The Interactive Fly

Zygotically transcribed genes

Immunity I

Immune recognition of microbial agents in Drosophila: Toll pathway and immune deficiency (Imd) pathway. The Toll and The immune deficiency (Imd) pathways control inducible immune responses to bacteria and fungi in Drosophila through systemic production of antimicrobial peptides (AMPs). In the Toll pathway, immune recognition activates a proteolytic cascade that culminates in the maturation of the cytokine Spätzle, ultimately leading to the nuclear translocation of the nuclear factor-κB (NF-κB) transcription factor Dif, to induce the expression of AMP genes such as Drosomycin. Activation of the Imd pathway leads to the nuclear translocation of the NF-κB transcription factor Relish to activate the expression of AMP genes such as Diptericin (see Buchon, 2014).

Buchon, N., Silverman, N. and Cherry, S. (2014). Immunity in Drosophila melanogaster--from microbial recognition to whole-organism physiology. Nat Rev Immunol 14: 796-810. PubMed ID: 25421701

Immune Response

  • Development of haemocytes and the lymph gland
  • Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms
  • Sequential activation of signaling pathways during innate immune responses
  • Immunodeficient Drosophila mutants: Constitutive expression of a single antimicrobial peptide can restore wild-type resistence to infection
  • The immune phenotype of three Drosophila leukemia models
  • Tissue- and ligand-specific sensing of gram-negative infection in Drosophila by PGRP-LC isoforms and PGRP-LE
  • Regulation of dual oxidase activity by the Galphaq-phospholipase Cbeta-Ca2+ pathway in Drosophila gut immunity
  • Interaction between familial transmission and a constitutively active immune system shapes gut microbiota in Drosophila melanogaster
  • Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila
  • A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity
  • SLC46 family transporters facilitate cytosolic innate immune recognition of monomeric peptidoglycans
  • Ecdysone mediates the development of immunity in the Drosophila embryo
  • Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster
  • Apoptosis in hemocytes induces a shift in effector mechanisms in the Drosophila immune system and leads to a pro-inflammatory state
  • Convergent balancing selection on an antimicrobial peptide in Drosophila
  • Complex coding and regulatory polymorphisms in a restriction factor determine the susceptibility of Drosophila to viral infection
  • The raspberry gene is involved in the regulation of the cellular immune response in Drosophila melanogaster
  • Leishmania amazonensis engages CD36 to drive parasitophorous vacuole maturation
  • Inhibition of phagocytic killing of Escherichia coli in Drosophila hemocytes by RNA chaperone Hfq
  • Transdifferentiation and proliferation in two distinct hemocyte lineages in Drosophila melanogaster larvae after wasp infection
  • Age and diet affect genetically separable secondary injuries that cause acute mortality following traumatic brain injury in Drosophila
  • The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors
  • Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis
  • NF-κB immunity in the brain determines fly lifespan in healthy aging and age-related neurodegeneration
  • Antimicrobial peptides extend lifespan in Drosophila
  • Functional screening of mammalian mechanosensitive genes using Drosophila RNAi library - Smarcd3/Bap60 is a mechanosensitive pro-inflammatory gene
  • The genetic architecture of defense as resistance to and tolerance of bacterial infection in Drosophila melanogaster
  • A test for Y-linked additive and epistatic effects on surviving bacterial infections in Drosophila melanogaster
  • The distinct function of Tep2 and Tep6 in the immune defense of Drosophila melanogaster against the pathogen Photorhabdus
  • Immune modulation by MANF promotes tissue repair and regenerative success in the retina
  • Bap180/Baf180 is required to maintain homeostasis of intestinal innate immune response in Drosophila and mice
  • Circulating immune cells mediate a systemic RNAi-based adaptive antiviral response in Drosophila
  • Proprotein convertase Furin1 expression in the Drosophila fat body is essential for a normal antimicrobial peptide response and bacterial host defense
  • The Drosophila Thioester containing Protein-4 participates in the induction of the cellular immune response to the pathogen Photorhabdus
  • The selective antifungal activity of Drosophila melanogaster Metchnikowin reflects the species-dependent inhibition of succinate-coenzyme Q reductase

    Immune Deficiency Pathway
  • Tissue-specific regulation of Drosophila NF-κB pathway activation by peptidoglycan recognition protein
  • Cytokine Diedel and a viral homologue suppress the IMD pathway in Drosophila
  • Small RNA-Seq analysis reveals microRNA-regulation of the Imd pathway during Escherichia coli infection in Drosophila
  • UbcD4, an ortholog of E2-25K/Ube2K, is essential for activation of the immune deficiency pathway in Drosophila
  • RNA interference directed against the Transglutaminase gene triggers dysbiosis of gut microbiota in Drosophila

    Toll Pathway
  • The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila
  • Toll receptor-mediated Hippo signaling controls innate immunity in Drosophila
  • MicroRNAs that contribute to coordinating the immune response in Drosophila melanogaster
    Genes involved in the immune response

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

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

    Sequential activation of signaling pathways during innate immune responses

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

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

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

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

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

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

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

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

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

    The signaling requirements for these differentially expressed transcripts were examined in mutant alleles of known Toll and Imd/Rel pathway components, reasoning that additional pathways might be uncovered by analyzing patterns that cannot be reconciled with expected signaling patterns. Flies homozygous for loss-of-function mutations in tube, key, or rel were infected with gram-negative and gram-positive bacteria, and expression profiles were generated for a 6 hr time point after infection. In addition, noninfected Tl10b, a gain-of-function allele of the receptor, and cact, a homolog of the inhibitory factor IkappaB, were used to monitor transcripts that are constitutively expressed in gain-of-function signaling mutants. The antimicrobial peptides dipt and drosomycin (drs) are representative targets for the Toll and Imd/Rel pathways, respectively. dipt induction is not detectable in the expression profiles in either a rel or key mutant background, whereas its expression is not affected in tube mutants. In contrast, drs relies on Tube to convey a Toll-dependent signal. Consistently, the expression profiles show that, in a tube mutant background, drs expression is diminished. These experiments showed that the analysis of mutant expression profiles can be used to deduce signaling requirements for distinct target groups (Boutros, 2002).

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

    Interestingly, some genes do not fit either pattern, suggesting that they are regulated by other pathways. One group of genes, including cytoskeletal factors such as actin88F, flightin, and tpnC41C, is induced in Tl10b, but not in cact, mutants. In contrast, totM and CG11501 are expressed at high levels in cact mutant flies but are not expressed in Tl10b mutant flies. In addition, these transcripts are highly inducible in a tube genetic background, but they are not inducible in key or rel. This may suggest that Toll, Tube, and Cact do not act in a linear pathway under all circumstances. Moreover, rel shows an expression pattern suggesting that it is regulated by both the Imd/Rel and Toll pathways. Thus, these results indicate that, in addition to the canonical Toll and Imd pathways, other signaling events and possibly signaling pathway branching contribute to the complex expression patterns after septic injury. Finally, there is a strong correlation between pathway requirement and temporal expression pattern. Whereas Toll targets are exclusively found in the sustained cluster, Imd/Rel targets are expressed early and transiently after septic injury. The two additional clusters with noncanonical patterns show temporal patterns distinct from either Toll or Imd pathways (Boutros, 2002).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    The immune phenotype of three Drosophila leukemia models

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    Antimicrobial peptides extend lifespan in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    SLC46 family transporters facilitate cytosolic innate immune recognition of monomeric peptidoglycans

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

    Ecdysone mediates the development of immunity in the Drosophila embryo

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

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

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

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

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

    Convergent balancing selection on an antimicrobial peptide in Drosophila

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

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

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

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

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

    Leishmania amazonensis engages CD36 to drive parasitophorous vacuole maturation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    From a pilot screen, one mutation was identified that was named picky eaterZ2–4761 (picky) because the mutants fail to phagocytose S. aureus particles, but can still phagocytose E. coli and Saccharomyces cerevisiae zymosan particles. Only 25% of picky mutant flies showed any phagocytosis response to S. aureus, but 83% of those same flies were still able to phagocytose E. coli. To investigate whether the recognition involved the difference in peptidoglycan between Gram+ and Gram bacteria, phagocytosis was examined of a B. subtilis-GFP strain. picky mutants are able to phagocytose B. subtilis. This finding suggests that the picky gene product is important for the specific recognition of the Lys-type peptidoglycans, typical of most Gram+ bacteria, but not found in Bacillus spp (Garver, 2006).

    Because picky flies appear to be impaired in the recognition of S. aureus for phagocytosis and survival, it was possible that the mutation might also affect activation of the Toll or Imd pathways. The induction of Drosomycin and Diptericin AMP genes is often used to assess activation of the Toll/Gram+ and Imd/Gram signaling pathways, respectively. The AMP responses in picky were compared with those of the known Toll pathway components, seml and Dif, and to an Imd pathway component, ird7. picky flies showed no induction of Drosomycin in response to S. aureus, E. coli, or Micrococcus luteus at either 2 or 24 h. All of the S. aureus-infected picky flies were dead at 24 h, so the Drosomycin expression in this sample was not assessed. seml and Dif both show some induction of Drosomycin at 2 and 24 h. In comparison, picky has a more consistent and dramatic effect on Drosomycin expression and likely encodes an essential component of the Toll pathway. In contrast, the expression of Diptericin in response to bacterial infection in picky mutants was higher than that seen in wild type. Therefore, the defect in picky appears to be selective for the Toll pathway. To determine where in the Toll pathway picky might lie, the picky mutation was crossed into a Toll10b gain of function mutant. picky was not able to suppress the constitutive expression of Drosomycin in a Toll10b mutant. This epistasis analysis places picky either upstream of Toll, which would be consistent with a role in bacterial recognition or in a parallel pathway (Garver, 2006).

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

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

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

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

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

    Toll receptor-mediated Hippo signaling controls innate immunity in Drosophila

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

    MicroRNAs that contribute to coordinating the immune response in Drosophila melanogaster

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


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    Zygotically transcribed genes

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