Toll-mediated airway homeostasis is essential for fly survival upon injection of RasV12-GFP oncogenic cells.

Toll signaling is well known for its pivotal role in the host response against the invasion of external pathogens. Here, we investigate the potential involvement of Toll signaling in the intersection between the host and oncogenic cells. We show that loss of myeloid differentiation factor 88 (Myd88) leads to drastic fly death after the injection of RasV12-GFP oncogenic cells. Transcriptomic analyses show that challenging flies with oncogenic cells or bacteria leads to distinct inductions of Myd88-dependent genes. We note that downregulation of Myd88 in the tracheal system accounts for fly mortality, and ectopic tracheal complementation of Myd88 rescues the survival defect in Myd88 loss-of-function mutants following RasV12-GFP injection. Further, molecular and genetic evidence indicate that Toll signaling modulates fly resistance to RasV12-GFP cells through mediating airway function in a rolled-dependent manner. Collectively, our data indicate a critical role of Toll signaling in tracheal homeostasis and host survival after the injection of oncogenic cells.

mthl1, a potential Drosophila homologue of mammalian adhesion GPCRs, is involved in antitumor reactions to injected oncogenic cells in flies.

Injection of OCs into adult male flies induces a strong transcriptomic response in the host flies featuring in particular genes encoding bona fide G coupled proteins, among which the gene for methuselah like 1 is prominent. The injection is followed after a 3-d lag period, by the proliferation of the oncogenic cells. We hypothesized that through the product of mthl1 the host might control, at least in part, this proliferation as a defense reaction. Through a combination of genetic manipulations of the mthl1 gene (loss of function and overexpression of mthl1), we document that indeed this gene has an antiproliferative effect. Parallel injections of primary embryonic Drosophila cells or of various microbes do not exhibit this effect. We further show that mthl1 controls the expression of a large number of genes coding for chemoreceptors and genes implicated in regulation of development. Of great potential interest is our observation that the expression of the mouse gene coding for the adhesion G-protein-coupled receptor E1 (Adgre1, also known as F4/80), a potential mammalian homologue of mthl1, is significantly induced by B16-F10 melanoma cell inoculation 3 d postinjection in both the bone marrow and spleen (nests of immature and mature myeloid-derived immune cells), respectively. This observation is compatible with a role of this GPCR in the early response to injected tumor cells in mice.

Single Cell Analysis of the Fate of Injected Oncogenic RasV12 Cells in Adult Wild Type Drosophila.

We have injected dish-cultured oncogenic RasV12 cells into adult male flies and analyzed by single cell transcriptomics their destiny within the host after 11 days. We identified in the preinjection samples and in the 11-day postinjection samples in all 16 clusters of cells, of which 5 disappeared during the experiment in the host. The other cell clusters expanded and expressed genes involved in the regulation of cell cycle, metabolism, and development. In addition, three clusters expressed genes related to inflammation and defense. Predominant among these were genes coding for phagocytosis and/or characteristic for plasmatocytes (the fly equivalent of macrophages). A pilot experiment indicated that the injection into flies of oncogenic cells, in which two of most strongly expressed genes had been previously silenced by RNA interference, into flies resulted in a dramatic reduction of their proliferation in the host flies as compared to controls. As we have shown earlier, the proliferation of the injected oncogenic cells in the adult flies is a hallmark of the disease and induces a wave of transcriptions in the experimental flies. We hypothesize that this results from a bitter dialogue between the injected cells and the host, while the experiments presented here should contribute to deciphering this dialogue.

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

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

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

Oncogenic RasV12 cells [A. Simcox et al., PLoS Genet 4, e1000142 (2008)] injected into adult males proliferated massively after a lag period of several days, and led to the demise of the flies after 2 to 3 wk. The injection induced an early massive transcriptomic response that, unexpectedly, included more than 100 genes encoding chemoreceptors of various families. The kinetics of induction and the identities of the induced genes differed markedly from the responses generated by injections of microbes. Subsequently, hundreds of genes were up-regulated, attesting to intense catabolic activities in the flies, active tracheogenesis, and cuticulogenesis, as well as stress and inflammation-type responses. At 11 d after the injections, GFP-positive oncogenic cells isolated from the host flies exhibited a markedly different transcriptomic profile from that of the host and distinct from that at the time of their injection, including in particular up-regulated expression of genes typical for cells engaged in the classical antimicrobial response of Drosophila.

Pharmacological modulation of nucleic acid sensors - therapeutic potential and persisting obstacles.

Nucleic acid sensors, primarily TLR and RLR family members, as well as cGAS-STING signalling, play a critical role in the preservation of cellular and organismal homeostasis. Accordingly, deregulated nucleic acid sensing contributes to the origin of a diverse range of disorders, including infectious diseases, as well as cardiovascular, autoimmune and neoplastic conditions. Accumulating evidence indicates that normalizing aberrant nucleic acid sensing can mediate robust therapeutic effects. However, targeting nucleic acid sensors with pharmacological agents, such as STING agonists, presents multiple obstacles, including drug-, target-, disease- and host-related issues. Here, we discuss preclinical and clinical data supporting the potential of this therapeutic paradigm and highlight key limitations and possible strategies to overcome them.

The Kinase IKKß Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila.

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKß and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKß and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKß to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.

Analysis of the Contribution of Hemocytes and Autophagy to Drosophila Antiviral Immunity.

UNLABELLED: Antiviral immunity in the model organism Drosophila melanogaster involves the broadly active intrinsic mechanism of RNA interference (RNAi) and virus-specific inducible responses. Here, using a panel of six viruses, we investigated the role of hemocytes and autophagy in the control of viral infections. Injection of latex beads to saturate phagocytosis, or genetic depletion of hemocytes, resulted in decreased survival and increased viral titers following infection with Cricket paralysis virus (CrPV), Flock House virus (FHV), and vesicular stomatitis virus (VSV) but had no impact on Drosophila C virus (DCV), Sindbis virus (SINV), and Invertebrate iridescent virus 6 (IIV6) infection. In the cases of CrPV and FHV, apoptosis was induced in infected cells, which were phagocytosed by hemocytes. In contrast, VSV did not trigger any significant apoptosis but we confirmed that the autophagy gene Atg7 was required for full virus resistance, suggesting that hemocytes use autophagy to recognize the virus. However, this recognition does not depend on the Toll-7 receptor. Autophagy had no impact on DCV, CrPV, SINV, or IIV6 infection and was required for replication of the sixth virus, FHV. Even in the case of VSV, the increases in titers were modest in Atg7 mutant flies, suggesting that autophagy does not play a major role in antiviral immunity in Drosophila Altogether, our results indicate that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in insects. IMPORTANCE: Phagocytosis and autophagy are two cellular processes that involve lysosomal degradation and participate in Drosophila immunity. Using a panel of RNA and DNA viruses, we have addressed the contribution of phagocytosis and autophagy in the control of viral infections in this model organism. We show that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in Drosophila This work brings to the front a novel facet of antiviral host defense in insects, which may have relevance in the control of virus transmission by vector insects or in the resistance of beneficial insects to viral pathogens.

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

Viruses are obligatory intracellular parasites that suffer strong evolutionary pressure from the host immune system. Rapidly evolving viral genomes can adapt to this pressure by acquiring genes that counteract host defense mechanisms. For example, many vertebrate DNA viruses have hijacked cellular genes encoding cytokines or cytokine receptors to disrupt host cell communication. Insect viruses express suppressors of RNA interference or apoptosis, highlighting the importance of these cell intrinsic antiviral mechanisms in invertebrates. Here, we report 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). We show that die mutant flies 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. Our results reveal an endogenous pathway that is exploited by insect viruses to modulate NF-κB signaling and promote fly survival during the antiviral response.

RACK1 controls IRES-mediated translation of viruses.

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

Drosophila C virus systemic infection leads to intestinal obstruction.

UNLABELLED: Drosophila C virus (DCV) is a positive-sense RNA virus belonging to the Dicistroviridae family. This natural pathogen of the model organism Drosophila melanogaster is commonly used to investigate antiviral host defense in flies, which involves both RNA interference and inducible responses. Although lethality is used routinely as a readout for the efficiency of the antiviral immune response in these studies, virus-induced pathologies in flies still are poorly understood. Here, we characterize the pathogenesis associated with systemic DCV infection. Comparison of the transcriptome of flies infected with DCV or two other positive-sense RNA viruses, Flock House virus and Sindbis virus, reveals that DCV infection, unlike those of the other two viruses, represses the expression of a large number of genes. Several of these genes are expressed specifically in the midgut and also are repressed by starvation. We show that systemic DCV infection triggers a nutritional stress in Drosophila which results from intestinal obstruction with the accumulation of peritrophic matrix at the entry of the midgut and the accumulation of the food ingested in the crop, a blind muscular food storage organ. The related virus cricket paralysis virus (CrPV), which efficiently grows in Drosophila, does not trigger this pathology. We show that DCV, but not CrPV, infects the smooth muscles surrounding the crop, causing extensive cytopathology and strongly reducing the rate of contractions. We conclude that the pathogenesis associated with systemic DCV infection results from the tropism of the virus for an important organ within the foregut of dipteran insects, the crop. IMPORTANCE: DCV is one of the few identified natural viral pathogens affecting the model organism Drosophila melanogaster. As such, it is an important virus for the deciphering of host-virus interactions in insects. We characterize here the pathogenesis associated with DCV infection in flies and show that it results from the tropism of the virus for an essential but poorly characterized organ in the digestive tract, the crop. Our results may have relevance for other members of the Dicistroviridae, some of which are pathogenic to beneficial or pest insect species.

Akirin2 is critical for inducing inflammatory genes by bridging IκB-ζ and the SWI/SNF complex.

Transcription of inflammatory genes in innate immune cells is coordinately regulated by transcription factors, including NF-κB, and chromatin modifiers. However, it remains unclear how microbial sensing initiates chromatin remodeling. Here, we show that Akirin2, an evolutionarily conserved nuclear protein, bridges NF-κB and the chromatin remodeling SWI/SNF complex by interacting with BRG1-Associated Factor 60 (BAF60) proteins as well as IκB-ζ, which forms a complex with the NF-κB p50 subunit. These interactions are essential for Toll-like receptor-, RIG-I-, and Listeria-mediated expression of proinflammatory genes including Il6 and Il12b in macrophages. Consistently, effective clearance of Listeria infection required Akirin2. Furthermore, Akirin2 and IκB-ζ recruitment to the Il6 promoter depend upon the presence of IκB-ζ and Akirin2, respectively, for regulation of chromatin remodeling. BAF60 proteins were also essential for the induction of Il6 in response to LPS stimulation. Collectively, the IκB-ζ-Akirin2-BAF60 complex physically links the NF-κB and SWI/SNF complexes in innate immune cell activation. By recruiting SWI/SNF chromatin remodellers to IκB-ζ, transcriptional coactivator for NF-κB, the conserved nuclear protein Akirin2 stimulates pro-inflammatory gene promoters in mouse macrophages during innate immune responses to viral or bacterial infection.

The chromatin regulator DMAP1 modulates activity of the nuclear factor B (NF-B) transcription factor Relish in the Drosophila innate immune response.

The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-B family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-B protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling.

Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge.

The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.

big bang gene modulates gut immune tolerance in Drosophila.

Chronic inflammation of the intestine is detrimental to mammals. Similarly, constant activation of the immune response in the gut by the endogenous flora is suspected to be harmful to Drosophila. Therefore, the innate immune response in the gut of Drosophila melanogaster is tightly balanced to simultaneously prevent infections by pathogenic microorganisms and tolerate the endogenous flora. Here we describe the role of the big bang (bbg) gene, encoding multiple membrane-associated PDZ (PSD-95, Discs-large, ZO-1) domain-containing protein isoforms, in the modulation of the gut immune response. We show that in the adult Drosophila midgut, BBG is present at the level of the septate junctions, on the apical side of the enterocytes. In the absence of BBG, these junctions become loose, enabling the intestinal flora to trigger a constitutive activation of the anterior midgut immune response. This chronic epithelial inflammation leads to a reduced lifespan of bbg mutant flies. Clearing the commensal flora by antibiotics prevents the abnormal activation of the gut immune response and restores a normal lifespan. We now provide genetic evidence that Drosophila septate junctions are part of the gut immune barrier, a function that is evolutionarily conserved in mammals. Collectively, our data suggest that septate junctions are required to maintain the subtle balance between immune tolerance and immune response in the Drosophila gut, which represents a powerful model to study inflammatory bowel diseases.

Broad RNA interference-mediated antiviral immunity and virus-specific inducible responses in Drosophila.

The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific.

Drosophila EYA regulates the immune response against DNA through an evolutionarily conserved threonine phosphatase motif.

Innate immune responses against DNA are essential to counter both pathogen infections and tissue damages. Mammalian EYAs were recently shown to play a role in regulating the innate immune responses against DNA. Here, we demonstrate that the unique Drosophila eya gene is also involved in the response specific to DNA. Haploinsufficiency of eya in mutants deficient for lysosomal DNase activity (DNaseII) reduces antimicrobial peptide gene expression, a hallmark for immune responses in flies. Like the mammalian orthologues, Drosophila EYA features a N-terminal threonine and C-terminal tyrosine phosphatase domain. Through the generation of a series of mutant EYA fly strains, we show that the threonine phosphatase domain, but not the tyrosine phosphatase domain, is responsible for the innate immune response against DNA. A similar role for the threonine phosphatase domain in mammalian EYA4 had been surmised on the basis of in vitro studies. Furthermore EYA associates with IKKß and full-length RELISH, and the induction of the IMD pathway-dependent antimicrobial peptide gene is independent of SO. Our data provide the first in vivo demonstration for the immune function of EYA and point to their conserved immune function in response to endogenous DNA, throughout evolution.

Pillars article: the dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996. 86: 973-983.

The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, spätzle regulatory gene cassette, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways.