz. To test this model, spi within the PSC was inactivated in larvae infected by parasitic wasps. This caused a strong suppression of the lamellocyte response; the few remaining L1 marker-positive cells are immature, as indicated by their relatively small cell size and their morphology. In addition, melanotic capsules that are indicative of extensive cellular immune response to parasitic infection do not develop in a spi mutant background during wasp infestation. Inactivation of spitz in the PSC did not affect the increase in ROS triggered by wasp infeststion. Thus spi does not regulate the ROS levels in the PSC; rather, wasp infection raises ROS levels, which leads to release of the s.Spi. Previous studies have shown that s.Spi production requires the function of the trafficking protein Star (S), and the protease Rhomboid (Rho1). This study found that the wasp-induced lamellocyte response and melanotic capsule formation are robustly suppressed on the loss of a single copy of Star. More importantly, parasite-induced immune challenge specifically upregulates Rho1 in the PSC by an as yet unidentified mechanism. These data establish that S and Rho1 are canonically required for processing and releasing the Spitz from the PSC (Sinenko, 2011).

Secreted Spitz is known to bind to EGFR and activate the Ras/Erk pathway. A dominant-negative form of EGFR (Egfr^DN) strongly suppresses the lamellocyte response induced by wasp infestation when it is expressed in the lymph gland and the circulating haemocytes using the pan-haemocyte HHLT Gal4 driver. This phenotype is virtually identical to that seen when spi^RNAi is expressed in the PSC using Antp-Gal4. In addition, compartment-specific drivers were used, and inactivation of the receptor in the cortical zone of the lymph gland and in circulating haemocytes (using lineage-traced Hml^Δ-Gal4 line) was found to prevent Hml-positive cells from becoming lamellocytes on wasp infestation. Importantly, it was also found that a small subset of lamellocytes does not express Hml in the wild-type background and consequently Egfr^DN is not expressed in these cells when Hml^Δ-Gal4 is used as a driver. These Hml⁻,L1⁺ lamellocytes are easily detectable in this genetic background and act as an internal control. Expression of an activated form of EGFR (Egfr^Act) in Hml⁺ haemocytes causes a robust increase in lamellocyte differentiaion. This is also consistent with previous work, which showed that activated Ras induces an increase in the total number of haemocytes, including lamellocytes. Finally, both loss of ND75 in the PSC and wasp infestation cause robust activation of Erk as evident by an increase in dpErk staining in circulating haemocytes including lamellocytes. This indicates that lamellocytes in circulation differentiate from precursor cells on activation of Spi/EGFR/Erk signalling (Sinenko, 2011).

PSC cells have two independent functions: they serve as a haematopoietic niche in the lymph gland, where they orchestrate the maintenance and proper differentiation of haematopoietic progenitors, and they regulate the cellular immune response by controlling lamellocyte differentiation in response to infection. The results presented in this study establish the mechanism for this latter function. Changes in oxidative status, caused by events of parasite invasion or ETC dysfunction, initiates a signal within this immunocompetent compartment causing the secretion of a cytokine ligand, Spitz, that induces differentiation of lamellocyte precursors in the circulatory system of the larva. The identified mechanism is consistent with previously reported studies in mammals, which have shown that mitochondrial ROS can trigger systemic signals that reinforce the innate immune response. These studies raise the possibility that specific populations of cells also exist in mammalian systems that sense oxidative stress due to infection and non-autonomously signal myeloid progenitors to initiate differentiation and enhance the immune response. Whether such populations are to be found within the haematopoietic niche as in Drosophila remains a speculation that can be tested in future studies (Sinenko, 2011).

A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity

A conserved interaction between RB proteins and the Condensin II protein CAP-D3 is important for ensuring uniform chromatin condensation during mitotic prophase (Longworth, 2008). The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. This study shows that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. The data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes (Longworth, 2012).

Recent studies have suggested that pRB family members may impact the organization of higher-order chromatin structures, in addition to their local effects on the promoters of individual genes (Longworth, 2010). Mutation of pRB causes defects in pericentric heterochromatin and RBF1 is necessary for uniform chromatin condensation in proliferating tissues of Drosophila larvae (Longworth, 2008). Part of the explanation for these defects is that RBF1 and pRB promote the localization of the Condensin II complex protein, CAP-D3 to DNA both in Drosophila and human cells (Longworth, 2008). Depletion of pRB from human cells strongly reduces the level of CAP-D3 associated with centromeres during mitosis and causes centromere dysfunction (Longworth, 2012).

Condensin complexes are necessary for the stable and uniform condensation of chromatin in early mitosis. They are conserved from bacteria to humans with at least two types of Condensin complexes (Condensin I and II) present in higher eukaryotes. Both Condensin I and II complexes contain heterodimers of SMC4 and SMC2 proteins that form an ATPase which acts to constrain positive supercoils. Each type of Condensin also contains three specific non-SMC proteins that, upon phosphorylation, stabilize the complex and promote ATPase activity. The kleisin CAPH and two HEAT repeat containing subunits, CAP-G and CAP-D2 are components of Condensin I, while the kleisin CAP-H2 and two HEAT repeat containing subunits, CAP-G2 and CAP-D3, are constituents of Condensin II (Longworth, 2012).

Given the well-established functions of Condensins during mitosis, and of RBF1 in G1 regulation, the convergence of these two proteins was unexpected. Nevertheless, mutant alleles in the non-SMC components of Condensin II suppress RBF1-induced phenotypes, and immunostaining experiments revealed that RBF1 displays an extensive co-localization with dCAP-D3 (but not with dCAP-D2) on the polytene chromatin of Drosophila salivary glands (Longworth, 2008). This co-localization occurs in cells that will never divide, suggesting that Condensin II subunits and RBF1 co-operate in an unidentified process in non-mitotic cells. In various model organisms, the mutation of non-SMC Condensin subunits has been associated with changes in gene expression raising the possibility that dCAP-D3 may affect some aspect of transcriptional regulation by RBF1. However, the types of RBF1-regulated genes that might be affected by dCAP-D3, the contexts in which this regulation becomes important, and the consequences of losing this regulation are all unknown (Longworth, 2012).

This study identified sets of genes that are dependent on both rbf1 and dCap-D3. The majority of genes that show altered expression in both rbf1 and dCap-D3 mutants (larvae or adults) are not genes involved in the cell cycle, DNA repair, proliferation, but are genes with cell type-specific functions and many are spaced within 10 kb of one another in 'gene clusters'. To better understand this mode of regulation, the effects were investigated of RBF1 and dCAP-D3 on one of the most highly misregulated clusters which includes genes coding for antimicrobial peptides (AMPs). AMPs are produced in many organs, and one of the major sites of production is in the fat body. Following production in the fat body, AMPs are subsequently dumped into the hemolymph where they act to destroy pathogens. RBF1 and dCAP-D3 are required for the transcriptional activation of many AMPs in the adult fly. Analysis of one such gene cluster shows that RBF1 and dCAP-D3 bind directly to this region and that they bind, in the fat body, to sites flanking the locus. RBF1 and dCAP-D3 are both necessary in the fat body for maximal and sustained induction of AMPs following bacterial infection, and RBF1 and dCAP-D3 deficient flies have an impaired ability to respond efficiently to bacterial infection. These results identify dCAP-D3 as an important transcriptional regulator in the fly. Together, the findings suggest that RBF1 and dCAP-D3 regulate the expression of clusters of genes in post-mitotic cells, and this regulation has important consequences for the health of the organism (Longworth, 2012).

The idea that dCAP-D3 and RBF1 could cooperate to promote tissue development and differentiation is supported by the fact that both proteins are most highly expressed in the late stages of the fly life cycle, and accumulate at high levels in the nuclei of specific cell types in adult tissues. As an illustration of the cell-type specific nature of RBF1/dCAP-D3-regulation this study shows that dCAP-D3 and RBF1 are both required for the constituive expression of a large set of AMP genes in fat body cells. The loss of this regulation compromises pathogen-induction of gene expression and has functional consequences for innate immunity. Interestingly, different sets of RBF1/dCAP-D3-dependent genes were evident in the gene expression profiles of mutant larvae and adults. Given this, and the fact that the gene ontology classification revealed multiple groups of genes, it is suggested that the targets of RBF1/dCAP-D3-regulation do not represent a single transcriptional program, but diverse sets of cell-type specific programs that need to be activated (or repressed) in specific developmental contexts (Longworth, 2012).

The changes in gene expression seen in the mutant flies suggest that RBF1 has a significant impact on the expression of nearly half of the dCAP-D3-dependent genes. This fraction is consistent with previous data showing partial overlap between RBF1 and dCAP-D3 banding patterns on polytene chromatin, and the finding that chromatin-association by dCAP-D3 is reduced, but not eliminated, in rbf1 mutant animals and RBF1-depeleted cells. Although it has been previously shown that RBF1 and dCAP-D3 physically associate with one another (Longworth, 2008), and the current studies illustrate the fact that they each bind to similar sites at a direct target, the molecular events that mediate the co-operation between RBF1 and dCAP-D3 remain unknown (Longworth, 2012).

These results represent the first published ChIP data for the CAP-D3 protein in any organism. Although only a small number of targets were examined, it is interesting to note that the dCAP-D3 binding patterns are different for activated and repressed genes. More specifically, dCAP-D3 binds to an area within the open reading frame of a gene which it represses. However, dCAP-D3 binds to regions which flank a cluster of genes that it activates. Whether or not this difference in binding is true for all dCAP-D3 regulated genes will require a more global analysis (Longworth, 2012).

Human Condensin non-SMC subunits are capable of forming subcomplexes in vitro that are separate from the SMC protein- containing holocomplex, but currently, the extent to which dCAP-D3 relies on the other members of the Condensin II complex remains unclear. It is noted that fat body cells contain polytene chromatin. Condensin II subunits have been shown to play a role in the organization of polytene chromatin in Drosophila nurse cells. Given that RB proteins physically interact with other members of the Condensin II complex (Longworth, 2008), it is possible that RBF1 and the entire Condensin II complex, including dCAP-D3, may be especially important for the regulation of transcription on this type of chromatin template (Longworth, 2012).

A potentially significant insight is that the genes that are deregulated in both rbf1 and dCap-D3 mutants tend to be present in clusters located within 10 kb of one another. This clustering effect seems to be a more general feature of regulation by dCAP-D3, which is enhanced by RBF1, since clustering was far more prevalent in the list of dCAP-D3 target genes than in the list of RBF1 target genes (Longworth, 2012).

These studies focussed on one of the most functionally related families of clustered target genes that were co-dependent on RBF1/dCAP-D3 for activation in the adult fly: the AMP family of genes. AMP loci represent 20% of the gene clusters regulated by RBF1 and dCAP-D3 in adults. ChIP analysis of one such region, a cluster of AMP genes at the diptericin locus, showed this locus to be directly regulated by RBF1 and dCAP-D3 in the fat body and revealed a pattern of RBF1 and dCAP-D3-binding that was very different from the binding sites typically mapped at E2F targets. Unlike the promoter-proximal binding sites typically mapped at E2F-regulated promoters, RBF1 and dCAP-D3 bound to two distant regions, one upstream of the promoter and one downstream of the diptericin B translation termination codon, a pattern that is suggestive of an insulator function. It is hypothesized that RBF1 and dCAP-D3 act to keep the region surrounding AMP loci insulated from chromatin modifiers and accessible to transcription factors needed for basal levels of transcription. The modEncode database shows binding sites for multiple insulator proteins, as well as GATA factor binding sites, at these regions. GATA has been previously implicated in transcriptional regulation of AMPs in the fly, and future studies of dCAP-D3 binding partners in Drosophila fat body tissue may uncover other essential activators. Additionally, the chromatin regulating complex, Cohesin, which exhibits an almost identical structure to Condensin, has been shown to promote looping of chromatin and to bind proteins with insulator functions. Therefore, it remains a possibility that Condensin II, dCAP-D3 may actually possess insulator function, itself. It is proposed that dCAP-D3 may be functioning as an insulator protein, both insulating regions of DNA containing clusters of genes from the spread of histone marks and possibly looping these regions away from the rest of the body of chromatin. This would serve to keep the region in a 'poised state' available for transcription factor binding following exposure to stimuli that would induce activation. In the case of AMP genes, which are made constituitively in specific organs at low levels, dCAP-D3 would bind to regions flanking a cluster, and loop the cluster away from the body of chromatin. Upon systemic infection, these clusters would be more easily accessible to transcription factors like NF-κB. If dCAP-D3 is involved in looping of AMP clusters, then it may also regulate interchromosomal looping which could bring AMP clusters on different chromosomes closer together in 3D space, allowing for a faster and more coordinated activation of all AMPs (Longworth, 2012).

AMP expression is essential for the ability of the fly to recover from bacterial infection. Experiments with bacterial pathogens show that RBF1 and dCAP-D3 are both necessary for induction and maintenance of the AMP gene, drosomycin following infection, but only dCAP-D3 is necessary for the induction of the diptericin AMP gene. Similarly, survival curves indicate, that while dCAP-D3 deficient flies die more quickly in response to both Gram positive and Gram negative bacterial infection, RBF1 deficient flies die faster only in response to Gram positive bacterial infection. The differences seen between RBF1 and dCAP-D3 deficient flies in diptericin induction cannot be attributed to functional compensation by the other Drosophila RB protein family member, RBF2, since results show that loss of RBF2 or both RBF2 and RBF1 do not decrease AMP levels following infection. Since results demonstrate that RBF1 binds most strongly to an AMP cluster prior to infection and regulates basal levels of almost all AMPs tested, it is hypothesized that RBF1 (and possibly RBF2) may be more important for cooperating with dCAP-D3 to regulate basal levels of AMPs. Reports have shown that basal expression levels of various AMPs are regulated in a gene-, sex-, and tissue-specific manner, and it is thought that constitutive AMP expression may help to maintain a proper balance of microbial flora and/or help to prevent the onset of infections. In support of this idea, one study in Drosophila which characterized loss of function mutants for a gene called caspar, showed that caspar mutants increased constitutive transcript levels of diptericin but not transcript levels following infection. This correlated with increased resistance to septic infection with Gram negative bacteria, proving that changes in basal levels of AMPs do have significant effects on the survival of infected flies. Additionally, disruption of Caudal expression, a protein which suppresses NF-κB mediated AMP expression following exposure to commensal bacteria, causes severe defects in the mutualistic interaction between gut and commensal bacteria. It is therefore possible that RBF1 and dCAP-D3 may help to maintain the balance of microbial flora in specific organs of the adult fly and/or be involved in a surveillance-type mechanism to prevent the start of infection. RBF1 deficient flies also exhibit defects in Drosomycin induction following Gram positive bacterial infection. Mutation to Drosophila GNBP-1, an immune recognition protein required to activate the Toll pathway in response to infection with Gram positive bacteria has been show to result in decreased Drosomycin induction and decreased survival rates, without affecting expression of Diptericin. Therefore, it is possible that inefficient levels of Drosomycin, a major downstream effector of the Toll receptor pathway, combined with decreased basal transcription levels of a majority of the other AMPs, would cause RBF1 deficient flies to die faster following infection with Gram positive S. aureus but not Gram negative P. aeruginosa (Longworth, 2012).

Some dCAP-D3 remains localized to DNA in RBF1 deficient flies and it is also possible that other proteins may help to promote the localization of dCAP-D3 to AMP gene clusters following infection. Given that dCAP-D3 regulates many AMPs including some that do not also depend on RBF1 for activation, and given that dCAP-D3 binding to an AMP locus increases with time after infection whereas RBF1 binding is at its highest levels at the start of infection, it may not be too surprising that dCAP-D3 showed a more pronounced biological role in pathogen assays involving two different species of bacteria (Longworth, 2012).

Remarkably, and perhaps unexpectedly, the levels of both RBF1 and dCAP-D3 impact the basal levels of human AMP transcripts, as well. This indicates that the mechanism of RBF1/dCAP-D3 regulation may not be unique to Drosophila. It is striking that many of the human AMP genes (namely, the defensins) are clustered together in a region that spans approximately 1 Mb of DNA. It seems telling that both the clustering of these genes, and a dependence on pRB and CAP-D3, is apparently conserved from flies to humans. The fact that dCAP-D3 and RBF1 dependent activation of Drosomycin was necessary for resistance to Gram positive bacterial infection in flies suggests the same could also be true for the human orthologs in human cells. Human AMPs expressed by epithelial cells, phagocytes and neutrophils are an important component of the human innate immune system. Human AMPs are often downregulated by various microbial pathogenicity mechanisms upon infection. They have also been reported to play roles in the suppression of various diseases and maladies including cancer and Inflammatory Bowel Disease. It is noted that the chronic or acute loss of Rb expression from MEFs resulted in an unexplained decrease in the expression of a large number of genes that are involved in the innate immune system. In humans, the bacterium, Shigella flexneri was recently shown to down regulate the host innate immune response by specifically binding to the LXCXE cleft of pRB, the same site that was previously shown to be necessary for CAP-D3 binding). An improved understanding of how RB and CAP-D3 regulate AMPs in human cells may provide insight into how these proteins are able to regulate clusters of genes, and may also open up new avenues for therapeutic targeting of infection and disease. Further studies of in differentiated human cells may identify additional sets of genes that are regulated by pRB and CAP-D3 (Longworth, 2012).

SLC46 family transporters facilitate cytosolic innate immune recognition of monomeric peptidoglycans

Tracheal cytotoxin (TCT), a monomer of DAP-type peptidoglycan from Bordetella pertussis, causes cytopathology in the respiratory epithelia of mammals and robustly triggers the Drosophila Imd pathway. PGRP-LE, a cytosolic innate immune sensor in Drosophila, directly recognizes TCT and triggers the Imd pathway, yet the mechanisms by which TCT accesses the cytosol are poorly understood. This study reports that CG8046, a Drosophila SLC46 family transporter, is a novel transporter facilitating cytosolic recognition of TCT, and plays a crucial role in protecting flies against systemic Escherichia coli infection. In addition, mammalian SLC46A2s promote TCT-triggered NOD1 activation in human epithelial cell lines, indicating that SLC46As is a conserved group of peptidoglycan transporter contributing to cytosolic immune recognition (Kiak, 2017).

Ecdysone mediates the development of immunity in the Drosophila embryo

Beyond their role in cell metabolism, development, and reproduction, hormones are also important modulators of the immune system. In the context of inflammatory disorders, systemic administration of pharmacological doses of synthetic glucocorticoids (GCs) is widely used as an anti-inflammatory treatment. However, not all actions of GCs are immunosuppressive, and many studies have suggested that physiological concentrations of GCs can have immunoenhancing effects. For a more comprehensive understanding of how steroid hormones regulate immunity and inflammation, a simple in vivo system is required. The Drosophila embryo has recently emerged as a powerful model system to study the recruitment of immune cells to sterile wounds and host-pathogen dynamics. This study investigated the immune response of the fly embryo to bacterial infections and found that the steroid hormone 20-hydroxyecdysone (20-HE) can regulate the quality of the immune response and influence the resolution of infection in Drosophila embryos (Tan, 2014).

Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster

In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. This study provides evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. Early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. It was also shown that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down-regulated and activation and translocation of transcription factor Relish is also affected (Verma, 2015).

Apoptosis in hemocytes induces a shift in effector mechanisms in the Drosophila immune system and leads to a pro-inflammatory state

Apart from their role in cellular immunity via phagocytosis and encapsulation, Drosophila hemocytes release soluble factors such as antimicrobial peptides, and cytokines to induce humoral responses. In addition, they participate in coagulation and wounding, and in development. To assess their role during infection with entomopathogenic nematodes, plasmatocytes and1 crystal cells, the two classes of hemocytes present in naive larvae were deleted by expressing proapoptotic proteins in order to produce hemocyte-free (Hml-apo, originally called Hemoless) larvae. Surprisingly, Hml-apo larvae are still resistant to nematode infections. When further elucidating the immune status of Hml-apo larvae, a shift was observed in immune effector pathways including massive lamellocyte differentiation and induction of Toll- as well as repression of imd signaling. This leads to a pro-inflammatory state, characterized by the appearance of melanotic nodules in the hemolymph and to strong developmental defects including pupal lethality and leg defects in escapers. Further analysis suggests that most of the phenotypes that were observed in Hml-apo larvae are alleviated by administration of antibiotics and by changing the food source indicating that they are mediated through the microbiota. Biochemical evidence identifies nitric oxide as a key phylogenetically conserved regulator in this process. Finally it was shown that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.

Long-term in vivo tracking of inflammatory cell dynamics within Drosophila pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle - the Drosophila pupa - offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. This study describes a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, a number of specific in vivo fluorescent markers is used for both the epithelium and innate immune cells. The effectiveness is demonstrated of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site (Weavers, 2018).

Convergent balancing selection on an antimicrobial peptide in Drosophila

Genes of the immune system often evolve rapidly and adaptively, presumably driven by antagonistic interactions with pathogens. Those genes encoding secreted antimicrobial peptides (AMPs), however, have failed to exhibit conventional signatures of strong adaptive evolution, especially in arthropods and often segregate for null alleles and gene deletions. Furthermore, quantitative genetic studies have failed to associate naturally occurring polymorphism in AMP genes with variation in resistance to infection. Both the lack of signatures of positive selection in AMPs and lack of association between genotype and immune phenotypes have yielded an interpretation that AMP genes evolve under relaxed evolutionary constraint, with enough functional redundancy that variation in, or even loss of, any particular peptide would have little effect on overall resistance. In stark contrast to the current paradigm, this study identified a naturally occurring amino acid polymorphism in the AMP Diptericin that is highly predictive of resistance to bacterial infection in Drosophila melanogaster. The identical amino acid polymorphism arose in parallel in the sister species D. simulans, by independent mutation with equivalent phenotypic effect. Convergent substitutions at the same amino acid residue have evolved at least five times across the Drosophila genus. The study hypothesizes that the alternative alleles are maintained by balancing selection through context-dependent or fluctuating selection. This pattern of evolution appears to be common in AMPs but is invisible to conventional screens for adaptive evolution that are predicated on elevated rates of amino acid divergence (Unckless, 2016).

Balancing selection drives the maintenance of genetic variation in Drosophila antimicrobial peptides

Genes involved in immune defense against pathogens provide some of the most well-known examples of both directional and balancing selection. Antimicrobial peptides (AMPs) are innate immune effector genes, playing a key role in pathogen clearance in many species, including Drosophila. Conflicting lines of evidence have suggested AMPs may be under directional, balancing or purifying selection. This study used both a linear model and control gene-based approach to show that balancing selection is an important force shaping AMP diversity in Drosophila. In D. melanogaster, this is most clearly observed in ancestral African populations. Furthermore, the signature of balancing selection is even more striking once background selection has been accounted for. Balancing selection also acts on AMPs in D. mauritiana, an isolated island endemic separated from D. melanogaster by about 4 million years of evolution. This suggests that balancing selection may be broadly acting to maintain adaptive diversity in Drosophila AMPs, as has been found in other taxa (Chapman, 2019).

Complex coding and regulatory polymorphisms in a restriction factor determine the susceptibility of Drosophila to viral infection

It is common to find that major-effect genes are an important cause of variation in susceptibility to infection. This study characterised natural variation in a gene called pastrel that explains over half of the genetic variance in susceptibility to the virus DCV in populations of Drosophila melanogaster. Extensive allelic heterogeneity was found, with a sample of seven alleles of pastrel from around the world conferring four phenotypically distinct levels of resistance. By modifying candidate SNPs in transgenic flies, this study showed that the largest effect is caused by an amino acid polymorphism that arose when an ancestral threonine was mutated to alanine, greatly increasing resistance to DCV. Overexpression of the ancestral susceptible allele provides strong protection against DCV, indicating that this mutation acted to improve an existing restriction factor. The pastrel locus also contains complex structural variation and cis-regulatory polymorphisms altering gene expression. Higher expression of pastrel was associated with increased survival after DCV infection. To understand why this variation is maintained in populations, genetic variation was investigated surrounding the amino acid variant that is causing flies to be resistant. No evidence was found of natural selection causing either recent changes in allele frequency or geographical variation in frequency, suggesting that this is an old polymorphism that has been maintained at a stable frequency. Overall, these data demonstrate how complex genetic variation at a single locus can control susceptibility to a virulent natural pathogen (Cao, 2017).

Identification of cis-regulatory sequences reveals potential participation of lola and Deaf1 transcription factors in Anopheles gambiae innate immune response

The innate immune response of Anopheles gambiae involves the transcriptional upregulation of effector genes. Therefore, the cis-regulatory sequences and their cognate binding factors play essential roles in the mosquito's immune response. However, the genetic control of the mosquito's innate immune response is not yet fully understood. To gain further insight on the elements, the factors and the potential mechanisms involved, an open chromatin profiling was carried out on A. gambiae-derived immune-responsive cells. This study reports the identification of cis-regulatory sites, immunity-related transcription factor binding sites, and cis-regulatory modules. A de novo motif discovery carried out on this set of cis-regulatory sequences identified immunity-related motifs and cis-regulatory modules. These modules contain motifs that are similar to binding sites for REL-, STAT-, lola- and Deaf1-type transcription factors. Sequence motifs similar to the binding sites for GAGA were found within a cis-regulatory module, together with immunity-related transcription factor binding sites. The presence of Deaf1- and lola-type binding sites, along with REL- and STAT-type binding sites, suggests that the immunity function of these two factors could have been conserved both in Drosophila and Anopheles gambiae (Perez-Zamorano, 2017).

The raspberry gene is involved in the regulation of the cellular immune response in Drosophila melanogaster

Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. This study shows that the Drosophila inosine 5'-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid (Kari, 2016).

Constitutive activation of cellular immunity underlies the evolution of resistance to infection in Drosophila

Organisms rely on inducible and constitutive immune defences to combat infection. Constitutive immunity enables a rapid response to infection but may carry a cost for uninfected individuals, leading to the prediction that it will be favoured when infection rates are high. When populations of Drosophila melanogaster were exposed to intense parasitism by the parasitoid wasp Leptopilina boulardi, they evolved resistance by developing a more reactive cellular immune response. Using single-cell RNA sequencing, this study found that immune-inducible genes had become constitutively upregulated. This was the result of resistant larvae differentiating precursors of specialized immune cells called lamellocytes that were previously only produced after infection. Therefore, populations evolved resistance by genetically hard-wiring the first steps of an induced immune response to become constitutive (Leitao, 2020).

The Effect of Developmental Pleiotropy on the Evolution of Insect Immune Genes

The pressure to survive ever-changing pathogen exposure explains the frequent observation that immune genes are among the fastest evolving in the genomes of many taxa, but an intriguing proportion of immune genes also appear to be under purifying selection. Though variance in evolutionary signatures of immune genes is often attributed to differences in gene-specific interactions with microbes, this explanation neglects the possibility that immune genes participate in other biological processes that could pleiotropically constrain adaptive selection. This study, analyzed available transcriptomic and genomic data from Drosophila melanogaster and related species to test the hypothesis that there is substantial pleiotropic overlap in the developmental and immunological functions of genes involved in immune signaling and that pleiotropy would be associated with stronger signatures of evolutionary constraint. The results suggest that pleiotropic immune genes do evolve more slowly than those having no known developmental functions and that signatures of constraint are particularly strong for pleiotropic immune genes that are broadly expressed across life stages. These results support the general yet untested hypothesis that pleiotropy can constrain immune system evolution, raising new fundamental questions about the benefits of maintaining pleiotropy in systems that need to rapidly adapt to changing pathogen pressures (Williams, 2023).

Leishmania amazonensis engages CD36 to drive parasitophorous vacuole maturation

Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. This work developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, a collection of RNAi-expressing flies were screened for anti-Leishmania defense factors. Notably, three CD36-like scavenger receptors (<croquemort, CG31741, and CG10345) were found that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection (Okuda, 2016).

Inhibition of phagocytic killing of Escherichia coli in Drosophila hemocytes by RNA chaperone Hfq

An RNA chaperone of Escherichia coli, called host factor required for phage Qbeta RNA replication (Hfq), forms a complex with small noncoding RNAs to facilitate their binding to target mRNA for the alteration of translation efficiency and stability. Although the role of Hfq in the virulence and drug resistance of bacteria has been suggested, how this RNA chaperone controls the infectious state remains unknown. The present study addressed this issue using Drosophila melanogaster as a host for bacterial infection. In an assay for abdominal infection using adult flies, an E. coli strain with mutation in hfq was eliminated earlier, whereas flies survived longer compared with infection with a parental strain. The same was true with flies deficient in humoral responses, but the mutant phenotypes were not observed when a fly line with impaired hemocyte phagocytosis was infected. The results from an assay for phagocytosis in vitro revealed that Hfq inhibits the killing of E. coli by Drosophila phagocytes after engulfment. Furthermore, Hfq seemed to exert this action partly through enhancing the expression of E. coli σ38, a stress-responsive sigma factor that was previously shown to be involved in the inhibition of phagocytic killing of E. coli, by a posttranscriptional mechanism. This study indicates that the RNA chaperone Hfq contributes to the persistent infection of E. coli by maintaining the expression of bacterial genes, including one coding for sigma38, that help bacteria evade host immunity (Shiratsuchi, 2016).

Duox and Jak/Stat signalling influence disease tolerance in Drosophila during Pseudomonas entomophila infection

Disease tolerance describes an infected host's ability to maintain health independently of the ability to clear microbe loads. The Jak/Stat pathway plays a pivotal role in humoral innate immunity by detecting tissue damage and triggering cellular renewal, making it a candidate tolerance mechanism. This study found that in Drosophila melanogaster infected with Pseudomonas entomophila disrupting ROS-producing dual oxidase (duox) or the negative regulator of Jak/Stat Socs36E, render male flies less tolerant. Another negative regulator of Jak/Stat, G9a - which has previously been associated with variable tolerance of viral infections - did not affect the rate of mortality with increasing microbe loads compared to flies with functional G9a, suggesting it does not affect tolerance of bacterial infection as in viral infection. These findings highlight that ROS production and Jak/Stat signalling influence the ability of flies to tolerate bacterial infection sex-specifically and may therefore contribute to sexually dimorphic infection outcomes in Drosophila (Prakash, 2023).

Transdifferentiation and proliferation in two distinct hemocyte lineages in Drosophila melanogaster larvae after wasp infection

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, this study developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. It was found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which were named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. These data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail (Anderl, 2016).

Blood cells are the central players in the cellular immune response, and evolutionarily conserved signaling pathways control their hematopoiesis. Three main types of blood cells or hemocytes have been described for Drosophila melanogaster; plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes, the main hemocyte type in healthy larvae, are professional phagocytes, and they are functionally similar to mammalian monocytes, macrophages, and neutrophils. Lamellocytes are formed in response to wasp infection, when they are needed for the encapsulation and killing of parasitoids. Finally, crystal cells, are required for the melanization of wounds. Together with lamellocytes, crystal cells probably also contribute to the melanization of capsules, a presumed effector mechanism of the immune defense (Anderl, 2016).

A variety of parasitoid hymenopteran wasp species, including the genus Leptopilina, deposit their eggs in the hemocoel of fly larvae. This triggers a melanotic encapsulation reaction that comprises a fixed sequence of events. After the egg is injected, a thin electron-dense layer of unknown material is deposited on the chorion. Then plasmatocytes attach to and spread on the egg. Several layers of lamellocytes encapsulate the egg, the capsule is sealed by septate junctions between the cells, and finally melanin is deposited by the action of the enzyme phenol oxidase. Both crystal cells and lamellocytes participate in the melanization reaction. Parasitoid wasp species, in turn, deploy several virulence strategies, which incapacitate the host's cellular immune system in different ways and visibly affect hemocytes (Anderl, 2016).

Drosophila larval hemocytes originate from two embryonic sources; the procephalic and the cardiogenic mesoderm anlagen. The cardiogenic anlage gives rise to two rows of hematopoietic organs, called lymph glands, which are situated on each side of the dorsal vessel. The paired primary lobes of the lymph glands consist of a medullary zone with progenitor cells, a cortical zone with differentiated hemocytes, and a posterior signaling center that supervises the maintenance and differentiation of progenitor cells. Prior to pupariation, the lymph glands disintegrate and release mature hemocytes. In response to wasp infection the primary lobes can disintegrate earlier and release differentiated plasmatocytes and lamellocytes. The hemocytes of procephalic origin give rise to the peripheral hemocyte population, which is distinct from the lymph glands. Peripheral hemocytes colonize a second hematopoietic compartment of sessile hemocyte islets, which are arranged in segments under the skin. These hemocytes proliferate in contact with peripheral neurons (Makhijani, 2011) and alternate between sessile positions in the islets and circulation in the open body cavity of larvae. In healthy larvae, all hemocytes are of procephalic origin until the onset of metamorphosis, when hemocytes are released from the lymph glands. Procephalic hemocytes also persist into adulthood. Although research during the past 15 years has highlighted the lymph glands as the source of lamellocytes, it was suggested already in 1957 that peripheral plasmatocytes give rise to lamellocytes. This idea was more recently corroborated by lineage tracing in three studies. After a wasp attack, a main fraction of the hemocytes participating in the encapsulation reaction originates from the peripheral population. Nevertheless, the origin of lamellocytes remains ambiguous, and the dynamics of the cellular immune system in the encapsulation reaction is unclear (Anderl, 2016).

Initially, hemocytes were classified by their morphology. The development of the enhancer trap system in Drosophila enabled the production of the first generation of genetic hemocyte markers. Later, hemocyte-specific antibodies provided pan-hemocyte antibodies as well as specific antibodies for the different hemocyte classes. These antibodies were also instrumental in the discovery of new hemocyte-specific proteins. Hemocyte-specific GAL4 constructs and fluorescent enhancer-reporter fusions further diversified the genetic toolbox, allowing the observation of hemocytes or specific hemocyte subclasses in vivo. Despite these advances, peripheral hemocytes are mainly counted with hemocytometers, which is labor-intensive and error-prone. So far, the use of flow cytometry in the differential cell counting and sorting of Drosophila hemocytes has been minimal (Anderl, 2016).

This study presents a combined approach of flow cytometry and microscopy to investigate the dynamics of hematopoiesis after a wasp infection. Advantage was taken of the previously developed enhancer-reporter constructs eater-GFP (here called eaterGFP), which is specific for plasmatocytes, and MSNF9MO-mCherry (msnCherry), which is specific for lamellocytes. Three species of the parasitoid wasp genus Leptopilina, each with different well-established effects on the immune response of Drosophila larvae, were chosen to better understand the origin of lamellocytes and the dynamics of the hemocyte compartments during the encapsulation reaction. This study shows that flow cytometry, combined with fluorescent enhancer-reporter constructs, is an effective way to distinguish different hemocyte classes. A wasp infection induces several novel hemocyte classes that belong to two major lineages, the plasmatocyte and the lamellocyte lineage. These lineages give rise to two types of lamellocytes and activated plasmatocytes in a demand-adapted response (Anderl, 2016).

The switch from steady-state to infection-induced hematopoiesis in Drosophila melanogaster is marked by the generation of a new blood cell type, the lamellocyte. Three different models for lamellocyte hematopoiesis have been proposed. Firstly, prohemocytes in the lymph glands self-renew and directly transform into lamellocytes that are released into the circulation. Secondly, prohemocytes from the lymph glands, or putative prohemocytes in the circulation, develop into plasmatocytes, which transdifferentiate via so-called podocytes into lamellocytes. Thirdly, peripheral plasmatocytes of procephalic origin transdifferentiate directly into lamellocytes. Nonetheless, the dynamics of lamellocyte hematopoiesis remain largely elusive. This study presents a two-lineage model for lamellocyte hematopoiesis, where one type of lamellocytes is generated from the plasmatocyte lineage, by direct transdifferentiation of plasmatocytes on the surface of the parasite, and the other from a designated lamellocyte lineage, with infection-induced lamelloblasts that differentiate into circulating lamellocytes (Anderl, 2016).

Lamelloblasts are characterized by the expression of the plasmatocyte markers eaterGFP and a phagocytosis receptor NimC1, albeit the eaterGFP expression level is ten times lower in lamelloblasts than in plasmatocytes. Therefore, it was first assumed that lamelloblasts might be generated from plasmatocytes by downregulating eaterGFP or via the cell division of plasmatocytes, which would dilute the GFP fluorescence. But several arguments speak against these ideas. Firstly, as GFP is a highly stable molecule with a half-life of 24 hours or more, it seems unlikely that downregulation would result in such a large difference in GFP expression after just one round of cell division. Secondly, lamelloblasts appear suddenly 8 h after infection, without the presence of obvious precursors among the circulating cells. The lamelloblast count increased from zero to more than 1000 cells in only six hours, whereas plasmatocyte numbers remained at a constant level. Producing this many lamelloblasts in one cell division would entirely deplete the plasmatocyte pool. Asymmetric cell division could in principle have generated cells with reduced levels of GFP expression, but mitotic plasmatocytes with unequal distribution of nuclear GFP expression were never observed. Furthermore, lamelloblasts are a uniform population of small cells with lower granularity than observed in plasmatocytes. Several other studies attribute similar features to prohemocytes. Taken together, these features establish lamelloblasts as a population that is clearly distinct from plasmatocytes and suggest a non-plasmatocyte origin for these cells (Anderl, 2016).

Several of the findings imply that lamelloblasts derive directly from sessile prohemocytes. Knocking down the cytokine Edin in the fat body reduced the number of lamelloblasts in the circulation. Furthermore, previous work found that sessile cells are not released into the circulation in response to a wasp infection in edin knockdown larvae. This indicates that the precursor cells of lamelloblasts likely reside in the sessile tissue. In addition, the Eater protein was originally described as a phagocytosis receptor on plasmatocytes, but recently it was shown that it is also required for the attachment of hemocytes to the sessile compartment (Bretscher, 2015). This might suggest that the low expression level of eaterGFP in lamelloblasts induces their release from the sessile islets. Markus (2009) found that cells expressing neither plasmatocyte nor lamellocyte antigens were lost from the sessile population after a wasp infection and that transplanting sessile hemocytes into recipient larvae triggered lamellocyte hematopoiesis in the transplanted cells. This shows that sessile hemocytes can be a source of lamellocytes (Anderl, 2016).

The relative contribution of lymph glands to circulating hemocytes during the immune response is still uncertain. Lymph glands release prohemocytes, plasmatocytes, and lamellocytes into the circulation, but only after the immune response against the wasp egg has already started. Furthermore, this study shows that even though the primary lymph gland lobes stay intact after a L. heterotoma infection, all hemocyte types of the lamellocyte lineage develop normally. Still, the lymph glands likely contribute to the population of lamellocytes or their precursors at later time points after an infection (Anderl, 2016).

This study confirmed that lamellocytes are terminally differentiated and non-mitotic, but nevertheless EdU-positive lamellocytes were detected after a wasp infection. Biosynthetically active cell types are known to undergo endocycles characterized by the uncoupling of DNA-replication from mitosis. Endoreplicating cells typically go through the S- and G1-phases of the cell cycle. Therefore 5-ethynyl-2'-deoxyuridine (EdU)-incorporation in lamellocytes could be due to endoreplication, but the absence of >S/G2/M-Green expression in lamellocytes indicates that DNA-synthesis is quiescent. Although it cannot be entirely excluded that lamellocytes endoreplicate, it is suggested that they originate from mitotically active precursor cells, namely lamelloblasts and prelamellocytes (Anderl, 2016).

The plasmatocyte lineage originates from procephalic plasmatocytes. At the time point when the wasp eggs hatch, activated plasmatocytes appear in large numbers. This suggests that plasmatocytes play an important role in the defense response, although they were not seen on the wasp egg. When plasmatocytes are activated they become more granular, grow in size, and accumulate cytoplasmic mCherry-positive foci. The granular mCherry fluorescence in activated plasmatocytes may indicate the phagocytosis of lamellocyte-derived material, but the expression of lamellocyte antigens might also signify the general activation of the immune system (Anderl, 2016).

An important question is how the hemocyte classes in Drosophila are related to the blood cells of other species. Unfortunately, the naming of Drosophila hemocytes is not congruent with the generally accepted terminology for other insect orders, including other dipterans. Drosophila plasmatocytes are structurally and functionally very similar to the professional phagocytes of other insects, usually called granulocytes or granular cells, and these cell types are considered homologous. On the other hand, general insect terminology reserves the term plasmatocyte for a granulocyte-like, but agranular, class of cells that actively participate in the encapsulation of parasites, much like the Drosophila lamellocytes. The observation that Drosophila lamellocytes actually originate from a group of round cells of low granularity, the lamelloblasts, suggests that the lamellocyte lineage may indeed be homologous to the plasmatocytes of other insects. Notably, hemocytes of lamellocyte morphology are only found among the Drosophila species of the melanogaster subgroup, although lamellocyte-like cells have been described in several other drosophilids. Instead of lamellocytes, some drosophilids have evolved other bizarre hemocyte types that participate in the encapsulation of parasitoid wasps, such as the hairy pseudopodocytes of the obscura group and the highly motile multinucleated giant hemocytes of the ananassae subgroup. In the drosophilid Zaprionus indianus and several Drosophila species, spindle- or thread-shaped nematocytes appear together with lamellocyte-like cells. A parsimonious interpretation of these observations is that an ancestral hemocyte class, specialized in the encapsulation of parasites, has undergone rapid and diversifying evolution in the Drosophila lineage (Anderl, 2016).

Homologies between Drosophila and human blood cells remain entirely speculative. Indeed, it is not unlikely that different types of blood cells evolved independently in vertebrates and arthropods. Still, the activation of plasmatocytes in the plasmatocyte lineage can be seen as an interesting analogy to the transformation of monocytes into macrophages (Anderl, 2016).

Peripheral plasmatocytes proliferate by self-renewal (Makhijan, 2011) and their numbers increase during larval development. Hemocyte numbers have also been shown to increase after a wasp infection. This increase has been linked to the release of cells from the sessile compartment and from the lymph glands. The current results show that the demand-adapted hematopoiesis of the lamellocyte and plasmatocyte lineages is reason for the increase in cell counts after a wasp infection. Moreover, hemocytes divide on the wasp egg and in the sessile compartment. Infection-induced mitosis has been observed in Anopheles gambiae, but this has not previously been demonstrated for immune-induced cell types in Drosophila. In mammals, on the other hand, demand-adapted hematopoiesis is a well described trait of the immune response and is characterized by the increase of cell numbers several-fold over the steady-state levels of blood cell production. Recently, subpopulations of tissue macrophages derived from embryonic cells were also found to divide in situ rather than being replenished by myelopoiesis. Taken together, the immune response after wasp infection is reminiscent of the demand-adapted hematopoiesis in mammals (Anderl, 2016).

Antibodies have been instrumental in defining blood cell populations in Drosophila larvae, where the expression of the P1/NimC1 antigen marks plasmatocyte identity and the expression of the L-antigens lamellocyte identity. eaterGFP and msnCherry have been introduced as specific markers for plasmatocytes and lamellocytes respectively. However, after immune activation, cells with plasmatocyte morphology express varying levels of L-antigens, indicating that they represent intermediate cell types. Similarly, this study shows that the expression of eaterGFP and msnCherry is not restricted to plasmatocytes or lamellocytes, but that cell populations expressing both reporter constructs exist. Available plasmatocyte and lamellocyte markers unambiguously define unchallenged plasmatocytes and fully differentiated lamellocytes, respectively, but because of the dynamic nature of the immune response a combination of reporter constructs, or the corresponding plasmatocyte and lamellocyte antibodies, have to be used in order to define the blood cell lineages (Anderl, 2016).

In conclusion, flow cytometry in combination with fluorescent hemocyte markers is an accurate and fast method for the differential counting of Drosophila blood cell populations from single larvae, and is potentially useful for the high throughput analysis of hemocyte phenotypes in genetic screening, or drug testing in vivo. Overall, these findings show that lamellocytes are generated in parallel by the transdifferentiation of plasmatocytes and de novo from lamelloblasts. However, the origin of lamelloblasts remains uncertain. The challenge is now to create appropriate genetic tools to track and experimentally manipulate individual hemocyte populations and to understand how the as yet elusive signals from different tissues, like the fat body and somatic muscles, integrate to shape a functional immune response (Anderl, 2016).

Metabolic control of cellular immune-competency by odors in Drosophila

Studies in different animal model systems have revealed the impact of odors on immune cells; however, any understanding on why and how odors control cellular immunity remained unclear. This study found that Drosophila employ an olfactory-immune cross-talk to tune a specific cell type, the lamellocytes, from hematopoietic-progenitor cells. Neuronally released GABA derived upon olfactory stimulation is utilized by blood-progenitor cells as a metabolite and through its catabolism, these cells stabilize Sima/HIFα protein. Sima capacitates blood-progenitor cells with the ability to initiate lamellocyte differentiation. This systemic axis becomes relevant for larvae dwelling in wasp-infested environments where chances of infection are high. By co-opting the olfactory route, the preconditioned animals elevate their systemic GABA levels leading to the upregulation of blood-progenitor cell Sima expression. This elevates their immune-potential and primes them to respond rapidly when infected with parasitic wasps. The present work highlights the importance of the olfaction in immunity and shows how odor detection during animal development is utilized to establish a long-range axis in the control of blood-progenitor competency and immune-priming (Madhwal, 2020).

Age and diet affect genetically separable secondary injuries that cause acute mortality following traumatic brain injury in Drosophila

Outcomes of traumatic brain injury (TBI) vary because of differences in primary and secondary injuries. Primary injuries occur at the time of a traumatic event, whereas secondary injuries occur later as a result of cellular and molecular events activated in the brain and other tissues by primary injuries. This study used a Drosophila melanogaster TBI model to investigate secondary injuries that cause acute mortality. By analyzing percent mortality within 24 hours of primary injuries, it was previously found that age at the time of primary injuries and diet afterward affect the severity of secondary injuries. This study shows that secondary injuries peaked in activity 1-8 hours after primary injuries. Additionally, it was demonstrated that age and diet activated distinct secondary injuries in a genotype-specific manner and that concurrent activation of age- and diet-regulated secondary injuries synergistically increased mortality. To identify genes involved in secondary injuries that cause mortality, genome-wide mRNA expression profiles were compared of uninjured and injured flies under age and diet conditions that had different mortalities. During the peak period of secondary injuries, innate immune response genes were the predominant class of genes that changed expression. Furthermore, age and diet affected the magnitude of the change in expression of some innate immune response genes, suggesting roles for these genes in inhibiting secondary injuries that cause mortality. These results indicate that the complexity of TBI outcomes is due in part to distinct, genetically controlled, age- and diet-regulated mechanisms that promote secondary injuries and that involve a subset of innate immune response gene (Katzenberger, 2016).

Functional analysis of PGRP-LA in Drosophila immunity

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. This paper presents a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, a genome-wide microarray analysis was conducted of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing these data to previous microarray studies, it is reported that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, these results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia (Gendrin, 2013).

This structural study predicts that the PGRP domain of PGRP-LA is unlikely to bind peptidoglycan by itself. Over-expression of PGRP-LAD isoform, but not of PGRP-LAC and PGRP-LAF, leads to the activation of Diptericin expression in absence of infection. The experiments placed PGRP-LAD upstream of the Dredd caspase and of the Tak1 MAP3K. The intracellular domain of PGRP-LAD contains a RHIM motif similar to that observed in PGRP-LC and PGRP-LE for which it is essential for Imd pathway activation. This suggests that the RHIM motif confers to PGRP-LAD the capacity to induce the Imd pathway. Studies involving short mutations in PGRP-LC and PGRP-LE reported that their RHIM motifs are not involved in any physical interaction with Imd, the downstream adaptor of the Imd pathway, but bind with Pirk, a negative regulator of the Imd pathway. Further analysis will be required to test whether the different PGRP-LA isoforms physically interacts with Pirk and/or with PGRP-LC. Collectively, this initial molecular characterization of PGRP-LA suggests a modulatory role of this PGRP in the Imd pathway (Gendrin, 2013).

Using a PGRP-LA-deficient line, PGRP-LA was shown to not be required for the systemic production of antimicrobial peptides in the adult. Consistent with this observation, mutations in PGRP-LA did not increase the susceptibility to systemic bacterial infection. This matches with the very low expression of PGRP-LA in the fat body. Of note, phagocytosis was also not affected in the PGRP-LA2A mutant. Consistently, previous studies on S2-cells did not reveal any role of PGRP-LA in the induction of antimicrobial peptides by peptidoglycan or Gram-negative bacteria (Choe, 2002; Ramet, 2002) or in the phagocytosis of Gram-negative or Gram-positive bacteria. All these data clearly indicate that PGRP-LA is not compulsory for the systemic activation of the Imd or Toll pathways, although a more specific role under a very specific condition or in response to a specific form of peptidoglycan could formally not be excluded (Gendrin, 2013).

Several studies have shown that the antimicrobial response of Drosophila exhibits major differences depending on the tissue. Notably, regulatory mechanisms controlling the antimicrobial response in barrier epithelia significantly differ from that involved in fat body-mediated systemic immune response. For instance, the expression of antimicrobial peptide genes (including Drosomycin) in the midgut or the tracheae relies only on the Imd pathway. In addition, it has recently been shown that PGRP-LE has a significant role in Imd pathway activation in the midgut while PGRP-LC is the main sensor of Gram-negative bacteria during systemic infection. These differences are probably a consequence of the necessity to maintain tight control on immune activation according to the level of exposure to bacteria or microbial products; while the hemocoel surrounding the fat body remains sterile, organs such as the digestive tract and tracheae are constantly in direct contact with the external environment. This raises the possibility that PGRP-LA has a subtler role in barrier epithelia where its expression is enriched. In support of this notion, microarray analysis revealed a lower expression of antimicrobial peptides in PGRP-LA2A tracheae of both Ecc15-infected and unchallenged larvae. The idea that PGRP-LA could establish the basal level of Imd pathway in unchallenged conditions is intriguing. These results were confirmed in RT-qPCR, but limitations due to the low and variable levels of antimicrobial gene expression in the tracheae and the gut in unchallenged conditions, when maintaining fly lines in autoclaved fly medium, did not allow confirmation of this hypothesis. Nevertheless, it was observed that the expression of several antimicrobial peptide genes was reduced in larval tracheae and adult guts of PGRP-LA2A mutants upon Ecc15 infection. A rescue experiment confirms that the phenotype is specifically linked to the PGRP-LA deletion and not to the genetic background. However, in normal laboratory conditions the PGRP-LA phenotype is not very strong and no infectious conditions were detected for which a contribution of PGRP-LA to adult survival was discernable (Gendrin, 2013).

The results support the notion that PGRP-LA positively regulates the antibacterial response in infected epithelia. However, subtle additional roles for PGRP-LA cannot be excluded, such as its participation in inter-organ communication by spreading immune signaling from epithelia to another tissue (e.g. between the gut and the tracheae). Such immune communication between tissues occurs between several epithelia and the fat body in Drosophila. However, no role of PGRP-LA could be discerned in the activation of the systemic response upon gut or genital infections (Gendrin, 2013).

The implication of several pattern-recognition receptors in the gut highlights the complexity of mechanisms underlying bacterial sensing in barrier epithelia. The conservation of PGRP-LA in mosquito (contrary to PGRP-LE or PGRP-LF) where it is also located in cluster with PGRP-LC suggests the conservation of its function in other insect species. The genomic organization of the PGRP-LA, LC, LF cluster is intriguing since the Imd-receptor gene PGRP-LC is flanked by both a positive (PGRP-LA) and a negative (PGRP-LF) regulator of the pathway. Future studies should elucidate the mechanisms by which PGRP-LA modulates the Imd pathway, notably to determine which PGRP-LA isoforms are involved. Another question to address will be the respective contributions of PGRP-LA, LC, and LE in the sensing of bacteria in the intestine. Thus, the current data add a layer of complexity to the mechanism regulating the Imd pathway and further investigation is needed to fully characterize the role of PGRP-LA (Gendrin, 2013).

The Drosophila tracheal immune response remains poorly characterized. In this study, a general analysis is presented of tracheal transcriptome variations after bacterial infection in larvae. The data reveal a major role of the Imd pathway, which controls the expression of half of the genes regulated upon infection and of most of the immunity-related genes, such as antimicrobial genes. This is in accordance with previous reports showing that this pathway controls the local production of antimicrobial peptide genes, in tracheae and the gut. It is noted that it also regulates genes involved in other cellular functions such as metabolism. Interestingly, this study observed that many genes encoding putative or characterized cuticle proteins are down-regulated upon infection. The shape of the tracheae is maintained by helicoidal thickenings of the intima called taenidiae. Therefore, the down-regulation of structural genes highlighted in the microarray suggests a remodeling of this structure upon infection. Consistent with this down-regulation, an apical-basal enlargement of the cells of the airway epithelium has been previously reported in regions of the tracheae exhibiting a strong immune response. This enlargement might be explained by a thinning of the cuticle and consequent loss of rigidity. Thus, infection with Ecc15 not only induces an immune and stress response, but also alters the metabolism and physiology of tracheae. Interestingly, microarray comparison of the immune response during systemic (fat body), gut, and tracheal immune response reveals that only a small group of common genes are induced, all regulated by the Imd pathway and encoding mainly antimicrobial peptides and other pathway components. These genes may therefore represent the 'core' of Imd pathway that are complemented by tissue-specific genes to achieve an optimal immune response (Gendrin, 2013).

The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. This study describes rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. It is proposed that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat (Neyen, 2016).

PGRP-LC has a clear role as the major signaling receptor sensing Gram-negative bacteria in flies, but its contribution to the resolution phase once bacteria are killed and release polymeric PGN has remained elusive. This study has uncovered a regulatory isoform of LC (rLC) that adjusts the immune response to the level of threat. rLC specifically downregulates IMD pathway activation in response to polymeric PGN, a hallmark of efficient bacterial killing. The data are consistent with a model whereby the presence of rLC leads to efficient endocytosis of LC and termination of signaling via the ESCRT pathway. Trafficking-mediated shutdown of LC-dependent signaling ensures that LC receptors are switched off once the balance is tipped in favor of ligands signifying dead bacteria, allowing Drosophila to terminate a successful immune response. Failure to do so results in over-signaling, leading to the death of the host despite bacterial clearance. Consistent with this model, defects were found in endosome maturation and in the formation of MVBs enhance immune activation and prevent immune resolution. In addition to regulating LC signaling via the ESCRT machinery, rLC can also inhibit LC signaling by forming signaling-incompetent rLC-LC heterodimers or rLC-rLC homodimers (Neyen, 2016).

Recent evidence from vertebrates also implicates the ESCRT machinery in suppressing spurious NF-κB activation: the TNFR superfamily member lymphotoxin-β receptor, which activates a signaling cascade that is functionally similar to IMD signaling, is degraded in an ESCRT-dependent manner in zebrafish and human cells. Thus ESCRT-mediated clearance of receptors upstream of NF-κB seems well conserved throughout evolution (Neyen, 2016).

Internalization of receptor-ligand complexes raises the question of whether peptidoglycan is fully degraded in the endolysosomal compartment or fragmented and released into the cytosol for sensing by PGRP-LE, as is the case for peptidoglycan sensing by cytoplasmic NOD2 receptors in mammalian cells. Mechanistic coupling of LC-dependent peptidoglycan endocytosis and PGRP-LE-dependent cytosolic sensing of exported peptidoglycan fragments would help explain the partial cooperation between the two receptors (Neyen, 2016).

Molecularly, rLC is characterized by a cytosolic PHD domain predicted to bind to phosphoinositides. The PHD domain targets rLC were found to be distinct membrane domains but it cannot be excluded that this localization relies on additional protein-protein interactions. Furthermore, the PHD domain also mediates binding of rLC to the cytosolic regulator Pirk and the ubiquitin ligase DIAP2. The combined capability to control membrane localization and to recruit downstream signaling modulators is reminiscent of the 'sorting-signaling adaptor paradigm' that is emerging for mammalian PRRs. Sorting adaptors are cytosolic signaling components with phosphoinositide-binding domains that are selectively recruited to defined subcellular locations and thereby shape the signaling output of the receptors they interact with. In vertebrate immune signaling, bacterial sensing modules (for example, TLRs), lipid-binding sorting modules (for example, TIRAP or TRIF) and signaling modules (for example, MyD88 and TRAM) are carried on separate molecules and assemble via transient interactions. Drosophila MyD88 combines sorting and signaling functions in a single molecule, bypassing the need for TIRAP. Notably, rLC merges features of sensing and signaling receptors and sorting adaptors into a single molecule. The fact that Drosophila rLC has no immediate homologs in vertebrates with PGN-sensing and PGN-signaling pathways suggests evolutionary uncoupling of sensing and sorting domains, possibly to increase the spectrum of signaling by combinatorial recruitment of adaptors to sensing receptors (Neyen, 2016).

Participation of a galactose-specific C-type lectin in Drosophila immunity

A galactose-specific C-type lectin has been purified from a pupal extract of Drosophila melanogaster. This lectin gene, named DL1 (Drosophila lectin 1; Lectin-galC1), is part of a gene cluster with the other two galactose-specific C-type lectin genes, named DL2 (Drosophila lectin 2) and DL3 (Drosophila lectin 3). These three genes are expressed differentially in fruit fly, but show similar haemagglutinating activities. The present study characterized the biochemical and biological properties of the DL1 protein. The recombinant DL1 protein bound to Escherichia coli and Erwinia chrysanthemi, but not to other Gram-negative or any other kinds of microbial strains that have been investigated. In addition, DL1 agglutinated E. coli and markedly intensified the association of a Drosophila haemocytes-derived cell line with E. coli. For in vivo genetic analysis of the lectin genes, this study also established a null-mutant Drosophila. The induction of inducible antibacterial peptide genes was not impaired in the DL1 mutant, suggesting that the galactose-specific C-type lectin does not participate in the induction of antibacterial peptides, but possibly participates in the immune response via the haemocyte-mediated mechanism (Tanji, 2006).

The genetic architecture of defense as resistance to and tolerance of bacterial infection in Drosophila melanogaster

Defense against pathogenic infection can take two forms: resistance and tolerance. Resistance is the ability of the host to limit a pathogen burden, whereas tolerance is the ability to limit the negative consequences of infection at a given level of infection intensity. Evolutionarily, a tolerance strategy that is independent of resistance could allow the host to avoid mounting a costly immune response and, theoretically, to avoid a coevolutionary arms race between pathogen virulence and host resistance. In order to understand the impact of tolerance on host defense and identify genetic variants that determine host tolerance, genetic variation in tolerance was defined as the residual deviation from a binomial regression of fitness under infection against infection intensity. A genome-wide association study (GWAS) was performed to map the genetic basis of variation in resistance to and tolerance of infection by the bacterium Providencia rettgeri. Positive genetic correlation was found between resistance and tolerance, and the level of resistance was highly predictive of tolerance. Thirty loci were identified that predict tolerance, many of which are in genes involved in the regulation of immunity and metabolism. RNAi was performed to confirm that a subset of mapped genes have a role in defense, including putative wound repair genes grainy head and debris buster. The results indicate that tolerance is not an independent strategy from resistance, but that defense arises from a collection of physiological processes intertwined with canonical immunity and resistance (Howick, 2017).

A test for Y-linked additive and epistatic effects on surviving bacterial infections in Drosophila melanogaster

Y and W chromosomes offer a theoretically powerful way for sexual dimorphism to evolve. Consistent with this possibility, Drosophila melanogaster Y-chromosomes can influence gene regulation throughout the genome; particularly immune-related genes. In order for Y-linked regulatory variation (YRV) to contribute to adaptive evolution it must be comprised of additive genetic variance, such that variable Ys induce consistent phenotypic effects within the local gene pool. The potential for Y-chromosomes to adaptively shape gram-negative and gram-positive bacterial defense was tested by introgressing Ys across multiple genetic haplotypes from the same population. No Y-linked additive effects on immune phenotypes were found, suggesting a restricted role for the Y to facilitate dimorphic evolution. A large magnitude Y was found by background interaction that induced rank order reversals of Y-effects across the backgrounds (i.e. sign epistasis). Thus, Y-chromosome effects appeared consistent within backgrounds, but highly variable among backgrounds. This large sign epistatic effect could constrain monomorphic selection in both sexes, considering that autosomal alleles under selection must spend half of their time in a male background where relative fitness values are altered. If the pattern described in this study is consistent for other traits or within other XY (or ZW) systems, then YRV may represent a universal constraint to autosomal trait evolution (Kutch, 2017).

The distinct function of Tep2 and Tep6 in the immune defense of Drosophila melanogaster against the pathogen Photorhabdus

Previous and recent investigations on the innate immune response of Drosophila have identified certain mechanisms that promote pathogen elimination. However, the function of Thioester-containing proteins (TEPs) in the fly still remains elusive. Recent work has shown the contribution of TEP4 in the antibacterial immune defense of Drosophila against non-pathogenic E. coli, and the pathogens Photorhabdus luminescens and P. asymbiotica. This study examined the function of Tep genes in both humoral and cellular immunity upon E. coli and Photorhabdus infection. While Tep2 is induced after Photorhabdus and E. coli infection; Tep6 is induced by P. asymbiotica only. Moreover, functional ablation of hemocytes results in significantly low transcript levels of Tep2 and Tep6 in response to Photorhabdus. This study shows that tep2 and tep6 loss-of-function mutants have prolonged survival against P. asymbiotica, tep6 mutants survive better the infection of P. luminescens, and both tep mutants are resistant to E. coli and Photorhabdus. A distinct pattern of immune signaling pathway induction was found in E. coli or Photorhabdus infected tep2 and tep6 mutants. Tep2 and Tep6 were shown to participate in the activation of hemocytes in Drosophila responding to Photorhabdus. Finally, inactivation of Tep2 or Tep6 affects phagocytosis and melanization in flies infected with Photorhabdus. These results indicate that distinct Tep genes might be involved in different yet crucial functions in the Drosophila antibacterial immune response (Shokal, 2017).

Immune modulation by MANF promotes tissue repair and regenerative success in the retina

Regenerative therapies are limited by unfavorable environments in aging and diseased tissues. A promising strategy to improve success is to balance inflammatory and anti-inflammatory signals and enhance endogenous tissue repair mechanisms. This study identified a conserved immune modulatory mechanism that governs the interaction between damaged retinal cells and immune cells to promote tissue repair. In damaged retina of flies and mice, platelet-derived growth factor (PDGF)-like signaling induced mesencephalic astrocyte-derived neurotrophic factor (MANF) in innate immune cells. MANF promoted alternative activation of innate immune cells, enhanced neuroprotection and tissue repair, and improved the success of photoreceptor replacement therapies. Thus, immune modulation is required during tissue repair and regeneration. This approach may improve the efficacy of stem-cell-based regenerative therapies (Neves, 2016).

This study has confirmed that MANF is expressed in fly innate immune cells (hemocytes) using immunohistochemistry of hemolymph smears from late 2nd instar larvae. In these smears, hemocytes were identified by Green Fluorescent Protein (GFP) expression driven by the hemocyte specific driver Hemolectin:Gal4 (HmlΔ:Gal4). MANF was also detected by immuno blot in the plasma fraction of the hemolymph, confirming its secretion. Consistent with the RNAseq data, Reverse Transcription and Real Time quantitative Polymerase Chain Reaction (RT-qPCR) analysis revealed that MANF mRNA levels were significantly higher in hemocytes from UV treated larvae compared to untreated controls, and that this induction was PvR dependent. Over-expression of Pvf-1 in the retina (using GMR:Gal4; Glass Multimer Reporter as a driver) was sufficient to induce MANF mRNA specifically in hemocytes, in the absence of damage, and was accompanied by a significant increase in MANF protein in the hemolymph (Neves, 2016).

Flies overexpressing MANF in hemocytes showed significant tissue preservation after UV exposure, even after PvR knock-down in hemocytes. This protective activity of hemocyte-derived MANF was further confirmed in two genetic models of retinal damage, in which degeneration is induced by retinal (GMR driven) over-expression of the pro-apoptotic gene grim or of mutant Rhodopsin (Rh1G69D) (Neves, 2016).

Null mutations in the manf gene are homozygous lethal at early 1st instar larval stages, yet MANF heterozygotes (which express significantly lower levels of MANF in hemocytes compared to wild-types) had a significantly increased tissue degeneration response to UV. This increase in tissue loss could be rescued by MANF over-expression in hemocytes and was recapitulated by hemocyte-specific knock-down of MANF (Neves, 2016).

The protective effect of hemocyte-derived MANF could be caused by direct neuroprotective activity of MANF on retinal cells, or could reflect an indirect effect of MANF on the microenvironment of the damaged retina. To distinguish between these possibilities, whether MANF could influence hemocyte phenotypes was tested. Hemocytes can acquire lamellocyte phenotypes, characterized by down-regulation of plasmatocyte markers (hemolectin, hemese) and expression of Atilla protein, during sterile wound healing. These phenotypes correlate with hemocyte activation and may influence tissue repair capabilities, and they were recapitulated in the UV damage paradigm. Over-expression of MANF in hemocytes in vivo or treatment of hemocytes in culture with human recombinant MANF (hrMANF) significantly increased the proportion of lamellocytes in hemocyte smears, as detected by Atilla expression. This correlated with a decrease in the proportion of cells expressing GFP driven by HmlΔ:Gal4 and a decrease in hml transcripts. Furthermore, MANF was necessary and sufficient to induce the Drosophila homolog of the mammalian M2 marker arginase1 (arg) in hemocytes, suggesting that these cells may be able to acquire phenotypes similar to alternative activation. Most MANF expressing hemocytes also expressed Arg, suggesting that there is an association between MANF expression and M2-like activation of hemocytes (Neves, 2016).

To test whether MANF's immune modulatory function is required for retinal repair, retinal tissue preservation was assessed in conditions in which hemocytes express and secrete high levels of MANF, but are unable to be activated in response to this signal. Such a condition was generated by overexpressing MANF in the absence of Kdel Receptors (KdelRs). In human cells, KdelRs modulate MANF secretion and cell surface binding. Intracellular KdelR prevents MANF secretion, while cell surface bound KdelR promotes binding of extracellular MANF. Knock-down of the one Drosophila KdelR homologue in hemocytes resulted in a significant induction of MANF transcripts and the detection of MANF protein in the hemolymph, suggesting that KdelR-depleted hemocytes secrete high levels of MANF. In these hemocytes, MANF-induced lamellocyte formation and Arg expression were significantly decreased. Hemocyte activation by extracellular MANF is thus impaired after KdelR knock-down. This genetic perturbation also resulted in a significant enhancement of UV-induced tissue loss, which could not be rescued by MANF over-expression. Thus, immune modulation by MANF is critical for tissue repair (Neves, 2016).

The results identify MANF as an evolutionarily conserved immune modulator that plays a critical role in the regulatory network mediating tissue repair in the retina. The ability of MANF to increase regenerative success in the mouse retina highlights the promise of modulating the immune environment as a strategy to improve regenerative therapies (Neves, 2016).

MANF has previously been described as a neurotrophic factor, and it may also exert a direct neuroprotective effect in the retina, yet the data suggest a more expansive role: because MANF cannot promote tissue repair in flies in which the hemocyte response to MANF is selectively ablated, or in mammalian retinas depleted of innate immune cells or containing macrophages that are unresponsive to MANF, it is proposed that MANF's role in promoting alternative activation of innate immune cells is central to its function in tissue repair. Further studies will be required to determine the specific contribution of alternative-activated macrophages in mediating these effects. While the data point to an important role of macrophages in mediating the effects it does not exclude the possibility that other cell types are involved in the process, nor that macrophages' functions other than polarization may influence the outcome of MANF's protective effects (Neves, 2016).

Clinically, MANF may thus have a distinct advantage over previously described neurotrophic factors in both improving survival of transplanted cells directly, as well as in promoting a microenvironment supportive of local repair and integration. Because integration efficiency correlates with the extent of vision restoration it can be anticipated that MANF supplementation will have an important impact in clinical settings (Neves, 2016).

Further studies involving tissue specific knockdown of MANF in mammals will be required to evaluate the relative contribution of different cellular and tissue sources for MANF in homeostatic and damage conditions. While this study found that MANF is strongly expressed in immune cells, MANF expression was also observed in other cell types, in agreement with previous reports (Neves, 2016).

Similarly, the molecular mechanism involved in MANF signaling remains elusive. To date, a signal transducing receptor for MANF has not been identified, although Protein kinase C (PKC) signaling has been described to be activated downstream of MANF. MANF can further negatively regulate NF-κB signaling in mammalian cells and loss of MANF in Drosophila results in the infiltration of pupal brains with cells resembling hemocytes with high Rel/NFκB activity, potentially representing pro-inflammatory, M1-like phenotypes. The identification of immune cells as a target for MANF in this study may accelerate the discovery of putative MANF receptors and downstream signaling pathways (Neves, 2016).

Because neurotoxic inflammation has been implicated in Parkinson's disease, it is possible that the protective effects of MANF in this context are also mediated by immune modulation, as this study has shown for retinal disease. Indeed, recent reports suggest that the MANF paralog, cerebral dopamine neurotrophic factor (CDNF), has an anti-inflammatory function in murine models of Parkinson's disease and in nerve regeneration after spinal cord injury. A recent study has further shown that loss of MANF leads to beta cell loss in the pancreas. Beta cell loss is a commonly associated with chronic inflammation, and it is thus tempting to speculate that MANF is broadly required in various contexts to aid conversion of pro-inflammatory macrophages into pro-repair anti-inflammatory macrophages. Future studies will clarify the role of MANF in resolving inflammation and promoting tissue repair not only in the retina and brain, but also in other tissues. A deeper understanding of MANF-mediated immune modulation and its impact on stem cell function, wound repair and tissue maintenance is thus expected to help in the development of effective regenerative therapies (Neves, 2016).

Circulating immune cells mediate a systemic RNAi-based adaptive antiviral response in Drosophila

Effective antiviral protection in multicellular organisms relies on both cell-autonomous and systemic immunity. Systemic immunity mediates the spread of antiviral signals from infection sites to distant uninfected tissues. In arthropods, RNA interference (RNAi) is responsible for antiviral defense. This study shows that flies have a sophisticated systemic RNAi-based immunity mediated by macrophage-like haemocytes. Haemocytes take up dsRNA from infected cells and, through endogenous transposon reverse transcriptases, produce virus-derived complementary DNAs (vDNA). These vDNAs template de novo synthesis of secondary viral siRNAs (vsRNA), which are secreted in exosome-like vesicles. Strikingly, exosomes containing vsRNAs, purified from haemolymph of infected flies, confer passive protection against virus challenge in naive animals. Thus, similar to vertebrates, insects use immune cells to generate immunological memory in the form of stable vDNAs that generate systemic immunity, which is mediated by the vsRNA-containing exosomes (Tassetto, 2017).

Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense

Invading pathogens provoke robust innate immune responses in Dipteran insects, such as Drosophila melanogaster. In a systemic bacterial infection, a humoral response is induced in the fat body. Gram-positive bacteria trigger the Toll signaling pathway, whereas gram-negative bacterial infections are signaled via the immune deficiency (IMD) pathway. This study shows that the RNA interference-mediated silencing of Furin1-a member of the proprotein convertase enzyme family-specifically in the fat body, results in a reduction in the expression of antimicrobial peptides. This, in turn, compromises the survival of adult fruit flies in systemic infections that are caused by both gram-positive and -negative bacteria. Furin1 plays a nonredundant role in the regulation of immune responses, as silencing of Furin2, the other member of the enzyme family, had no effect on survival or the expression of antimicrobial peptides upon a systemic infection. Furin1 does not directly affect the Toll or IMD signaling pathways, but the reduced expression of Furin1 up-regulates stress response factors in the fat body. This study also demonstrated that Furin1 is a negative regulator of the JAK/STAT signaling pathway, which is implicated in stress responses in the fly. In summary, these data identify Furin1 as a novel regulator of humoral immunity and cellular stress responses in Drosophila (Aittomaki, 2017).

The Drosophila Thioester containing Protein-4 participates in the induction of the cellular immune response to the pathogen Photorhabdus

The function of thioester-containing proteins (TEPs) in the immune defense of the fruit fly Drosophila melanogaster is yet mostly unexplored. Recent work has shown the involvement of TEP4 in the activation of humoral and phenoloxidase immune responses of Drosophila against the pathogenic bacteria Photorhabdus luminescens and Photorhabdus asymbiotica. This study investigated the participation of Tep4 in the cellular defense of Drosophila against the two pathogens. Significantly lower numbers of live and dead plasmatocytes are reported in the tep4 mutants compared to control flies in response to Photorhabdus infection. Fewer crystal cells were found in the control flies than in tep4 mutants upon infection with Photorhabdus. These results further suggest that Drosophila hemocytes constitute a major source for the transcript levels of Tep4 in flies infected by Photorhabdus. Finally, Tep4 was shown to participate in the phagocytic function in Drosophila adult flies. Collectively these data support the protective role for TEP4 in the cellular immune response of Drosophila against the entomopathogen Photorhabdus (Shokal, 2017).

The selective antifungal activity of Drosophila melanogaster Metchnikowin reflects the species-dependent inhibition of succinate-coenzyme Q reductase

Insect-derived antifungal peptides have a significant economic potential, particularly for the engineering of pathogen-resistant crops. However, the nonspecific antifungal activity of such peptides could result in detrimental effects against beneficial fungi, whose interactions with plants promote growth or increase resistance against biotic and abiotic stress. The antifungal peptide Metchnikowin (Mtk) from Drosophila melanogaster acts selectively against pathogenic Ascomycota, including Fusarium graminearum, without affecting Basidiomycota such as the beneficial symbiont Piriformospora indica. This study investigated the mechanism responsible for the selective antifungal activity of Mtk by using the peptide to probe a yeast two-hybrid library of F. graminearum cDNAs. Mtk was found to specifically target the iron-sulfur subunit (SdhB) of succinate-coenzyme Q reductase (SQR). A functional assay based on the succinate dehydrogenase (SDH) activity of mitochondrial complex II clearly demonstrated that Mtk inhibited the SDH activity of F. graminearum mitochondrial SQR by up to 52%, but that the equivalent enzyme in P. indica was unaffected. A phylogenetic analysis of the SdhB family revealed a significant divergence between the Ascomycota and Basidiomycota. SQR is one of the key targets of antifungal agents and Mtk is therefore proposed as an environmentally sustainable and more selective alternative to chemical fungicides (Moghaddam, 2017).

Insects are well protected against pathogens by an immunity-related arsenal of effector molecules including antimicrobial peptides (AMPs). Some AMPs are active against a broad spectrum of microbes, whereas the activity of others is restricted to certain types of bacteria or fungi. Insects produce a large number of antifungal peptides to protect them against fungal pathogens and parasites, and these peptides often interact with intracellular targets to inhibit key physiological processes such as DNA and protein synthesis, cell cycle progression and metabolic activity. AMPs therefore offer significant potential as leads for the development of drugs and biocides, but the mechanisms of action must first be understood. A small number of natural antifungal peptides have been characterized in this regard, including termicin from termites, heliomicin from the tobacco budworm Heliothis virescens , gallerimycin from larvae of the greater wax moth Galleria mellonella 8, and drosomycin and metchnikowin (Mtk) from Drosophila melanogaster. The 26-residue proline-rich linear peptide Mtk is induced following microbial infection in D. melanogaster and its activity is remarkably specific. Whereas the activity of other antifungal peptides can affect both pathogens and beneficial endophytes, Mtk acts specifically against pathogenic Ascomycota such as Fusarium graminearum and Blumeria graminis f. sp. hordei, but is inactive against beneficial endophytic Basidiomycota such as Piriformospora indica, when expressed in barley. This is particularly important because plants benefit from their mutual interactions with fungal endophytes. Although the precise basis of this specificity is not understood, previous observations suggest that Mtk inhibits the ability of susceptible fungi to suppress plant defense responses. A recent study revealed that Mtk interferes with F. graminearum cell wall biosynthesis by targeting the β(1,3)-glucanosyltransferase Gel1, which is responsible for β(1,3)-glucan chain elongation in the cell wall (Moghaddam, 2017).

AMPs often have multiple targets to reduce the likelihood of emerging microbial resistance. This study therefore sought additional Mtk intracellular targets by probing a yeast two-hybrid library of F. graminearum cDNAs with an artificial Mtk peptide, revealing a specific interaction with the iron-sulfur subunit (SdhB) of succinate-coenzyme Q reductase (SQR). The holoenzyme is a heterotetramer comprising two hydrophilic subunits (flavoprotein SdhA and iron-sulfur protein SdhB) and two hydrophobic subunits (SdhC and SdhD). SdhA contains the cofactor flavin adenine dinucleotide (FAD) and a succinate binding site, whereas SdhB contains three iron-sulfur clusters, and SdhC and SdhD are the membrane anchor subunits. SQR is a key enzyme in both the Krebs cycle (also known as the citric acid cycle or tricarboxylic acid cycle) and the electron transport chain, both of which are required for energy generation. In the Krebs cycle, SQR catalyzes the oxidation of succinate to fumarate via the reduction of ubiquinone to ubiquinol. The resulting electrons enter the respiratory chain complex III, reducing oxygen to water and thus providing the electrochemical gradient across the mitochondrial inner membrane which is needed for ATP synthesis (Moghaddam, 2017).

Therefore this study investigated whether Mtk can selectively inhibit mitochondrial SQR (complex II) activity in F. graminearum but not in P. indica, based on the hypothesis that the specific activity of Mtk against Ascomycota reflects the selective inhibition of this enzyme. A phylogenetic analysis of SdhB homologs in different Ascomycota and Basidiomycota was conducted to determine whether the enzyme has diverged in these two fungal phyla (Moghaddam, 2017).

Insect AMPs have often been shown to act against phytopathogenic fungi, and could therefore be suitable for expression as recombinant peptides in transgenic plants to reduce yield and quality losses. However, the nonspecific antifungal effects of many such peptides limit their plant protection applications because most crops establish beneficial mutualistic interactions with fungal endophytes, which promote growth by facilitating access to water and nutrients, or by inhibiting other pathogens. The deployment of AMPs with selective activity against pathogenic fungi offers an environmentally sustainable alternative to hazardous chemical fungicides, particularly given the emergence and spread of fungicide resistance. The proline-rich antifungal peptide Mtk from D. melanogaster acts selectively against phytopathogenic Ascomycota such as F. graminearum without affecting beneficial endophytic Basidiomycota such as P. indica. Recent findings indicate that this specific activity partly reflects the interaction between Mtk and the Ascomycota β(1,3)glucanosyltransferase Gel1, which is only distantly related to Gel homologs in the Basidiomycota (Moghaddam, 2017).

The specificity of Mtk against Ascomycota was considered in more detail by screening a yeast two-hybrid library of F. graminearum cDNAs using Mtk as the probe, which identified the iron-sulfur subunit of SQR (UniProtKB-I1RNM7) as an additional Mtk target. The F2H and BiFC systems were used to verify the interaction. The advantage of protein-protein interaction assays based on two-hybrid systems rather than in vitro biophysical or biochemical methods is their ability to detect weak or transient interactions. Some of these interactions need species-dependent post-translational modifications and/or particular cofactors, therefore verification in mammalian and/or plant cells is highly recommended. The F2H assay reported a positive interaction but only in a few cells, suggesting the ability of BHK-21 cells to support the interaction was limited. Potential explanations include the nuclear targeting of the bait and prey, forcing them to interact in a non-native compartment, or changes in conformation caused by the fluorescent protein tags, obscuring the interaction site. The BiFC assay was also unsatisfactory, which may reflect the non-optimal stoichiometry of the interacting components caused by differences in transformation efficiency by the separate Agrobacterium tumefaciens strains, or non-specific reassembly due to the prevention of translational read-through in the native Gateway cloning cassettes. Furthermore, background signals in BiFC assays can obscure weak interactions. The possibility cannot be excluded that the rare Mtk-SdhB interaction events in mammalian and tobacco cells indicate the lack of physical interaction. However, a functional assay demonstrated that Mtk reduces the SDH activity of F. graminearum SQR but not that of its P. indica homolog. Therefore, the absence of an expected physical interaction in the F2H and BiFC assays may reflect the requirement for additional components that are present in the S. cerevisiae strain used for the original Y2H assay but not in the BHK-21 or tobacco cells, bearing in mind that S. cerevisiae is also a member of the Ascomycota (Moghaddam, 2017).

The mitochondrial enzyme SQR, which contains three further subunits in addition to the iron-sulfur subunit SdhB, plays an important role in both the Krebs cycle and the mitochondrial electron transport chain, both of which are essential for oxidative phosphorylation. SQR catalyzes the oxidation of succinate to fumarate in the mitochondrial matrix, which results in the reduction of ubiquinone to ubiquinol in the mitochondrial inner membrane. SdhB, which is located between SdhA and the two transmembrane subunits SdhC and SdhD, contains three iron-sulfur clusters that are required for tunneling the electrons through the complex. Coenzyme Q accepts the electrons from complexes I and II, and carries them to complex III, from where they are diverted to reduce the ubiquinone pool. The reducing equivalents reduce superoxide anions that accumulate from either exogenous sources or the respiratory chain. In contrast to other dehydrogenases in the Krebs cycle, SQR does not transport the succinate-derived electrons to soluble NAD+ intermediates but to the ubiquinone pool of the respiratory chain as an electron sink to provide antioxidants in the mitochondrial inner membrane. This study found that Mtk diminishes the overall mitochondrial SDH activity by up to 52%, which may lead to (1) higher levels of reactive oxygen species such as superoxide due to the depleted ubiquinone pool, and (2) loss of the electron gradient, thus compromising the survival of the fungus. The inhibition of SdhB by Mtk may arrest the delivery of electrons required for the full reduction of ubiquinone to ubiquinol, and may therefore increase oxygen toxicity in the mitochondria. On the other hand, a deficient electrochemical gradient across the mitochondrial inner membrane would inhibit the generation of ATP, which is coupled to the oxidation of nicotinamide adenine dinucleotide (NADH)/FADH2 and the reduction of oxygen to water within the respiratory chain25. Therefore, the inhibition of SDH activity by Mtk would impose a high fitness cost on F. graminearum, explaining the resistance of transgenic barley plants expressing Mtk against this pathogen. The ability of Mtk to inhibit SdhB and thus perturb the Krebs cycle could also disturb the equilibrium between the malate-aspartate shuttle and reducing equivalents, further restricting electron delivery from the mitochondrial inner membrane to the electron transport chain. Mutations in the subunits of mitochondrial complex II therefore increase oxidative stress and decrease longevity (Moghaddam, 2017).

Interestingly, Mtk did not inhibit the SDH activity of mitochondrial SQR in P. indica, providing a further explanation for the selective activity of Mtk against Ascomycota11. Although it was not possible to comment on the relative activities of SQR enzymes in F. graminearum and P. indica, the mitochondrial respiratory activity in the latter is so high that it can be detected in colonized plant roots using the SDH assay. Previous studies have already revealed that the selective activity of Mtk is partly due to the specific interaction between Mtk and the Ascomycota β(1,3)-glucanosyltransferase Gel1, which is only distantly related to the equivalent enzyme in the Basidiomycota. The phylogenetic analysis described in this study also revealed a significant divergence between the Ascomycota and Basidiomycota SdhB homologs, which provides a mechanistic basis for the selective inhibition that was observed. Mtk is therefore a highly promising antifungal candidate that is active against pathogenic Ascomycota but not against beneficial endophytic Basidiomycota such as P. indica due to its phylum-specific activity against at least two distinct enzymes (Moghaddam, 2017).

SQRs are useful targets for fungicide development. SDH inhibitors (SDHIs) that occupy either the succinate-binding pocket (e.g. malonate) or the ubiquinone-binding pocket (e.g. carboxamides) are highly effective against diverse fungal species. Amide fungicides target SQR in the mitochondrial respiratory chain resulting in growth arrest and even cell death by disrupting the mitochondrial Krebs cycle and interfering with respiration. All SDHIs currently used for crop protection target the ubiquinone-binding pocket, which is structurally defined by the interface among the subunits SdhB, SdhC and SdhD. However, mutations in SQR subunits have conferred SDHI resistance in 14 fungal species thus far, highlighting the need for new antifungal agents with diverse mechanisms of action. Although F. graminearum remains susceptible to chemical SDHIs for the time being, mutations that reduce susceptibility have been identified in a number of pathogens including B. cinerea, Podosphaera xanthii, Alternaria spp. and Sclerotinia sclerotiorum. Mtk could help to prevent or at least delay the emergence of resistant fungal pathogens because resistance would require the simultaneous mutation of multiple targets (Moghaddam, 2017).

In conclusion, this study has shown that Mtk attacks F. graminearum using at least two distinct mechanisms. First the integrity of the cell wall is compromised when Mtk interacts with the β(1,3)glucanosyltransferase Gel1 to inhibit the synthesis of cell wall polymers, and then the general metabolic fitness of the fungus is targeted by inhibiting the SDH activity of mitochondrial SQR, resulting in suboptimal energy generation. The fungus suffers a loss of fitness due to the combined impact of a weak cell wall, compromised energy generation, oxidative stress and slow metabolism. Mtk represents an ideal lead for the development of environmentally sustainable fungicides due to its selectivity and multiple intracellular targets, which reduces the impact on beneficial fungi and discourages the emergence of resistant pathogens (Moghaddam, 2017).

The insect-derived antimicrobial peptide metchnikowin targets Fusarium graminearum beta(1,3)glucanosyltransferase Gel1, which is required for the maintenance of cell wall integrity

Antimicrobial peptides (AMPs) are essential components of the insect innate immune system. Their diversity provides protection against a broad spectrum of microbes and they have several distinct modes of action. Insect-derived AMPs are currently being developed for both medical and agricultural applications, and their expression in transgenic crops confers resistance against numerous plant pathogens. The antifungal peptide Metchnikowin (Mtk), which was originally discovered in the fruit fly Drosophila melanogaster, is of particular interest because it has potent activity against economically important phytopathogenic fungi of the phylum Ascomycota, such as Fusarium graminearum, but it does not harm beneficial fungi such as the mycorrhizal basidiomycete Piriformospora indica. To investigate the specificity of Mtk, the peptide was used to screen a F. graminearum yeast two-hybrid library. This revealed that Mtk interacts with the fungal enzyme beta(1,3)-glucanosyltransferase Gel1 (FgBGT), which is one of the enzymes responsible for fungal cell wall synthesis. The interaction was independently confirmed in a second interaction screen using mammalian cells. FgBGT is required for the viability of filamentous fungi by maintaining cell wall integrity. This study therefore paves the way for further applications of Mtk in formulation of bio fungicides or as a supplement in food preservation (Moghaddam, 2017a).

Glycosylation reduces the glycan-independent immunomodulatory effect of recombinant Orysata lectin in Drosophila S2 cells

Several plant lectins, or carbohydrate-binding proteins, interact with glycan moieties on the surface of immune cells, thereby influencing the immune response of these cells. Orysata, a mannose-binding lectin from rice, has been reported to exert immunomodulatory activities on insect cells. While the natural lectin is non-glycosylated, recombinant Orysata produced in the yeast Pichia pastoris (YOry) is modified with a hyper-mannosylated N-glycan. Since it is unclear whether this glycosylation can affect the YOry activity, non-glycosylated rOrysata was produced in Escherichia coli (BOry). In a comparative analysis, both recombinant Orysata proteins were tested for their carbohydrate specificity on a glycan array, followed by the investigation of the carbohydrate-dependent agglutination of red blood cells (RBCs) and the carbohydrate-independent immune responses in Drosophila melanogaster S2 cells. Although YOry and BOry showed a similar carbohydrate-binding profiles, lower concentration of BOry were sufficient for the agglutination of RBCs and BOry induced stronger immune responses in S2 cells. The data are discussed in relation to different hypotheses explaining the weaker responses of glycosylated YOry. In conclusion, these observations contribute to the understanding how post-translational modification can affect protein function, and provide guidance in the selection of the proper expression system for the recombinant production of lectins (Chen, 2021).

Cecropins contribute to Drosophila host defense against a subset of fungal and Gram-negative bacterial infection

Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding cecropins are strongly induced upon infection, pointing to their role in host-defense. In Drosophila, four cecropin genes clustered in the genome (CecA1, CecA2, CecB and CecC) are expressed upon infection downstream of the Toll and Imd pathways. This study generated a short deletion ΔCecA-C removing the whole cecropin locus. Using the ΔCecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, this study addressed the function of cecropins in the systemic immune response. ΔCecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing ΔCecA-C in flies already lacking ten other AMP genes revealed a role for cecropins in defense against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, this work provides the first genetic demonstration of a role for cecropins in insect host defense, and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line (ΔAMP14) that lacks fourteen immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond (Carboni, 2021).

Glial immune-related pathways mediate effects of closed head traumatic brain injury on behavior and lethality in Drosophila

In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, this study developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, TBI-dependent behavior and lethality were examined in mutants of the master immune regulator NF-κB, important for AMP induction: while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects (van Alphen, 2022).

This study has developed a straightforward and reproducible Drosophila model for closed head TBI where precisely controlled strikes are delivered to the head of individually restrained, unanesthetized flies. This TBI paradigm is validated by recapitulating many of the phenotypes observed in mammalian TBI models, including increased mortality, increased neuronal cell death, impaired motor control, decreased/fragmented sleep, and hundreds of TBI-induced changes to the transcriptome, including the activation of many AMPs, indicating a strong activation of the immune response. These results set the stage to leverage Drosophila genetic tools to investigate the role of the immune response as well as novel pathways in TBI pathology (van Alphen, 2022).

The single fly paradigm is a more valid Drosophila model for TBI that circumvents the lack of specificity of currently available models or the use of anesthesia. Both previous assays induce TBI by subjecting the whole fly to trauma, which makes it hard to distinguish whether observed phenotypes are a due to TBI or a consequence of internal injuries. A recently published method uses a pneumatic device to strike an anesthetized fly's head. This method is an improvement of earlier assays and results in increased mortality in a stimulus strength-dependent manner. However, it only shows a modest reduction in locomotor activity, without demonstrating any other TBI-related phenotypes such as neuronal cell death or immune activation. The dependence on CO2 anesthesia further impairs the usefulness of this assay, as prolonged behavioral impairments in Drosophila occur even after brief exposure to CO2 anesthesia. Additionally, anesthetics that are administered either during or shortly after TBI induction can offer neuroprotective effects and alter cognitive, motor, and histological outcomes in mammalian models of TBI as well as affecting mortality in a whole body injury model in flies. The Drosophila model allows study of how TBI affects behavior and gene expression without the confounding effects of anesthesia, making it a more valid model for TBI that occurs under natural conditions (van Alphen, 2022).

The force used in this study (8.34 N) is higher than the force used in the HIT assay (2.5 N). When designing the TBI paradigm, several commercially available solenoids were tested for their ability to induce TBI, and the one that gave the best results was used. A higher force may be needed because brain damage is caused by the direct impact of the solenoid to the fly head, where the fly head moves with the solenoid rather than full body injury or compression injuries used in the other Drosophila TBI assays. Although it cannot be excluded that the neck is not damaged in this assay, cell death was observed in the central brain and significant changes in glia after TBI were observed, suggesting that TBI does occur (van Alphen, 2022).

This study also elucidate, in an unbiased manner, the genomic response to TBI. Glial cells play an important role in immune responses in both mammals and Drosophila, and changes to glial morphology and function were reported in earlier Drosophila TBI models. Until now, profiling TBI-induced changes in gene expression have either been limited to a small number of preselected genes in both mammals and Drosophila or focused on whole brain tissue rather than individual cell types. Using TRAP in combination with RNA-seq, previously reported up-regulation of Attacin-C, Diptericin-B, and Metchnikowin was validated. Additionally, an acute, broad-spectrum immune response was detected, where AMPs and stress response genes are up-regulated 24 hours after TBI. These include antibacterial, antifungal, and antiviral peptides as well as peptides from the Tot family, which are secreted as part of a stress response induced by bacteria, UV, heat, and mechanical stress. Although an increase in the heatshock protein 70 family of stress response genes was reported earlier, this study detected a significant glial up-regulation only in Hsp70BC (van Alphen, 2022).

Three days after TBI, only Attacin-C, Diptericin A, and Metchnikowin are up-regulated. Seven days after TBI, AMPs or stress response genes are not detectably up-regulated. These findings match reports in mammalian TBI models, where inflammatory gene expression spikes shortly after TBI but mostly dies down during subsequent days. Using CRISPR deletions of AMP classes, this study demonstrates that most AMPs not only protect against microbes but are also crucial in promoting survival after TBI. The exception is Defensin, as loss of this peptide increases survival, indicating that the Drosophila innate immune response to TBI can have both beneficial and detrimental effects. While loss of AMPs may render flies more susceptible to TBI, the hypothesis that AMP induction after TBI actively plays a role in mediating TBI effects is favored (van Alphen, 2022).

Besides validating the Drosophila model with the detection of a strongly up-regulated immune response, several novel genes were detected among the total of 512 different glial genes that were either up- or down-regulated after TBI. Immune and stress response only make up 157 out of 512 differentially expressed glial genes. Genes involved in proteolysis and protein folding are a prominent portion (85/512) of these differentially expressed genes, yet their role in TBI is poorly understood. These results demonstrate that there are other candidate pathways that may modulate recovery, and Drosophila can be used as a first line screen to test their in vivo function and to disentangle beneficial from detrimental responses (van Alphen, 2022).

This study has successfully applied in vivo genetics to identify in vivo pathways important for TBI. Loss of master immune regulator NF-κB results in increased mortality after TBI, yet it protects against TBI-induced impairments in sleep and motor control. These findings align with previous reports showing links between sleep and the immune response in flies where NF-κB is required to alter sleep architecture after exposure to septic or aseptic injuries. It will be of interest to determine if NF-κB is required for TBI-induced cell death. One possibility is that sleep impairments can be a side effect of melanization, an invertebrate defense mechanism that requires dopamine as melanin precursor. If dopamine is up-regulated to create more melanin, decreased sleep would be a side effect. Consistent with this hypothesis, changes were observed in fumin and pale, which likely result in increased dopamine levels (van Alphen, 2022).

However, the role of sleep after injury is complex. Two recent studies demonstrated that sleep is increased after antennal transection and facilitates Wallerian degeneration and glia-mediated clearance of axonal debris, suggesting that different types of injury have different effects on sleep. Interestingly, sleep disturbances can increase the up-regulation of immune genes. Thus, it is possible that decreased sleep after TBI contributes to survival by stimulating the immune response. Some support is found for this hypothesis in the difference in TBI-induced changes to sleep in flies that survive 7 days of TBI versus flies that die within 7 days after TBI, where the survivors sleep significantly less for 4 days post-TBI and dying flies sleep is nearly unaffected. Additionally, immune response genes are up-regulated for up to 3 days after TBI, which correlates with the observed sleep impairments. Also, the engulfment receptor Draper, which mediates Wallerian degeneration, is not up-regulated in the glial TRAP-seq data, suggesting that Wallerian degeneration, and its accompanying increase in sleep, is not part of the response to dCHI (van Alphen, 2022).

TBI results in impaired climbing behavior that persists for up to 7 days, yet impairments to sleep disappear after a few days. Recently, it was shown that TBI through head compression results in impaired memory, as quantified through courtship conditioning, indicating that TBI also results in persistent memory defects (van Alphen, 2022).

Recently, it was shown that repressing neuronal NF-κB in a mouse model of TBI increases post-TBI mortality, as in the current studies, without reducing behavioral impairments, suggesting that nonneuronal NF-κB could underlie behavioral impairments after TBI. We demonstrate that behavioral responses to TBI (for example, sleep and geotaxis) are abolished in mutants of the transcription factor NF-κB Relish, which plays a central role in regulating stress-associated and inflammatory gene expression in both mammals and flies. Nonetheless, Relish null mutants show increased mortality after TBI, but none of the behavioral impairments observed in wild-type flies, indicating that these impairments might be a side effect of immune activation rather than direct injury. The demonstration of an in vivo role for TBI-regulated genes will be important for defining their function (van Alphen, 2022).

In summary, the dCHI assay recapitulates many of the physiological symptoms observed in mammals, demonstrating that fruit flies are a valid model to study physiological responses to TBI. Both a potent induction of immune pathways and a requirement for an immune master regulator was demonstrated in mediating TBI effects on behavior. This model now provides a platform to perform unbiased genetic screens to study how gene expression changes after TBI in unanesthetized, awake animals result in the long-term sequelae of TBI. These studies raise the possibility of rapidly identifying key genes and pathways that are neuroprotective for TBI, thereby providing a high-throughput approach that could facilitate the discovery of novel genes and therapeutics that offer better outcomes after TBI (van Alphen, 2022).

Heterochromatic silencing of immune-related genes in glia is required for BBB integrity and normal lifespan in drosophila

Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. This study reports that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, a series of lifespan rescue experiments was conducted and the results indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, this study showed that GLAD encoded a heterochromatin-associating protein that bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, these findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan (Shu, 2023).

Aging is thought to be associated with decreased immune functions (immune-paralysis) and increased pro-inflammatory activity (inflamm-aging). Several cellular processes are considered contributing factors to the age-related changes in immunity and inflammation. For example, (1) the adaptive immune responses decrease with age, leading to accumulation of pathogens and cellular stress, which activate inflammatory responses such as the JAK/STAT pathway; (2) senescent cells accumulate in aging, which secrete a range of inflammatory cytokines, and chemokines; (3) the microbial load and pathogen diversity that one is exposed to grow with age; and, (4) AMPs of the IMD pathway are upregulated in the brain of aged animals even when reared in germ-free conditions, suggesting a pathogen-independent mechanism underlying the age-associated immune overactivation in the nervous system during aging (Shu, 2023).

The findings of this study add a new paradigm for age-related dysregulation of the innate immunity-the decline of heterochromatic silencing of immune-related genes in fly glia. This study shows that the Drosophila gene GLAD encodes a BAH domain-containing protein, which is localized to the heterochromatin and binds to the promotors of an array of immune-related genes to keep them silenced. With age, GLAD expression and GLAD-mediated heterochromatic silencing decrease. Meanwhile, the innate immunity including the expression of AMP genes is increased in the brain of aged flies. A progressive heterochromatin loss also led to deregulation of the genes involved in immune responses in the gut of aged flies. Thus, it is possible that the heterochromatin-mediated immune silencing may be an underappreciated mechanism that is employed by other tissues and systems to keep immunity on a leash as well. In addition, it will be interesting to investigate how GLAD affects the cell number of glia in the fly brain in the future. This could not be done because the strong deleterious effect of RNAi-GLAD made it difficult to accurately assess the cell number of glia or the effect of GLAD on the cell cycle in vivo in this study (Shu, 2023).

It should be pointed out that, although this work focuses on glia and immunity, GLAD does not function 'only' in glia. Nor does it 'only' repress immune-related genes. With that said, the RNA-seq analysis of the TubGS > RNAi-GLAD fly heads indeed indicates that the most affected molecular pathways are enriched in immune and related functions. It is interesting to note that the immune genes in the Drosophila genome display a significant excess of clustering in the chromatin. And, some heterochromatin domains are enriched for genes involved in immunity in both fly and mammalian cells. Similarly, the genes of the GstD family are also clustered in the fly genome. Heterochromatin is spatially more complex and dynamic than previously thought, and a network of subdomains can regulate diverse heterochromatin functions. Thus, the chromosomal arrangement of the fly genome may facilitate the recruitment and repression of selected heterochromatin subdomains enriched for immune-related genes. In addition, several upstream PGRP genes (e.g., PGRP-SD and PGRP-SA) are upregulated in the TubGS > RNAi-GLAD flies, which may enhance the overactivation of the IMD and Toll immune pathways, thereby further boosting the upregulation of the downstream effector AMPs (Shu, 2023).

Widespread, age-dependent BBB disintegration is associated with physiological aging and may be an early pathological hallmark of several human diseases. In particular, the BBB is vulnerable to systemic overactivation of immune and inflammation in neurological disorders. This study shows that local overactivation of the innate immunity in fly glia, by either KD of GLAD or OE of Imd, is sufficient to cause BBB breakdown. With the leaky BBB, immune and inflammatory factors released from glia and immune cells may leak out of the nervous system and get into the circulation system (and vice versa), which may further augment local and systemic immune and inflammatory responses to accelerate aging. Indeed, glial KD of GLAD or OE of Imd leads to age-dependent brain degeneration and dramatically reduces longevity in flies. Of note, although the anatomy of the fly BBB is different from that of mammals, they are organized and function in a similar manner. For example, the mammalian BBB is formed by a tightly sealed monolayer of brain endothelial cells that are connected by tight junctions (TJs); whereas in the fly BBB, the SPGs are connected to each other by septate junctions (SJs). The SJs function as the paracellular barrier like the mammalian TJs and disruption of SJs impairs the fly BBB. Moreover, Claudin is a key component of the mammalian TJs and the fly SJs contains Claudin-like molecules, suggesting the evolutionary conserved molecular organization of the TJs and the SJs. Therefore, it would be interesting to investigate how the GLAD-regulated, age-associated effector AMPs and cytokines and their mammalian counterparts affect BBB permeability in the future, which may provide new mechanistic insights about immune overactivation and BBB disintegration in aging and neurodegenerative diseases (Shu, 2023).

Finally, Bahd1 is the putative mammalian homologue of GLAD, which encodes a core protein of the heterochromatin-repressive complex. BAHD1 silences IFN-stimulated genes and modulates innate immune defense in response to bacterial infection. The Bahd1 null mutation causes significant perinatal death in mice, whilst the Bahd1+/− heterozygotes exhibit anxiety-like behaviors suggesting a potential role of BAHD1 in the rodent nervous system. Future research on GLAD and BAHD1 is warranted to reveal the evolutionarily conserved and the species-specific mechanisms in heterochromatin-mediated immune gene silencing and how they impact on brain aging and longevity (Shu, 2023).

An anti-inflammatory transcriptional cascade conserved from flies to humans

Innate immunity is an ancestral process that can induce pro- and anti-inflammatory states. A major challenge is to characterize transcriptional cascades that modulate the response to inflammation. Since the Drosophila glial cells missing (Gcm) transcription factor has an anti-inflammatory role, this study explored its regulation and evolutionary conservation. The murine Gcm2 (mGcm2) gene was shown to be expressed in a subpopulation of aged microglia (chronic inflammation) and upon lysophosphatidylcholine (LPC)-induced central nervous system (CNS) demyelination (acute inflammation). Moreover, mGcm2 conditional knockout mice show an increased inflammatory phenotype upon aging or LPC injection, and hGCM2 is expressed in active demyelinating lesions of patients with multiple sclerosis. Finally, Drosophila Gcm expression is induced upon aging and acute challenge, and its overexpression decreases the inflammatory phenotype. Altogether, these data indicate that the inducible Gcm cascade is conserved from flies to humans and represents a potential therapeutic target in the control of the inflammatory response (Pavlidaki, 2022).

Ageing leads to reduced specificity of antimicrobial peptide responses in Drosophila melanogaster

Evolutionary theory predicts a late-life decline in the force of natural selection, possibly leading to late-life deregulations of the immune system. A potential outcome of such deregulations is the inability to produce specific immunity against target pathogens. This possibility was tested by infecting multiple Drosophila melanogaster lines (with bacterial pathogens) across age groups, where either individual or different combinations of Imd- and Toll-inducible antimicrobial peptides (AMPs) were deleted using CRISPR gene editing. A high degree of non-redundancy and pathogen-specificity of AMPs was demonstrated in young flies: in some cases, even a single AMP could confer complete resistance. However, ageing led to drastic reductions in such specificity to target pathogens, warranting the action of multiple AMPs across Imd and Toll pathways. Moreover, use of diverse AMPs either lacked survival benefits or even accompanied survival costs post-infection. These features were also sexually dimorphic: females required a larger repertoire of AMPs than males but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP-repertoire was accompanied with ageing-induced downregulation of negative-regulators of the Imd pathway and damage to renal function post-infection, as features of poorly regulated immunity. Overall, this study highlights the potentially non-adaptive role of ageing in producing less-specific AMP responses, across sexes and pathogens (Shit, 2022).

SPS1 deficiency-triggered PGRP-LC and Toll expression controls innate immunity in Drosophila S2 cells

Selenophosphate synthetase 1 (SPS1) is an essential gene for the cell growth and embryogenesis in Drosophila melanogaster. A previous study reported that SPS1 deficiency stimulates the expression of genes responsible for the innate immune system, including antimicrobial peptides (AMPs), in Drosophila S2 cells. However, the underlying mechanism has not been elucidated. This study investigated the immune pathways that control the SPS1-deficiency-induced expression of AMPs in S2 cells. It was found that the activation of AMP expression is regulated by both immune deficiency (IMD) and the Toll pathway. Double knockdown of the upstream genes of each pathway with SPS1 showed that the peptidoglycan recognition protein-LC (PGRP-LC) and Toll genes are targeted by SPS1 for regulating these pathways, respectively. It was also found that the IMD and Toll pathway regulate AMP expression by cross-talking. The levels of PGRP-LC and Toll mRNAs were upregulated upon Sps1 knockdown (6.46±0.36 and 3.2±0.45-fold, respectively n=3). Overexpression of each protein also upregulated AMPs. Interestingly, PGRP-LC overexpression upregulated AMP more than Toll overexpression. These data strongly suggest that SPS1 controls the innate immune system of D. melanogaster through regulating PGRP-LC and Toll expression (Woo, 2022).

Accessibility to peptidoglycan is important for the recognition of gram-positive bacteria in Drosophila

In Drosophila, it is thought that peptidoglycan recognition proteins (PGRPs) SA and LC structurally discriminate between bacterial peptidoglycans with lysine (Lys) or diaminopimelic (DAP) acid, respectively, thus inducing differential antimicrobial transcription response. This study finds that accessibility to PG at the cell wall plays a central role in immunity to infection. When wall teichoic acids (WTAs) are genetically removed from S. aureus (Lys type) and Bacillus subtilis (DAP type), thus increasing accessibility, the binding of both PGRPs to either bacterium is increased. PGRP-SA and PFRP-LC double mutant flies are more susceptible to infection with both WTA-less bacteria. In addition, WTA-less bacteria grow better in PGRP-SA/-LC double mutant flies. Finally, infection with WTA-less bacteria abolishes any differential activation of downstream antimicrobial transcription. These results indicate that accessibility to cell wall PG is a major factor in PGRP-mediated immunity and may be the cause for discrimination between classes of pathogens (Vaz, 2019).

In Drosophila, the generally accepted explanation for selective activation of immune pathways at the recognition level has been that PGRPs structurally discriminate between different types of PGs. This discrimination occurs on the basis of the amino acid present at position 3 of the stem peptide. Thus, PGRP-SA recognizes Lys (found in Gram-positive bacteria), while PGRP-LC interacts with DAP (found in Gram-negative bacteria and Gram-positive bacilli). The idea of structural discrimination at the level of the stem peptide resulted from a combination of observations, including PG binding assays, structural work, and in vivo infection experiments. However, there are several concerns with the experiments that support this Lys/DAP dichotomy or with the interpretation of the data. A summary of these follows (Vaz, 2019).

First, the PG binding experiments were conducted with a buffer that is often used to solubilize proteins, but that was very different from hemolymph (where interactions between PG and PGRPs take place). Second, the PG used for binding as well as infection experiments was quantified only by weight and not by the number of disaccharide GlcNAc-MurNAC added to the binding reaction. Third, the structural data available and previously described referred to the binding of monomeric PG, whereas PGRP-SA and PGRP-LCx are both able to bind polymeric PG and may do so in vivo. In the context of polymeric PG binding, the small difference of a carboxyl group on the Cε with d-chirality in DAP may not be as important for downstream signaling. Moreover, superposition of the two structures showed that the electrostatic potential at the surface of both PGRPs suggests that binding to both types of PG in solution is probable (Vaz, 2019).

Based on the published structure, the defining characteristic of the binding groove is the conformation of Arg413, which securely anchors DAP-type PG and is likely to impose a preference for the latter via increased conformational stability. This preference was verified with quantitative binding to Lys and DAP-type PG, in which the latter was bound in increased quantities by PGRP-LC. Nevertheless, PGRP-LC did bind Lys-type PG in vitro. Moreover, binding of PGRP-LC to Lys-type bacteria was recorded ex vivo, and this binding resulted in a robust AMP induction that was statistically higher than the one elicited through PGRP-SA, considered the bona fide receptor for Gram-positive bacteria. This indicated that the upstream binding preferences of PGRP-LC did not result in differences in transcriptional AMP induction (Vaz, 2019).

For PGRP-SA, replacement of the anchoring Arg413 from PGRP-LC with a threonine should leave an accommodating void that can flexibly bind via a water molecule network either types of PG. In this case, there should be no noticeable difference in binding affinity. Little difference was found between PGRP-SA binding to Lys PG compared with DAP-type PG (Vaz, 2019).

These results showed that when WTAs were removed, the binding of PGRPs to whole bacteria was significantly improved. This, however, was not reflected in binding assays to purified PG, showing that the increase in binding was about accessibility to PG on the bacterial cell wall, not about changing the WTA-less PG itself. This is consistent with the fact that purified PG from WTA-less bacteria and their parental strains had no significant differences in their HPLC profile analysis that could lead to an improved binding of PGRP-SA or PGRP-LCs (Vaz, 2019).

The above results show that when WTAs were removed from bacteria, the majority of the observed phenotype attributed to selective recognition and downstream signaling based on the bacterial type were either abolished or diminished. This was accompanied by PGRP binding to whole bacteria, irrespective of PG type, in which both PGRPs played a role in host survival and restricting pathogen growth. These results are in conflict with the general consensus of Lys/DAP discrimination and put forward an alternative explanation for differential immune triggering. It is proposed that at least for bacteria that have their PGs exposed to the host environment (Gram-positive bacteria and bacilli), accessibility to PG is a major factor restricting binding, recognition, and downstream signaling. Structural discrimination may still be important when monomeric PG fragments are the only means of 'accessing' PG (in Gram-negative bacteria) (Vaz, 2019).

A fundamental issue when considering pathogen recognition in innate immunity is how a small number of germline coded receptors sense the vast variability of bacterial pathogens. Adapting receptors to an evolutionary conserved bacterial molecule essential for bacterial survival but not present in the host (e.g., PG), has been considered a productive host strategy. However, it is believed that it would have been detrimental for the host to further specialize its non-enzymatic PGRP receptors to PG-stem peptide variants, as this would essentially decrease even more the effective number of possible productive host-pathogen recognition interactions, especially in an animal such as Drosophila, challenged by a wide variety of pathogens, each of which interacts rarely with it. Put it another way, Drosophila is not subject to the arms race-type tight co-evolution with its pathogens that Anopheles is, for example, but it is subject to diffuse co-evolution with an array of non-specialist pathogens (Vaz, 2019).

Evolutionary studies on Drosophila PGRPs (including SA and LC) have shown that there is no strong co-evolution interaction, and as such, no need for specialization. It is proposed that it was the accessibility to PGs on different bacterial cell wall structures that defined how PGRPs were able to bind to PG on whole bacteria and the specified differences in downstream signaling responses, instead of direct structural specialization, as previous studies suggested. Thse results indicate that even when structural preference exists (as in the case of PGRP-LC), differences in downstream signaling are equalized when accessibility to PG on the cell wall is increased. More work is needed to test whether accessibility is also critical for PGRP-mediated recognition of whole bacteria in mammals (Vaz, 2019).

Based on the published structure, the defining characteristic of the binding groove is the conformation of Arg413, which securely anchors DAP-type PG and is likely to impose a preference for the latter via increased conformational stability. This preference was verified with quantitative binding to Lys and DAP-type PG, in which the latter was bound in increased quantities by PGRP-LC. Nevertheless, PGRP-LC did bind Lys-type PG in vitro. Moreover, binding of PGRP-LC to Lys-type bacteria was recorded ex vivo, and this binding resulted in a robust AMP induction that was statistically higher than the one elicited through PGRP-SA, considered the bona fide receptor for Gram-positive bacteria. This indicated that the upstream binding preferences of PGRP-LC did not result in differences in transcriptional AMP induction (Vaz, 2019).

For PGRP-SA, replacement of the anchoring Arg413 from PGRP-LC with a threonine should leave an accommodating void that can flexibly bind via a water molecule network either types of PG. In this case, there should be no noticeable difference in binding affinity. Little difference was found between PGRP-SA binding to Lys PG compared with DAP-type PG (Vaz, 2019).

The results showed that when WTAs were removed, the binding of PGRPs to whole bacteria was significantly improved. This, however, was not reflected in binding assays to purified PG, showing that the increase in binding was about accessibility to PG on the bacterial cell wall, not about changing the WTA-less PG itself. This is consistent with the fact that purified PG from WTA-less bacteria and their parental strains had no significant differences in their HPLC profile analysis that could lead to an improved binding of PGRP-SA or PGRP-LC (Vaz, 2019).

Drosophila immunity: the Drosocin gene encodes two host defence peptides with pathogen-specific roles

Antimicrobial peptides (AMPs) are key to defence against infection in plants and animals. Use of AMP mutations in Drosophila has now revealed that AMPs can additively or synergistically contribute to defence in vivo. However, these studies also revealed high specificity, wherein just one AMP contributes an outsized role in combatting a specific pathogen. This study shows the Drosocin locus (CG10816) is more complex than previously described. In addition to its namesake peptide 'Drosocin', it encodes a second mature peptide from a precursor via furin cleavage. This peptide corresponds to the previously uncharacterized 'Immune-induced Molecule 7'. A polymorphism (Thr52Ala) in the Drosocin precursor protein previously masked the identification of this peptide, which this paper names 'Buletin'. Using mutations differently affecting Drosocin and Buletin, this studybshow that only Drosocin contributes to Drosocin gene-mediated defence against Enterobacter cloacae. Strikingly, it was observed that Buletin, but not Drosocin, contributes to the Drosocin gene-mediated defence against Providencia burhodogranariea, including an importance of the Thr52Ala polymorphism for survival. This study reveals that the Drosocin gene encodes two prominent host defence peptides with different specificity against distinct pathogens. This finding emphasizes the complexity of the Drosophila humoral response and demonstrates how natural polymorphisms can affect host susceptibility (Hanson, 2022).

Repeated truncation of a modular antimicrobial peptide gene for neural context

Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading pathogens. These genes commonly encode multiple products as post-translationally cleaved polypeptides. Recent studies have highlighted roles for AMPs in neurological contexts suggesting functions for these defence molecules beyond infection. During an immune study characterizing the antimicrobial peptide gene Baramicin, multiple Baramicin paralogs were uncovered in Drosophila melanogaster and other species, united by their N-terminal IM24 domain. Not all paralogs were immune-induced. In this study, through careful dissection of the Baramicin family's evolutionary history, it was found that paralogs lacking immune induction result from repeated events of duplication and subsequent truncation of the coding sequence from an immune-inducible ancestor. These truncations leave only the IM24 domain as the prominent gene product. Surprisingly, using mutation and targeted gene silencing it was demonstrated that two such genes are adapted for function in neural contexts in D. melanogaster. Enrichment in the head was found for independent Baramicin genes in other species. The Baramicin evolutionary history reveals that the IM24 Baramicin domain is not strictly useful in an immune context. This study thus provides a case study for how an AMP-encoding gene might play dual roles in both immune and non-immune processes via its multiple peptide products. As many AMP genes encode polypeptides, a full understanding of how immune effectors interact with the nervous system will require consideration of all their peptide products (Hanson, 2022).

Ecology-relevant bacteria drive the evolution of host antimicrobial peptides in Drosophila

Antimicrobial peptides are host-encoded immune effectors that combat pathogens and shape the microbiome in plants and animals. However, little is known about how the host antimicrobial peptide repertoire is adapted to its microbiome. This study characterized the function and evolution of the Diptericin antimicrobial peptide family of Diptera. Using mutations affecting the two Diptericins (Dpt) of Drosophila melanogaster, the specific role of DptA for the pathogen Providencia rettgeri and DptB for the gut mutualist Acetobacter. The presence of DptA- or DptB-like genes across Diptera correlates with the presence of Providencia and Acetobacter in their environment. Moreover, DptA- and DptB-like sequences predict host resistance against infection by these bacteria across the genus Drosophila. This study explains the evolutionary logic behind the bursts of rapid evolution of an antimicrobial peptide family and reveals how the host immune repertoire adapts to changing microbial environments (Hanson, 2023).

Defective phagocytosis leads to neurodegeneration through systemic increased innate immune signaling

In nervous system development, disease, and injury, neurons undergo programmed cell death, leaving behind cell corpses that are removed by phagocytic glia. Altered glial phagocytosis has been implicated in several neurological diseases including Alzheimer's disease. To untangle the links between glial phagocytosis and neurodegeneration, Drosophila mutants lacking the phagocytic receptor Draper were investigated. Loss of Draper leads to persistent neuronal cell corpses and age-dependent neurodegeneration. Whether the phagocytic defects observed in draper mutants lead to chronic increased immune activation that promotes neurodegeneration was investigate. It was found that the antimicrobial peptide Attacin-A is highly upregulated in the fat body of aged draper mutants and that the inhibition of the Immune deficiency (Imd) pathway in the glia and fat body of draper mutants led to reduced neurodegeneration. Taken together, these findings indicate that phagocytic defects lead to neurodegeneration via increased immune signaling, both systemically and locally in the brain (Elguero, 2023).

A suite of selective pressures supports the maintenance of alleles of a Drosophila immune peptide

The innate immune system provides hosts with a crucial first line of defense against pathogens. While immune genes are often among the fastest evolving genes in the genome, in Drosophila, antimicrobial peptides (AMPs) are notable exceptions. Instead, AMPs may be under balancing selection, such that over evolutionary timescales multiple alleles are maintained in populations. This study focused on the Drosophila antimicrobial peptide Diptericin A, which has a segregating amino acid polymorphism associated with differential survival after infection with the Gram-negative bacteria Providencia rettgeri. Diptericin A also helps control opportunistic gut infections by common Drosophila gut microbes, especially those of Lactobacillus plantarum. In addition to genotypic effects on gut immunity, strong sex-specific effects are also seen that are most prominent in flies without functional Diptericin A. To further characterize differences in microbiomes between different diptericin genotypes, 16S metagenomics was use to look at the microbiome composition. Both lab reared and wild caught flies were used for sequencing and overall composition was looked at as well as the differential abundance of individual bacterial families. Overall, flies were found that are homozygous serine for diptericin A are better equipped to survive a systemic infection from P. rettgeri, but in general homozygous arginine flies have a longer lifespan after being fed common gut commensals. These results suggest a possible mechanism for the maintenance of genetic variation of diptericin A through the complex interactions of sex, systemic immunity, and the maintenance of the gut microbiome (Mullinax, 2023)

Predicting structural features of selected flavonoids responsible for neuroprotection in a Drosophila model of Parkinson's disease

Nature-derived bioactive compounds have emerged as promising candidates for the prevention and treatment of diverse chronic illnesses, including neurodegenerative diseases. However, the exact molecular mechanisms underlying their neuroprotective effects remain unclear. Most studies focus solely on the antioxidant activities of natural products which translate to poor outcome in clinical trials. Current therapies against neurodegeneration only provide symptomatic relief, thereby underscoring the need for novel strategies to combat disease onset and progression. This study has employed an environmental toxin-induced Drosophila Parkinson's disease (PD) model as an inexpensive in vivo screening platform to explore the neuroprotective potential of selected dietary flavonoids. A specific group of flavonoids known as flavones displaying protection against paraquat (PQ)-induced neurodegenerative phenotypes was indentified involving reduced survival, mobility defects, and enhanced oxidative stress. Interestingly, the other groups of investigated flavonoids, namely, the flavonones and flavonols failed to provide protection indicating a requirement of specific structural features that confer protection against PQ-mediated neurotoxicity in Drosophila. Based on this screen, the neuroprotective flavones lack a functional group substitution at the C3 and contain α,β-unsaturated carbonyl group. Furthermore, flavones-mediated neuroprotection is not solely dependent on antioxidant properties through nuclear factor erythroid 2-related factor 2 (Nrf2) but also requires regulation of the immune deficiency (IMD) pathway involving NFκB and the negative regulator poor Imd response upon knock-in (Pirk). These data have identified specific structural features of selected flavonoids that provide neuroprotection against environmental toxin-induced PD pathogenesis that can be explored for novel therapeutic interventions (Maitra, 2023).

The NF-kappaB/Relish Activates miR-308 to Negatively Regulate Imd Pathway Immune Signaling in Drosophila

The strength and duration of the NF-κB signaling response must be tightly modulated to avoid an inadequate or excessive immune response. Relish, a core NF-7kappa;B transcription factor of the Drosophila Imd pathway, can control the expression of antimicrobial peptides, including Dpt and AttA, to defend against Gram-negative bacterial infections, but whether Relish may regulate miRNA expression to participate in the immune response remains unclear. In this study, taking advantage of Drosophila S2 cells and different overexpression/knockout/knockdown flies, it was first found that Relish could directly activate the expression of miR-308 to negatively regulate the immune response and promote the survival of Drosophila during Enterobacter cloacae infection. Second, the results demonstrated that Relish-mediated expression of miR-308 could suppress target gene Tab2 to attenuate the Drosophila Imd pathway signal during the middle and late stages of the immune response. Third, the dynamic expression patterns of Dpt, AttA, Relish, miR-308, and Tab2 was detected in wild-type flies after E. coli infection, which further revealed that the feedback regulatory loop of Relish-miR-308-Tab2 plays a crucial role in the immune response and homeostasis maintenance of the Drosophila Imd pathway. Overall, this study not only illustrates an important mechanism by which this Relish-miR-308-Tab2 regulatory axis can negatively control the Drosophila immune response and participate in homeostasis maintenance but also provides new insights into the dynamic regulation of the NF-κB/miRNA expression network of animal innate immunity (Yao, 2023).

The Relish/miR-275/Dredd mediated negative feedback loop is crucial to restoring immune homeostasis of Drosophila Imd pathway

The NF-κB/Relish, as a core transcription factor of Drosophila immune deficiency (Imd) pathway, activates the transcriptions of antimicrobial peptides (AMPs) to combat gram-negative bacterial infections, but its role in regulating miRNA expression during immune response has less been reported. This study describes a negative feedback loop of Imd signaling mediated by Relish/miR-275/Dredd that controls Drosophila immune homeostasis after Escherichia coli (E. coli) infection. The results demonstrate that Relish may directly activate the transcription of miR-275 via binding to its promoter in vitro and vivo, particularly miR-275 further inhibits the expression of Dredd through binding to its 3'UTR to negatively control Drosophila Imd immune response. Remarkably, the ectopic expression of miR-275 significantly reduces Drosophila lifespan. More importantly, this work uncovers a new mechanism by which Relish can flexibly switch its role to maintain Drosophila immune response and homeostasis during infection. Collectively, this study not only reveals the functional duality of Relish in regulating immune response of Drosophila Imd pathway, but also provides a new insight into the maintenance of animal innate immune homeostasis (Pan, 2023).

IMD signaling in the gut and the brain modulates Amyloid-beta-induced deficits in Drosophila

Evidence indicates accumulating Aβ peptides (see Drosophila Appl) in brain activates immune responses in neuronal and peripheral system, which may collaboratively influence pathogenesis of Alzheimer's disease (AD). This study aimed to investigate whether regulating intestinal innate immune signaling ameliorates Aβ-induced impairments in Drosophila melanogaster. Quantitative polymerase chain reaction (qPCR) was used to observe expression changes of innate immune responses related genes in brain and in gut under the circumstance of Aβ overexpressing in nerve system. Aversive olfactory conditioning and survival assay were used to investigate effects of modulating Attacin-A (AttA) and Dpitercin-A (DptA). Fluorometric assays of respiratory burst activity was introduced to explore whether reducing oxidative stress enables overexpressing intestinal AttA and DptA to reverse Aβ-induced deficits. In vivo genetic analysis revealed that accumulating Aβ42 in neurons modulates innate immune signaling of the IMD pathway both in the brain and in the gut. Increased expression levels of the intestinal AttA and DptA improved learning performance and extended the lifespan of Aβ42 flies. The administration of apramycin led to alleviations of Aβ-induced behavioral changes, indicating that gram-negative bacteria are associated with the development of Aβ-induced pathologies. Further analysis showed that the neural expression of Aβ42 increased oxidative stress in the gut, which disrupted intestinal integrity and decreased learning performance. In addition, increased levels of AMPs targeting gram-negative bacteria and antioxidants reduced oxidative stress in the gut and reversed Aβ-induced behavioral damage. These findings suggest that innate immune responses in the gut play a pivotal role in modulating Aβ-induced pathologies (Hsieh, 2023).

Stochastic variation in the initial phase of bacterial infection predicts the probability of survival in D. melanogaster

A central problem in infection biology is understanding why two individuals exposed to identical infections have different outcomes. This study has developed an experimental model where genetically identical, co-housed Drosophila given identical systemic infections experience different outcomes, with some individuals succumbing to acute infection while others control the pathogen as an asymptomatic persistent infection. Differences in bacterial burden at the time of death did not explain the two outcomes of infection. Inter-individual variation in survival stems from variation in within-host bacterial growth, which is determined by the immune response. A model was developed that captures bacterial growth dynamics and identifies key factors that predict the infection outcome: the rate of bacterial proliferation and the time required for the host to establish an effective immunological control. The results provide a framework for studying the individual host-pathogen parameters governing the progression of infection and lead ultimately to life or death (Duneau, 2017).

Mechanical stress to Drosophila larvae stimulates a cellular immune response through the JAK/STAT signaling pathway

Acute inflammation can cause serious tissue damage and disease in physiologically-challenged organisms. The precise mechanisms leading to these detrimental effects remain to be determined. This study utilized a reproducible means to induce cellular immune activity in Drosophila larvae in response to mechanical stress. That is, forceps squeeze-administered stress induces lamellocytes, a defensive hemocyte type that normally appears in response to wasp infestation of larvae. The posterior signaling center (PSC) is a cellular microenvironment in the larval hematopoietic lymph gland that is vital for lamellocyte induction upon parasitoid attack. However, the PSC was not required for mechanical stress-induced lamellocyte production. In addition, it was observed that mechanical injury caused a systemic expression of Unpaired3. This cytokine is both necessary and sufficient to activate the cellular immune response to the imposed stress. These findings provide new insights into the communication between injured tissues and immune system induction, using stress-challenged Drosophila larvae as a tractable model system (Tokusumi, 2018).

Endosymbiont-based immunity in Drosophila melanogaster against parasitic nematode infection

Associations between endosymbiotic bacteria and their hosts represent a complex ecosystem within organisms ranging from humans to protozoa. Drosophila species are known to naturally harbor Wolbachia and Spiroplasma endosymbionts, which play a protective role against certain microbial infections. Thhis study investigated whether the presence or absence of endosymbionts affects the immune response of Drosophila melanogaster larvae to infection by Steinernema carpocapsae nematodes carrying or lacking their mutualistic Gram-negative bacteria Xenorhabdus nematophila (symbiotic or axenic nematodes, respectively). The presence of Wolbachia alone or together with Spiroplasma was found to promote the survival of larvae in response to infection with S. carpocapsae symbiotic nematodes, but not against axenic nematodes. Wolbachia numbers are reduced in Spiroplasma-free larvae infected with axenic compared to symbiotic nematodes, and they are also reduced in Spiroplasma-containing compared to Spiroplasma-free larvae infected with axenic nematodes. It was further shown that S. carpocapsae axenic nematode infection induces the Toll pathway in the absence of Wolbachia, and that symbiotic nematode infection leads to increased phenoloxidase activity in D. melanogaster larvae devoid of endosymbionts. Finally, infection with either type of nematode alters the metabolic status and the fat body lipid droplet size in D. melanogaster larvae containing only Wolbachia or both endosymbionts. These results suggest an interaction between Wolbachia endosymbionts with the immune response of D. melanogaster against infection with the entomopathogenic nematodes S. carpocapsae. Results from this study indicate a complex interplay between insect hosts, endosymbiotic microbes and pathogenic organisms (Yadav, 2018).

Transcript analysis reveals the involvement of NF-kappaB transcription factors for the activation of TGF-beta signaling in nematode-infected Drosophila

The common fruit fly Drosophila melanogaster is a powerful model for studying signaling pathway regulation. Conserved signaling pathways underlying physiological processes signify evolutionary relationship between organisms and the nature of the mechanisms they control. This study explores the cross-talk between the well-characterized nuclear factor kappa B (NF-kappaB) innate immune signaling pathways and transforming growth factor beta (TGF-beta) signaling pathway in response to parasitic nematode infection in Drosophila. To understand the link between signaling pathways, a transcript-level analysis of different TGF-beta signaling components was performed following infection of immune-compromised Drosophila adult flies with the nematode parasites Heterorhabditis gerrardi and H. bacteriophora. The findings demonstrate the requirement of NF-kappaB transcription factors for activation of TGF-beta signaling pathway in Drosophila in the context of parasitic nematode infection. Significant decrease were observed in transcript level of glass bottom boat (gbb) and screw (scw), components of the bone morphogenic protein (BMP) branch, as well as Activinbeta (actbeta) which is a component of the Activin branch of the TGF-beta signaling pathway. These results are observed only in H. gerrardi nematode-infected flies compared to uninfected control. Also, this significant decrease in transcript level is found only for extracellular ligands. Future research examining the mechanisms regulating the interaction of these signaling pathways could provide further insight into Drosophila anti-nematode immune function against infection with potent parasitic nematodes (Patrnogic, 2019).

Participation of the serine protease Jonah66Ci in the Drosophila anti-nematode immune response

Serine proteases and serine protease homologs form the second largest gene family in the Drosophila melanogaster genome. Certain genes in the Jonah multi-gene family encoding serine proteases have been implicated in the fly antiviral immune response. This study reports the involvement of Jonah66Ci in the Drosophila immune defense against Steinernema carpocapsae nematode infection. Jonah66Ci is upregulated in response to symbiotic (carrying the mutualistic bacteria Xenorhabdus nematophila) or axenic (lacking Xenorhabdus) Steinernema nematodes and is expressed exclusively in the gut of Drosophila larvae. Inactivation of Jonah66Ci provides a survival advantage to larvae against axenic nematodes and results in differential expression of Toll and Imd pathway effector genes, specifically in the gut. Also, inactivation of Jonah66Ci increases the numbers of enteroendocrine and mitotic cells in the gut of uninfected larvae and infection with Steinernema nematodes reduces their numbers, whereas the numbers of intestinal stem cells are unaffected by nematode infection. Jonah66Ci knockdown further reduces nitric oxide levels in response to infection with Steinernema symbiotic nematodes. Finally, Jonah66Ci knockdown does not alter the feeding rates of uninfected Drosophila larvae, however infection with Steinernema axenic nematodes lowers larval feeding. In conclusion, this study reports that Jonah66Ci participates in maintaining homeostasis of certain physiological processes in Drosophila larvae in the context of Steinernema nematode infection. Similar findings will ultimately lead to an understanding the molecular and physiological mechanisms that take place during parasitic nematode infection in insects (Yadav, 2019).

Activin and BMP Signaling Activity Affects Different Aspects of Host Anti-Nematode Immunity in Drosophila melanogaster

Functional characterization of the interaction between TGF-β signaling activity and the mechanisms activated by the D. melanogaster immune response against parasitic nematode infection remains unexplored. This study investigated the participation of the TGF-β signaling branches, activin and bone morphogenetic protein (BMP), to host immune function against axenic or symbiotic Heterorhabditis bacteriophora nematodes (parasites lacking or containing their mutualistic bacteria, respectively). Using D. melanogaster larvae carrying mutations in the genes coding for the TGF-β extracellular ligands Daw and Dpp, this study analyzed the changes in survival ability, cellular immune response, and phenoloxidase (PO) activity during nematode infection. Infection with axenic H. bacteriophora decreases the mortality rate of dpp mutants, but not daw mutants. Following axenic or symbiotic H. bacteriophora infection, both daw and dpp mutants contain only plasmatocytes. Higher levels of Dual oxidase gene expression was detected in dpp mutants upon infection with axenic nematodes and Diptericin and Cecropin gene expression in daw mutants upon infection with symbiotic nematodes compared to controls. Finally, following symbiotic H. bacteriophora infection, daw mutants have higher PO activity relative to controls. Together, these findings reveal that while D. melanogaster Dpp/BMP signaling activity modulates the DUOX/ROS response to axenic H. bacteriophora infection, Daw/activin signaling activity modulates the antimicrobial peptide and melanization responses to axenic H. bacteriophora infection. Results from this study expand the current understanding of the molecular and mechanistic interplay between nematode parasites and the host immune system, and the involvement of TGF-β signaling branches in this process. Such findings will provide valuable insight on the evolution of the immune role of TGF-β signaling, which could lead to the development of novel strategies for the effective management of human parasitic nematodes (Ozakman, 2021).

Parasitic nematode secreted phospholipase A(2) suppresses cellular and humoral immunity by targeting hemocytes in Drosophila melanogaster

A key aspect of parasitic nematode infection is the nematodes' ability to evade and/or suppress host immunity. This immunomodulatory ability is likely driven by the release of hundreds of excretory/secretory proteins (ESPs) during infection. While ESPs have been shown to display immunosuppressive effects on various hosts, understanding of the molecular interactions between individual proteins released and host immunity requires further study. A recently discovered secreted phospholipase A2 (sPLA(2)) released from the entomopathogenic nematode (EPN) Steinernema carpocapsae was named Sc-sPLA(2). This study reports that Sc-sPLA(2) increased mortality of Drosophila melanogaster infected with Streptococcus pneumoniae and promoted increased bacterial growth. Furthermore, the data showed that Sc-sPLA(2) was able to downregulate both Toll and Imd pathway-associated antimicrobial peptides (AMPs) including drosomycin and defensin, in addition to suppressing phagocytosis in the hemolymph. Sc-sPLA(2) was also found to be toxic to D. melanogaster with the severity being both dose- and time-dependent. Collectively, these data highlighted that Sc-sPLA(2) possessed both toxic and immunosuppressive capabilities (Parks, 2023).

A high-sugar diet affects cellular and humoral immune responses in Drosophila

A high-sugar diet (HSD) induces Type 2 diabetes (T2D) and obesity, which severely threaten human health. The Drosophila T2D model has been constructed to study the mechanisms of insulin resistance, diet-induced cardiovascular diseases and other conditions. Innate immunity is the first line of defense against invading pathogens and parasites. However, few studies have focused on the relationship between a HSD and the innate immune response in Drosophila. In this study, flies were fed a high-sucrose diet, and defects were observed in the phagocytosis of latex beads and B. bassiana spores. The actin cytoskeleton was also disrupted in hemocytes from HSD-fed larvae. Furthermore, HSD induced the differentiation of lamellocytes in the lymph gland and circulating hemolymph, which rarely occurs in healthy bodies, via JNK signaling. In addition, the Toll and JNK pathways were excessively activated in the fat bodies of HSD-fed larvae, and a large number of dead cells were observed. Finally, HSD induced the aberrant activation of the innate immune system, including inflammation. Our results have established a connection between T2D and the innate immune response (Yu, 2018).

Fat body cells are motile and actively migrate to wounds to drive repair and prevent infection

Adipocytes have many functions in various tissues beyond energy storage, including regulating metabolism, growth, and immunity. However, little is known about their role in wound healing. This study used live imaging of fat body cells, the equivalent of vertebrate adipocytes in Drosophila, to investigate their potential behaviors and functions following skin wounding. Pupal fat body cells are not immotile, as previously presumed, but actively migrate to wounds using an unusual adhesion-independent, actomyosin-driven, peristaltic mode of motility. Once at the wound, fat body cells collaborate with hemocytes, Drosophila macrophages, to clear the wound of cell debris; they also tightly seal the epithelial wound gap and locally release antimicrobial peptides to fight wound infection. Thus, fat body cells are motile cells, enabling them to migrate to wounds to undertake several local functions needed to drive wound repair and prevent infections (Franz, 2018).

The data show that FBCs, Drosophila adipocytes, are recruited to wounds in pupae where they have multiple local roles in wound healing. The observation that FBCs are motile cells that actively migrate to wounds is unexpected and has not previously been made for adipocytes in any other organism. However, these findings raise the interesting question as to whether vertebrate adipocytes might also have the capacity to migrate. In that regard, a recent mammalian wound study found that adipocytes repopulate murine wounds, and suggested that some may have migrated from distant sites. It will be fascinating to discover whether some sub-populations of vertebrate adipocytes are indeed motile and whether they utilize similar migratory strategies to those highlighted in Drosophila FBCs (Franz, 2018).

The mode of motility that was observed for FBCs moving through the hemolymph to wounds is unusual, since it does not appear to involve the use of standard lamellipodia or blebs, utilized by most known migrating cells as they crawl in an adhesion-dependent fashion over substrates and through a milieu of extracellular matrix. Adhesion-independent migration has recently emerged as an alternative migration mode that has now been described for several other types of cells, including ameba, lymphocytes, and some cancer cells. Four models have been proposed for adhesion-independent migration: force transmission driven by 'chimneying' between two opposing substrate faces, the intercalation of lateral cell protrusions with gaps in the substrate, non-specific friction between cell and substrate, and swimming by noncyclic cell shape deformations. Only the last of these is entirely independent of any interactions with (or close proximity to) a solid substrate and hence best describes the observation of the migration of FBCs through hemolymph to wounds, since no significant interactions of these cells are seen with any substrate or other cells as they migrate. Similar to FBCs, several other cell types have been reported to migrate by swimming, when they are required to move through viscous fluid: amebae and neutrophils have been shown to swim when in viscous solution and lymphocytes are known to migrate using contraction waves when in suspension. However, the exact mechanism by which these swimming cells generate internal forces and how these forces are transduced to the extracellular environment to generate forward movement is still unknown. A recent study has shed some light on how internal forces are generated during another type of adhesion-independent migration; it showed that the migration of Walker carcinoma cells in confinement is driven by cyclical rearward flow of cortical actin that is coupled to the substrate through friction. This migration depends on the contractility of cortical actin at the rear of the cells. Moreover, rearward flow of cortical actin has also been described for the oscillatory behavior of detached cells and cell fragments, as well as for the stable-bleb cell migration of zebrafish germ layer progenitor cells. This is strikingly similar to the rearward peristaltic actin waves observed in FBCs migrating to wounds, suggesting that this could be the mechanism of force generation in FBCs also (Franz, 2018).

However, it still remains unclear how such an intracellular force might be transduced to the extracellular environment to drive forward movement of FBCs. It has previously been presumed that, while swimming works for large multicellular organisms, it cannot operate at the microscopic cell level, where viscous forces are many orders of magnitude higher than inertial forces and hence geometrically reciprocal cell shape changes may not generate propulsive forces. However, this view has been challenged and may only be true for simple Newtonian fluids, like water, which the hemolymph that FBCs swim through is clearly not. Moreover, swimming in a non-Newtonian fluid is thought to be possible if the cell shape changes of migrating cells are nonreciprocal, which might be true for FBCs migrating to wounds. It is also possible that FBCs, in addition to swimming, make use of other mechanisms to migrate. The hemolymph is relatively densely packed with cells including hemocytes and other FBCs, and FBCs are adjacent to the epithelium and muscle, depending on the location in the body. Although no contacts were observed, it is possible that the close proximity of FBCs with other cells and tissues en route to a wound might enable them to occasionally generate additional frictional forces like the ones reported for non-adherent Walker cells migrating in a confined microfluidics channel, which may also contribute to their swimming motility (Franz, 2018).

This study shows that FBCs play multiple local roles in driving wound repair and preventing wound infection. Some of these local functions might also partially extrapolate to the vertebrate wound scenario. Drosophila FBCs have long been known to systemically produce a variety of AMPs following infection and this study reveals that, during wound infection, FBCs migrate to wounds to release AMPs locally. A recent study has shown that mouse adipocytes are able to produce AMPs following bacterial skin infections. Hence, it would be interesting to examine whether mammalian adipocytes, like Drosophila FBCs, play a local role during wound healing in delivering AMPs to fight wound infection (Franz, 2018).

Given the finding that hemocytes and FBCs collaborate during the wound repair process to clear cell debris and fight infection, it is tempting to speculate that these two cell types communicate with each other during vertebrate wound healing also. Interestingly, in recent years several mammalian studies have uncovered complex interactions between adipocytes and macrophages in white adipose tissue (WAT), with important implications for tissue regeneration and disease. One example is obesity-induced inflammation and insulin resistance, where, upon overnutrition, the adipocytes in visceral WAT are thought to release chemokines to stimulate macrophage recruitment into fat tissue, leading to smoldering inflammation and subsequently insulin resistance. This is believed to be due to proinflammatory macrophages releasing cytokines that attenuate insulin signaling in various cell types, including adipocytes. In support of these mammalian reports, a recent study in the fly showed that animals fed a lipid-rich diet display reduced insulin sensitivity and lifespan, and both of these effects are mediated by hemocytes (Franz, 2018).

Thus interactions between adipocytes and immune cells appear to be key in many diseases, including type 2 diabetes, and it is believed that important insights into these links may be provided by future studies of the functional relationship and communication between FBCs and hemocytes during pupal wound repair in flies (Franz, 2018).

These studies in Drosophila pupae point to novel behaviors and functions for FBCs in Drosophila and open up genetic opportunities to further understanding of the important roles played by adipocytes in repair and regeneration (Franz, 2018).

The mode of expression divergence in Drosophila fat body is infection-specific

Transcription is controlled by interactions of cis-acting DNA elements with diffusible trans-acting factors. Changes in cis or trans factors can drive expression divergence within and between species, and their relative prevalence can reveal the evolutionary history and pressures that drive expression variation. Previous work delineating the mode of expression divergence in animals has largely used whole body expression measurements in one condition. Since cis-acting elements often drive expression in a subset of cell types or conditions, these measurements may not capture the complete contribution of cis-acting changes. This study quantified the mode of expression divergence in the Drosophila fat body, the primary immune organ, in several conditions, using two geographically distinct lines of D. melanogaster and their F1 hybrids. Expression was measured in the absence of infection and in infections with Gram-negative S. marcescens or Gram-positive E. faecalis bacteria, which trigger the two primary signaling pathways in the Drosophila innate immune response. The mode of expression divergence strongly depends on the condition, with trans-acting effects dominating in response to Gram-positive infection and cis-acting effects dominating in Gram-negative and pre-infection conditions. Expression divergence in several receptor proteins may underlie the infection-specific trans effects. Before infection, when the fat body has a metabolic role, there are many compensatory effects, changes in cis and trans that counteract each other to maintain expression levels. This work demonstrates that within a single tissue, the mode of expression divergence varies between conditions and suggests that these differences reflect the diverse evolutionary histories of host-pathogen interactions (Ramirez-Corona, 2021).

Inherent constraints on a polyfunctional tissue lead to a reproduction-immunity tradeoff

Single tissues can have multiple functions, which can result in constraints, impaired function, and tradeoffs. The insect fat body performs remarkably diverse functions including metabolic control, reproductive provisioning, and systemic immune responses. How polyfunctional tissues simultaneously execute multiple distinct physiological functions is generally unknown. Immunity and reproduction are observed to trade off in many organisms but the mechanistic basis for this tradeoff is also typically not known. This study investigated constraints and trade-offs in the polyfunctional insect fat body. Using single-nucleus sequencing, it was determined that the Drosophila melanogaster fat body executes diverse basal functions with heterogenous cellular subpopulations. The size and identity of these subpopulations are remarkably stable between virgin and mated flies, as well as before and after infection. However, as an emergency function, the immune response engages the entire tissue and all cellular subpopulations produce induce expression of defense genes. Reproductively active females who were given bacterial infection exhibited signatures of ER stress and impaired capacity to synthesize new protein in response to infection, including decreased capacity to produce antimicrobial peptides. Transient provision of a reversible translation inhibitor to mated females prior to infection rescued general protein synthesis, specific production of antimicrobial peptides, and survival of infection. The commonly observed tradeoff between reproduction and immunity appears to be driven, in D. melanogaster, by a failure of the fat body to be able to handle simultaneous protein translation demands of reproductive provisioning and immune defense. It is suggested that inherent cellular limitations in tissues that perform multiple functions may provide a general explanation for the wide prevalence of physiological and evolutionary tradeoffs (Gupta, 2022).

HP1a-mediated heterochromatin formation promotes antimicrobial responses against Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is a Gram-negative bacterium that causes severe infectious disease in diverse host organisms, including humans. Effective therapeutic options for P. aeruginosa infection are limited due to increasing multidrug resistance and it is therefore critical to understand the regulation of host innate immune responses to guide development of effective therapeutic options. The epigenetic mechanisms by which hosts regulate their antimicrobial responses against P. aeruginosa infection remain unclear. This study used Drosophila melanogaster to investigate the role of heterochromatin protein 1a (HP1a), a key epigenetic regulator, and its mediation of heterochromatin formation in antimicrobial responses against PA14, a highly virulent P. aeruginosa strain. Animals with decreased heterochromatin levels showed less resistance to P. aeruginosa infection. In contrast, flies with increased heterochromatin formation, either in the whole organism or specifically in the fat body-an organ important in humoral immune response-showed greater resistance to P. aeruginosa infection, as demonstrated by increased host survival and reduced bacterial load. Increased heterochromatin formation in the fat body promoted the antimicrobial responses via upregulation of fat body immune deficiency (imd) pathway-mediated antimicrobial peptides (AMPs) before and in the middle stage of P. aeruginosa infection. The fat body AMPs were required to elicit HP1a-mediated antimicrobial responses against P. aeruginosa infection. Moreover, the levels of heterochromatin in the fat body were downregulated in the early stage, but upregulated in the middle stage, of P. aeruginosa infection. These data indicate that HP1a-mediated heterochromatin formation in the fat body promotes antimicrobial responses by epigenetically upregulating AMPs of the imd pathway. This study provides novel molecular, cellular, and organismal insights into new epigenetic strategies targeting heterochromatin that have the potential to combat P. aeruginosa infection (Wu, 2022).

The genetic basis of variation in immune defense against Lysinibacillus fusiformis infection in Drosophila melanogaster

The genetic causes of phenotypic variation often differ depending on the population examined, particularly if the populations were founded by relatively small numbers of genotypes. Similarly, the genetic causes of phenotypic variation among similar traits (resistance to different xenobiotic compounds or pathogens) may also be completely different or only partially overlapping. Differences in genetic causes for variation in the same trait among populations suggests context dependence for how selection acts on those traits. Similarities in the genetic causes of variation for different traits, on the other hand, suggests pleiotropy which would also influence how natural selection shapes variation in a trait. This study characterized immune defense against a natural Drosophila pathogen, the Gram-positive bacterium Lysinibacillus fusiformis, in three different populations and found almost no overlap in the genetic architecture of variation in survival post infection. However, when comparing these results to a similar experiment with the fungal pathogen, B. bassiana, a convincing shared QTL peak was found for both pathogens. This peak contains the Bomanin cluster of Drosophila immune effectors. Loss of function mutants and RNAi knockdown experiments confirms a role of some of these genes in immune defense against both pathogens. This suggests that natural selection may act on the entire cluster of Bomanin genes (and the linked region under the QTL) or specific peptides for specific pathogens (Smith, 2023).

Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune function

During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. This study asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. Genetic or physiological activation of fat body Toll signaling was found to lead to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy phospholipid synthesis pathway induction is an impaired immune response to bacterial infection. These results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage (Martonez, 2020).

Bub1 facilitates virus entry through endocytosis in a model of Drosophila pathogenesis

In order to establish productive infection and dissemination, viruses usually evolve a number of strategies to hijack and/or subvert the host defense systems. However, host factors utilized by the virus to facilitate infection remain poorly characterized. Drosophila melanogaster deficient in budding uninhibited by benzimidazoles 1 (bub1), a highly conserved subunit of kinetochores complex regulating chromosome congression, became resistant to Drosophila C virus (DCV) infection evidenced in increased survival rates and reduced viral loads, compared to the wild type control. Mechanistic analysis further showed that Bub1 also functioned in the cytoplasm and was essentially involved in clathrin-dependent endocytosis of DCV and other pathogens, thus limiting pathogen entry. DCV infection potentially had strengthened the interaction between Bub1 and the clathrin adaptor on the cell membrane. Furthermore, the conserved function of Bub1 was as well verified in a mammalian cell line. Thus, these data demonstrated a previously unknown function of Bub1 that could be hijacked by pathogens to facilitate their infection and spread (Wang, 2018).

Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster

Non-coding RNAs have important roles in regulating physiology, including immunity. Transcriptome profiling of immune-responsive genes in Drosophila melanogaster was performed during a Gram-positive bacterial infection, concentrating on long non-coding RNA (lncRNA) genes. The gene most highly induced by a Micrococcus luteus infection was CR44404, named Induced by Infection (lincRNA-IBIN). lincRNA-IBIN is induced by both Gram-positive and Gram-negative bacteria in Drosophila adults and parasitoid wasp Leptopilina boulardi in Drosophila larvae, as well as by the activation of the Toll or the Imd pathway in unchallenged flies. Upon infection, lincRNA-IBIN is expressed in the fat body, in hemocytes and in the gut, and its expression is regulated by NF-kappaB signaling and the chromatin modeling brahma complex. In the fat body, overexpression of lincRNA-IBIN affected the expression of Toll pathway -mediated genes. Notably, overexpression of lincRNA-IBIN in unchallenged flies elevated sugar levels in the hemolymph by enhancing the expression of genes important for glucose retrieval. These data show that lncRNA genes play a role in Drosophila immunity and indicate that lincRNA-IBIN acts as a link between innate immune responses and metabolism (Valanne, 2019).

Lime is a new protein linking immunity and metabolism in Drosophila

he proliferation, differentiation and function of immune cells in vertebrates, as well as in the invertebrates, is regulated by distinct signalling pathways and crosstalk with systemic and cellular metabolism. This study has identified the Lime gene (Linking Immunity and Metabolism, CG18446) as one such connecting factor, linking hemocyte development with systemic metabolism in Drosophila. Lime is expressed in larval plasmatocytes and the fat body and regulates immune cell type and number by influencing the size of hemocyte progenitor populations in the lymph gland and in circulation. Lime mutant larvae exhibit low levels of glycogen and trehalose energy reserves and they develop low number of hemocytes. The low number of hemocytes in Lime mutants can be rescued by Lime overexpression in the fat body. It is well known that immune cell metabolism is tightly regulated with the progress of infection and it must be supported by systemic metabolic changes. This study demonstrated that Lime mutants fails to induce such systemic metabolic changes essential for the larval immune response. Indeed, Lime mutants are not able to sustain high numbers of circulating hemocytes and are compromised in the number of lamellocytes produced during immune system challenge, using a parasitic wasp infection model. It is therefore proposed the Lime gene as a novel functional link between systemic metabolism and Drosophila immunity (Mihajlovic, 2019).

Assessing the cellular immune response of the fruit fly, Drosophila melanogaster, using an in vivo phagocytosis assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. This study demonstrates an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance (Nazario-Toole, 2019).

Use of Clodronate Liposomes to Deplete Phagocytic Immune Cells in Drosophila melanogaster and Aedes aegypti

The innate immune system is the primary defense response to limit invading pathogens for all invertebrate species. In insects, immune cells are central to both cellular and humoral immune responses, however few genetic resources exist beyond Drosophila to study immune cell function. Therefore, the development of innovative tools that can be widely applied to a variety of insect systems is of importance to advance the study of insect immunity. This study has adapted the use of clodronate liposomes (CLD; a hydrophilic molecule that can be encapsulated within phospholipid bilayers) to deplete phagocytic immune cells in the vinegar fly, Drosophila melanogaster, and the yellow fever mosquito, Aedes aegypti. Through microscopy and molecular techniques, the depletion of phagocytic cell populations was validated in both insect species, and the integral role of phagocytes in combating bacterial pathogens demonstrated. Together, these data demonstrate the wide utility of CLD in insect systems to advance the study of phagocyte function in insect innate immunity (Kumar, 2021).

The Drosophila chemokine-like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons

Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, this study shows that the Drosophila chemokine-like protein Orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion function. Thus, these results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons (Ji, 2023).

Independent effects on cellular and humoral immune responses underlie genotype-by-genotype interactions between Drosophila and parasitoids

It is common to find abundant genetic variation in host resistance and parasite infectivity within populations, with the outcome of infection frequently depending on genotype-specific interactions. Underlying these effects are complex immune defenses that are under the control of both host and parasite genes. Extensive variation in Drosophila melanogaster's immune response was found against the parasitoid wasp Leptopilina boulardi. Some aspects of the immune response, such as phenoloxidase activity, are predominantly affected by the host genotype. Some, such as upregulation of the complement-like protein Tep1, are controlled by the parasite genotype. Others, like the differentiation of immune cells called lamellocytes, depend on the specific combination of host and parasite genotypes. These observations illustrate how the outcome of infection depends on independent genetic effects on different aspects of host immunity. These observations provide a physiological mechanism to generate phenomena like epistasis and genotype-interactions that underlie models of coevolution (Leitao, 2019).

Consequences of chronic bacterial infection in Drosophila melanogaster

Even when successfully surviving an infection, a host often fails to eliminate a pathogen completely and may sustain substantial pathogen burden for the remainder of its life. Using systemic bacterial infection in Drosophila melanogaster, this study characterize chronic infection by three bacterial species from different genera - Providencia rettgeri, Serratia marcescens, and Enterococcus faecalis-following inoculation with a range of doses. To assess the consequences of these chronic infections, the expression was determined of antimicrobial peptide genes, survival of secondary infection, and starvation resistance after one week of infection. While higher infectious doses unsurprisingly lead to higher risk of death, they also result in higher chronic bacterial loads among the survivors for all three infections. All three chronic infections caused significantly elevated expression of antimicrobial peptide genes at one week post-infection and provided generalized protection again secondary bacterial infection. Only P. rettgeri infection significantly influenced resistance to starvation, with persistently infected flies dying more quickly under starvation conditions relative to controls. These results suggest that there is potentially a generalized mechanism of protection against secondary infection, but that other impacts on host physiology may depend on the specific pathogen. It is proposed that chronic infections in D. melanogaster could be a valuable tool for studying tolerance of infection, including impacts on host physiology and behavior (Chambers, 2019).

Maternal priming of offspring immune system in Drosophila

Immune priming occurs when a past infection experience leads to a more effective immune response upon a secondary exposure to the infection or pathogen. In some instances, parents are able to transmit immune priming to their offspring, creating a subsequent generation with a superior immune capability, through processes that are not yet fully understood. Using a parasitoid wasp, which infects larval stages of Drosophila melanogaster, this study describes an example of an intergenerational inheritance of immune priming. This phenomenon is anticipatory in nature and does not rely on parental infection, but rather, when adult fruit flies are cohabitated with a parasitic wasp, they produce offspring that are more capable of mounting a successful immune response against a parasitic macro-infection. This increase in offspring survival correlates with a more rapid induction of lamellocytes, a specialized immune cell. RNA-sequencing of the female germline identifies several differentially expressed genes following wasp exposure, including the peptiodoglycan recognition protein-LB (PGRP-LB). Genetic manipulation of maternal PGRP-LB identifies this gene as a key element in this intergenerational phenotype (Bozler, 2019).

Iron sequestration by transferrin 1 mediates nutritional immunity in Drosophila melanogaster

This study used Drosophila as an in vivo model to investigate the role of transferrins in host defense. Systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires NF-kappaB immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. The iron transporter Transferrin 1 (Tsf1) was shown to be induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, it was discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, this study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. This in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy (Iatsenko, 2020).

The fliK Gene Is Required for the Resistance of Bacillus thuringiensis to Antimicrobial Peptides and Virulence in Drosophila melanogaster

Antimicrobial peptides (AMPs) are essential effectors of the host innate immune system and they represent promising molecules for the treatment of multidrug resistant microbes. A better understanding of microbial resistance to these defense peptides is thus prerequisite for the control of infectious diseases. In this study, using a random mutagenesis approach, the fliK gene, encoding an internal molecular ruler that controls flagella hook length, was identified as an essential element for Bacillus thuringiensis resistance to AMPs in Drosophila. Unlike its parental strain, that is highly virulent to both wild-type and AMPs deficient mutant flies, the fliK deletion mutant is only lethal to the latter's. In agreement with its conserved function, the fliK mutant is non-flagellated and exhibits highly compromised motility. However, comparative analysis of the fliK mutant phenotype to that of a fla mutant, in which the genes encoding flagella proteins are interrupted, indicate that B. thuringiensis FliK-dependent resistance to AMPs is independent of flagella assembly. As a whole, these results identify FliK as an essential determinant for B. thuringiensis virulence in Drosophila and provide new insights on the mechanisms underlying bacteria resistance to AMPs (Attieh, 2020).

Differences in post-mating transcriptional responses between conspecific and heterospecific matings in Drosophila

In many animal species, females undergo physiological and behavioral changes after mating. Some of these changes are driven by male-derived seminal fluid proteins, and are critical for fertilization success. Unfortunately, understanding of the molecular interplay between female and male reproductive proteins remains inadequate. This study analyzed the post-mating response in a Drosophila species that has evolved strong gametic incompatibility with its sister species; D. novamexicana females produce only ~1% fertilized eggs in crosses with D. americana males, compared to ~98% produced in within-species crosses. This incompatibility is likely caused by mismatched male and female reproductive molecules. In this study short-read RNA sequencing was used to examine the evolutionary dynamics of female reproductive genes and the post-mating transcriptome response in crosses within and between species. First, it was found that most female reproductive tract genes are slow-evolving compared to the genome average. Second, post-mating responses in con- and heterospecific matings are largely congruent, but heterospecific matings induce expression of additional stress-response genes. Some of those are immunity genes that are activated by the Imd pathway. Several genes in the JAK/STAT signaling pathway were identified that are induced in heterospecific, but not conspecific mating. While this immune response was most pronounced in the female reproductive tract, it was also detected in the female head and ovaries. These results show that the female's post-mating transcriptome-level response is determined in part by the genotype of the male, and that divergence in male reproductive genes and/or traits can have immunogenic effects on females (Ahmed-Braimah, 2020).

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry

Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (FACS) with mass spectrometry, making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms. SCMC was optimized to analyze hemocytes of the Drosophila innate immune system. Metal-conjugated antibodies (H2, H3, H18, L1, L4, and P1 at the cell surface, intracellular 3A5 and L2) and anti-IgM (L6 at the cell surface) were used to detect the levels of antigens, while anti-GFP was used to detect crystal cells in the immune induced samples. This study investigated the antigen expression profile of single cells and hemocyte populations in naive states, in immune induced states, in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hop^Tum) and carrying deficiency of a tumor suppressor l(3)mbn¹ gene, as well as in stem cell maintenance-defective hdc^Δ84) mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, and delineated the unique immunophenotype of Drosophila mutants. Subpopulations of L2(+)/P1(+) (l(3)mbn¹), L2(+)/L4(+)/P1(+) hop^Tum) transitional phenotype cells were identified in the tumorous strains and a subpopulation of L4(+)/P1(+) cells was identified upon immune induction. These results demonstrated for the first time that SCMC, combined with multidimensional bioinformatic analysis, represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila (Balog, 2021).

Drosophila melanogaster Y Chromosome Genes Affect Male Sensitivity to Microbial Infections

The genders of Drosophila melanogaster vary in their sensitivities to microbial pathogens. While many of the immunity-related genes are located on the X chromosome, the polymorphisms within the Y chromosome were also shown to affect the immunity of flies. This study investigated the necessity of individual genes on the Y chromosome (Y-genes) for male sensitivity to microbes. Several Y-genes were identified whose genetic inactivation either increases or decreases the sensitivity of males to gastrointestinal infections with fungal Saccharomyces cerevisiae and bacterial Serratia liquefaciens. Specifically, the loss of function mutations in fly kl-5 and Ppr-Y Y-genes lead to increased and decreased sensitivity of males to fungal challenge, respectively, compared to female sensitivity. In contrast, mutations in Drosophila Pp1-Y1, kl-5, kl-3, Ppr-Y, CCY, and FDY Y-genes lead to increased sensitivity of males to bacterial infection, compared to females. Moreover, while these Y-genes are necessary, the Y chromosome is not sufficient for the sensitivity of males to microbes, since the sensitivity of XXY females to fungal and bacterial challenges was not different from the sensitivity of wild-type female flies, compared to males. This study assigns a new immunity-related function to numerous Y-genes in D. melanogaster (Bartolo, 2021).

Immune Cell Production Is Targeted by Parasitoid Wasp Virulence in a Drosophila-Parasitoid Wasp Interaction

The interactions between Drosophila melanogaster and the parasitoid wasps that infect Drosophila species provide an important model for understanding host-parasite relationships. Following parasitoid infection, D. melanogaster larvae mount a response in which immune cells (hemocytes) form a capsule around the wasp egg, which then melanizes, leading to death of the parasitoid. Previous studies have found that host hemocyte load; the number of hemocytes available for the encapsulation response; and the production of lamellocytes, an infection induced hemocyte type, are major determinants of host resistance. Parasitoids have evolved various virulence mechanisms to overcome the immune response of the D. melanogaster host, including both active immune suppression by venom proteins and passive immune evasive mechanisms. This study identified a previously undescribed parasitoid species, Asobara sp. AsDen, which utilizes an active virulence mechanism to infect D. melanogaster hosts. Asobara sp. AsDen infection inhibits host hemocyte expression of msn, a member of the JNK signaling pathway, which plays a role in lamellocyte production. Asobara sp. AsDen infection restricts the production of lamellocytes as assayed by hemocyte cell morphology and altered msn expression. These findings suggest that Asobara sp. AsDen infection alters host signaling to suppress immunity (Trainor, 2021).

Natural selection has driven the recurrent loss of an immunity gene that protects Drosophila against a major natural parasite

Polymorphisms in immunity genes can have large effects on susceptibility to infection. To understand the origins of this variation, this study has investigated the genetic basis of resistance to the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster. Increased expression of the gene lectin-24A after infection by parasitic wasps was associated with a faster cellular immune response and greatly increased rates of killing the parasite. lectin-24A encodes a protein that is strongly up-regulated in the fat body after infection and localizes to the surface of the parasite egg. In certain susceptible lines, a deletion upstream of the lectin-24A has largely abolished expression. Other mutations predicted to abolish the function of this gene have arisen recurrently in this gene, with multiple loss-of-expression alleles and premature stop codons segregating in natural populations. The frequency of these alleles varies greatly geographically, and in some southern African populations, natural selection has driven them near to fixation. It is concluded that natural selection has favored the repeated loss of an important component of the immune system, suggesting that in some populations, a pleiotropic cost to lectin-24A expression outweighs the benefits of resistance (Arunkumar, 2023).

Regulatory regions in natural transposable element insertions drive interindividual differences in response to immune challenges in Drosophila

Variation in gene expression underlies interindividual variability in relevant traits including immune response. However, the genetic variation responsible for these gene expression changes remains largely unknown. Among the non-coding variants that could be relevant, transposable element insertions are promising candidates as they have been shown to be a rich and diverse source of cis-regulatory elements. This work used a population genetics approach to identify transposable element insertions likely to increase the tolerance of Drosophila melanogaster to bacterial infection by affecting the expression of immune-related genes. This study identified 12 insertions associated with allele-specific expression changes in immune-related genes. three of these insertions were experimentally validate including one likely to be acting as a silencer, one as an enhancer, and one with a dual role as enhancer and promoter. The direction in the change of gene expression associated with the presence of several of these insertions is consistent with an increased survival to infection. Indeed, for one of the insertions, it was shown that this is the case by analyzing both natural populations and CRISPR/Cas9 mutants in which the insertion is deleted from its native genomic context. It was shown that transposable elements contribute to gene expression variation in response to infection in D. melanogaster and that this variation is likely to affect their survival capacity. Because the role of transposable elements as regulatory elements is not restricted to Drosophila, transposable elements are likely to play a role in immune response in other organisms as well (Ullastres, 2021).

SUMOylation of Arginyl tRNA Synthetase Modulates the Drosophila Innate Immune Response

SUMO conjugation of a substrate protein can modify its activity, localization, interaction or function. A large number of SUMO targets in cells have been identified by Proteomics, but biological roles for SUMO conjugation for most targets remains elusive. The multi-aminoacyl tRNA synthetase complex (MARS) is a sensor and regulator of immune signaling. The proteins of this 1.2 MDa complex are targets of SUMO conjugation, in response to infection. Arginyl tRNA Synthetase (RRS), a member of the sub-complex II of MARS, is one such SUMO conjugation target. The sites for SUMO conjugation are Lys 147 and 383. Replacement of these residues by Arg (RRS (K147R,K383R)), creates a SUMO conjugation resistant variant (RRS (SCR)). Transgenic Drosophila lines for RRS (WT) and RRS (SCR) were generated by expressing these variants in a RRS loss of function (lof) animal, using the UAS-Gal4 system. The RRS-lof line was itself generated using CRISPR/Cas9 genome editing. Expression of both RRS (WT) and RRS (SCR) rescue the RRS-lof lethality. Adult animals expressing RRS (WT) and RRS (SCR) are compared and contrasted for their response to bacterial infection by gram positive M. luteus and gram negative Ecc15. This study finds that RRS (SCR), when compared to RRS (WT), shows modulation of the transcriptional response, as measured by quantitative 3' mRNA sequencing. This study uncovers a possible non-canonical role for SUMOylation of RRS, a member of the MARS complex, in host-defense (Nayak, 2021).

Glutamate metabolism directs energetic trade-offs to shape host-pathogen susceptibility in Drosophila

Individual hosts within populations often show inter-individual variation in their susceptibility to bacterial pathogen-related diseases. Utilizing Drosophila, this study highlighted that phenotypic variation in host-pathogen susceptibility within populations is driven by energetic trade-offs, facilitated by infection-mediated changes in glutamate metabolism. Furthermore, host-pathogen susceptibility is conditioned by life history, which adjusts immunometabolic sensing in muscles to direct vitamin-dependent reallocation of host energy substrates from the adipose tissue (i.e., a muscle-adipose tissue axis). Life history conditions inter-individual variation in the activation strength of intra-muscular NF-κB signaling. Limited intra-muscular NF-κB signaling activity allows for enhanced infection-mediated mitochondrial biogenesis and function, which stimulates glutamate dehydrogenase-dependent synthesis of glutamate. Muscle-derived glutamate acts as a systemic metabolite to promote lipid mobilization through modulating vitamin B enzymatic cofactor transport and function in the adipose tissue. This energy substrate reallocation improves pathogen clearance and boosts host survival. Finally, life history events that adjust energetic trade-offs can shape inter-individual variation in host-pathogen susceptibility after infection (Zhao, 2021).

Transcriptomic evidence for a trade-off between germline proliferation and immunity in Drosophila

Life-history theory posits that investment into reproduction might occur at the expense of investment into somatic maintenance, including immune function. If so, reduced or curtailed reproductive effort might be expected to increase immunity. In support of this notion, work in Caenorhabditis elegans has shown that worms lacking a germline exhibit improved immunity, but whether the antagonistic relation between germline proliferation and immunity also holds for other organisms is less well understood. This study reports that transgenic ablation of germ cells in late development or early adulthood in Drosophila melanogaster causes elevated baseline expression and increased induction of Toll and Imd immune genes upon bacterial infection, as compared to fertile flies with an intact germline. This study also identified immune genes whose expression after infection differs between fertile and germline-less flies in a manner that is conditional on their mating status. It is concluded that germline activity strongly impedes the expression and inducibility of immune genes and that this physiological trade-off might be evolutionarily conserved (Rodrigues, 2021).

Broad Ultrastructural and Transcriptomic Changes Underlie the Multinucleated Giant Hemocyte Mediated Innate Immune Response against Parasitoids

Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, this study showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. This study used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in Drosophila ananassae after parasitoid wasp infection. Dynamic structural changes were found, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, RNA sequencing was used to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. These results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response (Cinege, 2021).

Systemic innate immune response induces death of olfactory receptor neurons in Drosophila

Neural functions are known to decline during normal aging and neurodegenerative diseases. However, the mechanisms of functional impairment owing to the normal aging of the brain are poorly understood. It was previously reported that caspase-3-like protease, the protease responsible for inducing apoptosis, is activated in a subset of olfactory receptor neurons (ORNs), especially in Drosophila Or42b neurons, during normal aging. This study investigated the molecular mechanism underlying age-related caspase-3-like protease activation and cell death in Or42b neurons. Gene expression profiling of young and aged fly antenna showed that the expression of antimicrobial peptides was significantly upregulated, suggesting an activated innate immune response. Consistent with this observation, inhibition or activation of the innate immune pathway caused delayed or precocious cell death, respectively, in Or42b neurons. Accordingly, autonomous cell activation of the innate immune pathway in Or42b neurons is not likely required for their age-related death, whereas the systemic innate immune response induces caspase-3-like protease activation in Or42b neurons; this indicated that the death of these neurons is regulated non-cell autonomously. A possible link between the innate immune response and the death of olfactory neurons during normal aging is proposed (Takeuchi, 2021).

Pan-neuronal expression of human mutant huntingtin protein in Drosophila impairs immune response of hemocytes

Huntington's disease (HD) is a late-onset; progressive, dominantly inherited neurological disorder marked by an abnormal expansion of polyglutamine (poly Q) repeats in Huntingtin (HTT) protein. The pathological effects of mutant Huntingtin (mHTT) are not restricted to the nervous system but systemic abnormalities including immune dysregulation have been evidenced in clinical and experimental settings of HD. Indeed, mHTT is ubiquitously expressed and could induce cellular toxicity by directly acting on immune cells. However, it is still unclear if selective expression of mHTT exon1 in neurons could induce immune responses and hemocytes' function. This study intended to monitor perturbations in the hemocytes' population and their physiological functions in Drosophila, caused by pan-neuronal expression of mHTT protein. A measure of hemocyte count and their physiological activities caused by pan-neuronal expression of mHTT protein highlighted the extent of immune dysregulation occurring with disease progression. It was found that pan-neuronal expression of mHTT significantly alters crystal cells and plasmatocyte count in larvae and adults with disease progression. Interestingly, plasmatocytes isolated from diseased conditions exhibit a gradual decline in phagocytic activity ex vivo at progressive stages of the disease as compared to age-matched control groups. In addition, diseased flies displayed elevated reactive oxygen species (ROS) in circulating plasmatocytes at the larval stage and in sessile plasmatocytes of hematopoietic pockets at terminal stages of disease. These findings strongly implicate that neuronal expression of mHTT alone is sufficient to induce non-cell-autonomous immune dysregulation in vivo (Dhankhar, 2022).

A genetic screen in Drosophila reveals the role of fucosylation in host susceptibility to Candida infection

Candida infections constitute a blind spot in global public health as very few new anti-fungal drugs are being developed. Genetic surveys of host susceptibilities to such infections using mammalian models have certain disadvantages in that obtaining results is time-consuming owing to relatively long lifespans and these results have low statistical resolution because sample sizes are usually small. This paper reports a targeted genetic screening of 5698 RNAi lines encompassing 4135 Drosophila genes with human homologues, several of which were identified as important for host survival after Candida albicans infection. These include genes in a variety of functional classes encompassing gene expression, intracellular signalling, metabolism, and enzymatic regulation. Analysis of one of the screen hits, the infection-induced α-(1,3)-fucosylase FucTA, showed that N-glycan fucosylation has several targets among proteins involved in host defence supplying multiple avenues of investigation for the mechanistic analysis of host survival to systemic C. albicans infection (Glittenberg, 2022).

The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types

Mosquitoes are prolific vectors of human pathogens; a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. To determine whether Drosophila pathway-specific discrimination between pathogens is shared by mosquitoes, RNAseq was used to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, Ae. aegypti and An. gambiae were both found to mount a core response to all categories of infection. When the transcriptomes of mosquitoes infected with different types of bacteria were compared, it was observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, transcriptional signatures specific to bacterial infection and to fungal infection were identified. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi (Hixson, 2023).

Modulation of the cell membrane lipid milieu by peroxisomal beta-oxidation induces Rho1 signaling to trigger inflammatory responses

Phagocytosis, signal transduction, and inflammatory responses require changes in lipid metabolism. Peroxisome have key roles in fatty acid homeostasis and in regulating immune function. Drosophila macrophages lacking peroxisomes have perturbed lipid profiles, which reduce host survival after infection. Using lipidomic, transcriptomic, and genetic screens, we determine that peroxisomes contribute to the cell membrane glycerophospholipid composition necessary to induce Rho1-dependent signals, which drive cytoskeletal remodeling during macrophage activation. Loss of peroxisome function increases membrane phosphatidic acid (PA) and recruits RhoGAPp190 during infection, inhibiting Rho1-mediated responses. Peroxisome-glycerophospholipid-Rho1 signaling also controls cytoskeleton remodeling in mouse immune cells. While high levels of PA in cells without peroxisomes inhibit inflammatory phenotypes, large numbers of peroxisomes and low amounts of cell membrane PA are features of immune cells from patients with inflammatory Kawasaki disease and juvenile idiopathic arthritis. These findings reveal potential metabolic markers and therapeutic targets for immune diseases and metabolic disorders (Nath, 2022).

The circular RNA Edis regulates neurodevelopment and innate immunity

Circular RNAs (circRNAs) are widely expressed in eukaryotes. However, only a subset has been functionally characterized. This study identified and validated a collection of circRNAs in Drosophila, and showed that depletion of the brain-enriched circRNA Edis (circ_Ect4) causes hyperactivation of antibacterial innate immunity both in cultured cells and in vivo. Notably, Edis depleted flies display heightened resistance to bacterial infection and enhanced pathogen clearance. Conversely, ectopic Edis expression blocks innate immunity signaling. In addition, inactivation of Edis in vivo leads to impaired locomotor activity and shortened lifespan. Remarkably, these phenotypes can be recapitulated with neuron-specific depletion of Edis, accompanied by defective neurodevelopment. Furthermore, inactivation of Relish suppresses the innate immunity hyperactivation phenotype in the fly brain. Moreover, evidence is provided that Edis encodes a functional protein that associates with and compromises the processing and activation of the immune transcription factor Relish. Importantly, restoring Edis expression or ectopic expression of Edis-encoded protein suppresses both innate immunity and neurodevelopment phenotypes elicited by Edis depletion. Thus, this study establishes Edis as a key regulator of neurodevelopment and innate immunity (Xiong, 2022).

Transcriptome features of innate immune memory in Drosophila

Immune memory is the ability of organisms to elicit potentiated immune responses at secondary infection. Current studies have revealed that similar to adaptive immunity, innate immunity exhibits memory characteristics (called "innate immune memory"). Although epigenetic reprogramming plays an important role in innate immune memory, the underlying mechanisms have not been elucidated, especially at the individual level. This study established experimental systems for detecting innate immune memory in Drosophila melanogaster. Training infection with low-pathogenic bacteria enhanced the survival rate of the flies at subsequent challenge infection with high-pathogenic bacteria. Among low-pathogenic bacteria, Micrococcus luteus (Ml) and Salmonella typhimurium (St) exerted apparent training effects in the fly but exhibited different mechanisms of action. Ml exerted training effects even after its clearance from flies, while live St persisted in the flies for a prolonged duration. RNA sequencing (RNA-Seq) analysis revealed that Ml training enhanced the expression of the immune-related genes under the challenge condition but not under the non-challenge condition. In contrast, St training upregulated the expression of the immune-related genes independent of challenge. These results suggest that training effects with Ml and St are due to memory and persistence of immune responses, respectively. Furthermore, the gene involved in immune memory was souvhg, and a candidate gene, Ada2b, was identified which encodes a component of the histone modification complex. The Ada2b mutant suppressed Ml training effects on survival and disrupted the expression of some genes under the training + challenge condition. These results suggest that the gene expression regulated by Ada2b may contribute to innate immune memory in Drosophila (Fuse, 2022).

Trans-regulatory changes underpin the evolution of the Drosophila immune response

When an animal is infected, the expression of a large suite of genes is changed, resulting in an immune response that can defend the host. Despite much evidence that the sequence of proteins in the immune system can evolve rapidly, the evolution of gene expression is comparatively poorly understood. This study therefore investigated the transcriptional response to parasitoid wasp infection in Drosophila simulans and D. sechellia. Although these species are closely related, there has been a large scale divergence in the expression of immune-responsive genes in their two main immune tissues, the fat body and hemocytes. Many genes, including those encoding molecules that directly kill pathogens, have cis regulatory changes, frequently resulting in large differences in their expression in the two species. However, these changes in cis regulation overwhelmingly affected gene expression in immune-challenged and uninfected animals alike. Divergence in the response to infection was controlled in trans. It is argued that altering trans-regulatory factors, such as signalling pathways or immune modulators, may allow natural selection to alter the expression of large numbers of immune-responsive genes in a coordinated fashion (Ding, 2022).

Genetic dissection of innate immune memory in Drosophila melanogaster

P>Current studies have demonstrated that innate immunity possesses memory characteristics. Although the molecular mechanisms underlying innate immune memory have been addressed by numerous studies, genetic variations in innate immune memory and the associated genes remain unclear. This study explored innate immune memory in 163 lines of Drosophila melanogaster from the Drosophila Synthetic Population Resource. In this assay system, prior training with low pathogenic bacteria (Micrococcus luteus) increased the survival rate of flies after subsequent challenge with highly pathogenic bacteria (Staphylococcus aureus). This positive training effect was observed in most lines, but some lines exhibited negative training effects. Survival rates under training and control conditions were poorly correlated, suggesting that distinct genetic factors regulate training effects and normal immune responses. Subsequent quantitative trait loci analysis suggested that four loci containing 80 genes may be involved in regulating innate immune memory. Among them, Adgf-A, which encodes an extracellular adenosine deaminase-related growth factor, was shown to be associated with training effects. These findings help to elucidate the genetic architecture of innate immune memory in Drosophila and may provide insight for new therapeutic treatments aimed at boosting immunity (Tang, 2022).

Meta-Analysis of Immune Induced Gene Expression Changes in Diverse Drosophila melanogaster Innate Immune Responses

Organisms are commonly infected by a diverse array of pathogens and mount functionally distinct responses to each of these varied immune challenges. Host immune responses are characterized by the induction of gene expression, however, the extent to which expression changes are shared among responses to distinct pathogens is largely unknown. To examine this, meta-analysis was performed of gene expression data collected from Drosophila melanogaster following infection with a wide array of pathogens. Sixty-two genes were identified that are significantly induced by infection. While many of these infection-induced genes encode known immune response factors, 21 genes were identified that have not been previously associated with host immunity. Examination of the upstream flanking sequences of the infection-induced genes lead to the identification of two conserved enhancer sites. These sites correspond to conserved binding sites for GATA and nuclear factor κB (NFκB) family transcription factors and are associated with higher levels of transcript induction. Thirty-one genes were further identified with predicted functions in metabolism and organismal development that are significantly downregulated following infection by diverse pathogens. This study identifies conserved gene expression changes in Drosophila melanogaster following infection with varied pathogens, and transcription factor families that may regulate this immune induction (Waring, 2022).

A single mating is sufficient to induce persistent reduction of immune defense in mated female Drosophila melanogaster

In many species, female reproductive investment comes at a cost to immunity and resistance to infection. Mated Drosophila melanogaster females are more susceptible to bacterial infection than unmated females. Transfer of the male seminal fluid protein Sex Peptide reduces female post-mating immune defense. Sex Peptide is known to cause both short- and long-term changes to female physiology and behavior. While previous studies showed that females were less resistant to bacterial infection as soon as 2.5 h and as long as 26.5 h after mating, it is unknown whether this is a binary switch from mated to unmated state or whether females can recover to unmated levels of immunity. It is additionally unknown whether repeated mating causes progressive reduction in defense capacity. The immune defense of mated females when infected at 2, 4, 7, or 10 days after mating was compared to that of unmated females and no recovery of immune capacity was seen regardless of the length of time between mating and infection. Because D. melanogaster females can mate multiply, whether a second mating, and therefore a second transfer of seminal fluids, caused deeper reduction in immune performance was additionally tested. Females mated either once or twice before infection were found to survive at equal proportions, both with significantly lower probability than unmated females. It is concluded that a single mating event is sufficient to persistently suppress the female immune system. Interestingly, it was observed that induced levels of expression of genes encoding antimicrobial peptides (AMPs) decreased with age in both experiments, partially obscuring the effects of mating. Collectively, the data indicate that being reproductively active versus reproductively inactive are alternative binary states with respect to female D. melanogaster immunity. The establishment of a suppressed immune status in reproductively active females can inform understanding of the regulation of immune defense and the mechanisms of physiological trade-offs (Gordon, 2022).

Microbiome assembly on Drosophila body surfaces benefits the flies to combat fungal infections

In contrast to the well-characterized gut microbiomes, the composition and function of the insect body-surface microbiotas are still elusive and highly underexplored. This study reports the dynamic features of the Drosophila melanogaster surface microbiomes. It was found that the microbiomes assembled on fly surfaces could defend insects against fungal parasitic infections. The substantial increase of bacterial loads occurred within 10 days of fly eclosion, especially the expansion of Gilliamella species. The culturable bacteria such as Lactiplantibacillus plantarum could effectively inhibit fungal spore germinations, and the gnotobiotic addition of the isolated bacteria could substantially delay fungal infection of axenic flies. The fly tarsal segments were found to be largely accumulated with bacterial cells, which could accelerate cell dispersal onto different body parts to deter fungal spore germinations. These findings will facilitate future investigations of the surface microbiotas affecting insect physiologies (Hong, 2022).

microRNA-252 and FoxO repress inflammaging by a dual inhibitory mechanism on Dawdle-mediated TGF-beta pathway in Drosophila

Inflammaging refers to low-grade, chronically activated innate immunity that has deleterious effects on healthy lifespan. However, little is known about the intrinsic signaling pathway that elicits innate immune genes during aging. Using Drosophila melanogaster, the microRNA targetomes were profiled in young and aged animals and revealed Dawdle, an activin-like ligand of the TGF-7β pathway, as a physiological target of microRNA-252. microRNA-252 cooperates with Forkhead box O, a conserved transcriptional factor implicated in aging, to repress Dawdle. Unopposed Dawdle triggers hyperactivation of innate immune genes coupled with a decline in organismal survival. Using adult muscle tissues, single-cell sequencing analysis describes that Dawdle and its downstream innate immune genes are expressed in distinct cell types, suggesting a cell nonautonomous mode of regulation. The genetic cascade was determined by which Dawdle signaling leads to increased Kenny/IKKγ protein, which in turn activates Relish/NF-κB protein and consequentially innate immune genes. Finally, transgenic increase of microRNA-252 and Forkhead box O pathway factors in wild-type Drosophila extends lifespan and mitigates the induction of innate immune genes in aging. Together, it is proposed that microRNA-252 and Forkhead box O promote healthy longevity by cooperative inhibition on Dawdle-mediated inflammaging (Wu, 2022).

Mutations of gammaCOP Gene Disturb Drosophila melanogaster Innate Immune Response to Pseudomonas aeruginosa

Drosophila melanogaster (the fruit fly) is a valuable experimental platform for modeling host-pathogen interactions. It is also commonly used to define innate immunity pathways and to understand the mechanisms of both host tolerance to commensal microbiota and response to pathogenic agents. This study investigated how the host response to bacterial infection is mirrored in the expression of genes of Imd and Toll pathways when D. melanogaster strains with different γCOP genetic backgrounds are infected with Pseudomonas aeruginosa ATCC 27853. Using microarray technology, this study interrogated the whole-body transcriptome of infected versus uninfected fruit fly males with three specific genotypes, namely wild-type Oregon, γCOP(S057302)/TM6B and γCOP(14a)/γCOP(14a). While the expression of genes pertaining to Imd and Toll is not significantly modulated by P. aeruginosa infection in Oregon males, many of the components of these cascades are up- or downregulated in both infected and uninfected γCOP(S057302)/TM6B and γCOP(14a)/γCOP(14a) males. Thus, these results suggest that a γCOP genetic background modulates the gene expression profiles of Imd and Toll cascades involved in the innate immune response of D. melanogaster, inducing the occurrence of immunological dysfunctions in γCOP mutants (Chifiriuc, 2022).

Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster

In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. This study characterizes the Drosophila tuSz mutant strain, which mounts an aberrant immune response against its own fat body. This study demonstrates that this autoimmunity is the result of two mutations: 1) a mutation in the Glucosidase 1/GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals (Mortimer, 2021).

This work has investigated the Drosophila tuSz¹ mutant strain. tuSz¹ is a temperature-sensitive mutant, and at the restrictive temperature, posterior fat body tissue is melanotically encapsulated by hemocytes in a reaction similar to the antiparasitoid immune response. The tuSz¹ phenotype is caused by two tightly linked mutations: a nonconditional, dominant gain-of-function mutation in hop that leads to ectopic immune activation and a temperature-sensitive, recessive mutation in GCS1 that leads to loss of protein N-glycosylation of the ECM overlaying the posterior fat body. These data lead to a a proposal of a two-step model in which immune activation and the loss of SAMP presentation/recognition are both necessary for the breakdown of self-tolerance. In a naïve wild-type larva, neither condition is met and self-tolerance is maintained. In the case of the tuSz¹ mutant, the posterior fat body lacks appropriate ECM protein N-glycosylation and is targeted by constitutively activated hemocytes for encapsulation. This two-step model is also reflected in the hop^Tum mutant background, in which the simultaneous disruption of N-glycosylation in this immune-activated background results in tissue self-encapsulation similar to the tuSz¹ mutant (Mortimer, 2021).

Models describing the necessity for two independent signals in fly encapsulation responses. (A) Homeostasis is maintained in naïve wild-type larvae. (B) In tuSz¹ mutant larvae, immune cells are inappropriately activated by JAK-STAT pathway activation due to the hop^Sz gain-of-function mutation. The loss of protein N-glycosylation in posterior fat body tissue due to the GCS1^Sz mutation leads to loss of self-tolerance and tissue encapsulation. (C) In the model of self-tolerance described in a previous study, the coupled phenotypes of loss of cell integrity and loss of ECM integrity are sufficient to disrupt self-tolerance. (D) Immune cells are activated following parasitoid wasp infection, presumably due to the wound-mediated activation of JAK-STAT signaling. SAMP-presenting host tissues are protected from encapsulation, and wasp eggs may be targeted for encapsulation because they are missing the ECM N-glycosylation SAMP (Mortimer, 2021).

Interestingly, previous work also documented the necessity of at least two signals for self-encapsulation in Drosophila. In that case, both the loss of the ECM (with its glycosylated proteins) and the disrupted positional integrity of the underlying fat body cells (potentially mimicking a wound) were required for immune cells to become activated and encapsulate the self tissue. A similar loss of ECM and underlying cell integrity was also found in the classical melanotic tumor mutant tu(2)W. This model, in which at least two factors are required for self-encapsulation, may explain why the several classically described self-encapsulation mutants, unlike virtually all other types of visible Drosophila mutants, were never successfully mapped (Mortimer, 2021).

The disruption of either factor in the two-step model in isolation is not sufficient to cause self-encapsulation. This can be seen in parasitoid wasp infected larvae; the wounding associated with parasitoid infection leads to immune activation, but in the absence of SAMP disruption, the fly is able to specifically encapsulate the parasitoid egg while protecting against self-encapsulation. Conversely, while internal tissue damage in a naive larva does attract hemocyte interactions, in the absence of an immune stimulus, this does not lead to self-encapsulation, but rather the hemocytes attempt to repair the damaged tissue. That blood cells err on the side of fixing disrupted self tissue rather than treating it as pathogenic and encapsulating it unless another stress signal is also present suggests that flies may have evolved a multi-input system to safeguard against spurious encapsulation (Mortimer, 2021).

D. melanogaster immune responses have proven to be an excellent model for understanding the mechanisms underlying conserved innate immune responses, including those of humans. Findings on Drosophila self-tolerance may also be relevant to human innate self-tolerance. Indeed, data from a range of studies are consistent with the idea that protein glycosylation is a mediator of vertebrate immune responses, and cell-surface glycans have been proposed as candidate SAMPs for the innate immune response to distinguish healthy self tissues from aberrant or foreign tissues even if the mechanisms are not entirely understood. Protein-linked sugar groups should presumably fit this role well, as they can take on diverse combinations of sugar residues and branching patterns (Mortimer, 2021).

Protein N-glycosylation is a complex multistep process that begins with the addition of a presynthesized glycosyl precursor to the protein at an asparagine residue. This nascent glycan is then trimmed back to a core glycan structure, a process that is initiated by the activity of GCS1. The core glycan is then elaborated with the addition of multiple carbohydrate groups to give rise to a variety of final structures, with hybrid and complex type N-glycans among the most prevalent. Glycan elaboration begins with the activity of Mgat1, which leads to the production of hybrid type N-glycans. These hybrid N-glycans can be further processed by the α-mannosidase-I and -II family of enzymes to produce paucimannose N-glycans, which can serve as complex N-glycan precursors and are further elaborated by downstream enzymes to give rise to the final complex N-glycan structure. The current data suggest that disruption of any of the genes encoding key N-glycan-processing enzymes will be associated with the loss of self-tolerance in Drosophila. Similarly, the loss of the α-mannosidase-II (αM-II) gene in mice is linked with the development of an autoimmune phenotype that is likened to systemic lupus erythematosus. Like the tuSz¹ mutant, the αM-II mouse phenotype arises due to alterations in protein N-glycosylation in nonimmune tissues and is mediated by innate immune cells. Altered patterns of protein N-glycosylation are also observed in additional mouse models of autoimmune disease and have been linked to autoimmune disease in human patients (Mortimer, 2021).

The ECM is a conserved structure made up of numerous proteins, many of which are N-glycosylated, including laminin and collagen. A role for the ECM in mediating self-tolerance has been previously proposed: The encapsulation of self tissues in D. melanogaster is also observed following RNA interference (RNAi) knockdown of genes encoding the ECM proteins laminin and collagen, supporting the idea that SAMPs reside in the ECM. The role of the ECM in self-tolerance is further emphasized by tissue transplantation studies in Drosophila. Drosophila larvae are largely tolerant of conspecific tissue transplants, but this tolerance is abolished when tissues are first treated with collagenase to disrupt the ECM, leading to the specific encapsulation of treated tissues. Reactivity to ECM proteins is also associated with various forms of human autoimmune disease. Based on these data, it is proposed that the N-glycosylation of ECM proteins may serve as a conserved self-tolerance signal for innate immune mechanisms and that loss of ECM protein N-glycosylation may lead to loss of self-tolerance and, consequently, autoimmune disease in a diverse range of species (Mortimer, 2021).

An alternative means by which hosts can recognize pathogens is missing-self recognition. Instead of tracking pathogen diversity with numerous recognition receptors, as in nonself recognition, missing-self recognition does not rely on tracking pathogens at all, but only on specifically recognizing self and attacking tissues that lack the self signal. Further, unlike germ line-encoded forms of nonself recognition, missing-self recognition systems allow host species to respond to novel pathogen types that they have never encountered in their evolutionary history. Protein glycosylation plays an important role in the handful of identified missing-self recognition systems of vertebrates. The most well-known case of missing-self recognition involves the interaction between vertebrate NK cells and host MHC class I (MHCI) proteins. All vertebrate cells produce MHCI to display any possible antigens present in their cytoplasm to T cells, but intracellular pathogens often suppress host cell MHCI expression to prevent their molecules from being displayed. NK cells are lymphoid-type cells that induce cytolysis in infected host cells. In an uninfected state, recognition of properly glycosylated MHCI inhibits NK cell cytolysis of host cells, but in an infected state in which host cells are missing the MHCI self signal, the NK cell inhibitory receptors fail to recognize 'self,' and the infected host cells are lysed, an effective means of killing intracellular pathogens that have suppressed host MHC signaling (Mortimer, 2021).

As of yet, there are no examples of missing-self immune recognition systems in invertebrates, and it has been hypothesized that invertebrate immune systems rely largely on PRRs for nonself recognition of pathogens. Still, invertebrates do mount immune responses against a variety of inanimate objects like oil droplets, sterile nylon, and charged chromatography beads as well as tissue transplants from other insect species. All of these foreign bodies presumably lack distinct PAMPs, suggesting that invertebrates have some sort of missing-self recognition system. Additionally, while multiple antimicrobial PRRs have been identified in the Drosophila genome, PRRs targeting macroparasites like parasitoid wasps have not yet been discovered. The current model of self-recognition suggests that following parasitoid infection, activated immune cells assess all exposed tissue surfaces for the self-tolerance glycan signal and that the absence of this Drosophila SAMP on parasitoid wasp eggs might be the cue that targets them for melanotic encapsulation (Mortimer, 2021).

Role of neuronal and non-neuronal acetylcholine signaling in Drosophila humoral immunity

Acetylcholine (ACh) is one the major neurotransmitters in insects, whose role in mediating synaptic interactions between neurons in the central nervous system is well characterized. It also plays largely unexplored regulatory functions in non-neuronal tissues. This study demonstrates that ACh signaling is involved in the modulation of the innate immune response of Drosophila melanogaster. Knockdown of ACh synthesis or ACh vesicular transport in neurons reduced the activation of drosomycin (drs), a gene encoding an antimicrobial peptide, in adult flies infected with a Gram-positive bacterium. drs transcription was similarly affected in Drosophila α7 nicotinic acetylcholine receptor, nAChRalpha7 (D&al;pha;7) mutants, as well as in flies expressing in the nervous system a dominant negative form (Dα7(DN)) of this specific receptor subunit. Interestingly, Dα7(DN) elicited a comparable response when it was expressed in non-neuronal tissues and even when it was specifically produced in the hemocytes. Consistently, full activation of the drs gene required Dα7 expression in these cells. Moreover, knockdown of ACh synthesis in non-neuronal cells affected drs expression. Overall, these findings uncover neural and non-neural cholinergic signals that modulate insect immune defenses and shed light on the role of hemocytes in the regulation of the humoral immune response (Giordani, 2022).

Heightened immune surveillance in Drosophila melanogaster populations selected for faster development and extended longevity

Maximization of life-history traits is under constraints due to both, limitations of resource acquisition and the restricted pathways of resource allocation. Drosophila melanogaster has served as an excellent model organism to not only unravel various trade-offs among life history traits but also numerous aspects of host immune response. Drosophila larvae are semi-aquatic that live, feed and excrete inside the food source-often over-ripe fruits and vegetables that are rich in both commensal and pathogenic microbiota that can impact the larval survival. This study used six populations of D. melanogaster, three of which are selected for faster pre-adult development and extended adult longevity, and their three ancestral controls, to explore the impact of selection on the basal immune activity in the larval stage. The larvae from selected populations had nearly significantly upregulated plasmatocyte density, significantly higher percent phagocytosis, phagocytic index and higher transcript levels of Tep3, eater and NimC1. Selected populations also had significantly upregulated crystal cell number along with higher transcript of PPO2. Out of seven tested AMPs level, Drosomycin was significantly upregulated in selected populations while Drosocin was significantly higher in control populations. ROS levels were comparable in the selected and control populations. The results strongly suggest that enhanced basal immune activity during larval stage manages the faster development and could be responsible for comparable larval survival of selected and control populations (Shrivastava, 2023).

CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor

Entomopathogenic nematodes are widely used as biopesticides. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors. No protein receptors are known for any Tc toxins, which limits understanding of their specificity and pathogenesis. This study used genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. These findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens (Xu, 2023).

Neuron-derived neuropeptide Y fine-tunes the splenic immune responses

The nervous and immune systems are closely entwined to maintain the immune balance in health and disease. This study shows that LPS can activate suprarenal and celiac ganglia (SrG-CG) neurons and upregulate NPY expression in rats. Single-cell sequencing analysis revealed that knockdown of the NPY gene in SrG-CG altered the proliferation and activation of splenic lymphocytes. In a neuron and splenocyte coculture system and in vivo experiments, neuronal NPY in SrG-CG attenuated the splenic immune response. Notably, it was demonstrated that neuronal NPF in Drosophila exerted a conservative immunomodulatory effect. Moreover, numerous SNPs in NPY and its receptors were significantly associated with human autoimmune diseases, which was further supported by the autoimmune disease patients and mouse model experiments. Together, this study demonstrated that NPY is an ancient language for nervous-immune system crosstalk and might be utilized to alleviate inflammatory storms during infection and to modulate immune balance in autoimmune diseases (Yu, 2022).

It is well documented that sympathetic neurotransmitter norepinephrine (NE) is involved in the modulation of the LPS-elicited splenic immune response. This study revealed that SrG-CG neurons could be activated by LPS and then dramatically upregulated the expression of NPY. This elevated neuronal NPY crosstalks with the immunocytes in spleen and regulates the subsequent immune response. In this line, it has been reported that NPY is involved in modulating systemic inflammation during electroacupuncture. NE and NPY are stored in both small and large storage vesicles of sympathetic nerve terminals. The large vesicles contain both NE and NPY and can co-release both neurotransmitters upon stimulation, whereas the small vesicles only contain one of them. Notably, intravenous application of NPY followed by NE, dose-dependently modulate the NE-induced leukocytosis, suggesting an elegant cooperative regulation mechanism between these two neurotransmitters. Thus, it would be of great importance to utilize more accurate and real-time neurotransmitter detection tools to further explore the precise immune regulation mechanism by these two neurotransmitters (Yu, 2022).

Significant differentially expressed genes (DEGs) were observed in T cells and macrophages that express NPY1R, but not in B cells without NPY1R expression, suggesting that the neuronal NPY modulate splenic inflammation via NPY1R. Consistently, it has been demonstrated that NPY1R is expressed in T cells and macrophages and plays a critical role in inflammatory signaling. According to our single-cell sequencing analysis, NPY may directly act on the T cells and macrophages via NPY1R and subsequently inhibit the TNF signaling pathway, while NE stimulates T cells via β2-adrenergic receptor to secrete acetyl choline and thereby regulates the production of TNF-α and other inflammatory factors in macrophages. Thus, future research should be done to investigate the downstream immunomodulatory pathways of neuronal NPY on other immune cells after the activation macrophages and T cells (Yu, 2022).

Substantial evidence demonstrates that manipulation of the autonomic system can be a therapeutic option for the treatment of autoimmune diseases. Implantable vagus nerve-stimulating devices have been used for the treatment of Rheumatoid Arthritis (RA) and significantly alleviate the autoimmune response. However, the molecular mechanisms during this process-such as neurotransmitters mediating the regulation of the immune system by the nervous system and the role of the sympathetic neuron-NPY-inflammation pathway in the pathogenesis of autoimmune diseases-remain elusive. This study showed that knockdown of NPY in SrG-CG neurons in mouse CIA model caused higher clinical scores. Moreover, the NPY level in the autoimmune disease patients’ serum is significantly lower than that of the control individuals. Thus, it is hypothesized that neuronal dysfunction could disturb the well-balanced secretion of neuronal NPY, disrupt immune regulation, and may subsequently lead to the pathogenesis of autoimmune diseases. It would be of great importance to further investigate the causal effect of neuronal NPY in autoimmune diseases and the treatment of these diseases by manipulating NPY via the sympathetic pathway (Yu, 2022).

Thia study demonstrated that knockdown of NPY in SrG-CG neurons significantly increased the inflammatory cytokines in spleen during LPS treatment, whereas inhibition of NPY1R can promote cytokine production. These results suggested that NPY and NPY1R agonist may be exploited for pathogen infection-induced inflammation storm treatment. It has been shown that administration of NE and NPY or electroacupuncture to stimulate the sympathetic nerve have been employed as a potential therapeutic strategy for inflammatory storms and autoimmune diseases. To optimize the precise NPY pathway-based therapy, two desirable options have been proposed: (1) target delivery of the agonists using nanoparticles and (2) maintain a desired expression level of NPY or NPY1R via tissue-specific promoter by gene therapy (Chandrasekharan et al., 2013 ). These data suggest that NPY pathway-targeted therapy holds promise for future autoimmune diseases and inflammatory storm treatment (Yu, 2022).

Our data suggest that SrG-CG is activated during the immune response in rat and can precisely release NPY to target immunocytes to control inflammation. The serum NPY analysis from individuals with autoimmune diseases imply that the dysfunction of this pathway may lead to the pathogenesis of autoimmune diseases in humans. In other animals, such as fish and C. elegans, their NPY or NPY homologs have also been suggested to be inextricably linked to immune function (Gershkovich et al., 2019 ). The amino acid sequences of NPY and its homologs in different species are conserved in the amidated C terminus. These neuronal NPY/F homologs might be evolutionarily involved in the modulation of immune cells to produce different balanced types of cytokines and chemokines in different animals. These neuropeptides function as an ancient language, mediating the crosstalk between the nervous and immune systems. Compared with NPY/F derived from the paracrine system, neuronal NPY/F can be more precisely and rapidly released from prestored presynaptic vesicles to splenic target cells and can thereby specifically fine-tune the immunoreaction (Yu, 2022).

Taken together, these findings shed light on the crosstalk between the nervous and immune systems and might open another avenue for modulating inflammation during pathogen infection and autoimmune diseases (Yu, 2022).

Gut barrier defects, increased intestinal innate immune response, and enhanced lipid catabolism drive lethality in N -glycanase 1 deficient Drosophila

Intestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of infectious versus non-infectious mechanisms on animal health in the context of barrier dysfunction is not well understood. This study established that loss of Drosophila N -glycanase 1 (Pngl) leads to gut barrier defects, which cause starvation and increased JNK activity. These defects result in Foxo overactivation, which induces a hyperactive innate immune response and lipid catabolism, thereby contributing to lethality associated with loss of Pngl. Notably, germ-free rearing of Pngl mutants did not rescue lethality. In contrast, raising Pngl mutants on isocaloric, fat-rich diets improved animal survival in a dosage-dependent manner. These data indicate that Pngl functions in Drosophila larvae to establish the gut barrier, and that the immune and metabolic consequences of loss of Pngl are primarily mediated through non-infectious mechanisms (Pandey, 2023).

Inhibition of the MEK/ERK pathway suppresses immune overactivation and mitigates TDP-43 toxicity in a Drosophila model of ALS

TDP-43 is an important DNA/RNA-binding protein that is associated with age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, its pathomechanism is not fully understood. In a transgenic RNAi screen using Drosophila as a model, this study uncovered that knockdown (KD) of Dsor1 (the Drosophila MAPK kinase dMEK) suppressed TDP-43 toxicity without altering TDP-43 phosphorylation or protein levels. Further investigation revealed that the Dsor1 downstream gene rl (dERK) was abnormally upregulated in TDP-43 flies, and neuronal overexpression of dERK induced profound upregulation of antimicrobial peptides (AMPs). A robust immune overactivation was pbserved in TDP-43 flies, which could be suppressed by downregulation of the MEK/ERK pathway in TDP-43 fly neurons. Furthermore, neuronal KD of abnormally increased AMPs improved the motor function of TDP-43 flies. On the other hand, neuronal KD of Dnr1, a negative regulator of the Drosophila immune deficiency (IMD) pathway, activated the innate immunity and boosted AMP expression independent of the regulation by the MEK/ERK pathway, which diminished the mitigating effect of RNAi-dMEK on TDP-43 toxicity. Finally, this study showed that an FDA-approved MEK inhibitor trametinib markedly suppressed immune overactivation, alleviated motor deficits and prolonged the lifespan of TDP-43 flies, but did not exhibit a lifespan-extending effect in Alzheimer disease (AD) or spinocerebellar ataxia type 3 (SCA3) fly models. Together, these findings suggest an important role of abnormal elevation of the MEK/ERK signaling and innate immunity in TDP-43 pathogenesis and propose trametinib as a potential therapeutic agent for ALS and other TDP-43-related diseases (Yue, 2023).

No evidence for trans-generational immune priming in Drosophila melanogaster

Most organisms are under constant and repeated exposure to pathogens, leading to perpetual natural selection for more effective ways to fight-off infections. This could include the evolution of memory-based immunity to increase protection from repeatedly-encountered pathogens both within and across generations. There is mixed evidence for intra- and trans-generational priming in non-vertebrates, which lack the antibody-mediated acquired immunity characteristic of vertebrates. This work tested for trans-generational immune priming in adult offspring of the fruit fly, Drosophila melanogaster, after maternal challenge with 10 different bacterial pathogens. Focus was placed on natural opportunistic pathogens of Drosophila spanning a range of virulence from 10% to 100% host mortality. Mothers were infected via septic injury and tested for enhanced resistance to infection in their adult offspring, measured as the ability to suppress bacterial proliferation and survive infection. The mothers were categorized into four classes for each bacterium tested: those that survived infection, those that succumbed to infection, sterile-injury controls, and uninjured controls. No evidence was found for trans-generational priming by any class of mother in response to any of the bacteria (Radhika, 2023).

cGLRs are a diverse family of pattern recognition receptors in animal innate immunity

cGAS (cyclic GMP-AMP synthase; CG7194 in Drosophila) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates the protein STING and downstream immunity. This study discovered cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in animal innate immunity. Building on recent analysis in Drosophila, a bioinformatic approach was used to identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screen of 140 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of alternative nucleotide signals including isomers of cGAMP and cUMP-AMP. Using structural biology, this study explains how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Together these results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity (Li, 2023).

Adult and Larval Tracheal Systems Exhibit Different Molecular Architectures in Drosophila

Knowing the molecular makeup of an organ system is required for its in-depth understanding. This analyzed the molecular repertoire of the adult tracheal system of the fruit fly Drosophila melanogaster using transcriptome studies to advance knowledge of the adult insect tracheal system. Comparing this to the larval tracheal system revealed several major differences that likely influence organ function. During the transition from larval to adult tracheal system, a shift in the expression of genes responsible for the formation of cuticular structure occurs. This change in transcript composition manifests in the physical properties of cuticular structures of the adult trachea. Enhanced tonic activation of the immune system is observed in the adult trachea, which encompasses the increased expression of antimicrobial peptides. In addition, modulatory processes are conspicuous, in this case mainly by the increased expression of G protein-coupled receptors in the adult trachea. Finally, all components of a peripheral circadian clock are present in the adult tracheal system, which is not the case in the larval tracheal system. Comparative analysis of driver lines targeting the adult tracheal system revealed that even the canonical tracheal driver line breathless (btl)-Gal4 is not able to target all parts of the adult tracheal system. This study have uncovered a specific transcriptome pattern of the adult tracheal system and provide this dataset as a basis for further analyses of the adult insect tracheal system (Bossen, 2023).

Correlated responses in basal immune function in response to selection for fast development in Drosophila melanogaster

Often, immunity is invoked in the context of infection, disease and injury. However, an ever alert and robust immune system is essential for maintaining good health, but resource investment into immunity needs to be traded off against allocation to other functions. This examines the consequences of such a trade-off with growth by ascertaining various components of baseline innate immunity in two types of Drosophila melanogaster populations selected for fast development, in combination with either a long effective lifespan (FLJs) or a short effective lifespan (FEJs). Distinct immunological parameters were found to be constitutively elevated in both, FLJs and FEJs. Their ancestral control (JB) populations, and these constitutive elevated immunological parameters were associated with reduced insulin signalling and comparable total gut microbiota. The results bring into focus the inter-relationship between egg to adult development time, ecdysone levels, larval gut microbiota, insulin signalling, adult reproductive longevity and immune function. How changes in selection pressures operating on life-history traits can modulate different components of immune system is discussed (Shrivastava, 2023).

Immune defense in Drosophila melanogaster depends on diet, sex, and mating status

Immune defense is a complex trait that affects and is affected by many other host factors, including sex, mating, and dietary environment. This study used the agriculturally relevant fungal emtomopathogen, Beauveria bassiana, and the model host organism Drosophila melanogaster to examine how the impacts of sex, mating, and dietary environment on immunity are interrelated. The direction of sexual dimorphism in immune defense was shown to depend on mating status and mating frequency. It was also shown that post-infection dimorphism in immune defense changes over time and is affected by dietary condition both before and after infection. Supplementing the diet with protein-rich yeast improved post-infection survival but more so when supplementation was done after infection instead of before. The multi-directional impacts among immune defense, sex, mating, and diet are clearly complex, and while this study shines light on some of these relationships, further study is warranted. Such studies have potential downstream applications in agriculture and medicine (Rai, 2023).

A PI3K-calcium-Nox axis primes leukocyte Nrf2 to boost immune resilience and limit collateral damage

Phagosomal reactive oxygen species (ROS) are strategically employed by leukocytes to kill internalized pathogens and degrade cellular debris. Nevertheless, uncontrolled oxidant bursts could cause serious collateral damage to phagocytes or other host tissues, potentially accelerating aging and compromising host viability. Immune cells must, therefore, activate robust self-protective programs to mitigate these undesired effects, and yet allow crucial cellular redox signaling. This study dissected in vivo the molecular nature of these self-protective pathways, their precise mode of activation, and physiological effects. Drosophila embryonic macrophages activate the redox-sensitive transcription factor Nrf2 upon corpse engulfment during immune surveillance, downstream of calcium- and PI3K-dependent ROS release by phagosomal Nox. By transcriptionally activating the antioxidant response, Nrf2 not only curbs oxidative damage but preserves vital immune functions (including inflammatory migration) and delays the acquisition of senescence-like features. Strikingly, macrophage Nrf2 also acts non-autonomously to limit ROS-induced collateral damage to surrounding tissues. Cytoprotective strategies may thus offer powerful therapeutic opportunities for alleviating inflammatory or age-related diseases (Clemente, 2023).

Peroxiredoxins Play an Important Role in the Regulation of Immunity and Aging in Drosophila

Aberrant immune responses and chronic inflammation can impose significant health risks and promote premature aging. Pro-inflammatory responses are largely mediated via reactive oxygen species (ROS) and reduction-oxidation reactions. A pivotal role in maintaining cellular redox homeostasis and the proper control of redox-sensitive signaling belongs to a family of antioxidant and redox-regulating thiol-related peroxidases designated as peroxiredoxins (Prx). Recent studies in Drosophila have shown that Prxs play a critical role in aging and immunity. This study identified two important 'hubs', the endoplasmic reticulum (ER) and mitochondria, where extracellular and intracellular stress signals are transformed into pro-inflammatory responses that are modulated by the activity of the Prxs residing in these cellular organelles. This study found that mitochondrial Prx activity in the intestinal epithelium is required to prevent the development of intestinal barrier dysfunction, which can drive systemic inflammation and premature aging. Using a redox-negative mutant, this study demonstrated that Prx acts in a redox-dependent manner in regulating the age-related immune response. The hyperactive immune response observed in flies under-expressing mitochondrial Prxs is due to a response to abiotic signals but not to changes in the bacterial content. This hyperactive response, but not reduced lifespan phenotype, can be rescued by the ER-localized Prx (Odnokoz, 2023).

Macrophage-derived insulin antagonist ImpL2 induces lipoprotein mobilization upon bacterial infection

The immune response is an energy-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system. Although this interplay is fundamental for the function of the immune system, the underlying mechanisms remain elusive. The data of this study show that the pro-inflammatory polarization of Drosophila macrophages is coupled to the production of the insulin antagonist ImpL2 through the activity of the transcription factor HIF1α. ImpL2 production, reflecting nutritional demands of activated macrophages, subsequently impairs insulin signaling in the fat body, thereby triggering FOXO-driven mobilization of lipoproteins. This metabolic adaptation is fundamental for the function of the immune system and an individual's resistance to infection. This study demonstrated that analogically to Drosophila, mammalian immune-activated macrophages produce ImpL2 homolog IGFBP7 in a HIF1α-dependent manner and that enhanced IGFBP7 production by these cells induces mobilization of lipoproteins from hepatocytes. Hence, the production of ImpL2/IGFBP7 by macrophages represents an evolutionarily conserved mechanism by which macrophages alleviate insulin signaling in the central metabolic organ to secure nutrients necessary for their function upon bacterial infection (Krejxova, 2023).

Amendment of Altered Immune Response by Curcumin in Drosophila Model of Huntington's Disease

Though primarily classified as a brain disorder, surplus studies direct Huntington's disease (HD) to be a multi-system disorder affecting various tissues and organs, thus affecting overall physiology of host. Recently, it has been reported that neuronal expression of mutant huntingtin induces immune dysregulation in Drosophila and may pose chronic threat to challenged individuals. Therefore, the polyphenolic compound curcumin was tested to circumvent the impact of immune dysregulation in Drosophila model of HD. The present study examined the molecular basis underlying immune derangements and immunomodulatory potential of curcumin in HD. UAS-GAL4 system was used to imitate the HD symptoms in Drosophila, and the desired female progenies (elav > Httex1pQ25; control and elav > Httex1pQ93; diseased) were cultured on food mixed without and with 10 μM concentration of curcumin since early development. Effect of curcumin supplementation was investigated by monitoring the hemocytes' count and their functional abilities in diseased condition. Reactive oxygen species (ROS) level in cells was assessed by DHE staining and mitochondrial dysfunction was assessed by CMXros red dye. In addition, transcript levels of pro-inflammatory cytokines and anti-microbial peptides were monitored by qRT-PCR. Curcumin supplementation was found to effectively reduced higher crystal cell count and phenoloxidase activity in diseased flies. Interestingly, curcumin significantly managed altered plasmatocytes count, improved their phagocytic activity by upregulating the expression of key phagocytic receptors in HD condition. Moreover, substantial alleviation of ROS levels and mitochondria dysfunction was observed in plasmatocytes of diseased flies upon curcumin supplementation. Furthermore, curcumin administration effectively attenuated transcriptional expression of pro-inflammatory cytokines and AMPs in diseased flies. These results indicate that curcumin efficiently attenuates immune derangements in HD flies and may prove beneficial in alleviating complexities associated with HD (Dhankhar, 2023).

Perilipin 1 Deficiency Prompts Lipolysis in Lipid Droplets and Aggravates the Pathogenesis of Persistent Immune Activation in Drosophila

Lipid droplets (LDs) are highly dynamic intracellular organelles, which are involved in numerous of biological processes. However, the dynamic morphogenesis and functions of intracellular LDs during persistent innate immune responses remain obscure. This study induce long-term systemic immune activation in Drosophila through genetic manipulation. Then, the dynamic pattern of LDs is traced in the Drosophila fat body. Deficiency of Plin1, a key regulator of LDs' reconfiguration, blocks LDs minimization at the initial stage of immune hyperactivation but enhances LDs breakdown at the later stage of sustained immune activation via recruiting the lipase Brummer (Bmm, homologous to human ATGL). The high wasting in LDs shortens the lifespan of flies with high-energy-cost immune hyperactivation. Therefore, these results suggest a critical function of LDs during long-term immune activation and provide a potential treatment for the resolution of persistent inflammation (Wang, 2023).

Two clip-domain serine protease homologs, cSPH35 and cSPH242, act as a cofactor for prophenoloxidase-1 activation in Drosophila melanogaster

Insect phenoloxidases (POs) catalyze phenol oxygenation and o-diphenol oxidation to form reactive intermediates that kill invading pathogens and form melanin polymers. To reduce their toxicity to host cells, POs are produced as prophenoloxidases (PPOs) and activated by a serine protease cascade as required. In most insects studied so far, PPO activating proteases (PAPs) generate active POs in the presence of a high M(r) cofactor, comprising two serine protease homologs (SPHs) each with a Gly residue replacing the catalytic Ser of an S1A serine protease (SP). These SPHs have a regulatory clip domain at the N-terminus, like most of the SP cascade members including PAPs. In Drosophila, PPO activation and PO-catalyzed melanization have been examined in genetic analyses but it is unclear if a cofactor is required for PPO activation. This study produced the recombinant cSPH35 and cSPH242 precursors (see Drosophila Sph35 and Sph242), activated them with Manduca sexta PAP3, and confirmed their predicted role as a cofactor for Drosophila Prophenoloxidase 1 activation by MP2 (i.e., Sp7). The cleavage sites and mechanisms for complex formation and cofactor function are highly similar to those reported in M. sexta. In the presence of high M(r) complexes of the cSPHs, PO at a high specific activity of 260 U/μg was generated in vitro. To complement the in vitro analysis, hemolymph PO activity levels were measured in wild-type flies, cSPH35, and cSPH242 RNAi lines. Compared with the wild-type flies, only 4.4% and 18% of the control PO level (26 U/μl) was detected in the cSPH35 and cSPH242 knockdowns, respectively. Consistently, percentages of adults with a melanin spot at the site of septic pricking were 82% in wild-type, 30% in cSPH35 RNAi, and 53% in cSPH242 RNAi lines; the survival rate of the control (45%) was significantly higher than those (30% and 15%) of the two RNAi lines. These data suggest that Drosophila cSPH35 and cSPH242 are components of a cofactor for MP2-mediated PPO1 activation, which are indispensable for early melanization in adults (Jin, 2023).

Lipopolysaccharide -mediated resistance to host antimicrobial peptides and hemocyte-derived reactive-oxygen species are the major Providencia alcalifaciens virulence factors in Drosophila melanogaster

Bacteria from the genus Providencia are ubiquitous Gram-negative opportunistic pathogens, causing "travelers' diarrhea", urinary tract, and other nosocomial infections in humans. Some Providencia strains have also been isolated as natural pathogens of Drosophila melanogaster. This study investigated the virulence factors of a representative Providencia species-P. alcalifaciens. A P. alcalifaciens transposon mutant library was generated, and an unbiased forward genetics screen was performed in vivo for attenuated mutants. The screen uncovered 23 mutants with reduced virulence. The vast majority of them had disrupted genes linked to lipopolysaccharide (LPS) synthesis or modifications. These LPS mutants were sensitive to cationic antimicrobial peptides (AMPs) in vitro and their virulence was restored in Drosophila mutants lacking most AMPs. Thus, LPS-mediated resistance to host AMPs is one of the virulence strategies of P. alcalifaciens. Another subset of P. alcalifaciens attenuated mutants exhibited increased susceptibility to reactive oxygen species (ROS) in vitro and their virulence was rescued by chemical scavenging of ROS in flies prior to infection. Using genetic analysis, it was found that the enzyme Duox specifically in hemocytes is the source of bactericidal ROS targeting P. alcalifaciens. Consistently, the virulence of ROS-sensitive P. alcalifaciens mutants was rescued in flies with Duox knockdown in hemocytes. Therefore, these genes function as virulence factors by helping bacteria to counteract the ROS immune response. This reciprocal analysis of host-pathogen interactions between D. melanogaster and P. alcalifaciens identified that AMPs and hemocyte-derived ROS are the major defense mechanisms against P. alcalifaciens, while the ability of the pathogen to resist these host immune responses is its major virulence mechanism (Shaka, 2022)

The aim of this study was to dissect the host-pathogen interactions between Providencia and D. melanogaster. To achieve this aim, various genetic approaches were used that enabled determination of the contributions of both pathogen and host to the outcome of the infection. First, the responses of the fruit fly to Pa infection was characterized and, using mutant analysis, the Imd pathway and iron sequestration were identified as prominent defense mechanisms against Pa. Second, an unbiased forward genetics screen was performed using a transposon mutant library that were generated for this purpose, and Pa virulence factors necessary to infect the fly were identified. This mutant library has the potential to serve as a valuable resource for exploring the genetic basis for all Pa traits. Third, mutants of the major immune pathways in Drosophila were used, and they were infected with attenuated Pa mutants to identify pathogen virulence factors that allow the bacteria to respond to specific immune defenses and evade immune clearance. Thereby, this study dissected both sides of host-pathogen relationship in a Drosophila-Providencia model and provided the first insights into the molecular mechanisms of Pa virulence (Shaka, 2022)

To identify Pa virulence factors, an in vivo screen was performed which yielded 23 attenuated mutants. The majority of these mutants (15/23) had transposon insertions in genes involved in LPS biosynthesis and LPS modifications, pointing towards a vital role of intact LPS in Pa pathogenesis. This finding is consistent with a well-known role of LPS in host-pathogen interactions. At the mechanistic level, LPS protects Pa from Drosophila Imd pathway-dependent AMPs, particularly Drosocin. Consistent with this, Pa LPS mutants showed increased susceptibility in vitro to the cationic AMP polymyxin B and their virulence was restored in Relish and ΔAMP mutant flies deficient for Imd-dependent AMPs. The finding that Pa LPS mediates resistance to host AMPs complements numerous previous studies in diverse pathogens that reported a similar protective function of LPS against host innate defenses. Several studies that used Drosophila as an infection model also discovered LPS as an essential protective barrier against insect AMPs. For example, another study found that LPS O-antigen-deficient Serratia marcescens mutants were attenuated in wild-type flies but not in an Imd pathway mutant. A similar phenotype was reported for F. novicida mutants with affected LPS. These data demonstrate that a major determinant of virulence in several pathogens is the LPS-mediated ability to resist the systemic immune response. Additionally, LPS was shown to facilitate microbiota-host interactions. For instance, LPS biosynthesis mutants of Acetobacter fabarum, a Drosophila commensal, had a reduced ability to colonize the fruit fly intestine. While the mechanism behind this phenotype has not been investigated yet, increased sensitivity to intestinal AMPs is a likely reason, as shown for the human commensal Bacteroides thetaiotaomicron. Among the LPS mutants, ArnA (pmrA) (PL11H9) was found that encodes an enzyme that catalyzes the formation of modified arabinose UDP-L-4-formamido-arabinose (UDP-L-Ara4FN). The modified arabinose reduces the negative charge of lipid A and the binding of cationic AMPs. This is the most commonly observed LPS modification implicated in cationic AMP resistance. This modification is also crucial for Yersinia pestis resistance to the insect cecropin-like AMP cheopin (Shaka, 2022)

In addition to mutations affecting LPS, several were uncovered that disrupt lipoproteins, like OmpA (PL13H10), NlpI (PL7D10), and YbaY (PL5A4). While YbaY is poorly characterized, OmpA and NlpI were previously implicated in the virulence of different pathogens. Whereas OmpA contributes to virulence in various ways ranging from facilitating adhesion and invasion to conferring resistance to serum, NlpI function in virulence is less clear. The results suggest that all three lipoproteins mutants behave like LPS mutants–they are susceptible to polymyxin B and their virulence is rescued in an AMP mutant, indicating that their reduced virulence is due to an increased susceptibility to host AMPs. The mechanism behind this phenotype requires further investigation, however NlpI was shown to be essential for cell envelop integrity, which might contribute to increased sensitivity to AMPs. The screen uncovered two additional peptidoglycan-associated lipoproteins, TolB (PL2D4) and Pal (PL4B5), that are part of a multiprotein complex, the Tol-Pal system. It bridges between the peptidoglycan and the outer membrane and is important for proper structure and function of the outer membrane. Importantly, TolA and Pal are necessary for correct surface polymerization of O-antigen chains, likely explaining the sensitivity of tol and pal mutants to detergents and several antibiotics. Similar to the Pa tol and pal mutants, F. novicida mutants in these genes were attenuated in Drosophila infection and more sensitive to host AMPs (Shaka, 2022)

The second largest group of mutants with reduced virulence that were identified constitutes ROS-sensitive mutants. Since it was possible rescue the virulence of these mutants by chemical or genetic ROS scavenging, their attenuated virulence is likely due to an inability to resist host ROS produced in response to infection. Among such ROS-sensitive mutants, only the one lacking cytochrome oxidase (PL1A3) was previously shown to be required for virulence in other bacteria by enhancing the tolerance to oxidative stress. Some other genes, like dihydrolipoyl dehydrogenase (PL4F11) and typA (PL6B7), were also linked to virulence but not necessarily via ROS sensitivity. No previous evidence was found of the role of ATPase RavA stimulator ViaA (PL14C2) in virulence, however there seems to be a link to ROS response in E. coli. Therefore, further investigation of the identified genes is required to clarify their role in bacterial virulence and ROS sensitivity. While previous studies identified several sources of ROS in flies, including melanisation, hemocytes, Nox and, Duox, the results showed that Duox specifically in hemocytes is the major producer of ROS in case of Pa infection. Notably, in case of F. novicida melanisation played a prominent role as a source of ROS. An interesting avenue for future studies would be to understand the differences between Duox- and melanisation-derived ROS and their preferential activity against specific pathogens (Shaka, 2022)

The screen also identified several hypothetical proteins. Using ROS and polymyxin B sensitivity assays and rescue in AMP- and ROS-deficient flies, it was shown that PL4E6 and PL11H8 contribute to bacterial resistance to host AMPs, while PL6D10 is necessary to survive ROS exposure. Thus, with this approach a mechanism of virulence could be assigned to hypothetical proteins with unknown function. However, how those protein contribute to ROS or AMP sensitivity remains unknown (Shaka, 2022)

One Pa mutant (Sigma-E factor regulatory protein rseB, PL13C10), was identified that was not sensitive to ROS and polymyxin in vitro. However, the virulence of this mutant was rescued in Relish and AMP-deficient flies. Very likely the rescue phenotype could be due to sensitivity to additional antimicrobial peptides produced by flies. Such increased sensitivity to AMPs is possible given the role of Sigma-E factor in cell envelope integrity (Shaka, 2022)

Among all AMPs tested, Drosocin proved to be particularly important in controlling Pa infection. Consistent with the Pa LPS mutants, F. novicida mutants in LPS were particularly sensitive to Drosocin. Considering that Drosocin is known to bind bacterial LPS, alterations in LPS might promote Drosocin interactions with LPS and bacterial killing or make intracellular targets more accessible. A previous in vivo analysis of AMP specificity has shown that Drosocin plays a critical role in controlling Enterobacter cloacae infection. A recent study confirmed this finding, however additionally reported that the Drosocin gene encodes not one, but two AMPs: Drosocin and IM7 (newly named as Buletin). Buletin but not Drosocin contributes to host defense against Providencia burhodogranariea infection. Since the Drosocin mutant that was used lacked both Drosocin and Buletin and the Drosocin overexpression line similarly produced both peptides, it remains to be tested whether Drosocin or Buletin or both peptides together are involved in the defense against Pa (Shaka, 2022)

While in vivo experiments demonstrate that AMPs are the major Relish-regulated molecules controlling Pa LPS mutants, in vitro assays with synthesized Drosophila AMPs were not conclusive. None of the three Drosophila AMPs that were tested, Cecropin A, Cecropin B, and Diptericin B, showed activity against Pa. Considering the high specificity of some AMP-microbe interactions, it could be that the peptides that were tested have no effect on Pa. Indeed, based on in vivo results, Drosocin, which was not available for an in vitro test, is the primary AMP controlling Pa infection. Additionally, in vitro effects of AMPs can be different than in vivo effects of mutants or knockdowns for the same AMPs, suggesting that physiological context or interaction among peptides is important. Also, there are a number of technical reasons why in vitro assays may not reflect in vivo activities, including AMPs adhering to plastic assay plates, differences in salt concentrations or pH, stress on microbes, interactions among AMPs and between AMPs and other components of the immune system. These potential issues have to be considered when interpreting the results of in vitro antimicrobial tests performed with AMPs (Shaka, 2022)

Contrary to expectations, the screen did not hit any bacterial effectors, like toxins, that might be responsible for damaging the host. Since toxins are likely to be redundant, disruption of an individual toxin gene may not give a phenotype. Similarly, no mutants were identified in secretion systems, suggesting that Pa does not require effector translocation to infect Drosophila. The only toxin that was so far implicated in Pa pathogenesis is cytolethal distending toxin which blocks eukaryotic cell proliferation. Interestingly, Pa LPS was shown to cause epithelial barrier dysfunction by reducing occludin levels in Caco-2 cell monolayers and induced apoptosis in calf pulmonary artery endothelial cells. Thus, LPS might not only mediate resistance to host AMPs but also act as an effector-like molecule (Shaka, 2022)

By discovering the mechanisms of Pa resistance to host AMPs and ROS, this study opens the doors to potential strategies to exploit such Pa mechanisms and sensitize the pathogen to host defenses to improve infection treatment. To illustrate the feasibility of such an approach, polymyxin B treatment was used to disrupt Pa LPS in vivo, and it was found to be sufficient to improve Drosophila survival after infection. Such beneficial effect of polymyxin B required functional Imd pathway signalling and was independent of direct bactericidal activity, suggesting that disruption of the major barrier against AMPs sensitizes the pathogen to host defenses. These results suggest that affecting LPS function might be a useful strategy to treat Providencia infections, particularly those resistant to antibiotics (Shaka, 2022)

Sensitizing Pa to host ROS also appears to be an attractive anti-virulence strategy, considering that resistance to host ROS is one of the key Pa virulence mechanisms that was identified. Some compounds were shown to sensitize the pathogens to oxidative stress and immune clearance but in a species-specific manner. For example, 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC) treatment inhibits production of pyomelanin pigment and increases sensitivity of pyomelanogenic Pseudomonas aeruginosa strains to oxidative stress. Similarly, BPH-642 –cholesterol biosynthesis inhibitor, blocked biosynthesis of staphyloxanthin antioxidant pigment in S. aureus, resulting in increased immune clearance in a mouse infection model. However, to date there are no known compounds that would predispose Pa or generally any pathogen to ROS without being toxic to the host, thus limiting the development of ROS-potentiating anti-infectives (Shaka, 2022)

In summary, reciprocal analysis of interactions between D. melanogaster and P. alcalifaciens revealed that the host relies on Imd-dependent AMPs and hemocyte-derived ROS as major branches of immunity that are important for fighting infection with P. alcalifaciens. On the pathogen side, it was found that the ability to resist these host immune responses is the major virulence mechanism of P. alcalifaciens. Leveraging this knowledge has great potential to improve P. alcalifaciens infection treatment either by potentiating the host defenses or disrupting pathogen virulence (Shaka, 2022)

Tissue-specific regulation of Drosophila NF-κB pathway activation by peptidoglycan recognition protein

In Drosophila, peptidoglycan (PGN) is detected by PGN recognition proteins (PGRPs) that act as pattern recognition receptors. Some PGRPs such as PGRP-LB or PGRP-SCs are able to cleave PGN, therefore reducing the amount of immune elicitors and dampening immune deficiency (IMD) pathway activation. By generating PGRP-SC-specific mutants, this study reevaluated the roles of PGRP-LB, PGRP-SC1 and PGRP-SC2 during immune responses. These genes were shown to be expressed in different gut domains, and they follow distinct transcriptional regulation. Loss-of-function mutant analysis indicates that PGRP-LB is playing a major role in IMD pathway activation and bacterial load regulation in the gut, although PGRP-SCs are expressed at high levels in this organ. PGRP-SC2 is the main negative regulator of IMD pathway activation in the fat body. Accordingly, mutants for either PGRP-LB or PGRP-SC2 displayed a distinct susceptibility to bacteria depending on the infection route. Lastly, PGRP-SC1 and PGRP-SC2 are required in vivo for full Toll pathway activation by Gram-positive bacteria (Costechareyre, 2015).

Peptidoglycan (PGN) and PGN recognition proteins (PGRPs) are the main microbe-associated molecular patterns and pattern recognition receptors that regulate the antibacterial response in Drosophila, respectively. Some PGRP family members such as PGRP-LC, PGRP-SA, PGRP-SD or PGRP-LE have the ability to bind PGN and are therefore essential sentinels upstream of the two NF-κB-dependent Drosophila signaling cascades, called Toll and immune deficiency (IMD). Recognition of lysine (Lys)-type PGN by PGRP-SA is sufficient to trigger the Toll/Dorsal/Dif signaling, whereas detection of diaminopimelic (DAP)-type PGN (either membrane-associated via PGRP-LC or intracellularly via PGRP-LE) promotes IMD/Relish signaling activation (Costechareyre, 2015).

Biochemical experiments have demonstrated that other PGRP family members, such as PGRP-LB, PGRP-SB and PGRP-SC, are not only able to bind PGN but also display an amidasic activity that allows them to cleave PGN into smaller nonimmunogenic muropeptides. In the case of PGRP-LB, in vivo experiments have clearly shown that by degrading PGN, PGRP-LB provides a negative feedback regulation that allows a tight adjustment of the immune activation to the intensity of the infection. In the absence of PGRP-LB, flies overrespond to bacteria and eventually die for unknown reasons. Although the ability of PGRP-SC proteins to cleave PGN is clearly documented, published results on the in vivo role of PGRP-SC in immune system activation are difficult to reconcile into a coherent model (Costechareyre, 2015).

The PGRP-SC1 protein is coded by two genes, PGRP-SC1a and PGRP-SC1b, which both produce the same polypeptide. This amidase was initially identified as a scavenger receptor that, by cleaving Staphylococcus aureus Lys-type PGN, reduces its immune-stimulatory activity on the IMD pathway in cultured cells (Mellroth, 2003). Later on, using an RNAi-mediated approach, it was shown that simultaneous inactivation of PGRP-SC1a/PGRP-SC1b and PGRP-SC2 in the gut induces ectopic expression of immune-inducible genes in the fat body following Escherichia coli ingestion (Bischoff, 2006). This role of PGRP-SCs as negative regulators of IMD pathway activation was later confirmed using a deletion removing PGRP-SC1a/ PGRP-SC1b and PGRP-SC2 (Paredes, 2011). This study also revealed that PGRP-SC-dependent negative regulation takes place in the fat body during the systemic response and not in the gut itself. PGRP-SC1 was independently identified through an EMS genetic screen as a protein required for Toll pathway activation and for phagocytosis (Garver, 2006). Surprisingly, while the PGN-cleaving activity is required to mediate S. aureus phagocytosis, it is dispensable for Toll activation. Finally, a recent report proposed that by reducing IMD/Relish signaling in the gut, PGRP-SC2 is preventing commensal dysbiosis, stem cell hyperproliferation and epithelial dysplasia, and, in turn, prevents gut aging (Guo, 2014). The different conclusions drawn from these studies could be explained, at least partly, either by the different techniques used to inactivate the genes (RNAi, KO or EMS, for example) or by the fact that while some studies analyzed the effect of removing one PGRP-SC (PGRP-SC1 or PGRP-SC2), others described the phenotype of Drosophila mutant affecting both PGRP-SC1 and PGRP-SC2 (Costechareyre, 2015).

To clarify the respective role of PGRP-SCs and PGRP-LB in immune response modulation, this study generated specific KO for each of the PGRP-SC genes and analyzed their immune phenotypes. The results failed to identify any clear IMD-dependent function for PGRP-SC1, although its transcriptional induction is the highest of the entire genome after gut bacterial colonization. It was demonstrated that although PGRP-SC2 and PGRP-LB are both strong negative regulators of IMD, they act in different tissues. Whereas PGRP-LB is needed in the gut to cleave PGN and prevent both local gut activation and PGN dissemination into the hemolymph, PGRP-SC2 is mainly required in the fat body to control systemic immune response. Rescue experiments also show that PGRP-SC2 and PGRP-LB are not functionally equivalent. Finally, mutant phenotype analysis indicated that both PGRP-SC1 and PGRP-SC2 are positive regulators of the Toll signaling cascade (Costechareyre, 2015).

The results presented in this study demonstrate that PGRP-LB, PGRP-SC1 and PGRP-SC2 have different spatiotemporal expression patterns and play specific roles in regulating Drosophila immune responses. As far as the IMD pathway is concerned, PGRP-SC1a/PGRP-SC1b elimination did not provoke any modification in immune pathway activation. This was rather unexpected, since PGRP-SC1 is the Drosophila most induced gene following bacterial colonization. In contrast, the data showed that both PGRP-SC2 and PGRP-LB are strong dampeners of the IMD pathway. In accordance with previous work, it was demonstrated that PGRP-LB is the essential amidase in the gut. However, it remained unclear why the PGRP-SC2 amidase which is highly expressed in the gut has such a minor role in regulating IMD pathway activation or bacterial load in this organ. Some kind of functional redundancy could explain the lack of effect. However, previous work did not report a very strong IMD pathway up-regulation in the double PGRP-SC mutant. In addition, whereas PGRP-LB and PGRP-SC2 are both expressed in the Vtr, removing the PGRP-LB gene had a clear phenotype, speaking against functional redundancy between these two PGN-cleaving enzymes. In addition, using ectopic expression tools, it was possible to show that while ectopic PGRP-LB expression can rescue the PGRP-LB-mutant phenotype, PGRP-SC1 and PGRP-SC2 cannot. This clearly demonstrated that in addition to being expressed in different spatiotemporal patterns, amidases are not functionally equivalent. In this respect, it is interesting to note that PGRP-LB is functionally important in the gut and PGRP-SC2 in the circulating hemolymph. Indeed, it was shown previously that the mode of bacterial detection in the gut and in the fat body are different. While most enterocytes rely on the intracellular PGRP-LE for PGN detection, fat body cells detect PGN mainly via PGRP-LC. It is well possible that these two receptors are activated in vivo by different ligands. A possible model could be that PGRP-LB is preventing the production of PGRP-LE-activating ligands (such as TCT) whereas PGRP-SC2 is preventing accumulation of PGRP-LC ligands. Further experiments will be needed to test this hypothesis (Costechareyre, 2015).

Using PGRP-SC mutants, this study also showed that amidases are not only required to dampen the IMD pathway but also to facilitate Toll signaling activation. This indicated that, surprisingly, the action of amidases had opposite effects on Toll and IMD signaling activation. Since the activation of both pathways depends on PGN recognition by PGRP family members, one can postulate that while a PGRP-SC-digested DAP-type PGN will be a weaker IMD pathway activator and therefore probably a weak PGRP-LC ligand, a PGRP-SC-digested Lys-type PGN will be a good inducer of Toll signaling and therefore strongly recognized by PGRP-SA. This antagonistic effect correlates well with the fact that while the IMD cascade strongly needs to be down-regulated to prevent flies from dying of infection, this is not at all the case for the Toll pathway whose constitutive activation has no effect on the flies' viability but is probably more efficient to fight infection (Costechareyre, 2015).

The data presented in this study demonstrated the complexity and interdependence of the interactions that are occurring to adapt the immune responses towards bacteria entering the body cavity of Drosophila. Analyzing immune responses in Vtr, Cc and Pmg separately, it was demonstrated that different gut domains produce different amidase cocktails and display specific responses. However, gut dissection has shown that the gut can be anatomically subdivided into more than ten subdomains. This could potentially greatly increase the complexity of the regulation. In addition, one also cannot exclude the possibility that amidases are acting successively to degrade PGN. If such a PGN will be first cleaved by a given amidase before being a target for another PGN-cleaving enzyme, the interpretation of the mutant phenotype will be even more complicated. This could be the case for Ecc PGN that could first be modified in the gut lumen by PGRP-LB before being digested in the hemolymph by PGRP-SC2. One should also keep in mind that PGRPs with amidase activity are potentially secreted proteins and could therefore act distant from the site where they are produced. They could eventually travel together with the bacteria from one gut domain to another. Finally, the data showed that mutations in a given amidase can have opposite effects on the regulation of the two main signaling immune pathways, IMD and Toll. This could potentially be explained with two biological roles of PGRP-SC, an amidase-dependent and an amidase-independent function. Consistently, Garver (2006) demonstrated that a noncatalytic cysteine-serine PGRP-SC1a transgene is able to rescue a PGRP-SC1a mutant as far as Toll pathway activation is concerned. Knowing that some immune genes are specifically activated by one cascade whereas others depend on both signaling pathways, one should interpret the immunomodulation and immune phenotypes observed in amidase mutants with caution (Costechareyre, 2015).

Cytokine Diedel and a viral homologue suppress the IMD pathway in Drosophila

Insect viruses express suppressors of RNA interference or apoptosis, highlighting the importance of these cell intrinsic antiviral mechanisms in invertebrates. This study reports the identification and characterization of a family of proteins encoded by insect DNA viruses that are homologous to a 12-kDa circulating protein encoded by the virus-induced Drosophila gene diedel (die). die mutant flies were shown to have shortened lifespan and succumb more rapidly than controls when infected with Sindbis virus. This reduced viability is associated with deregulated activation of the immune deficiency (IMD) pathway of host defense and can be rescued by mutations in the genes encoding the homolog of IKKγ or IMD itself. These results reveal an endogenous pathway that is exploited by insect viruses to modulate NF-κB signaling and promote fly survival during the antiviral response (Lamiable, 2016).

Small RNA-Seq analysis reveals microRNA-regulation of the Imd pathway during Escherichia coli infection in Drosophila

Drosophila have served as a model for research on innate immunity for decades. However, knowledge of the post-transcriptional regulation of immune gene expression by microRNAs (miRNAs) remains rudimentary. Using small RNA-seq and bioinformatics analysis, this study identified 67 differentially expressed miRNAs in Drosophila infected with Escherichia coli compared to injured flies at three time-points. Twenty-one of these miRNAs were potentially involved in the regulation of Imd pathway-related genes. Strikingly, based on UAS-miRNAs line screening and Dual-luciferase assay, miR-9a and miR-981 both negatively regulated Drosophila antibacterial defenses and decreased the level of the antibacterial peptide, Diptericin. Taken together, these data support the involvement of miRNAs in the regulation of the Drosophila Imd pathway (Li, 2017).

UbcD4, an ortholog of E2-25K/Ube2K, is essential for activation of the immune deficiency pathway in Drosophila

Ubiquitination is a key regulatory mechanism in the immune deficiency (IMD) pathway in Drosophila. This study developed a simple immunoblot method to identify components involved in this pathway. Considering the emerging roles of ubiquitin-conjugating enzymes (E2s) in determining ubiquitin chain types and ubiquitination speed, a screen was performed for E2s required for IMD activation. UbcD4, in addition to the previously reported E2s Effete and Bendless, was shown to be required for activation of the IMD pathway. RNAi-mediated knockdown of the UbcD4 ortholog, E2-25K/Ube2K, inhibited TNFα- and LPS-mediated activation of the NF-κB pathway, implying that UbcD4 and E2-25K/Ube2K play a conserved role as positive regulators in both pathways (Park, 2015).

RNA interference directed against the Transglutaminase gene triggers dysbiosis of gut microbiota in Drosophila

Transglutaminase (TG) suppresses immune deficiency pathway-controlled antimicrobial peptides (IMD-AMPs), thereby conferring immune tolerance to gut microbes, and RNAi of the TG gene in flies has been shown to decrease the lifespan compared with non-TG-RNAi flies. In this study, analysis of the bacterial composition of the Drosophila gut by next-generation sequencing revealed that gut microbiota comprising one dominant genus of Acetobacter in non-TG-RNAi flies was shifted to that comprising two dominant genera of Acetobacter and Providencia in TG-RNAi flies. Four bacterial strains, including Acetobacter persici SK1 and Acetobacter indonesiensis SK2, Lactobacillus pentosus SK3, and Providencia rettgeri SK4, were isolated from the midgut of TG-RNAi flies. SK1 exhibited the highest resistance to the IMD-AMPs Cecropin A1 and Diptericin among the isolated bacteria. In contrast, SK4 exhibited considerably lower resistance against Cecropin A1, whereas SK4 exhibited high resistance to hypochlorous acid. The resistance of strains SK1-4 against IMD-AMPs in in vitro assays could not explain the shift of the microbiota in the gut of TG-RNAi flies. The lifespan was reduced in gnotobiotic flies that ingested both SK4 and SK1, concomitant with the production of reactive oxygen species and apoptosis in the midgut, whereas survival rate was not altered in gnotobiotic flies that mono-ingested either SK4 or SK1 (Sekihara, 2016).

Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus

This study used Drosophila as a model animal to investigate the molecular mechanisms of innate immunity. To combat orally transmitted pathogenic Gram-negative bacteria, the Drosophila gut is armed with the peritrophic matrix, which is a physical barrier composed of chitin and glycoproteins: the Duox system that produces reactive oxygen species (ROS), which in turn sterilize infected microbes, and the IMD pathway that regulates the expression of antimicrobial peptides (AMPs), which in turn control ROS-resistant pathogens. However, little is known about the defense mechanisms against Gram-positive bacteria in the fly gut. This study shows that the peritrophic matrix protects Drosophila against Gram-positive bacteria S. aureus. The few roles are described of ROS in response to the infection, and the IMD pathway was shown to be required for the clearance of ingested microbes, possibly independently from AMP expression. These findings provide a new aspect of the gut defense system of Drosophila, and helps to elucidate the processes of gut-microbe symbiosis and pathogenesis (Hori, 2017).

Allatostatin C modulates nociception and immunity in Drosophila

Bacterial induced inflammatory responses cause pain through direct activation of nociceptive neurons, and the ablation of these neurons leads to increased immune infiltration. This study investigated nociceptive-immune interactions in Drosophila and the role these interactions play during pathogenic bacterial infection. After bacterial infection, robust upregulation is found of ligand-gated ion channels and allatostatin receptors involved in nociception, which potentially leads to hyperalgesia. It was further found that Allatostatin-C Receptor 2 (AstC-R2) plays a crucial role in host survival during infection with the pathogenic bacterium Photorhabdus luminescens. Upon examination of immune signaling in AstC-R2 deficient mutants, it was demonstrated that Allatostatin-C Receptor 2 specifically inhibits the Immune deficiency pathway, and knockdown of AstC-R2 leads to overproduction of antimicrobial peptides related to this pathway and decreased host survival. This study provides mechanistic insights into the importance of microbe-nociceptor interactions during bacterial challenge. It is posited that Allatostatin C is an immunosuppressive substance released by nociceptors or Drosophila hemocytes that dampens IMD signaling in order to either prevent immunopathology or to reduce unnecessary metabolic cost after microbial stimulation. AstC-R2 also acts to dampen thermal nociception in the absence of infection, suggesting an intrinsic neuronal role in mediating these processes during homeostatic conditions. Further examination into the signaling mechanisms by which Allatostatin-C alters immunity and nociception in Drosophila may reveal conserved pathways which can be utilized towards therapeutically targeting inflammatory pain and chronic inflammation (Bachtel, 2018).

During bacterial challenge, the host immune response must be mounted in a tightly regulated and quantitatively precise manner. Overproduction of immune effectors results in immune-related pathophysiology, tissue damage, and metabolic cost whereas under-production of these effectors may permit bacterial expansion and subsequently bacterially derived damage. Recent studies have shown that bacteria can directly interact with nociceptive neurons, and that ablation of these neurons leads to increased lymph drainage during S. aureus infection most likely by suppressing immunomodulatory neuropeptide release. Thus, bacterial activation of nociceptive neurons may be a novel mechanism of immune control. This study represents the first attempt to characterize bacterially induced hyperalgesia and the effects of genes related to this process on host immunity in Drosophila melanogaster. This study provides support for a newly emerging idea that nociceptive neurons may be crucial to mounting an appropriate immune response during these infections (Bachtel, 2018).

This study investigated the gene kinetics, effect on noxious behavior, and immune consequences of nociceptive gene activation during microbial challenge. A robust upregulation was found of ligand gated ion channels (TRPA1 and ppk) and Allatostatin receptors (AstC-R1, AstC-R2, AstA-R1) upon microbial challenge, the homologs of these genes have been associated with hyperalgesia in mammalian systems. This study found that nociceptive gene activation differed temporally upon infection with E. coli as compared to pathogenic P. luminescens, and that bacterial load better correlated with nociceptive gene activation than immune activation (as measured by the IMD antimicrobial peptide encoding gene, Cecropin A1). Importantly, this correlation supports a recent paper demonstrating that S. aureus bacterial load better correlates with hyperalgesia than paw swelling (immune infiltration) in mice (Bachtel, 2018).

To determine whether the upregulation of these nociception-related genes contributed to hyperalgesia, immune and nociceptive knockdown fly mutants were generated for the genes upregulated, and changes to noxious heat sensitization were measured. Upon examining alterations to this behavior, it was found that AstC-R1 and AstC-R2 RNAi mutants displayed hyperalgesia whereas IMD and TRPA1 knockdown mutants showed robust hypoalgesia. These results are in agreement with previous studies demonstrating the importance of TRPA1 in noxious heat sensation. To determine whether it was possible to raise the noxious heat sensitivity of IMD mutants back to wild-type levels by infection with a bacterium, IMD knockdown flies were infected with a non-pathogenic strain of E. coli, and these mutants were found to display hyperalgesia, suggesting IMD activation contributes to, but is not necessary for hyperalgesia during bacterial infections. These results implicate NF-kappaB activation as a conserved mechanism of hyperalgesia in arthropod and mammalian lineages with the additional hyperalgesia seen upon infection of IMD knockdown mutants being attributed to Toll signaling or direct bacterial activation. Indeed, previous studies have found that a transcription factor downstream of IMD activation, Relish, alters thermal nociception as well (Bachtel, 2018).

Due to bacteria being able to potentially manipulate the expression of nociceptive genes in their favor, it was of interest to discover whether any of the nociception-related genes tested played a beneficial or detrimental role to the host during microbial challenge. To test this, each nociception-related gene was silenced ubiquitously in flies and their survival upon injection with the insect pathogen P. luminescens was measured. A trend towards decreased survival of AstC-R1 knockdown flies was found, and a significant decrease in survival was found upon knockdown of AstC-R2, suggesting a potential role for Allatostatin-C in modulating host immune processes during bacterial infection. However, when infecting AstC-R2 knockdown flies with the non-pathogen E. coli, no decreased survival was detected over 48 hours as compared to wild-type flies suggesting that this effect alone is not sufficient to cause death (Bachtel, 2018).

The mammalian homolog of Allatostatin is Somatostatin, which has documented effects in reducing systemic inflammation in mammalian systems, and thus whether knockdown of AstC-R2 leads to alterations in immune signaling that could contribute to the decreased survival was examined. A robust over-induction of IMD signaling was detected with a modest, but non-significant increase in Toll and decrease in Eiger as compared to wild-type flies, suggesting that AstC-R2 reduced IMD signaling independently of the Toll or Jak-Stat pathways respectively. Despite the robust upregulation of the IMD pathway, no changes were observed in bacterial load during P. luminescens infection of AstC-R2 knockdown flies as compared to wild-type controls. These results suggest that antimicrobial peptides related to this pathway are ineffective at controlling this pathogen. Indeed, recent reports have shown that an antimicrobial peptide-resistant sub-population of P. luminescens is responsible for the majority of the virulence during insect infection, and that P. luminescens is able to employ proteases that specifically degrade antimicrobial peptides, rendering them post-translationally ineffective (Bachtel, 2018).

By knocking down a receptor for Allatostatin C, which has dual role in inhibiting heat-driven nociception as well as inhibiting the IMD pathway during bacterial challenge, hyperactivation of this immune pathway, hyperalgesia, and reduced survival upon challenge with P. luminescens were observed. The hyperalgesia seen in AstC-R1 and AstC-R2 RNAi knockdown flies in the absence of bacterial challenge most likely is not due to dysregulation of the IMD pathway because similar basal transcript levels of Cecropin A1 were observed in AstC-R2 knockdown mutants as compared to wild-type flies. Indeed, AstC-R1 and AstC-R2 also share structural homology with mammalian opioid receptors. However, the reduced survival in AstC-R2 knockdown flies may be explained either directly or indirectly by over-activated IMD signaling and AstC-R2-IMD double knockdown mutants will be needed in order to confirm this hypothesis. Remarkably, the results recapitulate many of the findings found in a seminal study investigating the importance of somatostatin receptor 4 in the modulation of hyperalgesia and inflammation (Helyes, 2008). Therefore, Drosophila AstC-R2 may be more functionally similar to mammalian SSTR4 than previously perceived (Bachtel, 2018).

Due to the transcriptional upregulation of AstC-R1 and AstC-R2 during infection, it is likely that this upregulation reflects one mechanism of the host fine-tuning the immune response to prevent immune related damage from occurring as well as mediating avoidance behaviors while in a compromised state. Somatostatin regulatory circuits have been documented at sites of chronic inflammation where they have important roles in inhibiting pro-inflammatory cytokine production by macrophages and T-cells yet these processes have not been previously described in Drosophila. Interestingly, another neuropeptide that acts as a crucial component of this circuit by inhibiting somatostatin release is substance P, an additional molecule released from nociceptive neurons. Thus, immune manipulation during microbial challenge by nociceptive neurons is likely to be a well-orchestrated process that amplifies or suppresses pro-inflammatory cytokine production in a way to best ensure host survival (Bachtel, 2018).

The results imply that nociceptor-immune interactions during microbial infection in Drosophila may be more similar to mammalian systems than previously conceived (see Potential role for AstC-R2 in nociceptor-bacterial-immune interactions in Drosophila). This idea is supported by recent findings demonstrating that nociceptive neurons in flies are sensitive the proinflammatory cytokine Eiger, as well as bacterially derived lipopolysaccharides. Drosophila also possesses homologous genes for other immuno-modulatory substances released from nociceptors including substance P, CGRP and VIP (DTK, DH31, and Pdf respectively), yet their roles in pain sensation and immunity have not been characterized. Due to the wealth of transgenic lines available, quick developmental cycle and cheap cost of maintenance, Drosophila could prove to be a valuable tool in deciphering nociceptor-innate immune interactions in the future. Further studies into the interface of pain, immunity, and microbial challenge hold large promise for innovative treatments for inflammatory pain, auto-immune conditions, as well as potential explanations for host-tolerance of the gut microbiota (Bachtel, 2018).

Epstein-Barr Virus DNA Enhances Diptericin Expression and Increases Hemocyte Numbers in Drosophila melanogaster via the Immune Deficiency Pathway

Infection with the Epstein-Barr virus (EBV) is associated with several malignancies and autoimmune diseases in humans. The following EBV infection and establishment of latency, recurrences frequently occur resulting in potential viral DNA shedding, which may then trigger the activation of immune pathways. It has been demonstrated that levels of the pro-inflammatory cytokine IL-17, which is associated with several autoimmune diseases, are increased in response to EBV DNA injection in mice. Whether other pro-inflammatory pathways are induced in EBV DNA pathobiology remains to be investigated. The complexity of mammalian immune systems presents a challenge to studying differential activities of their intricate immune pathways in response to a particular immune stimulus. This study used Drosophila melanogaster to identify innate humoral and cellular immune pathways that are activated in response to EBV DNA. Injection of wild-type adult flies with EBV DNA induced the immune deficiency (IMD pathway resulting in enhanced expression of the antimicrobial peptide diptericin. Furthermore, EBV DNA increased the number of hemocytes in flies. Conditional silencing of the IMD pathway decreased diptericin expression in addition to curbing of hemocyte proliferation in response to challenge with EBV DNA. Comparatively, upon injecting mice with EBV DNA, enhanced expression of tumor necrosis factor-alpha (TNFalpha) was detected; this enhancement is rather comparable to IMD pathway activation in flies. This study hence indicates that D. melanogaster could possibly be utilized to identify immune mediators that may also play a role in the response to EBV DNA in higher systems (Sherri, 2018).

Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects

Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. This study shows that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline (Mondotte, 2020).

Two cGAS-like receptors induce antiviral immunity in Drosophila

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide (CDN) 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases, present in both prokaryotes and eukaryotes. In bacteria, these enzymes synthesize a range of cyclic oligonucleotide and have recently emerged as important regulators of phage infections. This study identified two novel cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. cGLR1 and cGLR2 activate Sting and NF-κB dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by dsRNA to produce the novel CDN 3'2'-cGAMP whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2' cGAMP in response to a yet unidentified stimulus. These data establish cGAS as the founding member of a family of receptors sensing different types of nucleic acids and triggering immunity through production of CDNs beyond 2'3'-cGAMP (Holleufer, 2021).

Evidence of Adaptive Evolution in Wolbachia-Regulated Gene DNMT2 and Its Role in the Dipteran Immune Response and Pathogen Blocking

Eukaryotic nucleic acid methyltransferase (MTase) proteins are essential mediators of epigenetic and epitranscriptomic regulation. DNMT2 belongs to a large, conserved family of DNA MTases found in many organisms, including holometabolous insects such as fruit flies and mosquitoes, where it is the lone MTase. Interestingly, despite its nomenclature, DNMT2 is not a DNA MTase, but instead targets and methylates RNA species. A growing body of literature suggests that DNMT2 mediates the host immune response against a wide range of pathogens, including RNA viruses. Curiously, although DNMT2 is antiviral in Drosophila, its expression promotes virus replication in mosquito species. Therefore this study sought to understand the divergent regulation, function, and evolution of these orthologs. The role is described of the Drosophila-specific host protein IPOD in regulating the expression and function of fruit fly DNMT2. Heterologous expression of these orthologs suggests that DNMT2's role as an antiviral is host-dependent, indicating a requirement for additional host-specific factors. Finally, this study identified and describes potential evidence of positive selection at different times throughout DNMT2 evolution within dipteran insects. Specific codons within each ortholog were identified that are under positive selection, and they were found to be restricted to four distinct protein domains, which likely influence substrate binding, target recognition, and adaptation of unique intermolecular interactions. Collectively, these findings highlight the evolution of DNMT2 in Dipteran insects and point to structural, regulatory, and functional differences between mosquito and fruit fly homologs (Bhattacharya, 2021).

Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila

The gut sets the immune and metabolic parameters for the survival of commensal bacteria. This study reports that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP, a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. Thise results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut (Bahuguna, 2022).

The animal gut accommodates a diverse array of bacteria, which assist in regulation of digestion, supply of nutrients and metabolites as well as in immune development. To reap benefits from these microbes, the host provides a symbiotic environment for sustaining them in the gut. The Drosophila gut and its bacteriome is used as a simpler model to study such host-microbe interactions. Although much less diverse compared to humans, the fly bacteriome is equally dynamic and changes with age and environmental conditions connected to reinfections during fly culture (Bahuguna, 2022).

The Drosophila intestinal epithelium is immunocompetent and upon enteric infection initiates innate immune responses via the NF-κB pathway IMD, mediating the production of antimicrobial peptides (AMPs) as well as the pathway centered on Dual Oxidase, the enzyme needed for the generation of Reactive Oxygen Species (ROS). However, it also preserves commensal bacteria, since transcription of AMPs is suppressed by Caudal and Nubbin, while bacterial-derived uracil is important for distinguishing pathogens from commensals (Bahuguna, 2022).

The evolutionary conserved Target of Rapamycin (TOR) pathway is a major pathway controlling cellular metabolism and growth. TOR balances lipid and glucose anabolism and catabolism in the cell through the activity of the TORC1 protein complex. TORC1 promotes protein synthesis primarily through phosphorylation of the eIF4E Binding Protein (4E-BP/Thor) and p70S6 Kinase 1 (S6K1). 4E-BP is a translational inhibitor, which binds and inhibits the activity of eIF4E an eukaryotic translation initiation factor responsible for the recruitment of 40s ribosomal subunit at the 5'-cap of mRNA. Phosphorylation of 4E-BP lowers its affinity towards eIF4E. This frees eIF4E, enabling it to promote cap-dependent translation. In the case of S6K1, activated S6K1 promotes protein synthesis by activating inducers of mRNA translation initiation whilst degrading inhibitors (Bahuguna, 2022).

Drosophila TOR has been extensively studied for its role in growth and development, using fly mutants or by treating flies with rapamycin. Rapamycin treatment in stress conditions, led to upregulation of 4E-BP activity resulting in an increase of whole-fly lipid reserves that could be used for the long-term survival of these stress conditions. In contrast, 4E-BP mutants were unable to preserve lipid stores and had thus compromised survival following starvation or oxidative stress. More broadly, the consensus is that in both Drosophila and mice, 4E-BP regulates fat levels in stress conditions like starvation and oxidative stress. During larval stages and when in food with poor nutritional value, the presence of the commensal Lactobacillus plantarum is important to sustain development through TOR, which is in turn crucial for sustaining this mutualistic relationship. The metabolic state of the gut is also influenced by dietary conditions and in its turn influences the bacteriome. Diet-dependent adaptations of the microbiota require NF-κB-dependent control of the translational regulator 4E-BP and this where TOR and NF-κB 'meet' (Bahuguna, 2022).

Drosophila has three NF-κB proteins namely, Relish, Dorsal and the Dorsal-related Immunity Factor (DIF). DIF is downstream of the Toll signalling pathway. Toll and Toll-like receptor (TLR) signalling is one of the most important evolutionary conserved mechanisms by which the innate immune system senses the invasion of pathogenic microorganisms. Unlike its mammalian counterparts however, Drosophila Toll is activated by an endogenous cytokine-like ligand, the Nerve Growth Factor homologue, Spz. Spz is processed to its active form by the Spz-Processing Enzyme (SPE). Two serine protease cascades converge on SPE: one triggered by bacterial or fungal serine proteases through the host serine protease Persephone and a second activated by host receptors that recognise bacterial or fungal cell wall. Prominent among these host receptors is the Peptidoglycan Recognition Protein-SA or PGRP-SA. PGRP-SA binds to peptidoglycan on the bacterial cell wall without structural preference but depending on accessibility and generates the downstream signal (Bahuguna, 2022).

When the recognition signal reaches the cell surface, it is communicated intracellularly via the Toll receptor and a membrane-bound receptor-adaptor complex including dMyd88, Tube (as an IRAK4 functional equivalent) and the Pelle kinase (as an IRAK1 functional homologue). Transduction of the signal culminates in the phosphorylation of the IκB homologue, Cactus probably by Pelle, leaving the NF-κB homologue DIF to move to the nucleus and regulate hundreds of target genes including antimicrobial peptides (AMPs) (Bahuguna, 2022).

In this study, evidence is presented that PGRP-SA is important for the preservation of commensal intestinal bacterial density. The results reveal that larvae and adults that are deficient in PGRP-SA or DIF have a significantly reduced commensal gut bacterial density. Inhibition of the activity of TOR by Rapamycin or TOR RNAi in enterocytes, restores bacterial density (but not diversity) in PGRP-SA or DIF mutant guts. However, flies mutants for PGRP-SA and deficient for 4EBP in enterocytes were unable to restore bacterial density upon Rapamycin treatment or TOR RNAi, demonstrating the important role of 4EBP. PGRP-SA mutants had increased intestinal fat stores that were restored to normal levels through Rapamycin or TOR-RNAi treatment in enterocytes. This restoration failed in PGRP-SA;4EBP double mutants indicating that 4EBP was crucial in regulating fat stores in the gut. Fat catabolism was important for gut bacterial restoration as flies deficient for PGRP-SA and treated with rapamycin were unable to restore bacterial density if the triglyceride lipase Brummer was knocked down in enterocytes. This mechanism gives an insight into how the host integrates immunity and metabolism to maintain commensal bacteria at the intestinal epithelium (Bahuguna, 2022).

In the absence of the bacterial receptor PGRP-SA from enterocytes, a reduction was observed in intestinal bacterial density. It was restored with the use of rapamycin, which targets TORC1 or by knocking-down TOR in enterocytes. This suggested that loss of the immune receptor PGRP-SA, generated a metabolic environment unfavourable for intestinal bacterial growth. The results indicated that at the centre of this relationship was 4E-BP, which is activated by Toll and suppressed by TORC1. In keeping with this, PGRP-SA^seml; NP1GAL4>4E-BPRNAi flies treated with rapamycin were unable to restore gut bacterial density. Intestinal lipid catabolism downstream of 4EBP was paramount for the maintenance of cultivable bacterial density because the loss of the lipase Bmm blocked restoration of gut bacteria after rapamycin treatment. Silencing of bmm in enterocytes caused intestinal lipid accumulation and prevented any restoration via rapamycin in PGRP-SA^seml flies (Bahuguna, 2022).

These results indicate that downstream of Toll, intestinal triglyceride levels were under 4E-BP control in enterocytes. Although the phenomenon of cultivable bacteriome reduction in PGRP-SA^seml flies was readily manifested in larvae and young flies, the results indicated that it was also there in older flies. Conventional fly rearing techniques ensure a steady stream of defaecation and re-introduction of bacteria over time. However, when food vials were changed rapidly re-infection was reduced and CFUs in 30-day old flies were significantly lower in PGRP-SA^seml than yw flies. Preservation of the bacteriome was dependent on PG recognition as the rescue of enteric CFUs in PGRP-SA^seml flies was only possible with PGRP-SA transgenes that had an intact PG binding ability. This indicated that bacterial sensing was the initial trigger point to activate the process (Bahuguna, 2022).

A working model is depicted A schematic model outlining the role of PGRP-SA/Toll/Dif in the retention of the gut bacteriome. PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. In turn, this keeps increased 4E-BP transcription/4E-BP protein phosphorylation in enterocytes, preserving a steady rate of intestinal lipid catabolism. The latter is important for maintaining normal density of commensal bacteria. It is hypothesised that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria. In keeping with this, stopping lipid catabolism by silencing the Bmm lipase in ECs resulted in accumulation of lipids and reduction of enteric CFUs. According to the model, bacteria should trigger lipid catabolism and 5-day old axenic flies showed a clear trend for lipid accumulation in their gut, but this was marginally not statistically significant. Studies with Vibrio cholera, have shown that intestinal acetate leads to deactivation of host insulin signalling and lipid accumulation in enterocytes, resulting in host lethality. Loss of PGRP-SA/Dif leads to a decrease in lifespan. Whether this is due to the long-term accumulation of lipids is an open question (Bahuguna, 2022).

PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. This increases 4E-BP transcription/4E-BP protein phosphorylation in enterocytes. 4EBP is important for maintaining normal density of commensal bacteria. It is hypothesized that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria (Bahuguna, 2022).

More work is needed to understand whether/how stored intestinal lipids maybe released to circulation, how commensal bacteria receive them and which component(s) of the bacteriome are recognised by PGRP-SA (Bahuguna, 2022).

SUMOylation of Jun fine-tunes the Drosophila gut immune response

Post-translational modification by the small ubiquitin-like modifier, SUMO can modulate the activity of its conjugated proteins in a plethora of cellular contexts. The effect of SUMO conjugation of proteins during an immune response is poorly understood in Drosophila. Previous work found that the transcription factor Jra, the Drosophila Jun ortholog and a member of the AP-1 complex is one such SUMO target. This study found that Jra is a regulator of the Pseudomonas entomophila induced gut immune gene regulatory network, modulating the expression of a few thousand genes, as measured by quantitative RNA sequencing. Decrease in Jra in gut enterocytes is protective, suggesting that reduction of Jra signaling favors the host over the pathogen. In Jra, lysines 29 and 190 are SUMO conjugation targets, with the JraK29R+K190R double mutant being SUMO conjugation resistant (SCR). Interestingly, a JraSCR fly line, generated by CRISPR/Cas9 based genome editing, is more sensitive to infection, with adults showing a weakened host response and increased proliferation of Pseudomonas. Transcriptome analysis of the guts of JraSCR and JraWT flies suggests that lack of SUMOylation of Jra significantly changes core elements of the immune gene regulatory network, which include antimicrobial agents, secreted ligands, feedback regulators, and transcription factors. Mechanistically, SUMOylation attenuates Jra activity, with the TFs, forkhead, anterior open, activating transcription factor 3 and the master immune regulator Relish being important transcriptional targets. This study implicates Jra as a major immune regulator, with dynamic SUMO conjugation/deconjugation of Jra modulating the kinetics of the gut immune response (Soory, 2022).

The Drosophila tumor necrosis factor receptor, Wengen, couples energy expenditure with gut immunity

It is well established that tumor necrosis factor (TNF) plays an instrumental role in orchestrating the metabolic disorders associated with late stages of cancers. However, it is not clear whether TNF/TNF receptor (TNFR) signaling controls energy homeostasis in healthy individuals. This study shows that the highly conserved Drosophila TNFR, Wengen (Wgn), is required in the enterocytes (ECs) of the adult gut to restrict lipid catabolism, suppress immune activity, and maintain tissue homeostasis. Wgn limits autophagy-dependent lipolysis by restricting cytoplasmic levels of the TNFR effector, TNFR-associated factor 3 (dTRAF3), while it suppresses immune processes through inhibition of the dTAK1/TAK1-Relish/NF-κB pathway in a dTRAF2-dependent manner. Knocking down dTRAF3 or overexpressing dTRAF2 is sufficient to suppress infection-induced lipid depletion and immune activation, respectively, showing that Wgn/TNFR functions as an intersection between metabolism and immunity allowing pathogen-induced metabolic reprogramming to fuel the energetically costly task of combatting an infection (Loudhaief, 2023).

Bioinformatic Analysis and Antiviral Effect of Periplaneta americana Defensins

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Due to the lack of an adaptive immune system, insects rely on innate immune mechanisms to fight against pathogenic infections. Two major innate immune pathways, Toll and IMD, orchestrate anti-pathogen responses by regulating the expression of antimicrobial peptide (AMP) genes. Although the antifungal or antibacterial function of AMPs has been well characterized, the antiviral role of AMPs in insects remains largely unclear. Periplaneta americana (P. americana), or the American cockroach, is used in traditional Chinese medicine as an antiviral agent; however, the underlying mechanism of action of P. americana extracts is unclear. A previous study showed that the P. americana genome encodes multiple antimicrobial peptide genes. Based on these data, five novel P. americana defensins (PaDefensins) are predicted, and their primary structure, secondary structure, and physicochemical properties were analyzed. The putative antiviral, antifungal, antibacterial, and anticancer activities suggested that PaDefensin5 is a desirable therapeutic candidate against viral diseases. As the first experimental evidence of the antiviral effects of insect defensins, this study also showed the antiviral effect of PaDefensin5 in Drosophila Kc cells and Drosophila embryos in vivo . In conclusion, results of both in silico predictions and subsequent antiviral experiments suggested PaDefensin5 a promising antiviral drug (Li, 2021).

The Drosophila Baramicin polypeptide gene protects against fungal infection

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. This study describes a novel Drosophila antifungal peptide gene that was named Baramicin A. BaraA was shown to encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, it was shown that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. This study has characterized a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response (Hanson, 2021).

The molecular architecture of Drosophila melanogaster defense against Beauveria bassiana explored through evolve and resequence and quantitative trait locus mapping

Little is known about the genetic architecture of antifungal immunity in natural populations. Using two population genetic approaches, Quantitative Trait Locus (QTL) Mapping and Evolve and Resequence (E&R), this study explored D. melanogaster immune defense against infection with the fungus Beauveria bassiana. Immune defense was highly variable both in the recombinant inbred lines from the Drosophila Synthetic Population Resource used for QTL mapping and in the synthetic outbred populations used in the E&R study. Survivorship of infection improved dramatically over just 10 generations in the E&R study, and continued to increase for an additional 9 generations, revealing a trade-off with uninfected longevity. Populations selected for increased defense against B. bassiana evolved cross resistance to a second, distinct B. bassiana strain but not to bacterial pathogens. The QTL mapping study revealed that sexual dimorphism in defense depends on host genotype, and the E&R study indicated that sexual dimorphism also depends on the specific pathogen to which the host is exposed. Both the QTL mapping and E&R experiments generated lists of potentially causal candidate genes, although these lists were non-overlapping (Shahrestani, 2021).

Ingestion of killed bacteria activates antimicrobial peptide genes in Drosophila melanogaster and protects flies from septic infection

Drosophila melanogaster possesses a sophisticated and effective immune system composed of humoral and cellular immune responses, and production of antimicrobial peptides (AMPs) is an important defense mechanism. Expression of AMPs is regulated by the Toll and IMD (immune deficiency) pathways. Production of AMPs can be systemic in the fat body or a local event in the midgut and epithelium. So far, most studies focus on systemic septic infection in adult flies and little is known about AMP gene activation after ingestion of killed bacteria. This study investigated activation of AMP genes in the wild-type w(1118), MyD88 and Imd mutant flies after ingestion of heat-killed Escherichia coli and Staphylococcus aureus. Ingestion of E. coli activated most AMP genes, including drosomycin and diptericin, in the first to third instar larvae and pupae, while ingestion of S. aureus induced only some AMP genes in some larval stages or in pupae. In adult flies, ingestion of killed bacteria activated AMP genes differently in males and females. Interestingly, ingestion of killed E. coli and S. aureus in females conferred resistance to septic infection by both live pathogenic Enterococcus faecalis and Pseudomonas aeruginosa, and ingestion of E. coli in males conferred resistance to P. aeruginosa infection. These results indicated that E. coli and S. aureus can activate both the Toll and IMD pathways, and systemic and local immune responses work together to provide Drosophila more effective protection against infection (Wen, 2019).

Regulation of the expression of nine antimicrobial peptide genes by TmIMD confers resistance against Gram-negative bacteria

Immune deficiency (IMD) is a death domain-containing protein that is essential for the IMD/NF-kappaB humoral and epithelial immune responses to Gram-negative bacteria and viruses in insects. In the immune signaling cascade, IMD is recruited together with FADD and the caspase DREDD after the mobilization of PGRP receptors. Activated IMD regulates the expression of effector antimicrobial peptides (AMP) that protect against invading microorganisms. To date, most studies of the IMD pathway, and the IMD gene in particular, have been restricted to Drosophila; few similar studies have been conducted in other model insects. This study cloned and functionally characterized an IMD homolog from the mealworm beetle Tenebrio molitor (TmIMD) and studied its role in host survival in the context of pathogenic infections. Phylogenetic analysis revealed the conserved caspase cleavage site and inhibitor of apoptosis (IAP)-binding motif (IBM). TmIMD expression was high in the hemocytes and Malpighian tubules of Tenebrio late-instar larvae and adults. At 3 and 6 hours' post-infection with Escherichia coli, Staphylococcus aureus, or Candida albicans, TmIMD expression significantly increased compared with mock-infected controls. Knockdown of the TmIMD transcript by RNAi significantly reduced host resistance to the Gram-negative bacterium E. coli and fungus C. albicans in a survival assay. Strikingly, the expression of nine T. molitor AMPs (TmTenecin1, TmTenecin2, TmTenecin4, TmDefensin2, TmColeoptericin1, TmColeoptericin2, TmAttacin1a, TmAttacin1b, and TmAttacin2) showed significant downregulation in TmIMD knockdown larvae challenged with E. coli. These results suggest that TmIMD is required to confer humoral immunity against the Gram-negative bacteria, E. coli by inducing the expression of critical transcripts that encode AMPs (Jo, 2019).

Adult Drosophila lack hematopoiesis but rely on a blood cell reservoir at the respiratory epithelia to relay infection signals to surrounding tissues

The use of adult Drosophila melanogaster as a model for hematopoiesis or organismal immunity has been debated. Addressing this question, an extensive reservoir of blood cells (hemocytes) was identified at the respiratory epithelia (tracheal air sacs) of the thorax and head. Lineage tracing and functional analyses demonstrate that the majority of adult hemocytes are phagocytic macrophages (plasmatocytes) from the embryonic lineage that parallels vertebrate tissue macrophages. Surprisingly, no sign of adult hemocyte expansion was observed. Instead, hemocytes play a role in relaying an innate immune response to the blood cell reservoir: through Imd signaling and the Jak/Stat pathway ligand Upd3, hemocytes act as sentinels of bacterial infection, inducing expression of the antimicrobial peptide Drosocin in respiratory epithelia and colocalizing fat body domains. Drosocin expression in turn promotes animal survival after infection. This work identifies a multi-signal relay of organismal humoral immunity, establishing adult Drosophila as model for inter-organ immunity (Sanchez Bosch, 2019).

Drosophila melanogaster has greatly promoted understanding of innate immunity and blood cell development, but the capacity of the adult animal as a model remains a matter of debate. Most studies reported lack of new blood cell production and increasing immunosenescence, while one publication claimed continued hematopoietic activity in adult Drosophila (Sanchez Bosch, 2019).

Drosophila blood cells, or hemocytes, emerge from two lineages that persist into the adult, showing parallels with the two myeloid systems in vertebrates. First, hemocytes originating in the embryo parallel vertebrate tissue macrophages, as they quickly differentiate into plasmatocytes (macrophage-like cells), and subsequently proliferate extensively, mainly in the hematopoietic pockets (HPs) of the larva (Gold, 2014; Gold, 2015; Makhijani, 2011; Makhijani, 2012). At least some of these plasmatocytes can further differentiate into other blood cell types such as crystal cells and, under immune challenge, lamellocytes. Second, hemocytes originating in the lymph gland (LG) also give rise to plasmatocytes, crystal cells, and lamellocytes, yet in the lymph gland they are predominantly generated from blood cell progenitors (prohemocytes). At the beginning of metamorphosis, hemocytes from both the hematopoietic pockets and the lymph gland enter the open circulatory system and intermix. The subsequent fate and capacity of adult blood cells has remained largely unclear. Accordingly, this study comprehensively investigated the hematopoietic capacity of the blood cell system in adult Drosophila. A second part of this study focused on the role of adult blood cells in the humoral immune response, identifying a system of organismal innate immunity that centers on the respiratory epithelia in Drosophila (Sanchez Bosch, 2019).

Historically, Drosophila has been instrumental in the discovery of innate immunity and Toll like receptor (TLR) signaling. Toll and the related immune deficiency (Imd) signaling are evolutionary conserved NFκB family pathways, studied in detail regarding their upstream activation by pathogens and other inputs, and downstream signal transduction components and mechanisms. Targets include antimicrobial peptides (AMPs), which have been investigated for their transcriptional gene regulation and functional properties. TLR signaling has been well established also in vertebrate systems for its roles in infection and inflammation. However, it has been far less understood how multiple tissues or organs communicate with each other to elicit local innate immune responses (Sanchez Bosch, 2019).

Addressing these questions, this study clarifies basic principles of the blood cell system in adult Drosophila and its role in multi-tissue organismal immunity. An extensive blood cell reservoir was identified at the respiratory epithelia and fat body, its dynamics were investigated and probed for various signs of hematopoiesis. A key role of adult blood cells is demonstrated as sentinels of bacterial infection that trigger a humoral response in their reservoir, i.e., the respiratory epithelia and colocalizing domains of the fat body. This response culminates in the expression of the AMP gene Drosocin, which is shown to be significant for animal survival after bacterial infection. This work identifies Imd signaling and Upd3 expression in hemocytes as required steps in this relay of organismal immunity, laying the foundation for the use of adult Drosophila to dissect additional mechanisms of multi-tissue innate immunity in the future (Sanchez Bosch, 2019).

This study discovered a central role for an extensive blood cell reservoir at the respiratory epithelia and fat body of adult Drosophila. The reservoir serves as major receptacle of blood cells and foreign particles, and in addition executes a local humoral immune response of Drosocin expression that promotes animal survival after bacterial infection. Both functions are tied together by hemocytes acting as sentinels of infection, that signal through the Imd pathway and Upd3 to induce Drosocin expression in the tissues of their surrounding reservoir, i.e., the respiratory epithelia and colocalizing domains of the fat body (Sanchez Bosch, 2019).

Historic literature on Drosophila and other insects focused on the adult heart as the site of hemocyte accumulation. It described clusters of hemocytes at the ostia of the heart as 'immune organ', locations where hemocytes and bacteria accumulate. More recently, adult blood cell production at the heart was proposed (Ghosh, 2015). Some studies described functions of hemocytes in other locations, such as at the ovaries or along the gut of adult flies. Taking a more global cryosectioning approach afforded the identification of the largest reservoir of hemocytes in adult Drosophila, which surrounds the respiratory epithelia and is lined by fat body of the thorax and head. It is concluded that hemocytes and particles are delivered to these areas by the streaming hemolymph, even though the detailed anatomy of the open circulatory system remains to be mapped in more detail. Hemocytes may be physically caught in these locations, or in addition may engage in active adhesion. The intimate relationship of hemocytes with the respiratory epithelia, hemolymph, and adjacent fat body may serve interconnected roles, (1) guarding the respiratory epithelia as a barrier to the environment through functions of hemocytes in both phagocytosis and the induction of humoral immunity, and (2) facilitating gas exchange of hemocytes and nearby immune tissues, which in turn may again benefit defense functions. The former may be particularly advantageous in the defense against fungal pathogens that invade Drosophila via the tracheal system as primary route of infection, such as the entomopathogenic fungus B. bassiana. Regarding the latter, a study in caterpillars described the association of hemocytes with trachea, proposing a function for the respiratory system to supply hemocytes with oxygen (Sanchez Bosch, 2019).

Drosophila adult blood cells derive from two lineages: one that originates in the embryo and resembles vertebrate tissue macrophages, and another that produces blood cells in the lymph gland through a progenitor-based mechanism. It is estimated that more than 60% of adult hemocytes derive from the embryonic lineage is surprising, considering past views that the majority of adult hemocytes would derive from the lymph gland. It places more importance on the Drosophila embryonic lineage of hemocytes and suggests additional parallels with tissue macrophages in vertebrates, which persist into adulthood and form a separate myeloid system independent of the progenitor-derived monocyte lineage. Future research will show whether the relative contribution of the two hemocyte lineages to the adult blood cell pool will be the same or different under conditions of stress and immune challenges (Sanchez Bosch, 2019).

Given that embryonic-lineage plasmatocytes are highly proliferative in the hematopoietic pockets of the larva, and lymph gland hemocyte progenitors and some lymph gland plasmatocytes proliferate during larval development, the absence of hemocyte proliferation in the adult may be surprising. Nevertheless, combining the broad evidence supporting lack of significant hematopoietic activity in adult Drosophila, and evidence that Srp in adult Drosophila is not a progenitor marker, the findings robustly contradict an adult hematopoiesis model. The findings further reveal important differences to embryonic development, where Srp is required for the specification of undifferentiated prohemocytes. This study shows that during maturation of the adult animal, hemocytes relocate to the respiratory epithelia and the heart, upon completion of cytolysis of larval fat body cells, thereby refuting claims of new blood cell production at the heart. Similarly, seemingly increased numbers of fluorescently labeled hemocytes following bacterial infection are likely based on infection-induced upregulation of hemocyte-specific genes and their respective enhancers including the reporter HmlΔ-GAL4, UAS-GFP. Enhanced hemocyte expression of Hemolectin (Hml) and other hemocyte-specific markers post-infection has been described previously (Sanchez Bosch, 2019).

Taken together, this broad evidence speaks to a lack of significant hematopoietic capacity of the blood cell system in adult Drosophila. The findings are in agreement with other studies that have reported a lack of hemocyte proliferation in adult Drosophila and functional immunosenescence in aging flies. Despite the scope of conditions tested, the possibility cannot be excluded that some other specific immune challenge or stress might exist that would be potent enough to trigger proliferation- or differentiation-based blood cell production in adult Drosophila. Likewise, it cannot be excluded that adult Drosophila may possess small numbers of proliferation- and/or differentiation-competent progenitors that may have persisted e.g. from the lymph gland posterior lobes; such cells might give rise to new differentiated hemocytes, although according to the current data they would remain insignificant in number (Sanchez Bosch, 2019).

Taking into account the short reproductive phase and relatively short life span of Drosophila, the adult fly may be sufficiently equipped with the pool of hemocytes that is produced in the embryo and larva. In fact, hemocytes do not seem essential for the immediate survival of adult flies: Drosophila ablated of hemocytes, and mutants devoid of hemocytes, survive to adulthood although they are more prone to, and succumb more rapidly to infection. A model is proposed that places emphasis on larval development as the sensitive phase for the expansion and regulation of the adult blood cell pool. In the larva, hemocytes of both the embryonic and lymph gland lineage integrate signals from a variety of internal and external stimuli to adapt to existing life conditions (Sanchez Bosch, 2019).

This work reveals a role for hemocytes in a local humoral immune response of the fat body and respiratory epithelia. Previous studies on hemocyte-ablated flies have reported increases in Defensin and IM1 expression. In contrast, this study finds a positive role for hemocytes in the induction of Drosocin in tissues that form the hemocyte reservoir, i.e., the respiratory epithelia and fat body domains of the head and thorax. The concept of hemocytes promoting AMP expression in other tissues is well established. A role for AMP expression in surface epithelia that interface with the environment was reported in a previous study, and Drosocin expression was described in embryonic and larval trachea and the abdominal tracheal trunks of adult Drosophila, albeit not in the respiratory epithelia (Sanchez Bosch, 2019).

In adult Drosophila, hemocytes tightly localize between the respiratory epithelia and fat body tissue that occupies the space toward to the cuticle exoskeleton. It is proposed that this close anatomical relationship facilitates rapid local signaling. Consistent with previous knowledge that Drosocin expression is lost in imd mutant backgrounds, this study found that hemocyte-autonomous Imd signaling is required, albeit not sufficient, to trigger the infection-induced Drosocin response. Likewise, the Imd pathway upstream receptor PGRP-LC is required in hemocytes, suggesting that DAP-type peptidoglycan recognition and initiation of Imd signaling are a critical step in triggering the Drosocin response. Transcriptional induction of upd3 by Imd signaling is supported by putative Rel binding sites identified in the upd3 genomic region, two of which are even fully conserved across seven Drosophilids including Drosophila melanogaster. The data suggest roles for hemocyte-expressed upd3, and corresponding Jak/Stat signaling in cells of the fat body and respiratory system, all of which are required albeit not sufficient. Overactivation of the pathway paradoxically suppresses Drosocin expression, and even temporally restricted expression of activated hopTumL in trachea was largely lethal, possibly indicating leaky expression of the transgene. Overall, it can only be speculated that the unexpected effects of Jak/Stat overactivation might be due to activation of some negative feedback loop or other complex signaling changes that remain a matter of future investigation (Sanchez Bosch, 2019).

Several reports provide precedent for a role of hemocyte-expressed Upd3 in the induction of immune responses in other target tissues. Following septic injury, upregulation of upd3 in hemocytes triggers induction of stress peptide genes of the turandot family including totA in fat body. Similarly, in response to injury, hemocyte-produced Upd3 induces Jak/Stat signaling in the fat body and gut. Under lipid-rich diet, upd3 is induced in hemocytes, causing impaired glucose homeostasis and reduced lifespan in adult Drosophila. In the larva, hemocyte-derived Upd2 and -3 activate Jak/Stat signaling in muscle, which are required for the immune response against parasitic wasps. However, in the Drosocin response around the reservoir of hemocytes, the data predict that additional signal/s and/or signaling pathway/s are needed to initiate Drosocin expression and potentially restrict its expression to defined fat body domains of the head and thorax. Additional events may include signaling through Toll or other signaling pathways in hemocytes and/or other tissues including the respiratory epithelia and fat body. Likewise, other types of signals may be required, such as reactive oxygen species (ROS) or nitric oxide (NO), which play roles in the relay of innate immune responses to infection and stress, or non-peptide hormones including ecdysone, which confers competence in the embryonic tracheal Drosocin response to bacterial infection and enhances humoral immunity under conditions of dehydration. Lastly, there could be requirement for additional processing to make bacterial ligands accessible for receptors in other tissues, as has been reported for Psidin, a lysosomal protein required in blood cells for degradation of engulfed bacteria and expression of Defensin in the fat body, although this mechanism may not be universal in all systems (Sanchez Bosch, 2019).

This work reveals an active role of endogenous Drosocin expression in survival after bacterial infection. Since the cloning of Drosocin and its classification as inducible antibacterial peptide, Drosocin has been studied for its transcriptional regulation, illustrating its induction under a variety of bacterial and other immune challenges. Drosocin structure and antimicrobial function have been studied in vitro and by overexpression from transgenes in Drosophila and in heterologous vertebrate systems. Consistent with the current findings, a recent study on CRISPR-based Drosocin null mutants reached similar conclusions regarding the requirement of endogenous Drosocin expression for animal survival following E. cloacae infection (Hanson, 2019). Expanding from these findings, this study reveals the anatomical features of Drosocin expression and its unique path of induction. In addition to Drosocin's role in animal survival after bacterial infection, the data suggest contribution of Drosocin to animal survival after injury through PBS injection. Injury has emerged as a factor that affects survival, a phenomenon for which the molecular mechanisms still remain to be determined. Alternatively, considering that the fly surface and living conditions are not sterile and survival experiments are performed over extended periods of time, it cannot be ruled out that PBS injections may have led to inadvertent infection with some low level contaminating microbes. A role for endogenous Drosocin levels in the antimicrobial response is strongly supported by independent data in the literature. Specifically, the minimum inhibitory concentration (MIC) of Drosocin against E. coli and E. cloacae was determined to be well within the range or below the endogenous concentration of Drosocin in the Drosophila hemolymph (MIC is 1 or 2 μM for the glycosylated forms, and 8 or 10 μM for the unglycosylated form, respectively, compared to 40 μM Drosocin in the Drosophila hemolymph (Sanchez Bosch, 2019).

In conclusion, this study revokes the use of adult Drosophila as effective model to study hematopoiesis, and establishes it as promising system for organismal immunity centering on the immune signaling relay at the reservoir of blood cells. At the evolutionary level, this model shows parallels with vertebrate immune cells of the lung and innate immune responses to bacterial infection. The Drosophila model opens countless avenues for exciting future research, e.g., to investigate additional molecular and cellular mechanisms in the immune signaling relay, the role and regulation of the system in the defense against pathogens that invade the trachea as natural route of infection, the use of the same axis by gram-positive or non-bacterial pathogens, and the induction of other AMPs and immune effector genes in the same axis of regulation (Sanchez Bosch, 2019).

Local Necrotic Cells Trigger Systemic Immune Activation via Gut Microbiome Dysbiosis in Drosophila

Necrotic cells elicit an inflammatory response through their endogenous factors with damage-associated molecular patterns. Blocking apoptosis in Drosophila wings leads to the necrosis-driven systemic immune response by unknown mechanisms. This study demonstrated that immune activation in response to necrotic cells is mediated by commensal gut microbiota. Removing the microbiome attenuates hyperactivation of the innate immune signaling IMD pathway in necrosis-induced flies. Necrotic cells in wings trigger Gluconobacter expansion in the gut. An isolated Gluconobacter sp. strain is sufficient for pathological IMD activation in necrosis-induced flies, while it is not inflammatory for control animals. In addition, bacterial colonization shifts the host metabolome and shortens the lifespan of necrosis-induced flies. This study shows that local necrosis triggers a pathological systemic inflammatory response through interaction between the host and the dysbiotic gut microbiome (Kosakamoto, 2020).

Origins of Metabolic Pathology in Francisella-Infected Drosophila

The origins and causes of infection pathologies are often not understood. Despite this, the study of infection and immunity relies heavily on the ability to discern between potential sources of pathology. Work in the fruit fly has supported the assumption that mortality resulting from bacterial invasion is largely due to direct host-pathogen interactions, as lower pathogen loads are often associated with reduced pathology, and bacterial load upon death is predictable. However, the mechanisms through which these interactions bring about host death are complex. This study shows that infection with the bacterium Francisella novicida leads to metabolic dysregulation and, using treatment with a bacteriostatic antibiotic, this pathology was shown to be the result of direct interaction between host and pathogen. Mutants of the immune deficiency immune pathway fail to exhibit similar metabolic dysregulation, supporting the idea that the reallocation of resources for immune-related activities contributes to metabolic dysregulation. Targeted investigation into the cross-talk between immune and metabolic pathways has the potential to illuminate some of this interaction (Vincent, 2020).

Downregulation of Perilipin1 by the Immune Deficiency Pathway Leads to Lipid Droplet Reconfiguration and Adaptation to Bacterial Infection in Drosophila

Lipid droplets (LDs), the highly dynamic intracellular organelles, are critical for lipid metabolism. Dynamic alterations in the configurations and functions of LDs during innate immune responses to bacterial infections and the underlying mechanisms, however, remain largely unknown. This study traced the time-course morphology of LDs in fat bodies of Drosophila after transient bacterial infection. Detailed analysis shows that perilipin1 (plin1), a core gene involved in the regulation of LDs, is suppressed by the immune deficiency signaling, one major innate immune pathway in Drosophila. During immune activation, downregulated plin1 promotes the enlargement of LDs, which in turn alleviates immune reaction-associated reactive oxygen species stress. Thus, the growth of LDs is likely an active adaptation to maintain redox homeostasis in response to immune deficiency activation. Therefore, this study provides evidence that plin1 serves as a modulator on LDs' reconfiguration in regulating infection-induced pathogenesis, and plin1 might be a potential therapeutic target for coordinating inflammation resolution and lipid metabolism (Wang, 2021).

Drosophila H2Av negatively regulates the activity of the IMD pathway via facilitating Relish SUMOylation

Insects depend on the innate immune response for defense against a wide array of pathogens. Central to Drosophila immunity are antimicrobial peptides (AMPs), released into circulation when pathogens trigger either of the two widely studied signal pathways, Toll or IMD. The Toll pathway responds to infection by Gram-positive bacteria and fungi while the IMD pathway is activated by Gram-negative bacteria. During activation of the IMD pathway, the NF-κB-like transcription factor Relish is phosphorylated and then cleaved, which is crucial for IMD-dependent AMP gene induction. This study shows that loss-of-function mutants of the unconventional histone variant H2Av upregulate IMD-dependent AMP gene induction in germ-free Drosophila larvae and adults. After careful dissection of the IMD pathway, it was found that Relish has an epistatic relationship with H2Av. In the H2Av mutant larvae, SUMOylation is down-regulated, suggesting a possible role of SUMOylation in the immune phenotype. Eventually it was demonstrated that Relish is mostly SUMOylated on amino acid K823. Loss of the potential SUMOylation site leads to significant auto-activation of Relish in vivo. Further work indicated that H2Av regulates Relish SUMOylation after physically interacting with Su(var)2-10, the E3 component of the SUMOylation pathway. Biochemical analysis suggested that SUMOylation of Relish prevents its cleavage and activation. These findings suggest a new mechanism by which H2Av can negatively regulate, and thus prevent spontaneous activation of IMD-dependent AMP production, through facilitating SUMOylation of the NF-κB like transcription factor Relish (Tang, 2021).

Selective autophagy controls innate immune response through a TAK1/TAB2/SH3PX1 axis

Selective autophagy is a catabolic route that turns over specific cellular material for degradation by lysosomes, and whose role in the regulation of innate immunity is largely unexplored. This study shows that the apical kinase of the Drosophila immune deficiency (IMD) pathway Tak1, as well as its co-activator Tab2, are both selective autophagy substrates that interact with the autophagy protein Atg8a. A role is presented for the Atg8a-interacting protein Sh3px1 in the downregulation of the IMD pathway, by facilitating targeting of the Tak1/Tab2 complex to the autophagy platform through its interaction with Tab2. These findings show the Tak1/Tab2/Sh3px1 interactions with Atg8a mediate the removal of the Tak1/Tab2 signaling complex by selective autophagy. This in turn prevents constitutive activation of the IMD pathway in Drosophila. This study provides mechanistic insight on the regulation of innate immune responses by selective autophagy (Tsapras, 2022a).

A yeast two-hybrid screening identifies novel Atg8a interactors in Drosophila

Macroautophagy/autophagy-related protein Atg8/LC3 is important for autophagosome biogenesis and required for selective degradation of various substrates. In a recent study, a yeast two-hybrid screening was performed to identify proteins that interact with Atg8a, the Drosophila homolog of Atg8/LC3. The screening identified several Atg8a-interacting proteins. These proteins include: i) proteins which have already been experimentally verified to bind Atg8a, such as Atg1, DOR, ref(2)P and key (Kenny); ii) proteins for which their mammalian homologs interact with Atg8-family members, like Ank2, Atg4, and Nedd4; and iii) several novel Atg8a-interacting proteins, such as trc/STK38 and Tak1. We showed that Tak1, as well as its co-activator, Tab2, both interact with Atg8a and are substrates for selective autophagic clearance. It was also determined that SH3PX1 interacts with Tab2 and is necessary for the effective regulation of the immune-deficiency (IMD) pathway. These findings suggest a mechanism for the regulatory interactions between Tak1-Tab2-SH3PX1 and Atg8a, which contribute to the fine-tuning of the IMD pathway (Tsapras, 2022b).

Atg2 Regulates Cellular and Humoral Immunity in Drosophila

Autophagy is a process that promotes the lysosomal degradation of cytoplasmic proteins and is highly conserved in eukaryotic organisms. Autophagy maintains homeostasis in organisms and regulates multiple developmental processes, and autophagy disruption is related to human diseases. However, the functional roles of autophagy in mediating innate immune responses are largely unknown. This study sought to understand how Atg2, an autophagy-related gene, functions in the innate immunity of Drosophila melanogaster. The results showed that a large number of melanotic nodules were produced upon inhibition of Atg2. In addition, inhibiting Atg2 suppressed the phagocytosis of latex beads, Staphylococcus aureus and Escherichia coli; the proportion of Nimrod C1 (one of the phagocytosis receptors)-positive hemocytes also decreased. Moreover, inhibiting Atg2 altered actin cytoskeleton patterns, showing longer filopodia but with decreased numbers of filopodia. The expression of AMP-encoding genes was altered by inhibiting Atg2. Drosomycin was upregulated, and the transcript levels of Attacin-A, Diptericin and Metchnikowin were decreased. Finally, the above alterations caused by the inhibition of Atg2 prevented flies from resisting invading pathogens, showing that flies with low expression of Atg2 were highly susceptible to Staphylococcus aureus and Erwinia carotovora carotovora 15 infections. In conclusion, Atg2 regulated both cellular and humoral innate immunity in Drosophila. We have identified Atg2 as a crucial regulator in mediating the homeostasis of immunity, which further established the interactions between autophagy and innate immunity (Qin, 2023).

Bacteria-Derived Peptidoglycan Triggers a Noncanonical Nuclear Factor-kappaB-Dependent Response in Drosophila Gustatory Neurons

Probing the external world is essential for eukaryotes to distinguish beneficial from pathogenic micro-organisms. If it is clear that the main part of this task falls to the immune cells, recent work shows that neurons can also detect microbes, although the molecules and mechanisms involved are less characterized. In Drosophila, detection of bacteria-derived peptidoglycan by pattern recognition receptors of the peptidoglycan recognition protein (PGRP) family expressed in immune cells triggers nuclear factor-κB (NF-κB)/immune deficiency (IMD)-dependent signaling. This study shows that one PGRP protein, called PGRP-LB, is expressed in bitter gustatory neurons of proboscises. In vivo calcium imaging in female flies reveals that the PGRP/IMD pathway is cell-autonomously required in these neurons to transduce the peptidoglycan signal. It was finally shown that NF-κB/IMD pathway activation in bitter-sensing gustatory neurons influences fly behavior. This demonstrates that a major immune response elicitor and signaling module are required in the peripheral nervous system to sense the presence of bacteria in the environment (Masuzzo, 2022).

Drosophila Relish Activating lncRNA-CR33942 Transcription Facilitates Antimicrobial Peptide Expression in Imd Innate Immune Response

Long noncoding RNAs (lncRNAs) are an emerging class of regulators that play crucial roles in regulating the strength and duration of innate immunity. However, little is known about the regulation of Drosophila innate immunity-related lncRNAs. In this study, it was first revealed that overexpression of lncRNA-CR33942 could strengthen the expression of the Imd pathway antimicrobial peptide (AMP) genes Diptericin (Dpt) and Attacin-A (AttA) after infection, and vice versa. Secondly, RNA-seq analysis of lncRNA-CR33942-overexpressing flies post Gram-negative bacteria infection confirmed that lncRNA-CR33942 positively regulated the Drosophila immune deficiency (Imd) pathway. Mechanistically, it was found that lncRNA-CR33942 interacts and enhances the binding of NF-κB transcription factor Relish to Dpt and AttA promoters, thereby facilitating Dpt and AttA expression. Relish could also directly promote lncRNA-CR33942 transcription by binding to its promoter. Finally, rescue experiments and dynamic expression profiling post-infection demonstrated the vital role of the Relish/lncRNA-CR33942/AMP regulatory axis in enhancing Imd pathway and maintaining immune homeostasis. This study elucidates novel mechanistic insights into the role of lncRNA-CR33942 in activating Drosophila Imd pathway and the complex regulatory interaction during the innate immune response of animals (Zhou, 2022).

Expression of antimicrobial peptides associated with different susceptibilities to environmental chemicals in Drosophila suzukii and Drosophila melanogaster

Drosophila suzukii is a serious agricultural pest. The evolved morphology of the female D. suzukii assists in penetrating the surface of fresh fruit and spawns eggs with its unique ovipositor. Conversely, Drosophila melanogaster, a taxonomically close species with D. suzukii, largely inhabits decaying and fermenting fruits and is consistently exposed to extensive environmental chemicals, such as 2-phenylethanol, ethanol, and acetic acid, produced by microorganisms. Considering the distinct habitats of the two flies, D. suzukii is thought to be more susceptible to environmental chemicals than D. melanogaster. This study investigated the significantly higher survival rate of D. melanogaster following exposure to 2-phenylethanol, ethanol, and acetic acid. A comparison of the expression of antimicrobial peptides (AMPs) between the two flies treated with chemicals established that AMPs were generally more abundantly induced in D. melanogaster than in D. suzukii, particularly in the gut and fat body. Among the AMPs, the induction of genes (Diptericin A, Diptericin B, and Metchnikowin), which are regulated by the immune deficiency (IMD) pathway, was significantly higher than that of Drosomycin, which belongs to the Toll pathway in chemical-treated D. melanogaster. A transgenic RNAi fly (D. melanogaster) with silenced expression of AMPs and Relish, a transcription factor of the IMD pathway, exhibited significantly reduced survival rates than the control fly. These results suggest that AMPs regulated by the IMD pathway play an important role in the chemical tolerance of D. melanogaster, and these flies are adapted to their habitats by physiological response (Kim, 2022).

Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. This study identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases (Shi, 2023).

Intestinal Immune Deficiency and Juvenile Hormone Signaling Mediate a Metabolic Trade-off in Adult Drosophila Females

A trade-off hypothesis pertains to the biased allocation of limited resources between two of the most important fitness traits, reproduction and survival to infection. This quid pro quo manifests itself within animals prioritizing their energetic needs according to genetic circuits balancing metabolism, germline activity and immune response. Key evidence supporting this hypothesis includes dipteran fecundity being compromised by systemic immunity, and female systemic immunity being compromised by mating. This study reveals a local trade-off taking place in the female Drosophila midgut upon immune challenge. Genetic manipulation of intestinal motility, permeability, regeneration and three key midgut immune pathways provides evidence of an antagonism between specific aspects of intestinal defense and fecundity. That is, juvenile hormone (JH)-controlled egg laying, lipid droplet utilization and insulin receptor expression are specifically compromised by the immune deficiency (Imd) and the dual oxidase (Duox) signaling in the midgut epithelium. Moreover, antimicrobial peptide (AMP) expression under the control of the Imd pathway is inhibited upon mating and JH signaling in the midgut. Local JH signaling is further implicated in midgut dysplasia, inducing stem cell-like clusters and gut permeability. Thus, midgut JH signaling compromises host defense to infection by reducing Imd-controlled AMP expression and by inducing dysplasia, while midgut signaling through the Imd and Duox pathways compromises JH-guided metabolism and fecundity (Shianiou, 2023).

CG4968 positively regulates the immune deficiency pathway by targeting Imd protein in Drosophila

Drosophila melanogaster relies solely on innate immunity to defend against various microbial pathogens. Although it is well-known that the adaptor protein Imd undergoes K63-linked ubiquitination to activate the downstream signaling cascades, its involvement with K48-linked ubiquitination and what is responsible for controlling this modification remain largely unknown. This study explored the immunological function of CG4968, which encodes a typical ovarian tumour-associated protease (OTU)-type deubiquitinase (Dub) in flies. In vitro and in vivo evidence demonstrated that CG4968 plays a positive role in governing the immune deficiency (IMD), but not the Toll innate immune response in an OTU domain-dependent manner. Mechanistically, it was found that CG4968 is associated with Imd to restrict its K48-linked ubiquitination, thereby contributing to its turnover. Collectively, this study uncovered a novel regulatory mechanism involving the K48-linked ubiquitination of Imd in Drosophila innate immunity (Li, 2023).

Antimicrobial peptides do not directly contribute to aging in Drosophila, but improve lifespan by preventing dysbiosis

Antimicrobial peptides (AMPs) are innate immune effectors first studied for their role in host defense. Recent studies have implicated these peptides in the clearance of aberrant cells and in neurodegenerative syndromes. In Drosophila, many AMPs are produced downstream of Toll and Imd NF-κB pathways upon infection. Upon aging, AMPs are upregulated, drawing attention to these molecules as possible causes of age-associated inflammatory diseases. However, functional studies overexpressing or silencing these genes have been inconclusive. Using an isogenic set of AMP gene deletions, this study investigated the net impact of AMPs on aging. Overall, no major effect of individual AMPs on lifespan was found, with the possible exception of Defensin. However, ΔAMP14 flies lacking seven AMP gene families display reduced lifespan. Increased bacterial load in the food of aged ΔAMP14 flies suggests their lifespan reduction is due to microbiome dysbiosis, consistent with a previous study. Moreover, germ-free conditions extends the lifespan of ΔAMP14 flies. Overall, these results do not point to an overt role of individual AMPs in lifespan. Instead, this study found that AMPs collectively impact lifespan by preventing dysbiosis during aging (Hanson, 2023).

Targeting Imd pathway receptor in Drosophila melanogaster and repurposing of phyto-inhibitors: structural modulation and molecular dynamics

Dysbiosis is a major cause of disease in an individual, generally initiated in the gastrointestinal tract. The gut, also known as the second brain, constitutes a major role in immune signaling. To study the immunity cascade, the Drosophila model was considered targeting the Imd pathway receptor (2F2L) located in the midgut. This receptor further initiates the immune signaling mechanism influenced by bacteria. To inhibit the Imd pathway, the crystal structure of Imd with PDB: 2F2L was considered for the screening of suitable ligand/inhibitor. In light of previous studies, repurposing of anti-diabetic ligands from the banana plant namely lupeol (LUP), stigmasterol (STI), β-sitosterol (BST) and umbelliferone (UMB) were screened. This study identifies the potential inhibitor along with the tracheal toxin (TCT), a major peptidoglycan constituent of microbes. The molecular docking and molecular dynamics simulation of complexes 2F2L-MLD, 2F2L- CAP, 2F2L-LUP, 2F2L-BST, 2F2L-STI and 2F2L-UMB elucidates the intermolecular interaction into the inhibitory property of ligands. The results of this study infer LUP and UMB as better ligands with high stability and functionality among the screened candidates. This study provides insights into the dysbiosis and its amelioration by plant-derived molecules. The identified drugs (LUP & UMB) will probably act as an inhibitor against microbial dysbiosis and other related pathogenesis (diabetes and diabetic neuropathy). Further, this study will widen avenues in fly biology research and which could be used as a therapeutic model in the rapid, reliable and reproducible screening of phytobiologics in complementary and alternative medicine for various lifestyle associated complications (Satapathy, 2020).

Persistent activation of the innate immune response in adult Drosophila following radiation exposure during larval development

This study investigates the role of the innate immune system in response to radiation exposure. It was shown that the innate immune response and NF-ĸB target gene expression is activated in the adult Drosophila brain following radiation exposure during larval development and that this response is sustained in adult flies weeks after radiation exposure. Preliminary data suggest that innate immunity is radioprotective during Drosophila development. Together these data suggest that activation of the innate immune response may be beneficial initially for survival following radiation exposure but result in long-term deleterious consequences, with chronic inflammation leading to impaired neuronal function and viability at later stages. This work lays the foundation for future studies of how the innate immune response is triggered by radiation exposure and its role in mediating the biological responses to radiation (Sudmeier, 2015).

RNA-binding protein Roquin negatively regulates STING-dependent innate immune response in Drosophila

Drosophila melanogaster utilizes innate immune response to defend against exogenous pathogens. The molecular regulation mechanism of the process is evolutionarily conserved. Research of the regulatory mechanisms of Drosophila innate immunity is greatly significant for understanding the modulation of the human innate immunity and the pathogenesis of related diseases. To explore novel regulators in the STING-dependent innate immune response in Drosophila, the double-stranded RNA-mediated gene expression silencing technique and the dual-luciferase reporter system were used in knockdown experiments on 9 genes encoding the ubiquitin ligase such as echinus (CG2904), usp16 (CG4165), smurf (CG4943), pellino (CG5212), usp47 (CG5486), diap2 (CG8293), dtraf2 (CG10961), roquin (CG16807) and usp10 (CG32479) in the S2 cells in vitro. The results suggested a negative correlation between CG16807 (roquin) and the STING signaling pathway. Further studies showed that over-expression of roquin in S2 cells significantly inhibited STING innate immune signaling. Meanwhile, Listeria infection experiments showed that knocking down of roquin markedly elevated the expression levels of anti-microbial peptides and inhibited the proliferation of Listeria, thus increasing the survival rates post pathogenic infection. Taken together, these results suggested that the RNA-binding protein Roquin negatively regulates the STING-dependent innate immune response in Drosophila. In view of the high correlation between Drosophila genes and human genes, this study provides a theoretical basis for further development of treatments for STING-related innate immune diseases in humans (Du, 2020)

A novel virus-induced cyclic dinucleotide, 2'3'-c-di-GMP, mediates STING-dependent antiviral immunity in Drosophila

In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP. This CDN binds to and activates the protein STING to trigger immunity. Two cGAS-like receptors (cGLRs) have been discovered that activate STING-dependent antiviral immunity and can produce 3'2'-cGAMP, in addition to 2'3'-cGAMP. This study explore dCDN-mediated immunity in 14 different Drosophila species covering 50 million years of evolution and reports that 2'3'-cGAMP and 3'2'-cGAMP fail to control infection by Drosophila C virus in D. serrata, D. sechellia and D. mojavensis. Using an accurate and sensitive mass spectrometry method, an unexpected diversity was discovered of CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including a novel CDN, 2'3'-c-di-GMP. 2'3'-c-di-GMP was shown to be the most potent STING agonist identified so far in D. melanogaster, and this molecule also activates a strong antiviral transcriptional response in D. serrata. These results shed light on the evolution of cGLRs in flies and provide a basis for the understanding of the function and regulation of this emerging family of PRRs in animal innate immunity (Cai, 2023).

Cross-species analysis of viral nucleic acid interacting proteins identifies TAOKs as innate immune regulators

The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. This study used an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. The family of TAO kinases (TAOK1, -2 and -3) was identified as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, this study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses (Pennemann, 2021).

Innate immune pathways act synergistically to constrain RNA virus evolution in Drosophila melanogaster

Host-pathogen interactions impose recurrent selective pressures that lead to constant adaptation and counter-adaptation in both competing species. This evolutionary arms-race was study, and the impact of the innate immune system on viral population diversity and evolution was assessed using Drosophila melanogaster as model host and its natural pathogen Drosophila C virus (DCV). Eight fly genotypes were isogenized, generating animals defective for RNAi, Imd and Toll innate immune pathways as well as pathogen-sensing and gut renewal pathways. Wild-type or mutant flies were then orally infected with DCV and the virus was serially passaged ten times via reinfection in naive flies. Viral population diversity was studied after each viral passage by high-throughput sequencing and infection phenotypes were assessed at the beginning and at the end of the evolution experiment. The absence of any of the various immune pathways studied increased viral genetic diversity while attenuating virulence. Strikingly, these effects were observed in a range of host factors described as having mainly antiviral or antibacterial functions. Together, these results indicate that the innate immune system as a whole and not specific antiviral defence pathways in isolation, generally constrains viral diversity and evolution (Mongelli, 2022).

Retrotransposon activation during Drosophila metamorphosis conditions adult antiviral responses

Retrotransposons are one type of mobile genetic element that abundantly reside in the genomes of nearly all animals. Their uncontrolled activation is linked to sterility, cancer and other pathologies, thereby being largely considered detrimental. This study reports that, within a specific time window of development, retrotransposon activation can license the host's immune system for future antiviral responses. mdg4 (also known as Gypsy) retrotransposon selectively becomes active during metamorphosis at the Drosophila pupal stage. At this stage, mdg4 activation educates the host's innate immune system by inducing the systemic antiviral function of the nuclear factor-κB protein Relish in a dSTING-dependent manner. Consequently, adult flies with mdg4, Relish or dSTING silenced at the pupal stage are unable to clear exogenous viruses and succumb to viral infection. Altogether, these data reveal that hosts can establish a protective antiviral response that endows a long-term benefit in pathogen warfare due to the developmental activation of mobile genetic elements (Wang, 2022).

Temperature and sex shape Zika virus pathogenicity in the adult Brat (cheesehead) brain: A Drosophila model for virus-associated neurological diseases

Severe neurological complications affecting brain growth and function have been well documented in newborn and adult patients infected by Zika virus (ZIKV), but the underlying mechanisms remain unknown. This study used a Drosophila melanogaster mutant, cheesehead (chs), with a mutation in the brain tumor (brat) locus that exhibits both aberrant continued proliferation and progressive neurodegeneration in the adult brain. This study reports that temperature variability is a key driver of ZIKV pathogenesis, thereby altering host mortality and causing motor dysfunction in a sex-dependent manner. Furthermore, it was shown that ZIKV is largely localized to the brat (chs) brain and activates the RNAi and apoptotic immune responses. These findings establish an in vivo model to study host innate immune responses and highlight the need of evaluating neurodegenerative deficits as a potential comorbidity in ZIKV-infected adults (Tafesh-Edwards 2023).

Investigating the outcomes of virus coinfection within and across host species

Interactions between coinfecting pathogens have the potential to alter the course of infection and can act as a source of phenotypic variation in susceptibility between hosts. This phenotypic variation may influence the evolution of host-pathogen interactions within host species and interfere with patterns in the outcomes of infection across host species. This study examine experimental coinfections of two Cripaviruses-Cricket Paralysis Virus (CrPV), and Drosophila C Virus (DCV)-across a panel of 25 Drosophila melanogaster inbred lines and 47 Drosophilidae host species. Interactions between these viruses were found to alter viral loads across D. melanogaster genotypes, with a ~3 fold increase in the viral load of DCV and a ~2.5 fold decrease in CrPV in coinfection compared to single infection, but little evidence was found of a host genetic basis for these effects. Across host species, no evidence was found of systematic changes in susceptibility during coinfection, with no interaction between DCV and CrPV detected in the majority of host species. These results suggest that phenotypic variation in coinfection interactions within host species can occur independently of natural host genetic variation in susceptibility, and that patterns of susceptibility across host species to single infections can be robust to the added complexity of coinfection (Imrie, 2023).

cGAS-like receptors sense RNA and control 3'2'-cGAMP signaling in Drosophila

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger 2'3'-cGAMP and controls activation of innate immunity in mammalian cells. Animal genomes typically encode multiple proteins with predicted homology to cGAS (CG7194 in Drosophila), but the function of these uncharacterized enzymes is unknown. This study shows that cGAS-like receptors (cGLRs) are innate immune sensors capable of recognizing divergent molecular patterns and catalyzing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signaling core shared with cGAS and a diversified primary ligand-binding surface modified with significant insertions and deletions. cGLR surface remodeling enables altered ligand specificity and use a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. Surprisingly, RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila Stimulator of Interferon Genes (cGLR1) in complex with 3'2'-cGAMP explains selective isomer recognition and this study demonstrates that 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, these results establish cGLRs as a diverse family of metazoan pattern recognition receptors (Slevik, 2021).

To define the function of cGAS-like enzymes in animals, wpredicted cGAS homologues were screened for suitability in structural analysis, and a 2.4 Å crystal structure was determined of the human protein MB21D2 (hMB21D2; encoded by C3orf59) and a 1.6 Å crystal structure was determined of a protein from the beetle species Tribolium castaneum (GenBank XP_969398.1). Despite divergence in the primary sequence, the hMB21D2 and T. castaneum XP_969398.1 structures each reveal close homology to human cGAS with a shared bi-lobed architecture, a caged nucleotidyltransferase core, a Gly-[Gly/Ser] activation loop and a putative catalytic triad. In human cGAS, the primary ligand-binding surface is a long groove on the back of the enzyme formed by the α-helix spine and a Zn-ribbon motif that is essential for recognition of double-stranded DNA. A conserved groove is present in both the hMB21D2 and the T. castaneum XP_969398.1 structures, but is notably distinguished by the absence of a Zn-ribbon and the insertion of a C-terminal α-helix in hMB21D2. It was hypothesized that the remodelling of this groove controls the detection of distinct ligands. The hMB21D2 surface is overall neutral with no obvious capacity to bind nucleic acids, and no enzymatic activity was detected with a panel of potential activating ligands. In contrast to hMB21D2, the surface of T. castaneum XP_969398.1 shares highly conserved basic residues with human cGAS (Fig. 1a) and we therefore tested this enzyme with candidate DNA and RNA ligands. It was observed that T. castaneum XP_969398.1 is activated to synthesize a nucleotide product upon recognition of double-stranded RNA (dsRNA). Despite exhibiting a clear difference in ligand specificity, analysis of all structures in the Protein Data Bank confirmed that T. castaneum XP_969398.1 is a close homologue of mammalian cGAS and is distinct from previously characterized RNA sensors including oligoadenylate synthase 1. Together, these results establish the existence of cGLRs in animals and demonstrate that remodelling of a primary ligand-binding surface enables the recognition of divergent molecular patterns (Slevik, 2021).

To identify additional cGLRs that respond to dsRNA, the T. castaneum cGLR (Tc-cGLR) sequence was used to search for predicted cGAS homologues in species related to the model organism D. melanogaster. 153 cGLR genes were identified across 42 species in the order Diptera, which cluster into distinct clades designated 1-5. Drosophila encode a remarkable number of cGLR genes, with individual species predicted to have between three and seven enzymes. In a systematic biochemical screen, 53 recombinant cGLR proteins were purified and tested, and active enzymes were identified from the species Lucilia cuprina, Drosophila eugracilis, Drosophila erecta and Drosophila simulans. Similar to Tc-cGLR, each active Diptera enzyme specifically responded to dsRNA, indicating that cGLR-based recognition of RNA is conserved across diverse insect species (Slevik, 2021).

The D. simulans enzyme identified in this screen shares 91% sequence identity with the protein product of the D. melanogaster gene CG12970. Analysis of recombinant D. melanogaster CG12970 protein revealed that it also synthesizes a nucleotide product specifically in the presence of dsRNA, and therefore this gene was named tcGAS-like Receptor 1 (Dm-cGLR1). To understand how dsRNA activates Drosophila cGLR1, the molecular determinants were determined for enzymatic activity in vitro. it was observed that D. simulans cGLR1 (Ds-cGLR1) and Dm-cGLR1 recognize dsRNAs longer than 30 bp with no preference for 5' RNA phosphorylation. Notably, activation of Ds-cGLR1 and Dm-cGLR1 requires dsRNA ligands that exceed the length of 21-23-bp RNA molecules commonly produced during RNA interference in Drosophila, suggesting specific avoidance of self-recognition. Similar to the formation of condensates observed with human cGAS recognition of dsDNA, Ds-cGLR1 selectively binds to dsRNA and forms a higher-order complex that is dependent on the length of dsRNA. Ectopic expression of Dm-cGLR1 or Ds-cGLR1 in human cells demonstrated that cGLR1 activity is sufficient to enable cellular dsRNA sensing and drive activation of a STING-dependent immune response. Dm-cGLR1 and Ds-cGLR1 signalling in cells required dsRNA stimulation, and mutations to the enzyme catalytic site disrupted downstream activation of STING. To understand how Drosophila cGLR1 engages dsRNA, interactions were modelled using the Tc-cGLR and human cGAS-DNA structures as a template and observed that charge-swap mutations to the conserved basic ligand-binding surface disrupted product synthesis in vitro and STING signalling in cells. Together, these data demonstrate that insect cGLRs and human cGAS use a shared mechanism of ligand detection and reveal that Dm-cGLR1 can function as a foreign RNA sensor (Slevik, 2021).

A role in sensing long dsRNA suggests that the function of Dm-cGLR1 is to control a downstream immune response in Drosophila. In human cells, cGAS synthesizes the nucleotide second messenger 2'3'-cGAMP, which contains a non-canonical 2'-5' phosphodiester linkage that is required for potent activation of immune signalling. To determine how Dm-cGLR1 controls cellular signalling, the nucleotide reaction product was purified for direct comparison to 2'3'-cGAMP. The Dm-cGLR1 product exhibited a C18 chromatography migration profile distinct from 2'3'-cGAMP and all previously known naturally occurring cyclic dinucleotide (CDN) signals. Production of this nucleotide signal was conserved in Diptera with Ds-cGLR1, Lc-cGLR and Deu-cGLR reactions, each synthesizing the same major reaction product. Using nucleobase-specific labelling and nuclease digestion of the Dm-cGLR1 product, a 3'−5' linkage connected to an adenosine phosphate and a protected 2'−5' linkage connected to a guanosine phosphate were determined, indicating a mixed-linkage cyclic GMP-AMP species. These findings were verified with comparative high-performance liquid chromatography and tandem mass spectrometry profiling against a chemically synthesized standard, and it was confirmed that the shared Diptera cGLR product is the novel isomer 3'2'-cGAMP (Slevik, 2021).

Dm-cGLR1 synthesizes 3'2'-cGAMP in a two-step reaction through production of the linear intermediate pppA[2'-5']pG and uses an opposite nucleobase reaction order compared with human cGAS2,3,19. Next mass spectrometry was used to analyse lysates expressing each recombinant dipteran cGLR from the screen. 3'2'-cGAMP was detected as a product of 15 cGLRs, including enzymes from each subgroup within clade 5 of the Diptera cGLR phylogeny. cGLRs clustered within clade 5 collectively represent 41 species, suggesting widespread conservation of 3'2'-cGAMP signalling in Diptera. The beetle enzyme Tc-cGLR synthesizes 2'3'-cGAMP, supporting that 2'3'-cGAMP is an ancestral signalling molecule in metazoans and that 3'2'-cGAMP signalling is a recent adaptation in flies. Insect and mammalian viruses encode 2'3'-cGAMP-specific nucleases named poxins that allow evasion of cGAS-STING immune responses. 3'2'-cGAMP was protected from cleavage by poxin, indicating that an isomeric switch in the specificity of phosphodiester linkage endows Drosophila with a signalling pathway resistant to a major form of viral immune evasion (Slevik, 2021).

Drosophila STING (dSTING) is known to function as a cyclic dinucleotide receptor in vivo, but an endogenous nucleotide second messenger has not been previously identified. Therefore an in vitro thermo-fluor binding assay was developed to analyse dSTING recognition of specific CDNs. dSTING preferentially formed a thermostable complex with 3'2'-cGAMP and exhibited no detectable complex formation with 2'3'-cGAMP or other CDNs in vitro. Using direct delivery of CDNs to permeabilized cells, it was confirmed that dSTING preferentially responds to 3'2'-cGAMP in the cellular environment. To define the mechanism of selective 3'2'-cGAMP recognition, a 2.0 Å crystal structure of the D. eugracilis STING (GenBank XP_017066673) CDN-binding domain was determined in complex with 3'2'-cGAMP. dSTING adopts a highly conserved V-shaped homodimeric architecture with a deep central pocket that binds to 3'2'-cGAMP. The dSTING-3'2'-cGAMP structure reveals a tightly 'closed' conformation with dSTING protomers positioned 36 Å apart, similar to the closed conformation of human STING bound to 2'3'-cGAMP5. Each nucleobase of 3'2'-cGAMP is stacked between dSTING Y164 and R234, and E257 specifically coordinates the 3'2'-cGAMP guanosine N2 position. In human STING, high-affinity recognition of 2'3'-cGAMP requires readout of the 2'-5' phosphodiester linkage by R232 in the β-strand lid. In dSTING, the equivalent R229 makes no contact with either phosphodiester bond. Instead, R229 is repositioned to extend outwards from the ligand-binding pocket by the deletion of a single lid residue and the formation of a salt bridge with E267 on the opposing protomer, explaining the diminished affinity of dSTING for 2'3'-cGAMP. In addition, a key asparagine substitution, N159, in dSTING extends across the binding pocket to coordinate the adenosine 3' OH in 3'2'-cGAMP and directly replaces the human STING S162 residue that contacts the guanosine 3' OH in 2'3'-cGAMP. A panel of dSTING-mutant proteins was tested and it was confirmed that mutations to each coordinating residue disrupt the formation of the dSTING-3'2'-cGAMP complex. The unique adaptations in the ligand-binding pocket observed in the dSTING-3'2'-cGAMP structure are widely conserved in Diptera and together explain a mechanism for how specific 3'2'-cGAMP-dependent signalling drives the activation of dSTING (Slevik, 2021).

To determine how Dm-cGLR1-3'2'-cGAMP-dSTING signalling controls immune responses in vivo, 3'2'-cGAMP was injected into D. melanogaster to directly monitor the dSTING response. 3'2'-cGAMP potently induced the expression of Sting and three other Sting-regulated genes (Srg) in a dose-dependent manner. Notably, 3'2'-cGAMP-dependent signalling through dSTING was significantly more potent than the response triggered by injection of the bacterial CDN signal 3'3'-c-di-GMP. Genetic mutations to Sting and the NF-κB homologue Relish ablated 3'2'-cGAMP-induced responses, demonstrating that signalling operates through a conserved dSTING-NF-7kappa;B pathway. Flies were challenged with viral infection and observed that 3'2'-cGAMP markedly suppressed the replication of two unrelated RNA viruses: Drosophila C virus (Dicistroviridae), a natural Drosophila pathogen, and vesicular stomatitis virus (Rhabdoviridae). 3'2'-cGAMP activation of antiviral immunity was strictly dependent on Sting and resulted in a response that significantly delayed pathogen-mediated mortality. Direct comparison of the protective effects against Drosophila C virus infection showed that the endogenous signal 3'2'-cGAMP exhibited greater antiviral potency than 2'3'-cGAMP. 3'2'-cGAMP more robustly suppressed RNA viral loads and extended animal survival, revealing that the dSTING antiviral signalling axis is preferentially activated by 3'2'-cGAMP in vivo. Together, these results demonstrate that 3'2'-cGAMP is an antiviral nucleotide second messenger in D. melanogaster and establish a cGLR-STING-NF-κB axis that protects animals from viral replication (Slevik, 2021).

Along with cGAS recognition of dsDNA, the discovery of animal cGLR dsRNA sensors establishes a diverse class of pattern recognition receptors conserved throughout metazoans. Divergent structural homologues of cGAS in humans and insects demonstrate that cGLRs constitute a rapidly evolving family of proteins in which remodelling of a primary binding surface enables the detection of diverse ligands. The mechanistic characterization of Drosophila cGLR1 activation shows that cGLRs function as direct sensors of pathogen-associated molecular patterns and synthesize distinct second messengers to control a conserved downstream signalling axis. Drosophila were previously thought to respond to foreign nucleic acid exclusively through RNA interference and direct cleavage of pathogen RNA. Drosophila cGLR1 reveals a parallel signalling system for sensing dsRNA and directing an inducible immune response through dSTING. Synthesis of the second messenger 3'2'-cGAMP by Drosophila cGLR1 and selective recognition by dSTING demonstrates that metazoans use CDNs beyond 2'3'-cGAMP as endogenous second messengers and highlights the evolutionary plasticity of cGLR signalling. Structural analysis also reveals that the human cGLR MB21D2 is competent for synthesis of nucleotide second messengers and has a remodelled ligand-binding groove that is probably adapted for detection of an unknown stimulus. Together with the known high frequency of hMB21D2 mutations in cancer, these results support a more extensive role for cGLR signalling in human biology. The existence of multiple unique cGLRs encoded within a single species suggests a model in which the cGLR signalling scaffold is harnessed to detect several distinct stimuli. In support of this conclusion, cGLR2 has been identified as a second functional cGLR in Drosophila (Holleufer, 2021), and it was demonstrated in vivo that cGLR1 and cGLR2 have discrete roles in Drosophila immunity. Together, these results define cGLRs as receptors in animal cells that are capable of detecting diverse pathogen-associated molecular patterns and dictating response to the foreign environment (Slevik, 2021).

Drosophila melanogaster relies on an evolutionarily conserved innate immune system to protect itself from a wide range of pathogens, making it a convenient genetic model to study various human pathogenic viruses and host antiviral immunity. This study explored for the first time the contribution of the Drosophila phenoloxidase (PO) system to host survival and defenses against Zika virus (ZIKV) infection by analyzing the role of mutations in the three prophenoloxidase (PPO) genes in female and male flies. Only PPO1 and PPO2 genes contribute to host survival and appear to be upregulated following ZIKV infection in Drosophila. Also, data suggesting that a complex regulatory system exists between Drosophila PPOs, potentially allowing for a sex-dependent compensation of PPOs by one another or other immune responses such as the Toll, Imd, and JAK/STAT pathways. Furthermore, this study showed that PPO1 and PPO2 are essential for melanization in the hemolymph and the wound site in flies upon ZIKV infection. These results reveal an important role played by the melanization pathway in response to ZIKV infection, hence highlighting the importance of this pathway in insect host defense against viral pathogens and potential vector control strategies to alleviate ZIKV outbreaks (Tafesh-Edwards, 2023).

Drosophila melanogaster as a Model System to Assess the Effect of Epstein-Barr Virus DNA on Inflammatory Gut Diseases

The Epstein-Barr virus (EBV) commonly infects humans and is highly associated with different types of cancers and autoimmune diseases. EBV has also been detected in inflamed gastrointestinal mucosa of patients suffering from prolonged inflammation of the digestive tract such as inflammatory bowel disease (IBD) with no clear role identified yet for EBV in the pathology of such diseases. Since immune-stimulating capabilities of EBV DNA has been reported in various models, this study investigated whether EBV DNA may play a role in exacerbating intestinal inflammation through innate immune and regeneration responses using the Drosophila melanogaster model. Inflamed gastrointestinal tracts were generated in adult fruit flies through the administration of dextran sodium sulfate (DSS), a sulfated polysaccharide that causes human ulcerative colitis- like pathologies due to its toxicity to intestinal cells. Intestinal damage induced by inflammation recruited plasmatocytes to the ileum in fly hindguts. EBV DNA aggravated inflammation by enhancing the immune deficiency (IMD) pathway as well as further increasing the cellular inflammatory responses manifested upon the administration of DSS. The study at hand proposes a possible immunostimulatory role of the viral DNA exerted specifically in the fly hindgut hence further developing understanding of immune responses mounted against EBV DNA in the latter intestinal segment of the D. melanogaster gut. These findings suggest that EBV DNA may perpetuate proinflammatory processes initiated in an inflamed digestive system. These findings indicate that D. melanogaster can serve as a model to further understand EBV-associated gastroinflammatory pathologies. Further studies employing mammalian models may validate the immunogenicity of EBV DNA in an IBD context and its role in exacerbating the disease through inflammatory mediators (Madi, 2021).

Dense time-course gene expression profiling of the Drosophila melanogaster innate immune response

Immune responses need to be initiated rapidly, and maintained as needed, to prevent establishment and growth of infections. At the same time, resources need to be balanced with other physiological processes. On the level of transcription, studies have shown that this balancing act is reflected in tight control of the initiation kinetics and shutdown dynamics of specific immune genes. To investigate genome-wide expression dynamics and trade-offs after infection at a high temporal resolution, an RNA-seq time course was performed on D. melanogaster with 20 time points post Imd stimulation. A combination of methods, including spline fitting, cluster analysis, and Granger causality inference, allowed detailed dissection of expression profiles, lead-lag interactions, and functional annotation of genes through guilt-by-association. Imd-responsive genes and co-expressed, less well characterized genes, with an immediate-early response and sustained up-regulation up to 5 days after stimulation were identified. In contrast, stress response and Toll-responsive genes, among which were Bomanins, demonstrated early and transient responses. This study further observed a strong trade-off with metabolic genes, which strikingly recovered to pre-infection levels before the immune response was fully resolved. This high-dimensional dataset enabled the comprehensive study of immune response dynamics through the parallel application of multiple temporal data analysis methods. The well annotated data set should also serve as a useful resource for further investigation of the D. melanogaster innate immune response, and for the development of methods for analysis of a post-stress transcriptional response time-series at whole-genome scale (Schlamp, 2021).

Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways

Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, a matching Drosophila model was established. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. These structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. These findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes (Wagner, 2021).

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, it was asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals (Jugder, 2021).

Protein Phosphatase 4 Negatively Regulates the Immune Deficiency-NF-kappaB Pathway during the Drosophila Immune Response

The evolutionarily conserved immune deficiency (IMD) signaling pathway shields Drosophila against bacterial infections. It regulates the expression of antimicrobial peptides encoding genes through the activation of the NF-κB transcription factor Relish. Tight regulation of the signaling cascade ensures a balanced immune response, which is otherwise highly harmful. Several phosphorylation events mediate intracellular progression of the IMD pathway. However, signal termination by dephosphorylation remains largely elusive. This study identifyied the highly conserved protein phosphatase 4 (PP4) complex as a bona fide negative regulator of the IMD pathway. RNA interference-mediated gene silencing of PP4-19c, PP4R2, and Falafel, which encode the catalytic and regulatory subunits of the phosphatase complex, respectively, caused a marked upregulation of bacterial-induced antimicrobial peptide gene expression in both Drosophila melanogaster S2 cells and adult flies. Deregulated IMD signaling is associated with reduced lifespan of PP4-deficient flies in the absence of any infection. In contrast, flies overexpressing this phosphatase are highly sensitive to bacterial infections. Altogether, these results highlight an evolutionarily conserved function of PP4c in the regulation of NF-κB signaling from Drosophila to mammals (Wehbe, 2021).

Activation of innate immunity during development induces unresolved dysbiotic inflammatory gut and shortens lifespan

An early-life inflammatory response is associated with risks of age-related pathologies. How transient immune signalling activity during animal development influences life-long fitness is not well understood. Using Drosophila as a model, this study found that activation of innate immune pathway Immune deficiency (Imd) signalling in the developing larvae increases adult starvation resistance, decreases food intake and shortens organismal lifespan. Interestingly, lifespan is shortened by Imd activation in the larval gut and fat body, whereas starvation resistance and food intake are altered by that in neurons. The adult flies that developed with Imd activation show sustained Imd activity in the gut, despite complete tissue renewal during metamorphosis. The larval Imd activation increases an immunostimulative bacterial species, Gluconobacter sp., in the gut microbiome, and this dysbiosis is persistent to adulthood. Removal of gut microbiota by antibiotics in the adult fly mitigates intestinal immune activation and rescues the shortened lifespan. This study demonstrates that early-life immune activation triggers long-term physiological changes, highlighted as an irreversible alteration in gut microbiota, prolonged inflammatory intestine and concomitant shortening of the organismal lifespan (Yamashita, 2021).

Analysis of Drosophila STING Reveals an Evolutionarily Conserved Antimicrobial Function

The vertebrate protein STING, an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response and the induction of type I interferon during pathogenic infection. This study showed that a STING ortholog (dmSTING) exists in Drosophila, which, similar to vertebrate STING, associates with cyclic dinucleotides to initiate an innate immune response. Following infection with Listeria monocytogenes, dmSTING activates an innate immune response via activation of the NF-kappaB transcription factor Relish, part of the immune deficiency (IMD) pathway. DmSTING-mediated activation of the immune response reduces the levels of Listeria-induced lethality and bacterial load in the host. Of significance, dmSTING triggers an innate immune response in the absence of a known functional cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) ortholog in the fly. Together, these results demonstrate that STING is an evolutionarily conserved antimicrobial effector between flies and mammals, and it comprises a key component of host defense against pathogenic infection in Drosophila (Martin, 2018).

Pathogenic infection of Drosophila induces the secretion of antimicrobial peptides by the fat body, an organ analogous to the mammalian liver, which accumulate in the hemolymph. Antimicrobial peptides are small, cationic molecules that are capable of killing bacteria and fungi. Like mammals, flies encode a number of pattern recognition receptors (PRRs) that recognize conserved pathogen motifs called pathogen-associated molecular patterns (PAMPs). The recognition of pathogens in Drosophila initiates a signaling cascade where one of the termination points is the induction of antimicrobial peptides (Martin, 2018).

As innate immunity is an ancient, evolutionarily conserved form of host defense, there is a high degree of similarity in the innate immune responses between flies and mammals. Mammalian PRRs consist of Toll-like receptors (TLR) and RIG-I-like receptors (RLR), among other families of PRRs. Activation of PRRs with their respective PAMPs leads to an innate immune response via the nuclear translocation of NF-κB or interferon (IFN) regulatory factor 3 (IRF3). This cascade culminates with the induction of hundreds of IRF-responsive genes, including IFN-β, a cytokine produced during the early stages of infection and the host defense response, which binds to the IFN-α/β receptor and induces IFN-stimulated gene expression. In Drosophila, two classical innate immune pathways function through either Toll or IMD (immune deficient). During Gram-positive bacteria infection, activation of peptidoglycan recognition protein SA (PGRP-SA), the serine protease Persephone, and Gram-negative binding protein 1 (GNBP1) lead to proteolytically processing of Spatzle and stimulation of the Toll receptor, which activates dMyD88, Tube, Pelle, and the NF-κB homolog DIF (dorsal-related immunity factor). The IMD pathway is stimulated by PGRP-LE and PGRP-LC, which recognize diaminopimelic acid (DAP) type peptidoglycan (PGN) on the surface of bacteria and activate autophagy or the IMD pathway through the NF-κB molecule Relish. Together, the Toll and IMD pathways make up two NF-κB pathways in Drosophila that function in the humoral response to pathogenic infection. Signaling through Drosophila NF-κB pathways is similar to the mammalian TLR pathways, both in pathway structure and the proteins involved in signaling. Antimicrobial peptide genes induced as part of the Toll and IMD pathways include Drosomycin (Drs), AttacinA (AttA), and CecropinA2 (CecA2), among others. Each pathway preferentially induces its own set of antimicrobial peptides, and mutations in the Toll-mediated NF-κB molecule DIF render flies susceptible and unable to induce Toll-mediated antimicrobial peptides during Gram-positive bacteria or fungal infections, while mutations in IMD or Relish render flies susceptible and unable to induce IMD-mediated antimicrobial peptides during Gram-negative bacteria infections. However, there is crosstalk between the Toll and IMD pathways, resulting in the sets of peptides being stimulated together. Ultimately, the fruit fly innate immune response must be fully functional for the proper secretion of antimicrobial peptides into the hemolymph to neutralize the pathogenic infection and curb mortality (Martin, 2018).

Another class of PAMPs in mammals that has not been extensively studied in Drosophila is one that recognizes cytosolic DNA or cyclic dinucleotides (CDNs). In mammals, these molecules trigger signaling pathways controlled by STING (stimulator of interferon genes) and lead to NF-κB and IRF3 activation and ultimately the induction of IFN-β. STING is a transmembrane protein that activates an innate immune response during viral or bacterial infection. STING activation in response to Listeria monocytogenes functions through CDNs, that are byproducts of Listeria infection known to induce IFN-β. Recent studies have also identified cyclic di-guanosine monophosphate (di-GMP) as a major signaling molecule in the Listeria life cycle that is able to activate STING. Indeed, during infection with Chlamydia trachomatis, bacterial CDNs directly activate STING to activate a type I IFN response. Cyclic GMP-AMP synthase (cGAS) signals upstream of STING by binding to cytosolic DNA, triggering cGAS to metabolize ATP and GTP into non-canonical cyclic-GMP-AMP (cGAMP) containing 2'-5' and 3'-5' mixed phosphodiester linkages, which are then able to activate STING. While the roles of STING and cGAS in sensing cytosolic nucleic acids have been comprehensively studied in mammalian immunity, less is known about their role in invertebrate immunity (Martin, 2018).

To date, the major nucleic acid sensors that have been identified in Drosophila are the Dicer proteins involved in the RNAi pathway. Dicer-2 is a pathogen-recognition receptor that senses viral nucleic acids and initiates the RNAi pathway to mount innate and antiviral responses to DNA viruses such as invertebrate iridescent virus 6 (IIV6) (Bronkhorst, 2012). Furthermore, Dicer-2 and the mammalian proteins MDA5 and RIG-I share sequence similarity at their RNA-binding helicase domains . While vertebrates sense cytosolic DNA with a variety of proteins, including IFI16, AIM2, and cGAS, only cGAS has an ortholog in Drosophila, namely CG7194. However, CG7194 lacks the zinc-ribbon domain and a positively charged N terminus, which are functionally important for DNA binding (Martin, 2018).

This study sought to identify a CDN-binding protein in Drosophila, and it was found that the Drosophila protein CG1667, henceforth referred to as dmSTING, is orthologous to the vertebrate STING protein. DmSTING retains its ability to bind cyclic di-GMP, leading to the induction of innate immune response genes. Knockdown of dmSTING during Listeria infection led to a loss of innate immune gene induction, increased bacterial burden, and consequent animal mortality. Conversely, overexpression of dmSTING led to increased antimicrobial peptide induction and activation of the Drosophila NF-κB homolog Relish. Interestingly, dmSTING was functional in mammalian cells, and it was able to induce mammalian NF-κB. Finally, epistasis analysis in flies indicated that dmSTING functioned predominantly through the IMD pathway and Relish to achieve antimicrobial peptide induction. Taken together, these results indicate that STING functions through an evolutionarily conserved host defense pathway, whose antimicrobial function, along with the RNAi, Toll, and JAK-STAT pathways, protects the invertebrate host against microbial infection (Martin, 2018).

In Drosophila, dmSTING is conserved at the amino acid level (22% identity and 57% similarity) with hsSTING, especially at regions that are crucial for binding to CDNs. Recent studies have performed evolutionary analyses to confirm that functional cGAS orthologs do not exist in insects (Martin, 2018).

Interestingly, a functional cGAS-STING pathway exists in the sea anemone Nematostella vectensis. However, purified sea anemone cGAS is not active in vitro but will function in human cells, suggesting that there are additional co-factors that are required for cGAS activity. Experiments using dsDNA virus infection in flies containing P elements in CG7194, a putative cGAS ortholog in Drosophila, further suggest the lack of a cGAS-STING axis in Drosophila since the induction of defense response genes or the dependence of dmSTING on survival to IIV6 infection was not observed. Rather, Drosophila Dicer-2, that contains a RIG-I-like helicase domain, activates an antiviral response to IIV6 through the RNAi pathway, and Dicer-2 plays a role in the defense response to both RNA and DNA virus infection in Drosophila through viral RNA sensing and subsequent degradation to protect the host. Taken together, the current results suggest that in Drosophila, STING senses CDNs, and in the absence of a functional cGAS molecule, bacterial CDNs directly lead to STING activation and a subsequent innate immune response. It is contended that dmSTING signals through the IMD pathway; however, gene expression analyses did show that some Toll-specific genes, including Relish, were less induced during Listeria infection when dmSTING was knocked down, likely due to synergism between the two pathways (Martin, 2018).

From an evolutionary standpoint, invertebrates utilize the RNAi pathway as a defense response to exogenous DNA and RNA encountered during viral infections, whereas RNAi plays less of a role, if any, in the defense response to viral infection in vertebrates. Rather, in vertebrates, antiviral immunity is mediated primarily through the RLR-MAVS axis for RNA viruses. Regarding DNA virus infection in vertebrate hosts, cGAS gained functionality in its ability to bind cytosolic DNA and metabolize CDNs as second messengers to activate STING and thus amplify antiviral immunity. As elegantly described by Kranzusch, 2015, while Nematostella vectensis contains a cGAS homolog (nv-cGAS) that stimulates STING signaling in human cells, nv-cGAS does not respond to dsDNA in vitro, also likely due to the absence of the zinc-ribbon domain and a positively charged N terminus, which are also lacking in CG7194. Like the STING homolog in N. vectensis, the role of dmSTING in innate immunity is to sense CDNs in the absence of amplification via cGAS. It should be noted that Kranzusch showed that insect STING orthologs, including dmSTING, did not associate with CDNs in their assay. However, in these experiments, a full-length dmSTING construct was used, containing the hydrophobic N-terminal transmembrane domains, which may inhibit CDN binding when the protein is not in its natural in vivo state. Crystal structures of dmSTING may be needed to uncover its precise interactions with CDNs (Martin, 2018).

During infection in mammals, bacteria such as Chlamydia generate CDNs that activate the innate immune response in a STING-dependent and cGAS-independent manner. Listeria secretes c-di-AMPs to induce an IFN response that stimulates the STING pathway. Additionally, Listeria generates c-di-GMP during its life cycle , which may be secreted or released intracellularly upon bacterial lysis to directly activate STING. However, the activation of STING in mice and the subsequent induction of IFN did not have an effect on Listeria load in the animals. In fact, the production of IFN during Listeria infection in mice is deleterious to survival, as type I IFN and IRF3 knockout mice are resistant to Listeria infection, since IFN promotes lymphocyte apoptosis. Conversely, an innate immune response in Drosophila to Listeria infection that induces antimicrobial peptides reduces Listeria-induced mortality and bacterial replication. Protection is mediated in part by the induction of IMD-mediated antimicrobial peptides such as Attacin, Cecropin, and Listericin. In the current experiments, STING-mediated induction of Attacin, Cecropin, and Listericin was observed during Listeria infection that was associated with decreased mortality and bacterial replication. Additionally, increased dmSTING-mediated Relish activation was observed, which in addition to inducing the antimicrobial peptides Attacin, Cecropin, and Listericin also positively regulates Zip3 and spirit (Martin, 2018).

A proposed mechanism by which dmSTING leads to the induction of antimicrobial peptides through the NF-κB homolog, Relish, is bolstered by the fact that dmSTING is able to induce mammalian NF-κ. The results indicate that dmSTING functions upstream of the Drosophila NF-κB ortholog Relish and likely also upstream of IMD, since knockdown of Relish and IMD in dmSTING-overexpressing flies resulted in decreased antimicrobial peptide induction during Listeria infection. Functional genomics and epistasis analysis indicates that the loss of dmSTING results in the loss of IMD signaling suggesting that dmSTING aids in inducing a defense response through the IMD signaling pathway. As compared to hsSTING, dmSTING is lacking 31 amino acids from its C terminus. The CTT in mammalian STING, which contain multiple phosphorylation sites, may have evolved to control the IRF family of transcription factors, since mammalian STING variants lacking these regions are unable to activate IRF3 but retain an ability to activate NF-κB. Additionally, the mammalian STING CTT may repress NF-κB activity, since significantly reduced mammalian NF-κB activity was observed when the CTT was appended onto dmSTING. Future experiments to assess the ability of dmSTING to activate NF-κB in other non-mammalian and invertebrate species would provide insight into how the STING-NF-κB signaling axis evolved (Martin, 2018).

In addition to the classical Toll, IMD, JAK-STAT, and RNAi pathways of innate immunity in Drosophila, autophagy and apoptosis play major roles in the defense response to infection. However, previous reports suggest that the pathway through which each function to induce a host defense response differs. For example, when chromosomal DNA escapes apoptotic degradation, there is induction of the IMD pathway, but not the Toll pathway. However, in response to viral infection, an antiviral state is induced via hemocyte-mediated phagocytosis of virions and, to a minor extent, autophagy through Atg7, independent of the canonical Toll, IMD, and JAK-STAT pathways. With regards to Listeria infection, both the Toll and IMD pathways are important to combat infection, as well as autophagy. While both PGRP-LC and -LE induce antimicrobial peptides in response to monomeric PGN stimulation , only PGRP-LE controls autophagy during Listeria infection. Since mammalian STING contributes to autophagy, it would be prudent to test the role of dmSTING in autophagy (Martin, 2018).

Further understanding of STING function in Drosophila, and more importantly, how it functions during pathogenic infection, will have an important impact on how methods are developed to target STING for therapeutic intervention, particularly with regards to insect vector-borne diseases. Additionally, studies of dmSTING may help in uncovering evolutionarily conserved mechanisms of autoimmunity, since STING activity must be kept under tight control to prevent autoimmune disease in humans (Martin, 2018).

Dietary Supplementation of Aspirin Promotes Drosophila Defense against Viral Infection

Aspirin, also known as acetylsalicylic acid, is widely consumed as a pain reliever and an anti-inflammatory as well as anti-platelet agent. Recently, studies using the animal model of Drosophila demonstrated that the dietary supplementation of aspirin renovates age-onset intestinal dysfunction and delays organismal aging. Nevertheless, it remains probable that aspirin plays functional roles in other biological activities, for instance antiviral defense reactions. Intriguingly, this study observed that the replications of several types of viruses were drastically antagonized in Drosophila macrophage-like S2 cells with the addition of aspirin. Further in vivo experimental approaches illustrate that adult flies consuming aspirin harbor higher resistances to viral infections with respect to flies without aspirin treatment. Mechanistically, aspirin positively contributes to the Drosophila antiviral defense largely through mediating the STING (stimulator of interferon genes) but not the IMD (immune deficiency) signaling pathway. Collectively, these studies uncover a novel biological function of aspirin in modulating Drosophila antiviral immunity and provide theoretical bases for exploring new antiviral treatments in clinical trials (Kong, 2023).

This study conducted both in vitro and in vivo experimental approaches to identify the antiviral role of aspirin. Compelling evidence evidence is provided showing that the dietary supplementation of aspirin not only lowers the mortality rate of Drosophila upon the infection of specific viruses, but also prevents the viral loads in the host flies. Of note is that the loss of sting or rel reverses the advantageous assessment of aspirin in the fly antiviral defense, implying that aspirin benefits Drosophila antiviral immunity in a STING-NF-kappaB-dependent manner. Thus, these studies demonstrate the functional role of aspirin against viral infection in vivo. Aspirin has long been well-known for its anti-inflammation and anti-platelet function, and widely utilized in medical treatments. It was lately shown to harbor an antiviral function, but these studies were mostly conducted in vitro systems. As a result, the prior objective of this research was to explore the potential involvement of aspirin in modulating the host antiviral defense reaction in vivo. To address this issue, attention was paid to the insect Drosophila, for which it is possible to establish the model of dietary consumption of aspirin and illustrating the anti-aging role of aspirin. As expected, the dietary supplementation of aspirin significantly enhanced the expressions of antiviral effectors and the survival of flies upon viral infections. In this regard, these studies not only confirm the antiviral role of aspirin that was suggested in the previous literature, but also provide for the first-time in vivo evidence to support this notion. Of interest is that aspirin enables the antagonization of several types of viruses, making it possible to become a broad antiviral drug in clinical medication (Kong, 2023).

How does aspirin benefit the Drosophila antiviral defense? Recent findings illustrated that aspirin down-regulates the K63 (63rd lysine)-linked ubiquitination of the Imd protein, thereby contributing to gut immune homeostasis and epithelial function. Therefore whether or not aspirin is involved in the fly antiviral reaction via modulating Imd was explored. Unexpectedly, dietary aspirin supplementation still enhanced the antiviral immunity in the imd LOF mutates. These observations encouraged further examination of the antiviral role of aspirin in flies including dif, hop, ago2, atg7, or sting LOF mutants, where the known antiviral pathways (Toll, Jak-STAT, RNAi, autophagy, and STING-NF-kappaB, respectively) were blocked separately. Intriguingly, the STING-NF-κappaB signal turned out to be responsible for the antiviral activity of aspirin in adult flies. Nevertheless, knowledge regarding how aspirin modulates the STING-NF-κappaB signaling pathway is still lacking. Based upon experience and knowledge with regard to the molecular mechanism by which aspirin mediates protein ubiquitination, one mechanism that is proposed is that the ubiquitination modification of a (some) key factor(s) in the STING-NF-κappaB signaling cascades is (are) regulated by aspirin (Kong, 2023).

Another question that needs to be addressed is that on which tissue/organ aspirin exerts its antiviral effect. Since experimental flies were raised with the dietary supplementation of aspirin, it is probable that aspirin functions in the digestive system to enhance fly metabolism, thereby contributing to the host antiviral response. In agreement with this, a previous transcriptomic analysis showed that aspirin can alter the expression of a series of genes involved in the metabolic pathway. On the other hand, aspirin may circulate into fat body (the main immune tissue/organ during systemic infection), where it upregulates STING-NF-kappaB activity. It would thus be worthwhile in the future to explore (1) the potential regulatory relationship between aspirin and the Sting protein; (2) the antiviral function of aspirin in a tissue/organ-specific manner, for instance with the use of flies with the gut or fat body-specific RNAi of sting (Kong, 2023).

These studies uncover the functional involvement of aspirin in the fly defense against several types of viruses. Even though the genetic evidence implies an intersection between aspirin and the STING-NF-κappaB signaling pathway, the underlying molecular mechanism is still not fully addressed. Further experimental approaches at the levels of molecular biology and biochemistry are required to identify the substrate of aspirin and to illustrate how aspirin modulates the Drosophila STING-NF-kappaB signaling cascades (Kong, 2023).

Verloren negatively regulates the expression of IMD pathway dependent antimicrobial peptides in Drosophila

Drosophila immune deficiency (IMD) pathway is similar to the human tumor necrosis factor receptor (TNFR) signaling pathway and is preferentially activated by Gram-negative bacterial infection. Recent studies highlighted the importance of IMD pathway regulation as it is tightly controlled by numbers of negative regulators at multiple levels. This study reports a new negative regulator of the IMD pathway, Verloren (Velo). Silencing of Velo led to constitutive expression of the IMD pathway dependent antimicrobial peptides (AMPs), and Escherichia coli stimulation further enhanced the AMP expression. Epistatic analysis indicated that Velo knock-down mediated AMP upregulation is dependent on the canonical members of the IMD pathway. The immune fluorescent study using overexpression constructs revealed that Velo resides both in the nucleus and cytoplasm, but the majority (~ 75%) is localized in the nucleus. It was also observed from in vivo analysis that Velo knock-down flies exhibit significant upregulation of the AMP expression and reduced bacterial load. Survival experiments showed that Velo knock-down flies have a short lifespan and are susceptible to the infection of pathogenic Gram-negative bacteria, P. aeruginosa. Taken together, these data suggest that Velo is an additional new negative regulator of the IMD pathway, possibly acting in both the nucleus and cytoplasm (Prakash, 2021).

Disparate regulation of IMD signaling drives sex differences in infection pathology in Drosophila melanogaster

Male and female animals exhibit differences in infection outcomes. One possible source of sexually dimorphic immunity is the sex-specific costs of immune activity or pathology, but little is known about the independent effects of immune- versus microbe-induced pathology and whether these may differ for the sexes. By measuring metabolic and physiological outputs in Drosophila melanogaster with wild-type and mutant immune responses, this study tested whether the sexes are differentially impacted by these various sources of pathology and identified a critical regulator of this difference. The sexes exhibit differential immune activity but similar bacteria-derived metabolic pathology. Female-specific immune-inducible expression of PGRP-LB, a negative regulator of the immune deficiency (IMD) pathway, enables females to reduce immune activity in response to reductions in bacterial numbers. In the absence of PGRP-LB, females are more resistant to infection, confirming the functional importance of this regulation and suggesting that female-biased immune restriction comes at a cost (Vincent, 2021).

Bap180/Baf180 is required to maintain homeostasis of intestinal innate immune response in Drosophila and mice

Immune homeostasis is a prerequisite to protective immunity against gastrointestinal infections. In Drosophila, immune deficiency (IMD) signalling (tumour necrosis factor receptor/interleukin-1 receptor, TNFR/IL-1R in mammals) is indispensable for intestinal immunity against invading bacteria. However, how this local antimicrobial immune response contributes to inflammatory regulation remains poorly defined. This study shows that flies lacking intestinal Bap180 (a subunit of the chromatin-remodelling switch/sucrose non-fermentable (SWI/SNF) complex) are susceptible to infection as a result of hyper-inflammation rather than bacterial overload. Detailed analysis shows that Bap180 is induced by the IMD-Relish response to both enteropathogenic and commensal bacteria. Upregulated Bap180 can feed back to restrain overreactive IMD signalling, as well as to repress the expression of the pro-inflammatory gene eiger (TNF), a critical step to prevent excessive tissue damage and elongate the lifespan of flies, under pathological and physiological conditions, respectively. Furthermore, intestinal targeting of Baf180 renders mice susceptible to a more aggressive infectious colitis caused by Citrobacter rodentium. Together, Bap180 and Baf180 serve as a conserved transcriptional repressor that is critical for the maintenance of innate immune homeostasis in the intestines (He, 2017).

The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila

From a forward genetic screen for phagocytosis mutants in Drosophila, a mutation was identified that affects peptidoglycan recognition protein (PGRP) SC1a and impairs the ability to phagocytose the bacteria Staphylococcus aureus, but not Escherichia coli and Bacillus subtilis. Because of the differences in peptidoglycan peptide linkages in these bacteria, the data suggest that the Drosophila gene PGRP-SC1a is necessary for recognition of the Lys-type peptidoglycan typical of most Gram⁺ bacteria. PGRP-SC1a mutants also fail to activate the Toll/NF-kappaB signaling pathway and are compromised for survival after S. aureus infection. This mutant phenotype is the first found for an N-acetylmuramoyl-L-alanine amidase PGRP that cleaves peptidoglycan at the lactylamide bond between the glycan backbone and the crosslinking stem peptides. By generating transgenic rescue flies that express either wild-type or a noncatalytic cysteine-serine mutant PGRP-SC1a, it was found that PGRP-SC1a amidase activity is not necessary for Toll signaling, but is essential for uptake of S. aureus into the host phagocytes and for survival after S. aureus infection. Furthermore, the PGRP-SC1a amidase activity can be substituted by exogenous addition of free peptidoglycan, suggesting that the presence of peptidoglycan cleavage products is more important than the generation of cleaved peptidoglycan on the bacterial surface for PGRP-SC1a mediated phagocytosis (Garver, 2006).

Host receptors must recognize a microbe before any immune response can begin. After recognition, signaling pathways are activated and result in effector responses such as induction of antimicrobial peptides (AMPs), melanization, and phagocytosis, which are important for controlling the infection. Much is known about the signaling pathways important for the AMP response in Drosophila, but less is known about the regulation of the other two responses. In Drosophila, if phagocytosis is blocked in a mutant that is already impaired for the AMP response, a normally innocuous Escherichia coli infection becomes lethal. Phagocytosis is also likely to be a first line of defense, because the response involves specific interaction between microbe and host receptors and occurs within half an hour, whereas the induction of AMPs occurs over several hours and AMPs can act on diverse groups of microbes (Garver, 2006).

The peptidoglycan recognition proteins (PGRPs) are critical receptors in the Drosophila immune response that are required for the recognition of peptidoglycan, a component of bacterial cell walls (Dziarski, 2004; Steiner, 2004), and for subsequent activation of AMP gene expression (Choe, 2002; Werner, 2003). PGRPs were first characterized in the moths Bombyx mori and Trichoplusia ni and proposed to be receptors that can trigger immune responses. PGRPs have also been identified in mammals, and mutant PGRP mice have been generated, but the most comprehensive characterization of PGRPs has been performed in Drosophila (Garver, 2006).

Drosophila has 13 PGRP genes, six long (L) forms with four that are predicted to reside in the plasma membrane, and seven short forms (S) that are all predicted to be secreted. PGRPs share homology with N-acetylmuramoyl-L-alanine amidases, which cleave peptidoglycan at the lactylamide bond between the glycan backbone and the stem peptides. Some PGRPs, such as PGRP-LC, -LE -SA, and -SD, lack a critical cysteine in the catalytic pocket and are not able to cleave peptidoglycan. PGRP-LC, -LE, and -SA have been demonstrated to bind peptidoglycan and are necessary for expression of AMP genes, supporting the hypothesis that PGRPs directly recognize bacteria and activate immune responses. The identification of mutations in PGRP-SA (seml) and PGRP-LC (ird7 or totem) indicated that these genes are necessary for activation of the two signaling pathways regulating AMP gene expression, the Toll pathway that responds to Gram⁺ bacteria and fungi and the Imd pathway that responds to Gram^– bacteria. Drosophila uses PGRP-SA and PGRP-LC to distinguish between Gram⁺ and Gram^– PGN for activation of the Toll and Imd signaling pathways (Leulier, 2003). Gram⁺ PGN and Gram^– PGN differ in the stem peptide portion; typical Gram⁺ bacteria have a lysine as the third amino acid, whereas Gram^– bacteria and the Gram⁺ Bacillus have a diaminopimelic acid (DAP) in that position. Two other noncatalytic PGRPs, PGRP-LE and PGRP-SD, also play a role in activation of the Imd and Toll pathways, respectively. PGRP-LC, PGRP-LE double mutants show a more dramatic phenotype to Bacillus and Gram^– bacterial infection than either mutation alone, suggesting that PGRP-LE acts with PGRP-LC in the recognition of Gram^– DAP-type peptidoglycan for the activation of the Imd pathway. PGRP-SD may be playing a similar role with PGRP-SA for activation of the Gram⁺/Toll pathway (Garver, 2006 and references therein).

Catalytic PGRPs, such as PGRP-SC1a and -SC1b, include this cysteine residue in the active site, and are potent enzymes that cleave peptidoglycan between the N-acetylmuramic acid of the backbone and the L-alanine in the stem peptide. After digestion with PGRP-SC1b, staphylococcal peptidoglycan exhibits less activation of the AMP genes in a Drosophila blood cell line, so it was hypothesized that catalytic PGRPs may act as scavengers to limit an inflammatory response to free peptidoglycan (Mellroth, 2003). This may not be absolute, since PGRP-SC1b-digested Gram^– peptidoglycan is still able to activate the AMP response in S2 cells, albeit at a higher dose. However, it was of interest to examine the role of these genes in vivo (Garver, 2006).

In a genetic screen for phagocytosis mutants, a novel mutant, picky was identified. picky flies fail to phagocytose S. aureus particles but can phagocytose E. coli, Bacillus subtilis, and Saccharomyces cerevisiae zymosan particles. picky mutants have defects in the activation of the Toll pathway. picky maps to the location of the PGRP-SC1a, -SC1b, and -SC2 genes, and picky flies express significantly less PGRP-SC1a. The picky defects can be rescued by expression of PGRP-SC1a, indicating that PGRP-SC1a is important for phagocytosis and activation of AMP responses. These results differ from the prevailing model of catalytic PGRPs as scavengers and suggest that in vivo, PGRP-SC1a is required for initiating immune pathways. By comparing a wild-type PGRP-SC1a with a cysteine-serine mutant PGRP-SC1a for rescue of picky phenotypes, it was found that the catalytic activity is essential for phagocytosis of live S. aureus, but not for activation of the Toll pathway (Garver, 2006).

To identify genes important for phagocytosis, a collection of ethylmethane sulfonate-induced adult viable Drosophila mutants was screened using an in vivo phagocytosis assay. To determine what a mutant might look like, the PGRP-LC (ird7) and PGRP-SA (seml) mutants were examined. PGRP-LC has been reported to be a recognition receptor for phagocytosis of E. coli in S2 cells. However, this study contradicted another report citing that ird7 blood cells can phagocytose bacteria (Choe, 2002). The current study found that ird7 mutants are able to phagocytose both E. coli and S. aureus particles. This finding suggested that phagocytosis of Gram^– bacteria may require other receptors in addition to PGRP-LC in vivo. In contrast, it was found that seml mutants are specifically impaired in their ability to phagocytose S. aureus but not E. coli. 94% of the seml mutant flies were able to phagocytose E. coli, but only 25% were able to phagocytose S. aureus. This finding suggests that PGRP-SA may be important for recognition of Gram⁺ bacteria for phagocytosis in addition to its role in activating the Toll pathway. In contrast, flies with mutations affecting other Toll pathway components, spatzle (the Toll ligand) and Dif (an NF-kappaB) were still able to phagocytose S. aureus, indicating that the phagocytosis defect is not a secondary effect from loss of Toll signaling (Garver, 2006).

From a pilot screen, one mutation was identified that was named picky eater^Z2–4761 (picky) because the mutants fail to phagocytose S. aureus particles, but can still phagocytose E. coli and Saccharomyces cerevisiae zymosan particles. Only 25% of picky mutant flies showed any phagocytosis response to S. aureus, but 83% of those same flies were still able to phagocytose E. coli. To investigate whether the recognition involved the difference in peptidoglycan between Gram⁺ and Gram^– bacteria, phagocytosis was examined of a B. subtilis-GFP strain. picky mutants are able to phagocytose B. subtilis. This finding suggests that the picky gene product is important for the specific recognition of the Lys-type peptidoglycans, typical of most Gram⁺ bacteria, but not found in Bacillus spp (Garver, 2006).

Because picky flies appear to be impaired in the recognition of S. aureus for phagocytosis and survival, it was possible that the mutation might also affect activation of the Toll or Imd pathways. The induction of Drosomycin and Diptericin AMP genes is often used to assess activation of the Toll/Gram⁺ and Imd/Gram^– signaling pathways, respectively. The AMP responses in picky were compared with those of the known Toll pathway components, seml and Dif, and to an Imd pathway component, ird7. picky flies showed no induction of Drosomycin in response to S. aureus, E. coli, or Micrococcus luteus at either 2 or 24 h. All of the S. aureus-infected picky flies were dead at 24 h, so the Drosomycin expression in this sample was not assessed. seml and Dif both show some induction of Drosomycin at 2 and 24 h. In comparison, picky has a more consistent and dramatic effect on Drosomycin expression and likely encodes an essential component of the Toll pathway. In contrast, the expression of Diptericin in response to bacterial infection in picky mutants was higher than that seen in wild type. Therefore, the defect in picky appears to be selective for the Toll pathway. To determine where in the Toll pathway picky might lie, the picky mutation was crossed into a Toll^10b gain of function mutant. picky was not able to suppress the constitutive expression of Drosomycin in a Toll^10b mutant. This epistasis analysis places picky either upstream of Toll, which would be consistent with a role in bacterial recognition or in a parallel pathway (Garver, 2006).

The peptidoglycan recognition protein family is important for allowing the Drosophila immune response to distinguish between Gram⁺ and Gram^– bacteria for the activation of specific AMP signaling pathways (Choe, 2002; Werner, 2003). The current data indicate that phagocytosis also relies on PGRPs to distinguish between Gram⁺ and Gram^– bacterial peptidoglycan. A catalytic PGRP, PGRP-SC1a, is essential for the phagocytosis of S. aureus and the activation of AMP responses. The fact that PGRP-SC1a mutants have a striking phagocytosis defect indicates that it is absolutely required and that other PGRPs are not able to substitute for its loss of function. PGRP-SC1a differs from the existing PGRP mutants in that it has catalytic activity that is essential for efficient phagocytosis and ultimately limiting the infection process. Hence, noncatalytic PGRPs, such as PGRP-SA, may not be able to substitute for PGRP-SC1a function. Because PGRP-SC1a is likely present in the hemolymph, it may be acting as an opsonin to bind bacteria, with the PGRP-bacterial complex afterwards being recognized by transmembrane phagocytosis receptors that complete the uptake into the phagocyte. An alternative model is that PGRP-SC1a may generate peptidoglycan cleavage products that function as immune modulators to stimulate phagocytic activity (Garver, 2006).

For S. aureus, PGRP-SC1a is necessary for recognition of bacteria for both the phagocytosis and AMP responses. However, for E. coli, PGRP-SC1a is similar to PGRP-LC in that it is not necessary for recognition for phagocytosis, but is necessary for the activation of an AMP response. This finding raises the interesting issue of the role PGRPs play in recognizing Gram^– bacteria. In Gram^– bacteria, the peptidoglycan is buried under an outer membrane, so it has been proposed that the small amounts of PGN that are shed may be sufficient to trigger the AMP response (Leulier, 2003). Because the cell wall PGN is not easily accessible, it makes sense that the PGRPs are not playing a direct role in recognition for phagocytosis. An alternate possibility is that the exposure of bacterial peptidoglycan occurs after the initial uptake into phagocytes, and at that later point, is then recognized by PGRP-LC and PGRP-SC1a to trigger AMP responses. There is precedence for intracellular recognition in the phagosome in mammalian immune responses, with studies indicating that mammalian short PGRPs function in the phagosome to inhibit bacterial growth and the observation that Toll-like receptor recognition of bacteria in the phagosome can influence the rate of phagosome maturation. A related possibility is that some basal level of activation of PGRP-SC1a and the Toll pathway may be required for any expression of Drosomycin. picky mutants show much lower expression of Drosomycin than wild type in the absence of infection, so perhaps an induction may not be detectable (Garver, 2006).

It was also found that seml mutants are defective in the phagocytosis of S. aureus but not E. coli. Recent reports have indicated that the processing of peptidoglycan into monomers, lactyltetrapeptides, or muropeptides, can yield potent activators of the AMP response for both the Imd and the Toll pathway. It was further suggested for the Toll pathway that processing of peptidoglycan may be a prerequisite step upstream of PGRP-SA/seml recognition and activation of the Toll AMP pathway. This model would be consistent with catalytic PGRPs, such as PGRP-SC1a, playing a supporting role in the processing of peptidoglycan for initiating recognition events (Garver, 2006).

Since many PGRPs are clearly required for activation of immune effector responses, the issue arises as to the specificity of PGRPs for their substrates. Work from several groups indicates that alternative splice variants (in PGRP-LC) or small differences in amino acid sequences (from PGRP-LB and human PGRP-Ialpha structural analyses) may result in the ability of different PGRPs to have distinct recognition potential. It will be interesting to explore the range of bacterial types recognized by the 17 Drosophila PGRP proteins and to determine how subtle differences in recognition may be reflected by specific amino acid sequences. There may also be genetic interactions or antagonism in their function, so using the genetic tools available in Drosophila may be necessary for ultimately determining both their unique and redundant roles (Garver, 2006).

This work demonstrates the utility of a forward genetic approach to understanding the recognition of pathogen types for phagocytosis and activation of AMP responses. It is hoped that identification of genes important for recognition of bacteria in Drosophila should increase understanding of how the process works the human immune system (Garver, 2006).

Beneficial commensal bacteria promote Drosophila growth by downregulating the expression of peptidoglycan recognition proteins

Commensal bacteria are known to promote host growth. Such effect partly relies on the capacity of microbes to regulate the host's transcriptional response. However, these evidences mainly come from comparing the transcriptional response caused by commensal bacteria with that of axenic animals, making it difficult to identify the animal genes that are specifically regulated by beneficial microbes. This study employed Drosophila melanogaster associated with Lactiplantibacillus plantarum to understand the host genetic pathways regulated by beneficial bacteria and leading to improved host growth. It was shown that microbial benefit to the host relies on the downregulation of peptidoglycan-recognition proteins. Specifically, it is reported that bacterial proliferation triggers the lower expression of PGRP-SC1 in larval midgut, which ultimately leads to improved host growth and development. This study helps elucidate the mechanisms underlying the beneficial effect exerted by commensal bacteria, defining the role of immune effectors in the relationship between Drosophila and its gut microbes (Gallo, 2022).

Toll receptor-mediated Hippo signaling controls innate immunity in Drosophila

The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. This study reports an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. These results elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings (Liu, 2016).

MicroRNAs that contribute to coordinating the immune response in Drosophila melanogaster

Small noncoding RNAs called microRNAs (miRNAs) have emerged as post-transcriptional regulators of gene expression related to host defences. This study used Drosophila melanogaster to explore the contribution of individual or clusters of miRNAs in countering systemic C. albicans infection. From a total of 72 tested, six miRNAs allelic mutant backgrounds were identified that modulate the survival response to infection and ability to control pathogen number. These mutants also exhibit dysregulation of the Toll pathway target transcripts Drosomycin (Drs) and Immune-Induced Molecule 1 (IM1). These are characteristics of defects in Toll signalling, and consistent with this, dependency for one of the miRNA mutants on the NF-κB homologue Dif was demonstrated. Changes were quantified in the miRNA expression profile over time in response to three pathogen types, and 13 mature miRNA forms affected by pathogens were identified that stimulate Toll signalling. To complement this, a genome-wide map is provided of potential NF-κB sites in proximity to miRNA genes. Finally, systemic C. albicans infection was demonstrated to contribute to a reduction in the total amount of Branch-Chained Amino Acids, which is miRNA-regulated. Overall, these data reveal a new layer of miRNA complexity regulating the fly response to systemic fungal infection (Atilano, 2017).

Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps

Members of the thioester-containing protein (TEP) family contribute to host defence in both insects and mammals. However, their role in the immune response of Drosophila is elusive. This study addresses the role of TEPs in Drosophila immunity by generating a mutant fly line, referred to as TEPq Delta , lacking the four immune-inducible TEPs, TEP1, 2, 3 and 4. Survival analyses with TEPq Delta flies reveal the importance of these proteins in defence against entomopathogenic fungi, Gram-positive bacteria and parasitoid wasps. These results confirm that TEPs are required for efficient phagocytosis of bacteria, notably for the two Gram-positive species tested, Staphylococcus aureus and Enterococcus faecalis. Furthermore, TEPq Delta flies have reduced Toll pathway activation upon microbial infection, resulting in lower expression of antimicrobial peptide genes. Epistatic analyses suggest that TEPs function upstream or independently of the serine protease ModSP at an initial stage of Toll pathway activation. Collectively, this study brings new insights into the role of TEPs in insect immunity. It reveals that TEPs participate in both humoral and cellular arms of immune response in Drosophila. In particular, it shows the importance of TEPs in defence against Gram-positive bacteria and entomopathogenic fungi, notably by promoting Toll pathway activation (Dostalova, 2017).

The Toll pathway underlies host sexual dimorphism in resistance to both Gram-negative and Gram-positive bacteria in mated Drosophila

This study used Drosophila melanogaster to assess and dissect sexual dimorphism in the innate response to systemic bacterial infection. Both virgin and mated females are more susceptible than mated males to most, but not all, infections. The lower resistance of females to infection with Providencia rettgeri, a Gram-negative bacterium that naturally infects D. melanogaster was investigated. Females were found to have a higher number of phagocytes than males, and ablation of hemocytes does not eliminate the dimorphism in resistance to P. rettgeri, so the observed dimorphism does not stem from differences in the cellular response. The Imd pathway is critical for the production of antimicrobial peptides in response to Gram-negative bacteria, but mutants for Imd signaling continued to exhibit dimorphism even though both sexes showed strongly reduced resistance. Instead, it was found that the Toll pathway is responsible for the dimorphism in resistance. The Toll pathway is dimorphic in genome-wide constitutive gene expression and in induced response to infection. Toll signaling is dimorphic in both constitutive signaling and in induced activation in response to P. rettgeri infection. The dimorphism in pathway activation can be specifically attributed to Persephone-mediated immune stimulation, by which the Toll pathway is triggered in response to pathogen-derived virulence factors. It was additionally found that, in absence of Toll signaling, males become more susceptible than females to the Gram-positive Enterococcus faecalis. This reversal in susceptibility between male and female Toll pathway mutants compared to wildtype hosts highlights the key role of the Toll pathway in D. melanogaster sexual dimorphism in resistance to infection. Altogether, these data demonstrate that Toll pathway activity differs between male and female D. melanogaster in response to bacterial infection (Duneau, 2017).

The serine protease homolog spheroide is involved in sensing of pathogenic Gram-positive bacteria

In Drosophila, recognition of pathogens such as Gram-positive bacteria and fungi triggers the activation of proteolytic cascades and the subsequent activation of the Toll pathway. This response can be achieved by either detection of pathogen associated molecular patterns or by sensing microbial proteolytic activities ("danger signals"). Previous data suggested that certain serine protease homologs (serine protease folds that lack an active catalytic triad) could be involved in the pathway. This study generated a null mutant of the serine protease homolog spheroide (sphe). These mutant flies are susceptible to Enterococcus faecalis infection and unable to fully activate the Toll pathway. Sphe is required to activate the Toll pathway after challenge with pathogenic Gram-Positive bacteria. Sphe functions in the danger signal pathway, downstream or at the level of Persephone (Patrnogic, 2017).

The circulating protease Persephone is an immune sensor for microbial proteolytic activities upstream of the Drosophila toll pathway

Microbial or endogenous molecular patterns as well as pathogen functional features can activate innate immune systems. Whereas detection of infection by pattern recognition receptors has been investigated in details, sensing of virulence factors activities remains less characterized. In Drosophila, genetic evidences indicate that the serine protease Persephone belongs to a danger pathway activated by abnormal proteolytic activities to induce Toll signaling. However, neither the activation mechanism of this pathway nor its specificity has been determined. This study identified a unique region in the pro-domain of Persephone that functions as bait for exogenous proteases independently of their origin, type, or specificity. Cleavage in this bait region constitutes the first step of a sequential activation and licenses the subsequent maturation of Persephone to the endogenous cysteine cathepsin 26-29-p. These results establish Persephone itself as an immune receptor able to sense a broad range of microbes through virulence factor activities rather than molecular patterns (Issa, 2018).

Short-Form Bomanins mediate humoral immunity in Drosophila

The Bomanins are a family of a dozen secreted peptides that mediate the innate response governed by the Drosophila Toll receptor. It was recently shown that deleting a cluster of 10 Bom genes blocks Toll-mediated defenses against a range of fungi and gram-positive bacteria. This study characterize the activity of individual Bom family members. Evidence is provided that the Boms overlap in function and that a single Bom gene encoding a mature peptide of just 16 amino acids can act largely or entirely independent of other family members to provide phenotypic rescue in vivo. It was further demonstrate that the Boms function in Drosophila humoral immunity, mediating the killing of the fungal pathogen Candida glabrata in an in vitro assay of cell-free hemolymph. In addition, the level of antifungal activity both in vivo and in vitro were found to be linked to the level of Bom gene expression. Although Toll dictates expression of the antimicrobial peptides (AMPs) Drosomycin and Metchnikowin, no evidence was found that Boms act by modifying the expression of the mature forms of these antifungal AMPs (Lindsay, 2018).

Dual comprehensive approach to decipher the Drosophila Toll pathway, ex vivo RNAi screenings and immunoprecipitation-mass spectrometry

The Drosophila Toll pathway is involved in embryonic development, innate immunity, and cell-cell interactions. However, compared to the mammalian Toll-like receptor innate immune pathway, its intracellular signaling mechanisms are not fully understood. A series of ex vivo genome-wide RNAi screenings was performed to identify genes required for the activation of the Toll pathway. This study has conducted an additional genome-wide RNAi screening using the overexpression of Tube, an adapter molecule in the Toll pathway, and has performed a co-immunoprecipitation assay to identify components present in the dMyd88-Tube complex. Based on the results of these assays, a bioinformatic analysis was performed, and candidate molecules and post-translational modifications are described that could be involved in Drosophila Toll signaling (Kanoh, 2018).

HSP70/DNAJA3 chaperone/cochaperone regulates NF-kappaB activity in immune responses

Nuclear factor kappa B (NF-kappaB) controls the transcription of various genes in response to immune stimuli. A previous study revealed that the Droj2/DNAJA3 cochaperone contributes to the NF-kappaB pathway in Drosophila and humans. In general, the cochaperone is associated with the 70-kDa heat shock protein (HSP70) chaperone and the complex supports the folding of diverse target proteins. The cochaperone/chaperone functions in the NF-kappaB pathway, however, are not clearly understood. This study reports that HSP70 proteins are involved in activating canonical NF-kappaB signaling during immune responses. In human cultured cells, HSP70 inhibitor destabilized the IKKbeta/IkappaBalpha/NF-kappaB p65 complex and dampened the phosphorylation of NF-kappaB p65 in response to flagellin stimulation. HSPA1A and HSPA8 were identified as the HSP70 family proteins that physically interact with DNAJA3, and established their requirement for the phosphorylation of NF-kappaB p65. Furthermore, as in flies with knockdown of Droj2, flies with knockdown of Hsc70-4, a Drosophila homolog of HSPA8, were more susceptible to infection. These results suggest that the chaperone/cochaperone complex regulates NF-kappaB immune signaling in an evolutionarily conserved manner (Kumada, 2019).

More than black or white: Melanization and toll share regulatory serine proteases in Drosophila

The melanization response is an important defense mechanism in arthropods. This reaction is mediated by phenoloxidases (POs), which are activated by complex extracellular serine protease (SP) cascades. This study investigated the role of SPs in the melanization response using compound mutants in D. melanogaster; phenotypes were discovered that were previously concealed in single-mutant analyses. Two SPs, Hayan and Sp7, were found to activate the melanization response in different manners: Hayan is required for blackening wound sites, whereas Sp7 regulates an alternate melanization reaction responsible for the clearance of Staphylococcus aureus. Evidence is presented that Sp7 is regulated by SPs activating the Toll NF-kappaB pathway, namely ModSP and Grass. Additionally, a role was revealed for the combined action of Hayan and Psh in propagating Toll signaling downstream of pattern recognition receptors activating either Toll signaling or the melanization response (Dudzic, 2019).

The Daisho Peptides Mediate Drosophila Defense Against a Subset of Filamentous Fungi

Fungal infections, widespread throughout the world, affect a broad range of life forms, including agriculturally relevant plants, humans, and insects. In defending against fungal infections, the fruit fly Drosophila melanogaster employs the Toll pathway to induce a large number of immune peptides. Some have been investigated, such as the antimicrobial peptides (AMPs) and Bomanins (Boms); many, however, remain uncharacterized. This study examined the role in innate immunity of two related peptides, Daisho1 and Daisho2 (formerly IM4 and IM14, respectively), found in hemolymph following Toll pathway activation. By generating a CRISPR/Cas9 knockout of both genes, Deltadaisho, this study found that the Daisho peptides are required for defense against a subset of filamentous fungi, including Fusarium oxysporum, but not other Toll-inducible pathogens, such as Enterococcus faecalis and Candida glabrata. Analysis of null alleles and transgenes revealed that the two daisho genes are each required for defense, although their functions partially overlap. Generating and assaying a genomic epitope-tagged Daisho2 construct, interaction was detected in vitro of Daisho2 peptide in hemolymph with the hyphae of F. oxysporum. Together, these results identify the Daisho peptides as a new class of innate immune effectors with humoral activity against a select set of filamentous fungi (Cohen, 2020).

Delayed host mortality and immune response upon infection with P. aeruginosa persister cells

Chronic infections are a heavy burden on healthcare systems worldwide. Persister cells are thought to be largely responsible for chronic infection due to their tolerance to antimicrobials and recalcitrance to innate immunity factors. Pseudomonas aeruginosa is a common and clinically relevant pathogen that contains stereotypical persister cells. Despite their importance in chronic infection, there have been limited efforts to study persister cell infections in vivo. Drosophila melanogaster has a well-described innate immune response similar to that of vertebrates and is a good candidate for the development of an in vivo model of infection for persister cells. Similar to what is observed in other bacterial strains, in this work it was found that infection with P. aeruginosa persister cells resulted in a delayed mortality phenotype in Caenorhabditis elegans, Arabidopsis thaliana, and D. melanogaster compared to infection with regular cells. An in-depth characterization of infected D. melanogaster found that bacterial loads differed between persister and regular cells' infections during the early stages. Furthermore, hemocyte activation and antimicrobial peptide expression were delayed/reduced in persister infections over the same time course, indicating an initial suppression of, or inability to elicit, the fly immune response. Overall, these findings support the use of D. melanogaster as a model in which to study persister cells in vivo, where this bacterial subpopulation exhibits delayed virulence and an attenuated immune response (Hastings, 2023).

Intramacrophage ROS Primes the Innate Immune System via JAK/STAT and Toll Activation

Tissue injury is one of the most severe environmental perturbations for a living organism. When damage occurs in adult Drosophila, there is a local response of the injured tissue and a coordinated action across different tissues to help the organism overcome the deleterious effect of an injury. This study shows a change in the transcriptome of hemocytes at the site of tissue injury, with pronounced activation of the Toll signaling pathway. Induction of the cytokine upd-3 and Toll receptor activation occur in response to injury alone, in the absence of a pathogen. Intracellular accumulation of hydrogen peroxide in hemocytes is essential for upd-3 induction and is facilitated by the diffusion of hydrogen peroxide through a channel protein Prip. Importantly, hemocyte activation and production of reactive oxygen species (ROS) at the site of a sterile injury provide protection to flies on subsequent infection, demonstrating training of the innate immune system (Chakrabarti, 2020).

Differential Requirements for Mediator Complex Subunits in Drosophila melanogaster Host Defense Against Fungal and Bacterial Pathogens

The humoral immune response to bacterial or fungal infections in Drosophila relies largely on a transcriptional response mediated by the Toll and Immune deficiency NF-κB pathways. Antimicrobial peptides are potent effectors of these pathways and allow the organism to attack invading pathogens. Dorsal-related Immune Factor (DIF), a transcription factor regulated by the Toll pathway, is required in the host defense against fungal and some Gram-positive bacterial infections. The Mediator complex is involved in the initiation of transcription of most RNA polymerase B (PolB)-dependent genes by forming a functional bridge between transcription factors bound to enhancer regions and the gene promoter region and then recruiting the PolB pre-initiation complex. Mediator is formed by several modules that each comprises several subunits. The Med17 subunit of the head module of Mediator has been shown to be required for the expression of Drosomycin, which encodes a potent antifungal peptide, by binding to DIF. Thus, Mediator is expected to mediate the host defense against pathogens controlled by the Toll pathway-dependent innate immune response. This study first focused on the Med31 subunit of the middle module of Mediator and find that it is required in host defense against Aspergillus fumigatus, Enterococcus faecalis, and injected but not topically-applied Metarhizium robertsii. Thus, host defense against M. robertsii requires Dif but not necessarily Med31 in the two distinct infection models. The induction of some Toll-pathway-dependent genes is decreased after a challenge of Med31 RNAi-silenced flies with either A. fumigatus or E. faecalis, while these flies exhibit normal phagocytosis and melanization. This study further tested most Mediator subunits using RNAi by monitoring their survival after challenges to several other microbial infections known to be fought off through DIF. The host defense against specific pathogens involves a distinct set of Mediator subunits with only one subunit for C. glabrata or Erwinia carotovora carotovora, at least one for M. robertsii or a somewhat extended repertoire for A. fumigatus (at least eight subunits) and E. faecalis (eight subunits), with two subunits, Med6 and Med11 being required only against A. fumigatus. Med31 but not Med17 is required in fighting off injected M. robertsii conidia. Thus, the involvement of Mediator in Drosophila innate immunity is more complex than expected (Huang, 2021).

Increasing adult density compromises survival following bacterial infections in Drosophila melanogaster

The density-dependent prophylaxis hypothesis predicts that risk of pathogen transmission increases with increase in population density, and in response to this, organisms mount a prophylactic immune response when exposed to high density. This prophylactic response is expected to help organisms improve their chances of survival when exposed to pathogens. Alternatively, organisms living at high densities can exhibit compromised defense against pathogens due to lack of resources and density associated physiological stress; the crowding stress hypothesis. This study housed adult Drosophila melanogaster flies at different densities and measured the effect this has on their post-infection survival and resistance to starvation. Flies housed at higher densities show greater mortality after being infected with bacterial pathogens, while also exhibiting increased resistance to starvation. These results are more in line with the density-stress hypothesis that postulates a compromised immune system when hosts are subjected to high densities (Das, 2022).

Enteric bacterial infection in Drosophila induces whole-body alterations in metabolic gene expression independently of the immune deficiency signaling pathway

When infected by intestinal pathogenic bacteria, animals initiate both local and systemic defence responses. These responses are required to reduce pathogen burden and also to alter host physiology and behavior to promote infection tolerance, and they are often mediated through alterations in host gene expression. This study used transcriptome profiling to examine gene expression changes induced by enteric infection with the Gram-negative bacteria Pseudomonas entomophila in adult female Drosophila. Infection was found to induce a strong upregulation of metabolic gene expression, including gut and fat body-enriched genes involved in lipid transport, lipolysis, and beta-oxidation, as well as glucose and amino acid metabolism genes. Furthermore, the classic innate immune deficiency (Imd)/Relish/NF-KappaB pathway were not required for, and in some cases limits, these infection-mediated increases in metabolic gene expression. Enteric infection with Pseudomonas entomophila downregulates the expression of many transcription factors and cell-cell signaling molecules, particularly those previously shown to be involved in gut-to-brain and neuronal signaling. Moreover, as with the metabolic genes, these changes occurred largely independent of the Imd pathway. Together, this study identifies many metabolic, signaling, and transcription factor gene expression changes that may contribute to organismal physiological and behavioral responses to enteric pathogen infection (Deshpande, 2022).

Drosophila immune priming to Enterococcus faecalis relies on immune tolerance rather than resistance

Innate immune priming increases an organism's survival of a second infection after an initial, non-lethal infection. Drosophila melanogaster and an insect-derived strain of Enterococcus faecalis were used to study transcriptional control of priming. In contrast to other pathogens, the enhanced survival in primed animals does not correlate with decreased E. faecalis load. Further analysis shows that primed organisms tolerate, rather than resist infection. Using RNA-seq of immune tissues, many genes were found to be upregulated in only primed flies, suggesting a distinct transcriptional program in response to initial and secondary infections. In contrast, few genes continuously express throughout the experiment or more efficiently re-activate upon reinfection. Priming experiments in immune deficient mutants revealed Imd is largely dispensable for responding to a single infection but needed to fully prime. Together, this indicates the fly's innate immune response is plastic-differing in immune strategy, transcriptional program, and pathway use depending on infection history (Cabrera, 2023).

Role of Rab5 early endosomes in regulating Drosophila gut antibacterial response

Interactions between prokaryotes and eukaryotes require a dialogue between microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs). In Drosophila, bacterial peptidoglycan is detected by PGRP receptors. While the components of the signaling cascades activated upon PGN/PGRP interactions are well characterized, little is known about the subcellular events that translate these early signaling steps into target gene transcription. Using a Drosophila enteric infection model, this study showed that gut-associated bacteria can induce the formation of intracellular PGRP-LE aggregates which colocalized with the early endosome marker Rab5. Combining microscopic and RNA-seq analysis, it was demonstrated that RNAi inactivation of the endocytosis pathway in the Drosophila gut affects the expression of essential regulators of the NF-κB response leading not only to a disruption of the immune response locally in the gut but also at the systemic level. This work sheds new light on the involvement of the endocytosis pathway in the control of the gut response to intestinal bacterial infection (Joshi, 2023).

Protein Restriction Effects on Healthspan and Lifespan in Drosophila melanogaster Are Additive With a Longevity-Promoting Diet

Aging of the organism is associated diminished response to external stimuli including weakened immune function, resulting in diseases that impair health and lifespan. Several dietary restriction modalities have been reported to improve health and lifespan in different animal models, but it is unknown whether any of the lifespan-extending dietary treatments could be combined to achieve an additive effect. This study investigated the effects of halving amino acids components in the HUNTaa diet, a synthetic medium known to extend lifespan in Drosophila. Dietary restriction by halving the entire amino acid components (DR group) could further extend lifespan and improve resistance to oxidative stress, desiccation stress, and starvation than flies on HUNTaa diet alone (wt group). Transcriptome analysis of Drosophila at 40, 60, and 80 days of age revealed that genes related to cell proliferation and metabolism decreased with age in the wt group, whereas background stimulus response and amino acid metabolism increased with age. However, these trends differed in the DR group, that is, the DR flies had downregulated stress response genes, including reduced background immune activation. Infection experiments demonstrated that these flies survived longer after feeding infection with Serratia marcescens and Enterococcus faecalis, suggesting that these flies had stronger immune function, and therefore reduced immune senescence. These results demonstrated that halving the entire amino acid components in the HUNTaa diet further extended health and lifespan and suggested that lifespan-extending diet and dietary restriction treatment could be combined to achieve additive beneficial results (Zhang, 2023).

Evolution of Toll, Spatzle and MyD88 in insects: the problem of the Diptera bias

Arthropoda, the most numerous and diverse metazoan phylum, has species in many habitats where they encounter various microorganisms and, as a result, mechanisms for pathogen recognition and elimination have evolved. The Toll pathway, involved in the innate immune system, was first described as part of the developmental pathway for dorsal-ventral differentiation in Drosophila. This study evaluated the diversity of Toll pathway gene families in 39 Arthropod genomes, encompassing 13 different Insect Orders. Through computational methods, this study sheds some light into the evolution and functional annotation of protein families involved in the Toll pathway innate immune response. The data indicates that: 1) intracellular proteins of the Toll pathway show mostly species-specific expansions; 2) the different Toll subfamilies seem to have distinct evolutionary backgrounds; 3) patterns of gene expansion observed in the Toll phylogenetic tree indicate that homology based methods of functional inference might not be accurate for some subfamilies; 4) Spatzle subfamilies are highly divergent and also pose a problem for homology based inference; 5) Spatzle subfamilies should not be analyzed together in the same phylogenetic framework; 6) network analyses seem to be a good first step in inferring functional groups in these cases. It was specifically shown that understanding Drosophila's Toll functions might not indicate the same function in other species. These results show the importance of using species representing the different orders to better understand insect gene content, origin and evolution. More specifically, in intracellular Toll pathway gene families the presence of orthologues has important implications for homology based functional inference. Also, the different evolutionary backgrounds of Toll gene subfamilies should be taken into consideration when functional studies are performed, especially for TOLL9, TOLL, TOLL2_7, and the new TOLL10 clade. The presence of Diptera specific clades or the ones lacking Diptera species show the importance of overcoming the Diptera bias when performing functional characterization of Toll pathways (Lima, 2021).

lncRNA-CR46018 positively regulates the Drosophila Toll immune response by interacting with Dif/Dorsal

The Toll signaling pathway is highly conserved from insects to mammals. Drosophila is a model species that is commonly used to study innate immunity. Although many studies have assessed protein-coding genes that regulate the Toll pathway, it is unclear whether long noncoding RNAs (lncRNAs) play regulatory roles in the Toll pathway. This study evaluated the expression of the lncRNA CR46018 in Drosophila. These results showed that this lncRNA was significantly overexpressed after infection of Drosophila with Micrococcus luteus. A CR46018-overexpressing Drosophila strain was then constructed; it was expected that CR46018 overexpression would enhance the expression of various antimicrobial peptides downstream of the Toll pathway, regardless of infection with M. luteus. RNA-seq analysis of CR46018-overexpressing Drosophila after infection with M. luteus showed that upregulated genes were mainly enriched in Toll and Imd signaling pathways. Moreover, bioinformatics predictions and RNA-immunoprecipitation experiments showed that CR46018 interacted with the transcription factors Dif and Dorsal to enhance the Toll pathway. During gram-positive bacterial infection, flies overexpressing CR46018 showed favorable survival compared with flies in the control group. Overall, this current work not only reveals a new immune regulatory factor, lncRNA-CR46018, and explores its potential regulatory model, but also provides a new perspective for the effect of immune disorders on the survival of Drosophila melanogaster (Zhou, 2021).

Injury-induced inflammatory signaling and hematopoiesis in Drosophila

This study explores mechanisms by which stress caused by acute injury affects blood cell development and inflammatory response in Drosophila. Similar to their mammalian myeloid counterparts, these cells are predisposed to sense and react to sterile injury at distant sites. Upon sterile injury, a breach of epidermis sets up a reactive oxygen species-based signal that bypasses the pathogen-sensing apparatus of septic immune challenge, but merges downstream to activate Toll. A number of autonomous and nonautonomous signaling pathways follow in a sequence and are mapped temporally by the appearance of their corresponding molecular phenotypes. A cell-type that fights deposited parasitic wasp eggs appears with sterile injury without the immune challenge, perhaps in anticipation, because in nature injury is usually followed by infection (Evans, 2022).

The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins

Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. Mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection (Xu, 2022).

SPS1 deficiency-triggered PGRP-LC and Toll expression controls innate immunity in Drosophila S2 cells

Selenophosphate synthetase 1 (SPS1) is an essential gene for the cell growth and embryogenesis in Drosophila melanogaster. It has been reported that SPS1 deficiency stimulates the expression of genes responsible for the innate immune system, including antimicrobial peptides (AMPs), in Drosophila S2 cells. However, the underlying mechanism has not been elucidated. This study investigated the immune pathways that control the SPS1-deficiency-induced expression of AMPs in S2 cells. It was found that the activation of AMP expression is regulated by both immune deficiency (IMD) and the Toll pathway. Double knockdown of the upstream genes of each pathway with SPS1 showed that the peptidoglycan recognition protein-LC (PGRP-LC) and Toll genes are targeted by SPS1 for regulating these pathways. It was also found that the IMD and Toll pathway regulate AMP expression by cross-talking. The levels of PGRP-LC and Toll mRNAs were upregulated upon Sps1 knockdown (6.±0.36 and 3.2±0.45-fold, respectively, n=3). Overexpression of each protein also upregulated AMPs. Interestingly, PGRP-LC overexpression upregulated AMP more than Toll overexpression. These data strongly suggest that SPS1 controls the innate immune system of D. melanogaster through regulating PGRP-LC and Toll expression (Yoo, 2022).

Infection increases activity via Toll dependent and independent mechanisms in Drosophila melanogaster

Host behavioural changes are among the most apparent effects of infection. 'Sickness behaviour' can involve a variety of symptoms, including anorexia, depression, and changed activity levels. Using a real-time tracking and behavioural profiling platform, this study shows that in Drosophila melanogaster, several systemic bacterial infections cause significant increases in physical activity and that the extent of this activity increase is a predictor of survival time in some lethal infections. Using multiple bacteria and D. melanogaster immune and activity mutants, this study showed that increased activity is driven by at least two different mechanisms. Increased activity after infection with Micrococcus luteus, a Gram-positive bacterium rapidly cleared by the immune response, strictly requires the Toll ligand spatzle. In contrast, increased activity after infection with Francisella novicida, a Gram-negative bacterium that cannot be cleared by the immune response, is entirely independent of both Toll and the parallel IMD pathway. The existence of multiple signalling mechanisms by which bacterial infections drive increases in physical activity implies that this effect may be an important aspect of the host response (Vincent, 2022).

SUMOylation of Dorsal attenuates Toll/NF-kappaB signaling

In Drosophila, Toll/NF-kappaB signaling plays key roles in both animal development and in host defense. The activation, intensity, and kinetics of Toll signaling are regulated by posttranslational modifications such as phosphorylation, SUMOylation, or ubiquitination that target multiple proteins in the Toll/NF-kappaB cascade. This study has generated a CRISPR-Cas9 edited Dorsal (DL) variant that is SUMO conjugation resistant. Intriguingly, embryos laid by dl^SCR mothers overcome dl haploinsufficiency and complete the developmental program. This ability appears to be a result of higher transcriptional activation by DL^SCR. In contrast, SUMOylation dampens DL transcriptional activation, ultimately conferring robustness to the dorso-ventral program. In the larval immune response, dl^SCR animals show an increase in crystal cell numbers, stronger activation of humoral defense genes, and high cactus levels. A mathematical model that evaluates the contribution of the small fraction of SUMOylated DL (1-5%) suggests that it acts to block transcriptional activation, which is driven primarily by DL that is not SUMO conjugated. These findings define SUMO conjugation as an important regulator of the Toll signaling cascade, in both development and host defense. These results broadly suggest that SUMO attenuates DL at the level of transcriptional activation. Furthermore, it is hypothesized that SUMO conjugation of DL may be part of a Ubc9-dependent mechanism that restrains Toll/NF-kappaB signaling (Hegde, 2022).

A Toll-dependent Bre1/Rad6-cact feedback loop in controlling host innate immune response

The Toll signaling pathway was initially identified for its involvement in the control of early embryogenesis. It was later shown to be also part of a major innate immune pathway controlling the expression of anti-microbial peptides in many eukaryotes including humans; cactus, the essential negative regulator of this pathway in flies, was found to be induced in parallel to the Toll-dependent activation process during immune defenses. This study was interested in the mechanisms of this dual effect and provides evidence that upon pathogenic stimuli, Dorsal, one of the transcription factors of the fly Toll pathway, can induce the expression of the E3 ligase Bre1. It was further shown that Bre1 complexes with the E2 Rad6 to mono-ubiquitinate histone H2B and to promote the transcription of cactus to achieve homeostasis of the Toll immune response. These studies characterize a Toll signal-dependent regulatory machinery in governing the Toll pathway in Drosophila (Cai, 2022).

Partner of neuropeptide bursicon homodimer pburs mediates a novel antimicrobial peptide Ten3LP via Dif/Dorsal2 in Tribolium castaneum

Bursicon is a cystine knot family neuropeptide, composed of two subunits, bursicon (burs) and partner of burs (pburs). The subunits can form heterodimers to regulate cuticle tanning and wing maturation and homodimers to signal different biological functions in innate immunity, midgut stem cell proliferation and energy homeostasis, and reproductive physiology in the model insects Drosophila melanogaster or Tribolium castaneum. This study reports on the role of the pburs homodimer in signaling innate immunity in T. castaneum larvae. Through transcriptome analysis a set of immune-related genes were identified that respond to pburs RNAi. Treating larvae with recombinant-pburs protein led to up-regulation of antimicrobial peptide (AMP) genes in vivo and in vitro. The upregulation of most AMP genes was dependent on the NF-κB transcription factor Relish. Most importantly, a novel AMP was discovered, Tenecin 3-like peptide (Ten3LP), regulated by pburs via NF-kappaB transcription factor Dorsal-related immunity factor (Dif)/Dorsal2, but not Relish. Ten3LP RNAi was conduction, recombinant Ten3LP protein was synthesized for microbial inhibition assays, and Ten3LP was functionally characterized as an AMP specific for fungi and Gram-positive bacteria. Expression of Ten3LP is activated by pburs via the Toll pathway. These findings identify new molecular targets for development of potential antibiotics for treating microbial infections and perhaps for RNAi based pest management technology (Li, 2023).

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spatzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, it was revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, it was found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage (Nakano, 2023).

A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. This study demonstrates that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways (Huang, 2023).

Endoplasmic reticulum-associated protein degradation contributes to Toll innate immune defense in Drosophila melanogaster

In Drosophila, the endoplasmic reticulum-associated protein degradation (ERAD) is engaged in regulating pleiotropic biological processes, with regard to retinal degeneration, intestinal homeostasis, and organismal development. The extent to which it functions in controlling the fly innate immune defense, however, remains largely unknown. This study shows that blockade of the ERAD in fat bodies antagonizes the Toll but not the IMD innate immune defense in Drosophila. Genetic approaches further suggest a functional role of Me31B in the ERAD-mediated fly innate immunity. Moreover, evidence is provided that silence of Xbp1 other than PERK or Atf6 partially rescues the immune defects by the dysregulated ERAD in fat bodies. Collectively, this study uncovers an essential function of the ERAD in mediating the Toll innate immune reaction in Drosophila (Zhu, 2022).

Involved in Toll Pathway-Dependent Antimicrobial Gene Induction in the Red Flour Beetle, Tribolium castaneum (Coleoptera; Tenebrionidae)

Insects rely only on their innate immune system to protect themselves from pathogens. Antimicrobial peptide (AMP) production is the main immune reaction in insects. In Drosophila melanogaster, the reaction is regulated mainly by the Toll and immune deficiency (IMD) pathways. Spaetzle proteins, activated by immune signals from upstream components, bind to Toll proteins, thus, activating the Toll pathway, which in turn, induces AMP genes. Previous studies have shown the difference in immune systems related to Toll and IMD pathways between D. melanogaster and Tribolium castaneum. In T. castaneum, nine Toll and seven spaetzle (spz) genes were identified. To extend understanding of AMP production by T. castaneum, functional assays of Toll and spaetzle genes related to Toll-pathway-dependent AMP gene expression were conducted in T. castaneum under challenge with bacteria or budding yeast. The results revealed that Toll3 and Toll4 double-knockdown and spz7 knockdown strongly and moderately reduced the Toll-pathway-dependent expression of AMP genes, respectively. Moreover, Toll3 and Toll4 double-knockdown pupae more rapidly succumbed to entomopathogenic bacteria than the control pupae, but spz7 knockdown pupae did not. The results suggest that Toll3 and Toll4 play a large role in Toll-pathway-dependent immune reactions, whereas spz7 plays a small part (Kato, 2023).

Drosophila Gut Immune Pathway Suppresses Host Development-Promoting Effects of Acetic Acid Bacteria

The physiology of most organisms, including Drosophila, is heavily influenced by their interactions with certain types of commensal bacteria. Acetobacter and Lactobacillus, two of the most representative Drosophila commensal bacteria, have stimulatory effects on host larval development and growth. However, how these effects are related to host immune activity remains largely unknown. This study shows that the Drosophila development-promoting effects of commensal bacteria are suppressed by host immune activity. Mono-association of germ-free Drosophila larvae with Acetobacter pomorum stimulated larval development, which was accelerated when host immune deficiency (IMD) pathway genes were mutated. This phenomenon was not observed in the case of mono-association with Lactobacillus plantarum. Moreover, the mutation of Toll pathway, which constitutes the other branch of the Drosophila immune pathway, did not accelerate A. pomorum-stimulated larval development. The mechanism of action of the IMD pathway-dependent effects of A. pomorum did not appear to involve previously known host mechanisms and bacterial metabolites such as gut peptidase expression, acetic acid, and thiamine, but appeared to involve larval serum proteins. These findings may shed light on the interaction between the beneficial effects of commensal bacteria and host immune activity (Lee, 2023).

Drosophila Ectoderm-expressed 4 modulates JAK/STAT pathway and protects flies against Drosophila C virus infection

Sterile alpha and HEAT/Armadillo motif-containing protein (SARM) is conserved in evolution and negatively regulates TRIF-dependent Toll signaling in mammals. The SARM protein from Litopenaeus vannamei and its Drosophila orthologue Ectoderm-expressed (Ect4/SARM) are also involved in immune defense against pathogen infection. However, the functional mechanism of the protective effect remains unclear. This study shows that Ect4 is essential for the viral load in flies after a Drosophila C virus (DCV) infection. Viral load is increased in Ect4 mutants resulting in higher mortality rates than wild-type. Overexpression of Ect4 leads to a suppression of virus replication and thus improves the survival rate of the animals. Ect4 is required for the viral induction of STAT-responsive genes, TotA and TotM. Furthermore, Ect4 interacts with Stat92E, affecting the tyrosine phosphorylation and nuclear translocation of Stat92E in S2 cells. Altogether, this study identifies the adaptor protein Ect4 of the Toll pathway contributes to resistance to viral infection and regulates JAK/STAT signaling pathway (Huang, 2023).

LncRNA-CR11538 Decoys Dif/Dorsal to Reduce Antimicrobial Peptide Products for Restoring Drosophila Toll Immunity Homeostasis

Avoiding excessive or insufficient immune responses and maintaining homeostasis are critical for animal survival. Although many positive or negative modulators involved in immune responses have been identified, little has been reported to date concerning whether the long non-coding RNA (lncRNA) can regulate Drosophila immunity response. Firstly, this study discovered that the overexpression of lncRNA-CR11538 can inhibit the expressions of antimicrobial peptides Drosomycin (Drs) and Metchnikowin (Mtk) in vivo, thereby suppressing the Toll signaling pathway. Secondly, the results demonstrate that lncRNA-CR11538 can interact with transcription factors Dif/Dorsal in the nucleus based on both subcellular localization and RIP analyses. Thirdly, the findings reveal that lncRNA-CR11538 can decoy Dif/Dorsal away from the promoters of Drs and Mtk to repress their transcriptions by ChIP-qPCR and dual luciferase report experiments. Fourthly, the dynamic expression changes of Drs, Dif, Dorsal and lncRNA-CR11538 in wild-type flies (w(1118)) at different time points after M. luteus stimulation disclose that lncRNA-CR11538 can help Drosophila restore immune homeostasis in the later period of immune response. Overall, this study reveals a novel mechanism by which lncRNA-CR11538 serves as a Dif/Dorsal decoy to downregulate antimicrobial peptide expressions for restoring Drosophila Toll immunity homeostasis, and provides a new insight into further studying the complex regulatory mechanism of animal innate immunity (Zhou, 2021).

Interaction of lncRNA-CR33942 with Dif/Dorsal Facilitates Antimicrobial Peptide Transcriptions and Enhances Drosophila Toll Immune Responses

The Drosophila Toll signaling pathway mainly responds to Gram-positive (G(+)) bacteria or fungal infection, which is highly conserved with mammalian TLR signaling pathway. Although many positive and negative regulators involved in the immune response of the Toll pathway have been identified in Drosophila, the roles of long noncoding RNAs (lncRNAs) in Drosophila Toll immune responses are poorly understood to date. In this study, the results demonstrate that lncRNA-CR33942 is mainly expressed in the nucleus and upregulated after Micrococcus luteus infection. Especially, lncRNA-CR33942 not only modulates differential expressions of multiple antimicrobial peptide genes but also affects the Drosophila survival rate during response to G(+) bacterial infection based on the transiently overexpressing and the knockdown lncRNA-CR33942 assays in vivo. Mechanically, lncRNA-CR33942 interacts with the NF-κB transcription factors Dorsal-related immunity factor/Dorsal to promote the transcriptions of antimicrobial peptides drosomycin and metchnikowin, thus enhancing Drosophila Toll immune responses. Taken together, this study identifies lncRNA-CR33942 as a positive regulator of Drosophila innate immune response to G(+) bacterial infection to facilitate Toll signaling via interacting with Dorsal-related immunity factor/Dorsal. It would be helpful to reveal the roles of lncRNAs in Toll immune response in Drosophila and provide insights into animal innate immunity (Zhou, 2022).

TOR signaling is required for host lipid metabolic remodelling and survival following enteric infection in Drosophila

When infected by enteric pathogenic bacteria, animals need to initiate local and whole-body defence strategies. While most attention has focused on the role innate immune anti-bacterial responses, less is known about how changes in host metabolism contribute to host defence. Using Drosophila as a model system, this study identified induction of intestinal target-of-rapamycin (TOR) kinase signaling as a key adaptive metabolic response to enteric infection. Enteric infection induces both local and systemic induction of TOR independently of the IMD innate immune pathway, and TOR functions together with IMD signaling to promote infection survival. These protective effects of TOR signaling are associated with re-modelling of host lipid metabolism. Thus, TOR is required to limit excessive infection-mediated wasting of host lipid stores by promoting an increase in the levels of gut- and fat body-expressed lipid synthesis genes. These data supports a model in which induction of TOR represents a host tolerance response to counteract infection-mediated lipid wasting in order to promote survival (Deshpande, 2022).

The influence of immune activation on thermal tolerance along a latitudinal cline

Global change is shifting both temperature patterns and the geographic distribution of pathogens, and infection has already been shown to substantially reduce host thermal performance, potentially placing populations at greater risk that previously thought. But what about individuals that are able to successfully clear an infection? While the direct damage a pathogen causes will likely lead to reductions in host's thermal tolerance, the response to infection often shares many underlying pathways with the general stress response, potentially acting as a buffer against subsequent thermal stress. By exposing Drosophila melanogaster to heat-killed bacterial pathogens, this study investigated how activation of a host's immune system can modify any response to both heat and cold temperature stress. In a single focal population, it was found that immune activation can improve a host's knockdown times during heat shock, potentially offsetting some of the damage that would subsequently arise as an infection progresses. Conversely, immune activation had a detrimental effect on CT(max), and did not influence lower thermal tolerance as measured by chill coma recovery time. However, it was also found that the influence of immune activation on heat knockdown times is not generalizable across an entire cline of locally adapted populations. Instead, immune activation led to signals of local adaptation to temperature being lost, erasing the previous advantage that populations in warmer regions had when challenged with heat stress. These results suggest that activation of the immune system may help buffer individuals against the detrimental impact of infection on thermal tolerance; however, any response will be population specific and potentially not easily predicted across larger geographic scales, and dependent on the form of thermal stress faced by a host (Hector, 2020).

Immune Control of Animal Growth in Homeostasis and Nutritional Stress in Drosophila

A large body of research implicates the brain and fat body (liver equivalent) as central players in coordinating growth and nutritional homeostasis in multicellular animals. In this regard, an underlying connection between immune cells and growth is also evident, although mechanistic understanding of this cross-talk is scarce. This study explored the importance of innate immune cells in animal growth during homeostasis and in conditions of nutrient stress. Drosophila larvae lacking blood cells eclose as small adults and show signs of insulin insensitivity. Moreover, when exposed to dietary stress of a high-sucrose diet (HSD), these animals are further growth retarded than normally seen in regular animals raised on HSD. In contrast, larvae carrying increased number of activated macrophage-like plasmatocytes show no defects in adult growth when raised on HSD and grow to sizes almost comparable with that seen with regular diet. These observations imply a central role for immune cell activity in growth control. Mechanistically, these findings reveal a surprising influence of immune cells on balancing fat body inflammation and insulin signaling under conditions of homeostasis and nutrient overload as a means to coordinate systemic metabolism and adult growth. This work integrates both the cellular and humoral arm of the innate immune system in organismal growth homeostasis, the implications of which may be broadly conserved across mammalian systems as well (Preethi, 2020).

Immune Receptor Signaling and the Mushroom Body Mediate Post-ingestion Pathogen Avoidance

In spite of the positive effects of bacteria on health, certain species are harmful, and therefore, animals must weigh nutritional benefits against negative post-ingestion consequences and adapt their behavior accordingly. This study used Drosophila to unravel how the immune system communicates with the brain, enabling avoidance of harmful foods. Using two different known fly pathogens, mildly pathogenic Erwinia carotovora (Ecc15) and highly virulent Pseudomonas entomophila (Pe), preference behavior was analyzed in naive flies and after ingestion of either of these pathogens. Although survival assays confirmed the harmful effect of pathogen ingestion, naive flies preferred the odor of either pathogen to air and also to harmless mutant bacteria, suggesting that flies are not innately repelled by these microbes. By contrast, feeding assays showed that, when given a choice between pathogenic and harmless bacteria, flies-after an initial period of indifference-shifted to a preference for the harmless strain, a behavior that lasted for several hours. Flies lacking synaptic output of the mushroom body (MB), the fly's brain center for associative memory formation, lost the ability to distinguish between pathogenic and harmless bacteria, suggesting this to be an adaptive behavior. Interestingly, this behavior relied on the immune receptors PGRP-LC and -LE and their presence in octopaminergic neurons. A model is postulated wherein pathogen ingestion triggers PGRP signaling in octopaminergic neurons, which in turn relay the information about the harmful food source directly or indirectly to the MB, where an appropriate behavioral output is generated (Kobler, 2020).

Bombardier Enables Delivery of Short-Form Bomanins in the Drosophila Toll Response

Toll mediates a robust and effective innate immune response across vertebrates and invertebrates. In Drosophila melanogaster, activation of Toll by systemic infection drives the accumulation of a rich repertoire of immune effectors in hemolymph, including the recently characterized Bomanins, as well as the classical antimicrobial peptides (AMPs). This study report the functional characterization of a Toll-induced hemolymph protein encoded by the bombardier (CG18067) gene. Using the CRISPR/Cas9 system to generate a precise deletion of the bombardier transcriptional unit, this study found that Bombardier is required for Toll-mediated defense against fungi and Gram-positive bacteria. Assaying cell-free hemolymph, it was found that the Bomanin-dependent candidacidal activity is also dependent on Bombardier, but is independent of the antifungal AMPs Drosomycin and Metchnikowin. Using mass spectrometry, it was demonstrated that deletion of bombardier results in the specific absence of short-form Bomanins from hemolymph. In addition, flies lacking Bombardier exhibited a defect in pathogen tolerance that was traced to an aberrant condition triggered by Toll activation. These results leadn to a model in which the presence of Bombardier in wild-type flies enables the proper folding, secretion, or intermolecular associations of short-form Bomanins, and the absence of Bombardier disrupts one or more of these steps, resulting in defects in both immune resistance and tolerance (Lin, 2019).

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Zygotically transcribed genes

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