InteractiveFly: GeneBrief

Peptidoglycan recognition protein LA: Biological Overview | References

Tissue homeostasis is achieved by balancing stem cell maintenance, cell proliferation and differentiation, as well as the purging of damaged cells. Elimination of unfit cells maintains tissue health: however, the underlying mechanisms driving competitive growth when homeostasis fails, for example, during tumorigenesis, remain largely unresolved. Using a Drosophila intestinal model, this study found that tumor cells outcompete nearby enterocytes (ECs) by influencing cell adhesion and contractility. This process relies on activating the immune-responsive Relish/NF-κB pathway to induce EC delamination and requires a JNK-dependent transcriptional upregulation of the peptidoglycan recognition protein PGRP-LA. Consequently, in organisms with impaired PGRP-LA function, tumor growth is delayed and lifespan extended. This study identifies a non-cell-autonomous role for a JNK/PGRP-LA/Relish signaling axis in mediating death of neighboring normal cells to facilitate tumor growth. It is proposed that intestinal tumors 'hijack' innate immune signaling to eliminate enterocytes in order to support their own growth (Zhou, 2021).

The intestinal epithelium separates the organism from the environment and plays essential roles in nutrient uptake and immune and regenerative processes. Intestinal renewal requires dynamic regulation of cell-cell contacts between enterocytes, and this is achieved by highly proliferative stem cells, proper differentiation, and cell loss by cell extrusion and apoptosis. Dysregulation of cell death in the intestinal epithelium can lead to pathologies such as intestinal bowel diseases and cancer (Zhou, 2021).

Studies in Drosophila have made important contributions toward an understanding of intestinal homeostasis, innate immunity, and aging. In adult flies, intestinal stem cells (ISCs) self-renew and produce progenitor cells called enteroblasts (EBs). These EBs can differentiate into either enteroendocrine cells (EEs) or enterocytes (ECs). The intestinal epithelium undergoes rapid stem cell division and differentiation to continuously replace damaged ECs and ensure tissue integrity and homeostasis, similar to mammalian intestines. Previous studies have shown that bacterial infection induces ISC proliferation and elimination of damaged ECs, thereby leading to remodeling of the intestinal epithelium. Enteric infection also triggers the evolutionarily conserved NF-ΚB pathway through the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), leading to the production of antimicrobial peptides (AMPs) for host immune defense (Zhou, 2021).

In addition to the role of the NF-κB pathway in AMP production in different cell types, several studies have identified non-immune functions. For example, constitutive activation of NF-κB reduces animal lifespan, NF-κB activity has been implicated in age-related neurodegenerative diseases, and NF-κB regulates Mef2 to coordinate its immune functions with metabolism. Further evidence links NFκB to Ras/MAPK and JAK/STAT signaling pathways. This allows for the proper balance of immune responses with cell growth and proliferation. Moreover, it has been recently reported that the NF-κB pathway in Drosophila is involved in infection-induced EC shedding, which facilitates maintenance of barrier function during intestinal regeneration (Zhou, 2021).

The Drosophila BMP2/4 homolog Decapentaplegic (Dpp) is involved in multiple developmental processes. The Dpp signal is transduced by the type I receptor Thickveins (Tkv) and type II receptor Punt that phosphorylate Drosophila Smad transcriptional factors such as Mothers against Dpp (Mad), Medea (Med), and the coregulator Schnurri (Shn) to regulate gene expression. Inactivation of BMP signaling components in the Drosophila intestine leads to intestinal tumor formation resembling juvenile polyposis syndrome (JPS). In humans, loss of BMP signaling leads to JPS, which has been associated with increased risks of developing gastrointestinal cancer (Zhou, 2021).

Cell replenishment and rearrangement are common mechanisms to sustain tissue homeostasis, which is also essential for development. Tissue growth requires dynamic cell rearrangements including cell elimination by mechanical competition. For example, epithelial cells can be eliminated by cell extrusion to maintain tissue homeostasis. Recent studies also suggested that tumor cells outcompete and eliminate their neighboring cells to clear space for their expansion. However, the underlying mechanisms and how tumor cells eliminate normal cells in the tumor microenvironment remain largely unknown (Zhou, 2021).

This study demonstrates a critical role for mechanical competition in the tumor microenvironment to promote tumorigenesis. Mechanistically, it was shown that tumor induces DE-cadherin- and myosin-dysregulation-associated mechanical stresses to nearby ECs. These processes trigger the ROCK-associated JNK signaling and subsequent activation of PGRP-LA/NF-κrB/ Relish in surrounding ECs to regulate the expression of pro- apoptotic genes and thereby promote cell delamination and apoptosis. The dying ECs then induce paracrine JAK/STAT signaling to trigger regeneration and further promote tumorigenesis. Importantly, tumors with associated activation of JNK/ PGPR-LA/Relish cascades can be inhibited by preventing apoptosis or by administering ROCK inhibitors. These results thus establish a tumor-cell-driven inflammatory feedback mechanism for competitive growth (Zhou, 2021).

This study demonstrates a non-cell-autonomous feedback mechanism that facilitates tumor development. First, tumor growth outcompetes its microenvironment by inducing mechanical forces. This triggers stress related ROCK/JNK signaling and induces EC elimination through activation of PGRP-LA/Relish signaling and downstream pro-apoptotic genes. Subsequently, dying ECs produce cytokines that activate JAK/STAT signaling in tumor cells to further promote tumor growth, thereby establishing a positive amplification loop between the tumor and its microenvironment (Zhou, 2021).

Cells undergoing rapid proliferation will push on their neighbors, which leads to local increase in mechanical pressures and triggers cell delamination. Previous studies have shown that the expansion of tumor cells triggers cell competition, which involves mechanical interactions. For instance, ectopic expression of Ras oncogene drives mechanical competitive growth and induces delamination of nearby wild-type cells in Drosophila. Hence, these data are in line with the current interpretation that mechanical competition also drives tumor competitive growth in the Drosophila intestine. In the epithelium, the mechanical modulation of surface tension is regulated by the actomyosin complex, counterbalanced by the Cadherin-dependent cell-cell adhesions. This study shows that tumors interact with their microenvironment and trigger ROCK/ JNK related cell death. A similar mechanism was observed in mammalian Madin-Darbin canine kidney (MDCK) cells, which, when deficient in the polarity gene scribble, are eliminated by mechanical cell competition, a process that requires the activation of the ROCK-p38-p53 pathway (Zhou, 2021).

The intestinal epithelium requires homeostatic mechanisms to counterbalance stem cell division and elimination of damaged or unfit cells. Dysregulation of either of these homeostatic programs can lead to tumor development. This study found that tumor cells induce immune-responsive PGRP- LA/Relish signaling for cell delamination and apoptosis. The role of NF-κB in the regulation of apoptosis has been discussed before for Drosophila Imd and mammalian TNFR1 pathways, which share key components to regulate NF-κB-related immune response and caspase-dependent apoptosis. Several studies have shown that tumor cells displace the nearby ECs through activation of Hippo and JNK signaling for tumor progression. In addition, JNK acts in parallel with NF-κB to control EC shedding during intestinal regeneration. In mammals, intestinal TNFR1 signaling is also required for EC detachment and apoptosis (Zhou, 2021).

Previous studies revealed a role for NF-κB signaling in cell death of outcompeted cells during development. In Drosophila wing discs, Myc-induced cell competition triggers Imd/Relish-related activation of the pro-apoptotic gene Hid for cell death. The Toll-signaling transcription factors Dorsal and Dif have been suggested to be required for Minute-induced cell competition by inducing Reaper-dependent apoptosis of outcompeted cells. However, further evidence showed axenic conditions abolished Toll-inhibition-induced competitive growth in the outcompeted cells. This suggested that infection contributes to Toll pathway inhibition induced cell competition. The current experiments indicate that axenic conditions failed to abolish the Imd activation in the tumor-surrounding ECs, suggesting a tumor-associated role of Imd/Relish induced EC cell death. Furthermore, a recent study discovered that cells with growth advantages, such as high protein synthesis, induce NF-κB-dependent autophagy to eliminate neighboring unfit cells in developing tissues. NF-κB and its upstream activating receptor are also required for salivary gland degradation through autophagy. Eye disc tumors also trigger a cell-autonomous feedback loop to promote proliferation by activation of JNK, Yki, and JAK/STAT signaling. In a distinct organ with a high rate of turnover, the data suggest that the NF-κB/Rel-dependent EC cell death cooperates with compensatory stem cell proliferation through the non-cell-autonomous activation of JNK and JAK/ STAT signaling for tumor progression (Zhou, 2021).

PGRPs are known as immune modulators of NF-κB signaling through binding and recognizing bacterial peptidoglycans. Several PGRPs have been implicated in other important biological processes beyond immunity such as host-microbe homeostasis, systemic inflammatory response, tissue integrity, and aging. PGRPs contain a RIP RHIM domain which has been proposed to activate NF-κB signaling. However, unlike in mammals, the Drosophila RHIMs may not be required for cell death. Consistently, it was found that the PGRP-LAD containing the RHIM domain does not induce cell death. However, PGRP-LAF lacking RHIM drives EC delamination and apoptosis. Previous studies suggested a regulatory role of PGRP-LA in controlling NF-κB activity rather than binding to peptidoglycan. In this study, PGRP-LA depletion extends the lifespan of tumor-bearing flies. However, the expression of PGRP-LA is low in the intestine during normal homeostasis, suggesting PGRP-LA may not be involved in normal aging and intestinal homeostasis. Whether the programed cell death pathways impact metazoan lifespan remains unknown, and this requires further research (Zhou, 2021).

This study revealed that tumor induces cytoplasmic enrichment of DE-cadherin::GFP and activates p-Myosin signal in nearby ECs with elongated cell morphology. The DE-cadherin reporter and p-Myosin staining have been previously used as mechano-transduction sensors in Drosophila. The results therefore suggest that the tumor induces DE-cadherin- and myosin-dysregulation-associated mechanical stresses. However, changes in mechanical tension in cells adjacent to tumor cells is a rapid process and more evidence will be required to illustrate mechanical competition, e.g., by monitoring mechanosensors in live tissue. Unfortunately, this remains technically difficult in the Drosophila intestine because of constraints on live imaging and a lack of molecular markers. Moreover, how cells sense and respond to mechanical stress in the context of tumor growth requires further investigations (Zhou, 2021).

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).

Gut microbiota is essential in PGRP-LA regulated immune protection against Plasmodium berghei infection

Malaria remains to be one of the deadliest infectious diseases and imposes substantial financial and social costs in the world. Mosquitoes rely on the immune system to control parasite infection. Peptidoglycan recognition proteins (PGRPs), a family of pattern-recognition receptors (PRR), are responsible for initiating and regulating immune signaling pathways. PGRP-LA is involved in the regulation of immune defense against the Plasmodium parasite, however, the underlying mechanism needs to be further elucidated. The spatial and temporal expression patterns of pgrp-la in Anopheles stephensi were analyzed by qPCR. The function of PGRP-LA was examined using a dsRNA-based RNA interference strategy. Western blot and periodic acid schiff (PAS) staining were used to assess the structural integrity of peritrophic matrix (PM). The expression of pgrp-la in An. stephensi was induced in the midgut in response to the rapid proliferating gut microbiota post-blood meal. Knocking down of pgrp-la led to the downregulation of immune effectors that control gut microbiota growth. The decreased expression of these immune genes also facilitated P. berghei infection. However, such dsLA treatment did not influence the structural integrity of PM. When gut microbiota was removed by antibiotic treatment, the regulation of PGRP-LA on immune effectors was abolished and the knock down of pgrp-la failed to increase susceptibility of mosquitoes to parasite infection. It is concluded that PGRP-LA regulates the immune responses by sensing the dynamics of gut microbiota. A mutual interaction between gut microbiota and PGRP-LA contributes to the immune defense against Plasmodium parasites in An. stephensi (Gao, 2020).

Peptidoglycan recognition protein genes and their roles in the innate immune pathways of the red flour beetle, Tribolium castaneum

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Functional Toll and IMD innate immune pathways exist in the model beetle, Tribolium castaneum while the beetle's pathways have broader specificity in terms of microbial activation than that of Drosophila. To elucidate the molecular basis of this broad microbial activation, focus was placed on potential upstream sensors of the T. castaneum innate immune pathways, peptidoglycan recognition proteins (PGRPs). Phenotype analyses utilizing RNA interference-based comprehensive gene knockdown followed by bacterial challenge suggested: PGRP-LA functions as a pivotal sensor of the IMD pathway for both Gram-negative and Gram-positive bacteria; PGRP-LC acts as an IMD pathway-associated sensor mainly for Gram-negative bacteria; PGRP-LE also has some roles in Gram-negative bacterial recognition of the IMD pathway. On the other hand, no clear phenotype changes were obtained by gene knockdown of short-type PGRP genes, probably because of highly inducible nature of these genes. These results may collectively account for the promiscuous bacterial activation of the T. castaneum innate immune pathways at least in part (Koyama, 2015).

Search PubMed for articles about Drosophila PGRP-LA

Choe, K. M., Werner, T., Stoven, S., Hultmark, D. and Anderson, K. V. (2002). Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296(5566): 359-362. PubMed ID: 11872802

Gao, L., Song, X. and Wang, J. (2020). Gut microbiota is essential in PGRP-LA regulated immune protection against Plasmodium berghei infection. Parasit Vectors 13(1): 3. PubMed ID: 31907025

Gendrin, M., Zaidman-Remy, A., Broderick, N. A., Paredes, J., Poidevin, M., Roussel, A. and Lemaitre, B. (2013). Functional analysis of PGRP-LA in Drosophila immunity. PLoS One 8(7): e69742. PubMed ID: 23922788

Ramet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B. and Ezekowitz, R. A. (2002). Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416(6881): 644-648. PubMed ID: 11912489

Zhou, J., Valentini, E. and Boutros, M. (2021). Microenvironmental innate immune signaling and cell mechanical responses promote tumor growth. Dev Cell 56(13): 1884-1899. PubMed ID: 34197724

date revised: 1 February 2022

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