Post-translational modifications such as ubiquitination play a key role in regulation of inflammatory nuclear factor-κB (NF-κB) signalling. The Drosophila IκB kinase γ (IKKγ) Kenny is a central regulator of the Drosophila Imd pathway responsible for activation of the NF-κB Relish. We found the Drosophila E3 ligase and HOIL-1L interacting protein (HOIP) orthologue linear ubiquitin E3 ligase (LUBEL) to catalyse formation of M1-linked linear ubiquitin (M1-Ub) chains in flies in a signal-dependent manner upon bacterial infection. Upon activation of the Imd pathway, LUBEL modifies Kenny with M1-Ub chains. Interestingly, the LUBEL-mediated M1-Ub chains seem to be targeted both directly to Kenny and to K63-linked ubiquitin chains conjugated to Kenny by DIAP2. This suggests that DIAP2 and LUBEL work together to promote Kenny-mediated activation of Relish. We found LUBEL-mediated M1-Ub chain formation to be required for flies to survive oral infection with Gram-negative bacteria, for activation of Relish-mediated expression of antimicrobial peptide genes and for pathogen clearance during oral infection. Interestingly, LUBEL is not required for mounting an immune response against systemic infection, as Relish-mediated antimicrobial peptide genes can be expressed in the absence of LUBEL during septic injury. Finally, transgenic induction of LUBEL-mediated M1-Ub drives expression of antimicrobial peptide genes and hyperplasia in the midgut in the absence of infection. This suggests that M1-Ub chains are important for Imd signalling and immune responses in the intestinal epithelia, and that enhanced M1-Ub chain formation is able to drive chronic intestinal inflammation in flies.

As several diseases have been linked to dysbiosis of the human intestinal microflora, manipulation of the microbiota has emerged as an exciting new strategy for potentially treating and preventing diseases. However, the human microbiota consists of a plethora of different species, and distinguishing the impact of a specific bacterial species on human health is challenging. In tackling this challenge, the fruit fly Drosophila melanogaster, with its far simpler microbial composition, has emerged as a powerful model for unraveling host-microbe interactions. To study the interplay between the resident commensal microbiome and the host, flies can be made germ-free, or axenic. To elucidate the impact of specific bacteria, axenic flies can then be re-introduced to specific microbial species. In this unit, we provide a step-by-step protocol on how to rear Drosophila melanogaster under axenic conditions and confirm the axenity of flies. © 2018 by John Wiley & Sons, Inc.

Methionine 1 (M1)-linked ubiquitination plays a key role in the regulation of inflammatory nuclear factor-jB (NF-jB) signalling and is important for clearance of pathogen infection in Drosophila melanogaster. M1-linked ubiquitin (M1-Ub) chains are assembled by the linear ubiquitin E3 ligase (LUBEL) in flies. Here, we have studied the role of LUBEL in sterile inflammation induced by different types of cellular stresses. We have found that the LUBEL catalyses formation of M1-Ub chains in response to hypoxic, oxidative and mechanical stress conditions. LUBEL is shown to be important for flies to survive low oxygen conditions and paraquat-induced oxidative stress. This protective action seems to be driven by stress-induced activation of the NF-jB transcription factor Relish via the immune deficiency (Imd) pathway. In addition to LUBEL, the intracellular mediators of Relish activation, including the transforming growth factor activating kinase 1 (Tak1), Drosophila inhibitor of apoptosis (IAP) Diap2, the IjB kinase c (IKKc) Kenny and the initiator caspase Death-related ced-3/Nedd2-like protein (Dredd), but not the membrane receptor peptidoglycan recognition protein (PGRP)-LC, are shown to be required for sterile inflammatory response and survival. Finally, we showed that the stress-induced upregulation of M1-Ub chains in response to hypoxia, oxidative and mechanical stress is also induced in mammalian cells and protects from stress-induced cell death. Taken together, our results suggest that M1-Ub chains are important for NF-jB signalling in inflammation induced by stress conditions often observed in chronic inflammatory diseases and cancer.Abbreviations AMP, antimicrobial peptide; daGal4, daughterless-Gal4; DAMP, danger-associated molecular pattern; Diap, Drosophila inhibitor of apoptosis; Dif, Dorsal-related immunity factor; Dredd, death-related ced-3/Nedd2-like protein; DUB, deubiquitinating enzyme; Fadd, fas-associated death domain; HIF, hypoxia-inducible factor; HOIL-1L, heme-oxidised iron-responsive element-binding protein 2 ubiquitin ligase-1L; HOIP, HOIL-1interacting protein; IKK, IjB kinase; Imd, immune deficiency; LDD, linear ubiquitin chain-determining domain; LUBAC, linear ubiquitin chain assembly complex; LUBEL, linear ubiquitin E3 ligase; NF-jB, nuclear factor j-light-chain enhancer of activated B cells; NZF, npl4 zinc finger; PAMP, pathogen-associated molecular pattern; PGRP, peptidoglycan recognition protein; PIM, PUB-interacting motif; PNG, peptide N-glycanase; PRR, pattern-recognition receptor; PUB, UBA or UBX-containing protein; RBR, RING in between RING; RING, really interesting new gene; SAMP, stress-associated molecular pattern; SHARPIN, SH3 and multiple ankyrin repeat domains protein (SHANK)-associated RBCK1 homology (RH) domain-interacting protein; Sima, similar; Tab2, transforming growth factor-b-activated kinase 1 binding protein 2; Tak1, transforming growth factor activating kinase; TGF, transforming growth factor; TNF, tumour necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; TUB...

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An intact cell death machinery is not only crucial for successful embryonic development and tissue homeostasis, but participates also in the defence against pathogens and contributes to a balanced immune response. Centrally involved in the regulation of both cell death and inflammatory immune responses is the evolutionarily conserved family of cysteine proteases named caspases. The Drosophila melanogaster genome encodes for seven caspases, several of which display dual functions, participating in apoptotic signalling and beyond. Among the Drosophila caspases, the caspase-8 homologue Dredd has a well-characterised role in inflammatory signalling activated by bacterial infections, and functions as a driver of NF-κB-mediated immune responses. Regarding the other Drosophila caspases, studies focusing on tissue-specific immune signalling and host-microbe interactions have recently revealed immunoregulatory functions of the initiator caspase Dronc and the effector caspase Drice. The aim of this review is to give an overview of the signalling cascades involved in the Drosophila humoral innate immune response against pathogens and of their caspase-mediated regulation. Furthermore, the apoptotic role of caspases during antibacterial and antiviral immune activation will be discussed.

The Drosophila IAP protein, Diap2, is a key mediator of NF-κB signalling and innate immune responses. Diap2 is required for both local immune activation, taking place in the epithelial cells of the gut and trachea, and for mounting systemic immune responses in the cells of the fat body. We have found that transgenic expression of Diap2 leads to a spontaneous induction of NF-κB target genes, inducing chronic inflammation in the Drosophila midgut, but not in the fat body. Drice is a Drosophila effector caspase known to interact and form a stable complex with Diap2. We have found that this complex formation induces its subsequent degradation, thereby regulating the amount of Diap2 driving NF-κB signalling in the intestine. Concordantly, loss of Drice activity leads to accumulation of Diap2 and to chronic intestinal inflammation. Interestingly, Drice does not interfere with pathogen-induced signalling, suggesting that it protects from immune responses induced by resident microbes. Accordingly, no inflammation was detected in transgenic Diap2 flies and Drice-mutant flies reared in axenic conditions. Hence, we show that Drice, by restraining Diap2, halts unwanted inflammatory signalling in the intestine.

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AbstractThe Drosophila IAP protein, DIAP2, is a key mediator of NF-κB signalling and is required for immune activation, both locally in the intestinal epithelia and during systemic, fat body-induced responses. We have found that transgenic expression of DIAP2 induces inflammation in the intestine, but not in the fat body, indicating a need for regulating DIAP2 in microbiotic environments. We describe the Drosophila caspase drICE, a known interaction partner of DIAP2, as a regulator of DIAP2 and NF-κB signalling in the intestinal epithelium. drICE acts by cleaving, and interfering with DIAP2’s ability to ubiquitinate and, thereby, activate mediators of the NF-κB pathway. The drICE-cleaved form of DIAP2 is, moreover, unable to induce inflammation during basal conditions in the intestine. Interestingly, cleavage of DIAP2 does not interfere with pathogen-induced signalling, suggesting that drICE protects from immune responses induced by resident microbes. Accordingly, the negative regulatory effect of drICE is lost when rearing flies axenic. Hence, we show that drICE halts unwanted inflammatory signalling in the intestine by forming an inhibitory complex with DIAP2, interfering with the ability of DIAP2 to induce downstream signalling and NF-κB target gene activation.