InteractiveFly: GeneBrief

Sting: Biological Overview | References

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).

Drosophila STING protein has a role in lipid metabolism

Stimulator of interferon genes (STING) plays an important role in innate immunity by controlling type I interferon response against invaded pathogens. This work describes a previously unknown role of STING in lipid metabolism in Drosophila. Flies with STING deletion are sensitive to starvation and oxidative stress, have reduced lipid storage and downregulated expression of lipid metabolism genes. Drosophila STING was found to interact with lipid synthesizing enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). ACC and FASN also interact with each other, indicating that all three proteins may be components of a large multi-enzyme complex. The deletion of Drosophila STING leads to disturbed ACC localization and decreased FASN enzyme activity. Together, these results demonstrate a previously undescribed role of STING in lipid metabolism in Drosophila (Akhmetova, 2021).

STimulator of INterferon Genes (STING) is an endoplasmic reticulum (ER)-associated transmembrane protein that plays an important role in innate immune response by controlling the transcription of many host defense genes. The presence of foreign DNA in the cytoplasm signals a danger for the cell. This DNA is recognized by specialized enzyme, the cyclic GMP-AMP synthase (cGAS), which generates cyclic dinucleotide (CDN) signaling molecules. CDNs bind to STING activating it (Wu, 2013; Burdette, 2011), and the following signaling cascade results in NF-κB- and IRF3-dependent expression of immune response molecules such as type I interferons (IFNs) and pro-inflammatory cytokines. Bacteria that invade the cell are also known to produce CDNs that directly activate STING pathway . Additionally, DNA that has leaked from the damaged nuclei or mitochondria can also activate STING signaling and inflammatory response, which, if excessive or unchecked, might lead to the development of autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis (Akhmetova, 2021).

STING homologs are present in almost all animal phyla. This protein has been extensively studied in mammalian immune response; however, the role of STING in the innate immunity of insects has been just recently identified (Hua, 2018; Goto, 2018; Liu, 2018; Martin, 2018). Fruit fly D. melanogaster STING homolog is encoded by the CG1667 gene, referred to as dSTING. dSTING displays anti-viral and anti-bacterial effects that however are not all encompassing. Particularly, it has been shown that dSTING-deficient flies are more susceptible to Listeria infection due to the decreased expression of antimicrobial peptides (AMPs) – small positively charged proteins that possess antimicrobial properties against a variety of microorganisms (Martin, 2018). However, no effect has been observed during Escherichia coli or Micrococcus luteus infections (Goto, 2018). dSTING has been shown to attenuate Zika virus infection in fly brains (Liu, 2018) and participate in the control of infection by two picorna-like viruses (DCV and CrPV) but not two other RNA viruses FHV and SINV or dsDNA virus IIV6 (Goto, 2018; Martin, 2018). All these effects are linked to the activation of NF-κB transcription factor Relish (Akhmetova, 2021).

The immune system is tightly linked with metabolic regulation in all animals, and proper re-distribution of the energy is crucial during immune challenges. In both flies and humans, excessive immune response can lead to a dysregulation of metabolic stores. Conversely, the loss of metabolic homeostasis can result in weakening of the immune system. The mechanistic links between these two important systems are integrated in Drosophila fat body. Similarly to mammalian liver and adipose tissue, insect fat body stores excess nutrients and mobilizes them during metabolic shifts. The fat body also serves as a major immune organ by producing AMPs during infection. There is an evidence that the fat body is able to switch its transcriptional status from 'anabolic' to 'immune' program. The main fat body components are lipids, with triacylglycerols (TAGs) constituting approximately 90% of the stored lipids. Even though most of the TAGs stored in fat body comes from the dietary fat originating from the gut during feeding, de novo lipid synthesis in the fat body also significantly contributes to the pool of storage lipids (Akhmetova, 2021).

Maintaining lipid homeostasis is crucial for all organisms. Dysregulation of lipid metabolism leads to a variety of metabolic disorders such as obesity, insulin resistance and diabetes. Despite the difference in physiology, most of the enzymes involved in metabolism, including lipid metabolism, are evolutionarily and functionally conserved between Drosophila and mammals. Major signaling pathways involved in metabolic control, such as insulin system, TOR, steroid hormones, FOXO, and many others, are present in fruit flies. Therefore, it is not surprising that Drosophila has become a popular model system for studying metabolism and metabolic diseases. With the availability of powerful genetic tools, Drosophila has all the advantages to identify new players and fill in the gaps in understanding of the intricacies of metabolic networks (Akhmetova, 2021).

This work describes a novel function of dSTING in lipid metabolism. Flies with a deletion of dSTING were found to be sensitive to the starvation and oxidative stress. Detailed analysis reveals that dSTING deletion results in a significant decrease in the main storage metabolites, such as TAG, trehalose, and glycogen. Two fatty-acid biosynthesis enzymes were identified – acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) – as the interacting partners for dSTING. Moreover, this study also found that FASN and ACC interacted with each other, indicating that all three proteins might be components of a large complex. Importantly, dSTING deletion leads to the decreased FASN activity and defects in ACC cellular localization suggesting a direct role of dSTING in lipid metabolism of fruit flies (Akhmetova, 2021).

STING plays an important role in innate immunity of mammals, where activation of STING induces type I interferons (IFNs) production following the infection with intracellular pathogens. However, recent studies showed that the core components of STING pathway evolved more than 600 million years ago, before the evolution of type I IFNs. This raises the question regarding the ancestral functions of STING. In this study it was found that STING protein is involved in lipid metabolism in Drosophila. The deletion of Drosophila STING (dSTING) gene rendered flies sensitive to the starvation and oxidative stress. These flies have reduced lipid storage and downregulated expression of lipid metabolism genes. It was further shown that dSTING interacted with the lipid synthesizing enzymes ACC and FASN suggesting a possible regulatory role in the lipid biosynthesis. In the fat body, main lipogenic organ in Drosophila, dSTING co-localized with both ACC and FASN in a cortical region of the ER. dSTING deletion resulted in the disturbed ACC localization in fat body cells and greatly reduced the activity of FASN in the in vitro assay (Akhmetova, 2021).

Importantly, it was also observed that ACC and FASN interacted with each other. Malonyl-CoA, the product of ACC, serves as a substrate for the FASN reaction of fatty acid synthesis. Enzymes that are involved in sequential reactions often physically interact with each other and form larger multi-enzyme complexes, which facilitate the substrate channeling and efficient regulation of the pathway flux. There are several evidences of the existence of the multi-enzyme complex involved in fatty acid biosynthesis. ACC, ACL (ATP citrate lyase), and FASN physically associated in the microsomal fraction of rat liver. Moreover, in the recent work, a lipogenic protein complex including ACC, FASN, and four more enzymes was isolated from the oleaginous fungus Cunninghamella bainieri. It is possible that a similar multi-enzyme complex exists in Drosophila and other metazoan species, and it would be of great interest to identify its other potential members (Akhmetova, 2021).

How does STING exert its effect on lipid synthesis? Recently, the evidence has emerged for the control of the de novo fatty acid synthesis by two small effector proteins – MIG12 and Spot14. MIG12 overexpression in livers of mice increased total fatty acid synthesis and hepatic triglyceride content. It has been shown that MIG12 protein binds to ACC and facilitates its polymerization thus enhancing the activity of ACC. For Spot14, both the activation and inhibition of de novo lipogenesis have been reported, depending upon the tissue type and the cellular context. Importantly, there is an evidence that all four proteins – ACC, FASN, MIG12, and Spot14 – exist as a part of a multimeric complex. It is plausible to suggest that Drosophila STING plays a role similar to MIG12 and/or Spot14 in regulating fatty acid synthesis. It is proposed that dSTING might 'anchor' ACC and FASN possibly together with other enzymes at the ER membrane. The resulting complex facilitates fatty acid synthesis by allowing for a quicker transfer of malonyl-CoA product of ACC to the active site of FASN. In dSTINGΔ mutants, ACC loses its association with some regions of the ER resulting in the weakened interaction between ACC and FASN. Less FASN immunoprecipitated with ACC in dSTINGΔ mutants compared to control flies, and the opposite effect was found in flies expressing GFP-tagged dSTING (Akhmetova, 2021).

It has been shown that de novo synthesis of fatty acids continuously contributes to the total fat body TAG storage in Drosophila. It is hypothesized that the reduced fatty acid synthesis due to the lowered FASN enzyme activity in dSTINGΔ deletion mutants might be responsible for the decreased TAG lipid storage and starvation sensitivity phenotypes. Sensitivity to oxidative stress might also be explained by the reduced TAG level. Evidences exist that the lipid droplets (consisting mainly of TAGs) provide protection against reactive oxygen species. Furthermore, flies with ACC RNAi are found to be sensitive to the oxidative stress (Akhmetova, 2021).

In addition to its direct role in ACC/FASN complex activity, STING might also affect a phosphorylation status of ACC and/or FASN. Both proteins are known to be regulated by phosphorylation/dephosphorylation. In mammals, STING is an adaptor protein that transmits an upstream signal by interacting with kinase TBK1 (TANK-binding kinase 1). When in a complex with STING, TBK1 activates and phosphorylates IRF3 allowing its nuclear translocation and transcriptional response. It is possible that in Drosophila, STING recruits a yet unidentified kinase that phosphorylates ACC and/or FASN thereby changing their enzymatic activity (Akhmetova, 2021).

Drosophila STING itself could also be regulated by the lipid- synthesizing complex. STING palmitoylation was recently identified as a posttranslational modification necessary for STING signaling in mice. In this way, palmitic acid synthesized by FASN might participate in the regulation of dSTING possibly providing a feedback loop (Akhmetova, 2021).

The product of ACC – malonyl-CoA – is a key regulator of the energy metabolism. During lipogenic conditions, ACC is active and produces malonyl-CoA, which provides the carbon source for the synthesis of fatty acids by FASN. In dSTING knockout, FASN activity is decreased and malonyl-CoA is not utilized and builds up in the cells. Malonyl-CoA is also a potent inhibitor of carnitine palmitoyltransferase CPT1, the enzyme that controls the rate of fatty acid entry into the mitochondria, and hence is a key determinant of the rate of fatty acid oxidation. Thus, a high level of malonyl-CoA results in a decreased fatty acid utilization for the energy. This might explain the down-regulation of lipid catabolism genes that was observed in dSTINGΔ mutants. A reduced fatty acid oxidation in turn shifts cells to the increased reliance on glucose as a source of energy. Consistent with this notion, an increased glucose level was observed in fed dSTINGΔ mutant flies, as well as increased levels of phosphoenolpyruvate (PEP). PEP is produced during glycolysis, and its level was shown to correlate with the level of glucose. A reliance on glucose for the energy also has a consequence of reduced incorporation of glucose into trehalose and glycogen for storage, and therefore, lower levels of these storage metabolites, which was observed. To summarize, based on the current findings, a model is presented in (see Model of dSTING deletion effect on Drosophila metabolism). Yhis suggests a direct involvement of dSTING in the regulation of lipid metabolism (Akhmetova, 2021).

Based on the data, dSTING interacts with lipid synthesizing enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In the absence of dSTING, the activity of FASN is reduced which results in decreased de novo fatty acid synthesis and triglyceride (TAG) synthesis. Low TAG level in turn lead to sensitivity to starvation and oxidative stress. Reduced FASN activity in dSTING mutants also results in ACC product malonyl-CoA build-up in the cells leading to the inhibition of the fatty acid oxidation in mitochondria. Reduced fatty acid oxidation shifts cells to the increased reliance on glucose as a source of energy resulting in reduced glycogen and trehalose levels in dSTING mutants. Palmitic acid synthesized by FASN might participate in the regulation of dSTING via palmitoylation possibly providing a feedback loop (Akhmetova, 2021).

Recent studies show that in mammals, the STING pathway is involved in metabolic regulation under the obesity conditions. The expression level and activity of STING were upregulated in livers of mice with high-fat diet-induced obesity. STING expression was increased in livers from nonalcoholic fatty liver disease (NAFLD) patients compared to control group. In nonalcoholic steatohepatitis mouse livers, STING mRNA level was also elevated. Importantly, STING deficiency ameliorated metabolic phenotypes and decreased lipid accumulation, inflammation, and apoptosis in fatty liver hepatocytes (Akhmetova, 2021).

Despite the accumulating evidences, the exact mechanism of STING functions in metabolism is not completely understood. The prevailing hypothesis is that the obesity leads to a mitochondrial stress and a subsequent mtDNA release into the cytoplasm, which activates cGAS-STING pathway. The resulting chronic sterile inflammation is responsible for the development of NAFLD, insulin resistance, and type 2 diabetes. In this case, the effect of STING on metabolism is indirect and mediated by inflammation effectors. The data presented in the current study strongly suggest that in Drosophila, STING protein is directly involved in lipid metabolism by interacting with the enzymes involved in a lipid biosynthesis. This raises the question if the observed interaction is unique for Drosophila or it is also the case for mammals. Future work is needed to elucidate the evolutionary aspect of STING role in metabolism. Understanding the relationships between STING and lipid metabolism may provide insights into the mechanisms of the obesity-induced metabolism dysregulation and thereby suggest novel therapeutic strategies for metabolic diseases (Akhmetova, 2021).

STING controls energy stress-induced autophagy and energy metabolism via STX17

The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. This study reports that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 (Syntaxin 17) interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, this study demonstrates a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17 (Rong, 2022).

This study demonstrates the crucial role of STING in glucose metabolism through its negative regulation in energy stress-induced autophagy. The connection between autophagy and the cGAS-STING pathway has been intensively investigated with a focus on pathogens, DNA, or cyclic dinucleotides-induced autophagy through a non-canonical pathway. This study found that STING also plays a crucial role in energy stress-induced autophagy, especially in autophagosome-lysosome fusion through its interaction with the autophagic SNARE STX17. In the unstressed conditions, STING physically interacts with STX17 and sequesters it at ER. This interaction is disrupted by STING activation (DNA treatment etc) or autophagy stimuli (energy stress, etc.), which leads to STX17 translocation to autophagosomes, assembly of autophagic SNARE complex, and promotion of autophagosomal fusion with lysosomes. STING-regulated energy stress-induced autophagy has at least two effects, to facilitate elimination of DNA and microbes in immune cells and to boost energy metabolism in non-immune cells (Rong, 2022).

STX17 is also important for autophagosome biogenesis. How the functions of STX17 in autophagy initiation and autophagosome-lysosome fusion is differentiated is an interesting question. It is proposed that STX17 phosphorylation by TBK1, as described by Kumar (2019), likely separates these two functions. TBK1-phosphorylated STX17 translocates from Golgi to mPAS, which is not controlled by STING, while the portion of STX17 that is not phosphorylated by TBK1 interacts with STING at ER/ERGIC (endoplasmic-reticulum-Golgi intermediate compartment), and this interaction is disrupted by autophagic stress likely through TBK1-independent regulatory events, which leads to translocation of this portion of STX17 from ER/ERGIC to complete autophagosomes. These observations nicely reconcile the different functions of STX17 in autophagy initiation and maturation (Rong, 2022).

STING-regulated energy stress-induced autophagy is different from previously reported STING-mediated non-canonical autophagy in several aspects: (1) different membrane trafficking pathways are utilized. Triggered by PAMPs (pathogen-associated molecular patterns), STING translocates to single bilayer membrane vesicles positive for LC3, but these STING-LC3 positive vesicles are negative for STX17; (2) PAMPs-triggered STING-mediated autophagy is independent of BECN1, ULK1, and Atg9a. STX17 neither localizes to STING-positive vesicles nor is it required for STING trafficking and degradation; (3) PAMPs-induced non-canonical autophagy is compromised when STING is absent; while canonical autophagy is further activated in the absence of STING given that more STX17 is released from ER; (4) PAMPs-induced STING-dependent autophagy activation is limited to immune cells, but STING-regulated canonical autophagy functions broadly in both immune and non-immune cells (Rong, 2022).

STING is a crucial regulator in the cancer-immunity cycle, and activation of STING represents a promising strategy for cancer therapy. This study suggests, in addition to immunity regulation, activation of STING also promotes energy stress-induced autophagy by releasing STX17 from ER. How autophagy activation contributes to STING mediates signaling remained to be investigated. At least, this study indicates that STING might play an unexpected broader role in energy metabolism due to its regulation of energy stress-induced autophagy. Autophagy has been implicated in a broad spectrum of human diseases, and STING also expresses and functions in non-immune tissues, suggesting that the regulatory effect of STING on autophagy might contribute to the pathogenesis of autophagy-related diseases and immune functions (Rong, 2022).

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).

Two cGAS-like receptors induce antiviral immunity in Drosophila

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 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 that is present in both prokaryotes and eukaryotes. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections. This study identified two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. cGLR1 and cGLR2 activate Sting- and NF-kappaB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. These data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP (Holleufer, 2021).

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).

The STING pathway does not contribute to behavioural or mitochondrial phenotypes in Drosophila Pink1/parkin or mtDNA mutator models

Mutations in PINK1 and Parkin/PRKN cause the degeneration of dopaminergic neurons in familial forms of Parkinson's disease but the precise pathogenic mechanisms are unknown. The PINK1/Parkin pathway has been described to play a central role in mitochondrial homeostasis by signalling the targeted destruction of damaged mitochondria, however, how disrupting this process leads to neuronal death was unclear until recently. An elegant study in mice revealed that the loss of Pink1 or Prkn coupled with an additional mitochondrial stress resulted in the aberrant activation of the innate immune signalling, mediated via the cGAS/STING pathway, causing degeneration of dopaminergic neurons and motor impairment. Genetic knockout of Sting was sufficient to completely prevent neurodegeneration and accompanying motor deficits. To determine whether Sting plays a conserved role in Pink1/parkin related pathology, genetic interactions between Sting and Pink1/parkin were tested in Drosophila. Surprisingly, it was found that loss of Sting, or its downstream effector Relish, was insufficient to suppress the behavioural deficits or mitochondria disruption in the Pink1/parkin mutants. Thus, it is concluded that phenotypes associated with loss of Pink1/parkin are not universally due to aberrant activation of the STING pathway (Lee, 2020).

2'3'-cGAMP triggers a STING- and NF-kappaB-dependent broad antiviral response in Drosophila

An ortholog of STING has been reported to regulate infection by picorna-like viruses in Drosophila In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. This study showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in Atg7 and AGO2, genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. These results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates (Cai, 2020).

Stimulator of interferon genes (STING) provides insect antiviral immunity by promoting Dredd caspase-mediated NF-kappaB activation

The antiviral cGMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway is well characterized in mammalian cells. However, whether this pathway also plays a role in insect antiviral immunity is unknown. This study found that cGAMP is produced in silkworm (Bombyx mori) cells infected with nucleopolyhedrovirus (NPV). In searches for STING-related sequences, BmSTING, a potential cGAMP sensor was identified in B. mori. BmSTING overexpression effectively inhibits NPV replication in silkworm larvae, whereas dsRNA-mediated BmSTING knockdown resulted in higher viral load. Cleavage and nuclear translocation of BmRelish, a NF-kappaB-related transcription factor, was also observed when BmSTING was overexpressed and was enhanced by cGAMP stimulation or viral infection of B. mori larvae. Moreover, a caspase-8-like protein (BmCasp8L) was identified as a BmSTING-interacting molecule and as a suppressor of BmSTING-mediated BmRelish activation. Interestingly, cGAMP stimulation decreased BmCasp8L binding to BmSTING and increased BmRelish activity. Of note, an interaction between death-related ced-3/Nedd2-like caspase (BmDredd) and BmSTING promoted BmRelish cleavage for efficient antiviral signaling and protection of insect cells from viral infection. These findings have uncovered BmSTING as a critical mediator of antiviral immunity in the model insect B. mori and have identified several BmSTING-interacting proteins that control antiviral defenses (Hua, 2018).

The kinase IKKbeta regulates a STING- and NF-kappaB-dependent antiviral response pathway in Drosophila

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. This study showed that two components of the IMD pathway, the kinase dIKKβ and the transcription factor Relish, were required to control infection by two picorna-like viruses. A set of genes induced by viral infection and regulated by dIKKβ and Relish included an ortholog of STING. dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKβ to regulate expression of Nazo, an antiviral factor. These data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity (Goto, 2018).

Inflammation-induced, STING-dependent autophagy restricts Zika virus infection in the Drosophila brain

The emerging arthropod-borne flavivirus Zika virus (ZIKV) is associated with neurological complications. Innate immunity is essential for the control of virus infection, but the innate immune mechanisms that impact viral infection of neurons remain poorly defined. Using the genetically tractable Drosophila system, this study shows that ZIKV infection of the adult fly brain leads to NF-kappaB-dependent inflammatory signaling, which serves to limit infection. ZIKV-dependent NF-kappaB activation induces the expression of Drosophila stimulator of interferon genes (dSTING) in the brain. dSTING protects against ZIKV by inducing autophagy in the brain. Loss of autophagy leads to increased ZIKV infection of the brain and death of the infected fly, while pharmacological activation of autophagy is protective. These data suggest an essential role for an inflammation-dependent STING pathway in the control of neuronal infection and a conserved role for STING in antimicrobial autophagy, which may represent an ancestral function for this essential innate immune sensor (Liu, 2018).

Search PubMed for articles about Drosophila Sting

Akhmetova, K., Balasov, M. and Chesnokov, I. (2021). Drosophila STING protein has a role in lipid metabolism. Elife 10. PubMed ID: 34467853

Bronkhorst, A. W., van Cleef, K. W., Vodovar, N., Ince, I. A., Blanc, H., Vlak, J. M., Saleh, M. C. and van Rij, R. P. (2012). The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery. Proc Natl Acad Sci U S A 109(51): E3604-3613. PubMed ID: 23151511

Burdette, D. L., Monroe, K. M., Sotelo-Troha, K., Iwig, J. S., Eckert, B., Hyodo, M., Hayakawa, Y. and Vance, R. E. (2011). STING is a direct innate immune sensor of cyclic di-GMP. Nature 478(7370): 515-518. PubMed ID: 21947006

Cai, H., Holleufer, A., Simonsen, B., Schneider, J., Lemoine, A., Gad, H. H., Huang, J., Huang, J., Chen, D., Peng, T., Marques, J. T., Hartmann, R., Martins, N. E. and Imler, J. L. (2020). 2'3'-cGAMP triggers a STING- and NF-kappaB-dependent broad antiviral response in Drosophila. Sci Signal 13(660). PubMed ID: 33262294

Du, B. B., Liu, L. and Zhu, Y. Y. (2020). RNA-binding protein Roquin negatively regulates STING-dependent innate immune response in Drosophila. Yi Chuan 42(12): 1201-1210. PubMed ID: 33509784

Goto, A., Okado, K., Martins, N., Cai, H., Barbier, V., Lamiable, O., Troxler, L., Santiago, E., Kuhn, L., Paik, D., Silverman, N., Holleufer, A., Hartmann, R., Liu, J., Peng, T., Hoffmann, J. A., Meignin, C., Daeffler, L. and Imler, J. L. (2018). The Kinase IKKbeta Regulates a STING- and NF-kappaB-Dependent Antiviral Response Pathway in Drosophila. Immunity 49(2): 225-234 e224. PubMed ID: 30119996

Holleufer, A., Winther, K. G., Gad, H. H., Ai, X., Chen, Y., Li, L., Wei, Z., Deng, H., Liu, J., Frederiksen, N. A., Simonsen, B., Andersen, L. L., Kleigrewe, K., Dalskov, L., Pichlmair, A., Cai, H., Imler, J. L. and Hartmann, R. (2021). Two cGAS-like receptors induce antiviral immunity in Drosophila. Nature 597(7874): 114-118. PubMed ID: 34261128

Hua, X., Li, B., Song, L., Hu, C., Li, X., Wang, D., Xiong, Y., Zhao, P., He, H., Xia, Q. and Wang, F. (2018). Stimulator of interferon genes (STING) provides insect antiviral immunity by promoting Dredd caspase-mediated NF-kappaB activation. J Biol Chem 293(30): 11878-11890. PubMed ID: 29875158

Kong, F., Qadeer, A., Xie, Y., Jin, Y., Li, Q., Xiao, Y., She, K., Zheng, X., Li, J., Ji, S. and Zhu, Y. (2023). Dietary Supplementation of Aspirin Promotes Drosophila Defense against Viral Infection. Molecules 28(14). PubMed ID: 37513173

Kranzusch, P. J., Wilson, S. C., Lee, A. S., Berger, J. M., Doudna, J. A. and Vance, R. E. (2015). Ancient Origin of cGAS-STING Reveals Mechanism of Universal 2',3' cGAMP Signaling. Mol Cell 59(6): 891-903. PubMed ID: 26300263

Kumar S., Gu, Y., Abudu, Y. P., Bruun, J. A., Jain, A., Farzam, F., Mudd, M., Anonsen, J. H., Rusten, T. E., Kasof, G., Ktistakis, N., Lidke, K. A., Johansen, T., Deretic, V. (2019). Phosphorylation of Syntaxin 17 by TBK1 Controls Autophagy Initiation. Dev Cell49(1):130-144 e136. PubMed ID: 32060339

Li, D., Xie, L., Qiao, Z., Mai, S., Zhu, J., Zhang, F., Chen, S., Li, L., Shen, F., Qin, Y., Yao, H., He, S. and Ma, F. (2021). STING-mediated degradation of IFI16 negatively regulates apoptosis by inhibiting p53 phosphorylation at serine 392. J Biol Chem 297(2): 100930. PubMed ID: 34216619

Liu, Y., Gordesky-Gold, B., Leney-Greene, M., Weinbren, N. L., Tudor, M. and Cherry, S. (2018). Inflammation-Induced, STING-Dependent Autophagy Restricts Zika Virus Infection in the Drosophila Brain. Cell Host Microbe 24(1): 57-68 e53. PubMed ID: 29934091

Martin, M., Hiroyasu, A., Guzman, R. M., Roberts, S. A. and Goodman, A. G. (2018). Analysis of Drosophila STING Reveals an Evolutionarily Conserved Antimicrobial Function. Cell Rep 23(12): 3537-3550. PubMed ID: 29924997

Rong Y., Zhang, S., Nandi, N., Wu, Z., Li, L., Liu, Y., Wei, Y., Zhao, Y., Yuan, W., Zhou, C., Xiao, G., Levine, B., Yan, N., Mou, S., Deng, L., Tang, Z., Liu, X., Kramer, H., Zhong, Q. (2022). STING controls energy stress-induced autophagy and energy metabolism via STX17. J Cell Biol 221(7). PubMed ID: 34811497

Wu, J., Sun, L., Chen, X., Du, F., Shi, H., Chen, C. and Chen, Z. J. (2013). Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339(6121): 826-830. PubMed ID: 23258412

date revised: 2 December 2021

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