Drosophila Immunity Research Articles

Progress and Prospect of Dscam Gene in Drosophila Immunity

The cell adhesion molecule (Dscam) gene in Down syndrome is an extraordinary example of diversity: by combining alternative splicing exons, thousands of subtypes can be generated from just one gene. So far, this diversity of the gene has only been found in insects and crustaceans, and the basic part of neural connections has been well characterized as Drosophila melanogaster. More than a decade ago, evidence from Drosophila melanogaster indicated that the Dscam gene is involved in insect immune defense. However, we are still far from fully understanding the functions of Dscam in parasites and pathogens, as well as its full correlation with the immune system. In this article, we first briefly introduce the immune mechanism of fruit flies and the current research status of Dscam. Finally, the future research prospects of Dscam are summarized and proposed, with the aim of providing a multi perspective perspective to further study the relationship between this gene and immune defense in detail.

The circular RNA circATP8B(2) regulates ROS production and antiviral immunity in Drosophila

We identified and validated a collection of circular RNAs (circRNAs) in Drosophila melanogaster. We show that depletion of the pro-viral circRNA circATP8B(2), but not its linear siblings, compromises viral infection both in cultured Drosophila cells and invivo. In addition, circATP8B(2) is enriched in the fly gut, and gut-specific depletion of circATP8B(2) attenuates viral replication in an oral infection model. Furthermore, circATP8B(2) depletion results in increased levels of reactive oxygen species (ROS) and enhanced expression of dual oxidase (Duox), which produces ROS. Genetic and pharmacological manipulations of circATP8B(2)-depleted flies that reduce ROS levels rescue the viral replication defects elicited by circATP8B(2) depletion. Mechanistically, circATP8B(2) associates with Duox, and circATP8B(2)-Duox interaction is crucial for circATP8B(2)-mediated modulation of Duox activity. In addition, Gαq, a G protein subunit required for optimal Duox activity, acts downstream of circATP8B(2). We conclude that circATP8B(2) regulates antiviral defense by modulating Duox expression and Duox-dependent ROS production.

Mitochondrial perturbation in immune cells enhances cell-mediated innate immunity in Drosophila

BackgroundMitochondria participate in various cellular processes including energy metabolism, apoptosis, autophagy, production of reactive oxygen species, stress responses, inflammation and immunity. However, the role of mitochondrial metabolism in immune cells and tissues shaping the innate immune responses are not yet fully understood. We investigated the effects of tissue-specific mitochondrial perturbation on the immune responses at the organismal level. Genes for oxidative phosphorylation (OXPHOS) complexes cI-cV were knocked down in the fruit fly Drosophila melanogaster, targeting the two main immune tissues, the fat body and the immune cells (hemocytes).ResultsWhile OXPHOS perturbation in the fat body was detrimental, hemocyte-specific perturbation led to an enhanced immunocompetence. This was accompanied by the formation of melanized hemocyte aggregates (melanotic nodules), a sign of activation of cell-mediated innate immunity. Furthermore, the hemocyte-specific OXPHOS perturbation induced immune activation of hemocytes, resulting in an infection-like hemocyte profile and an enhanced immune response against parasitoid wasp infection. In addition, OXPHOS perturbation in hemocytes resulted in mitochondrial membrane depolarization and upregulation of genes associated with the mitochondrial unfolded protein response.ConclusionsOverall, we show that while the effects of mitochondrial perturbation on immune responses are highly tissue-specific, mild mitochondrial dysfunction can be beneficial in immune-challenged individuals and contributes to variation in infection outcomes among individuals.

The role of immunological signaling in disrupted proteostasis in the gut‐brain axis

AbstractBackgroundThe pathophysiological mechanism underlying the progressive neurodegenerative process in Alzheimer’s disease (AD) is not well understood. Hence, there is no disease‐modifying treatment. It is then imperative to identify the triggering molecular factors of the disease and the mechanisms supporting molecular spreading of toxic assemblies. Our search for these key clues in the periphery is founded on the fact that peripheral dysfunction, particularly in the gastrointestinal (GI) system, can arise decades before the onset of the classic symptoms in neurodegenerative disorders. In fact, the risk of AD increases significantly in patients with inflammatory bowel disease (IBD) when compared with the non‐IBD population. Furthermore, Tau accumulates in Crohn’s Disease guts and in sigmoid colon of AD and FTD patients. Thus, we are investigating the physiological and molecular mechanisms that support Tau spreading in the prodromal stages, particularly, along the gut‐brain axis.MethodTo test whether corrupted proteins expressed in the gut will spread their pathology to the brain, we employed the Gal4/UAS system and expressed wild‐type or mutant human Tau in selected populations of gut epithelium cells. We then monitor behavior on aging flies expressing Tau in the gut epithelium using Drosophila activity monitors and analyzed their circadian patterns of activity along with several sleep parameters.ResultWe observed that flies selectively expressing full‐length‐2N4R human Tau in enteroendocrine cells of the gut exhibited a significant impairment in their daily activity pattern that specifically affected their behavior during the light phase (day) but not during the dark period (night). Interestingly, these flies also showed an increased sleepiness during the day without affecting their nocturnal sleep pattern.ConclusionOur results support the presence of spreading mechanisms for Tau from gut epithelial cells to the brain; hence, affecting the neuronal physiology that sustain daily activity patterns. We are now investigating the pathways and mechanisms that pathological Tau in the gut employs to target these brain regions responsible for the behavioral phenotypes: transsynaptic spreading or humoral signaling via the innate immune system. We are dissecting the 3 major pathways of innate immunity in Drosophila (Imd‐Toll‐TNF) for their role in disease progression and the involvement of their target antimicrobial peptides.

The adaptor protein 14-3-3zeta modulates intestinal immunity and aging in Drosophila

The proteins that coordinate the complex transcriptional networks of aging have not been completely documented. Protein 14-3-3zeta is an adaptor protein that coordinates signaling and transcription factor networks, but its function in aging is not fully understood. Here, we showed that the protein expression of 14-3-3zeta gradually increased during aging. High levels of 14-3-3zeta led to shortened lifespan and imbalance of intestinal immune homeostasis in Drosophila, but the decrease in 14-3-3zeta protein levels by RNAi was able to significantly promote the longevity and intestinal immune homeostasis of fruit flies. Importantly, we demonstrate that adult-onset administration of TIC10, a compound that reduces the aging-related AKT and extracellular signal-regulated kinase (ERK) signaling pathways, rescues the shortened lifespan of 14-3-3zeta-overexpressing flies. This funding suggests that 14-3-3zeta plays a critical role in regulating the aging process. Our study elucidates the role of 14-3-3zeta in natural aging and provides the rationale for subsequent 14-3-3zeta-based antiaging research.

CGAS-like receptors: back to the future

Recent studies have characterized ancient forms of cyclic GMP-AMP (cGAMP) synthase (cGAS)-like receptors (cGLRs) in bacterial and Drosophila immunity. Using bioinformatics and biochemical screening, Li et al. recently constructed and characterized >3000 cGLRs to reveal conserved mechanisms of nucleic acid sensing across animal immunity.

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). Our recent studies in Drosophila have shown that Prxs play a critical role in aging and immunity. We 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. Here, we 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, we 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.

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. In this study, we 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.

Methotrexate suppresses psoriatic skin inflammation by inhibiting muropeptide transporter SLC46A2 activity

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here, we focused on Slc46a2, which was highly expressed in mammalian epidermal keratinocytes, and showed that it was critical for the delivery of diaminopimelic acid (DAP)-muropeptides and activation of NOD1 in keratinocytes, whereas the related transporter Slc46a3 was critical for delivering the NOD2 ligand MDP to keratinocytes. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, whereas methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively, these studies define SLC46A2 as a transporter of NOD1-activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

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. Despite clinical relevance and extensive use in Drosophila immunity research, little is known about Providencia virulence mechanisms and the corresponding insect host defenses. To close this knowledge gap, we investigated the virulence factors of a representative Providencia species-P. alcalifaciens which is highly virulent to fruit flies and amenable to genetic manipulations. We generated a P. alcalifaciens transposon mutant library and performed an unbiased forward genetics screen in vivo for attenuated mutants. Our 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, we 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. Our 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. Thus, our work revealed a host-pathogen conflict mediated by ROS and AMPs.

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. Here, we show 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 we name ‘Buletin’. Using mutations differently affecting Drosocin and Buletin, we show that only Drosocin contributes to Drosocin gene-mediated defence against Enterobacter cloacae. Strikingly, we 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. Our 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.

Jumu is required for the activation of JAK/STAT in Drosophila lymph gland development and epidermal wounds

The regulatory mechanism of hematopoiesis and innate immunity in Drosophila is highly similar to that in mammals, and Drosophila has become a suitable model to understand vertebrate hematopoiesis and the immune response. JAK-STAT signaling pathway components are widely conserved during evolution, and contribute to hematopoiesis and multiple tissue damage and immune responses. Here, we demonstrate that Stat92E is widely expressed in the lymph gland, and the loss of jumu inhibits the maintenance of the JAK/STAT pathway in the CZ and MZ but not in the PSC of the lymph gland. Furthermore, we found that clean puncture wounding of the larval epidermis can lead to the activation of JAK/STAT signaling and the generation of lamellocytes, and Jumu is required for the activation of JAK/STAT in response to epidermal wounds.

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. Here, we report 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. We also identify immune genes whose expression after infection differs between fertile and germline‐less flies in a manner that is conditional on their mating status. We conclude that germline activity strongly impedes the expression and inducibility of immune genes and that this physiological trade‐off might be evolutionarily conserved.

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. In this study, we firstly discover 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, our results demonstrate that lncRNA-CR11538 can interact with transcription factors Dif/Dorsal in the nucleus based on both subcellular localization and RIP analyses. Thirdly, our 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 (w1118) 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, our 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.

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. Here we show 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, we 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 we 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. Our 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.

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 prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-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. Our 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.

Proteomics of purified lamellocytes from Drosophila melanogaster HopTum-l identifies new membrane proteins and networks involved in their functions

In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the HopTum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity.

PGRP-LB: An Inside View into the Mechanism of the Amidase Reaction.

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.

The Impact of Age on Response to Infection in Drosophila.

This review outlines the known cellular pathways and mechanisms involved in Drosophila age-dependent immunity to pathogenic microorganisms such as bacteria and fungi. We discuss the implication of host signaling pathways such as the Toll, Immune Deficiency (IMD), Janus kinase signal transducer and activator of transcription (JAK/STAT), and Insulin/Insulin Growth Factor/Target of Rapamycin (IIS/TOR) on immune function with aging. Additionally, we review the effects that factors such as sexual dimorphism, environmental stress, and cellular physiology exert on age-dependent immunity in Drosophila. We discuss potential tradeoffs between heightened immune function and longevity in the absence of infection, and we provide detailed tables outlining the various assays and pathogens used in the cited studies, as well as the age, sex, and strains of Drosophila used. We also discuss the overlapping effects these pathways and mechanisms have on one another. We highlight the great utility of Drosophila as a model organism and the importance of a greater focus on age-dependent antiviral immunity for future studies.

The S1A protease family members CG10764 and CG4793 regulate cellular immunity in Drosophila.

In nature, Drosophila melanogaster larvae are infected by parasitoid wasps and mount a cellular immune response to this infection. Several conserved signaling pathways have been implicated in coordinating this response, however our understanding of the integration and regulation of these pathways is incomplete. Members of the S1A serine protease family have been previously linked to immune functions, and our findings suggest roles for two S1A family members, CG10764 and CG4793 in the cellular immune response to parasitoid infection.