Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine

IntroductionStilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1).MethodsTo study how stilbenoid compounds affect inflammatory signaling in vivo, we have utilized the fruit fly, Drosophila melanogaster, as a model system. To induce intestinal inflammation in the fly, we have fed flies with the intestinal irritant dextran sodium sulphate (DSS).ResultsWe found that DSS induces severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. We have taken advantage of the DSS-model to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, we have identified PS and PSMME to be transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila. We have also computationally predicted the putative antagonist binding sites of these compounds at Drosophila TrpA1.DiscussionTaken together, we show that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila

Regulation of Host-Microbe Interactions and Inflammatory Signalling in Drosophila melanogaster

In order to eliminate harmful pathogens, while allowing beneficial microbes to persist, inflammatory signalling in the intestine needs to be carefully regulated. Overactive immune signalling can lead to chronic inflammation and an inflammatory environment is known to promote cancer development. The NF-κB family of transcription factors is a master regulator of inflammatory signalling and aberrant NF-κB signalling is characteristic for chronic inflammatory diseases, such as Crohn’s disease and ulcerative colitis. As the inflammatory response constitutes a complex network in mammalian cells, we take advantage of using Drosophila melanogaster, with a far simpler immune system, as a model organism when studying inflammatory signalling. The aim of this thesis is to elucidate the regulation of intestinal inflammation during basal conditions and to advance the use of Drosophila as a platform for studying host-microbe interactions. In order to investigate inflammatory regulation in the intestine, the fly microbiome needs to be manipulated. We, hence, started out by optimising a detailed protocol for rearing flies germ-free, or axenic. By carefully optimising the dechorionation of Drosophila embryos, sterile fly husbandry and validation of germ-free flies, we were able to successfully rear flies axenic in standard equipped laboratories. To further explore different avenues of modifying the microbiome, we, in a cross-disciplinary effort, designed antimicrobial mesoporous silica nanoparticles and characterised their antimicrobial properties. By using Drosophila, we were able to demonstrate in vivo antimicrobial activity of the designed particle against Escherichia coli, thereby, strengthening the use of Drosophila as a model in nanomedicine and drug development. Finally, to elucidate the regulation of inflammatory signalling in the intestine, we investigated the cellular regulation of Drosophila inhibitor of apoptosis 2 (Diap2), a potent inducer of NF-κB. We found a new role of the Drosophila caspase interleukin 1β-converting enzyme (Drice) as a regulator of inflammatory signalling in the fly gut. Drice acts by inducing the degradation of Diap2, thereby halting downstream NF-κB signalling. By studying the inflammatory phenotypes of the major immunological organs of the fly, we found that Drice acts specifically in the intestine, restraining inflammatory responses induced by commensal bacteria. In summary, the work in this thesis presents a new mode of inflammatory regulation in the Drosophila gut, and highlights the versatility of the fruit fly as a model organism. Due to well-conserved signalling pathways between mammals and Drosophila, research performed in the fly aids in understanding human inflammatory disease development and the interplay between human health, the microbiome and inflammatory signalling.För att främja utvecklingen av symbiotiska förhållanden mellan mikrobiomet och värdorganismen men samtidigt skydda organismen mot sjukdomsframkallande bakterier, måste tarmens immunsignalering regleras noga. En överaktiv inflammationssignalering kan leda till kronisk inflammation och inflammationshärdar har visats gynna utvecklingen av cancerceller. NF-κB-transkriptionsfaktorer är nyckelkomponenter vid aktiveringen av inflammation och en rubbad NF-κB-signalering är kännetecknande för kroniska inflammationssjukdomar så som Crohns sjukdom och ulcerös kolit. Eftersom den inflammatoriska responsen utgör ett komplext nätverk i däggdjursceller, använder vi Drosophila melanogaster, eller bananflugan, med ett mycket enklare immunförsvar, som modellorganism vid inflammationsstudier. Målet med denna avhandling är att belysa de mekanismer som reglerar inflammationssignaleringen i tarmen under basala förhållanden och att främja användningen av Drosophila som en modell för att studera samverkan mellan värdorganismen och mikrobiomet. För att studera hur inflammation regleras i tarmen måste flugans mikrobiom manipuleras. Vi började därmed med att optimera ett detaljerat protokoll som beskriver hur bananflugan kan odlas i sterila förhållanden, eller axeniskt. Genom att optimera dechorioneringen av flugembryon, upprätthållandet av flugor i sterila förhållanden, samt valideringen av axeniska flugor, lyckades vi erhålla sterila flugor i standardutrustade laboratorium. För att utforska andra sätt att manipulera flugans mikrobiom utvecklade vi i ett tvärvetenskapligt samarbete antimikrobiella mesoporösa kiseldioxid nanopartiklar och studerade deras antimikrobiella egenskaper. Med hjälp av Drosophila som modell kunde vi demonstrera antimikrobiell in vivo aktivitet hos nanopartiklarna mot Escherichia coli och därmed stärka rollen av Drosophila som modell vid utvecklingen av nanomedicin. Slutligen, för att utreda den cellulära regleringen av inflammation i flugans tarm på proteinnivå studerade vi hur proteinet Drosophila inhiberare av apoptos2 (Diap2), en stark inducerare av NF-κB, regleras under basala förhållanden. Vi fann en ny roll för kaspaset Drosophila interleukin-1βkonverterande enzym (Drice) i moduleringen av inflammation och Diap2 i flugans tarm. Genom att inducera nedbrytningen av Diap2 hindrar Drice fortskridningen av den inflammatoriska signaleringen aktiverad av kommensaler. Vi har dessutom kunnat påvisa att Drice fungerar specifikt i tarmen och att avsaknad av Drice leder till kronisk tarminflammation, hyperproliferation och dysbios av tarmens mikroflora. Sammanfattningsvis presenterar denna avhandling en ny kaspasmedierad mekanism vid regleringen av inflammation i bananflugans tarm och demonstrerar mångsidigheten hos Drosophila som modellorganism vid studier beträffande mikrobiomet. Tack vare evolutionärt bevarade signaleringsräckor hos Drosophila och däggdjur bidrar forskning som erhållits i flugan till att förstå utvecklingen av inflammatoriska sjukdomar i människan samt samverkan mellan inflammation, mikrobiomet och människans välmående

M1-linked ubiquitination facilitates NF-κB activation and survival during sterile inflammation

Methionine 1 (M1)-linked ubiquitination plays a key role in the regulation of inflammatory nuclear factor-κB (NF-κB) 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-κB 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 IκB kinase γ (IKKγ) 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-κB signalling in inflammation induced by stress conditions often observed in chronic inflammatory diseases and cancer.publishedVersionPeer reviewe

DataSheet_1_Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine.pdf

IntroductionStilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1).MethodsTo study how stilbenoid compounds affect inflammatory signaling in vivo, we have utilized the fruit fly, Drosophila melanogaster, as a model system. To induce intestinal inflammation in the fly, we have fed flies with the intestinal irritant dextran sodium sulphate (DSS).ResultsWe found that DSS induces severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. We have taken advantage of the DSS-model to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, we have identified PS and PSMME to be transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila. We have also computationally predicted the putative antagonist binding sites of these compounds at Drosophila TrpA1.DiscussionTaken together, we show that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila.</p