Experimental colitis ORIGINAL ARTICLE TLR-independent anti-inflammatory function Gut: first published as 10.1136/gutjnl-2014-308323 on 11 March 2015. Downloaded from of intestinal epithelial TRAF6 signalling prevents DSS-induced colitis in mice Katerina Vlantis,1,2,3 Apostolos Polykratis,1,2,3 Patrick-Simon Welz,1,2,3,4 Geert van Loo,5,6 Manolis Pasparakis,1,2,3 Andy Wullaert1,2,3,7,8 ▸ Additional material is ABSTRACT published online only. To view Objective The gut microbiota modulates host Significance of this study please visit the journal online fl (http://dx.doi.org/10.1136/ susceptibility to intestinal in ammation, but the cell gutjnl-2014-308323). types and the signalling pathways orchestrating this bacterial regulation of intestinal homeostasis remain What is already known on this subject? poorly understood. Here, we investigated the function of ▸ fi Studies in germfree mice showed that the gut For numbered af liations see intestinal epithelial toll-like receptor (TLR) responses in end of article. microbiota has a protective role during dextran the dextran sodium sulfate (DSS)-induced mouse model sodium sulfate (DSS) colitis in mice. Correspondence to of colitis. ▸ Gene targeting in mice showed that the toll- Dr Andy Wullaert, Department Design We applied an in vivo genetic approach like receptor (TLR) signalling molecule, MyD88, of Medical Protein Research, allowing intestinal epithelial cell (IEC)-specific deletion of mediates protective effects during DSS colitis. VIB; Department of ▸ Biochemistry, Ghent University, the critical TLR signalling adaptors, MyD88 and/or TIR- Bone marrow transplant experiments showed Technologiepark 927, 9052 domain-containing adapter-inducing interferon-β (TRIF), that MyD88/TRIF-mediated TLR signalling acts Ghent, Belgium; as well as the downstream ubiquitin ligase TRAF6 in in non-haematopoietic cells to protect mice [email protected] order to reveal the IEC-intrinsic function of these TLR from DSS colitis. Dr Manolis Pasparakis, CECAD signalling molecules during DSS colitis. ▸ fi Research Center, Institute for Distinct studies applying cell-type-speci c genetic Genetics, University of Results Mice lacking TRAF6 in IECs showed modulation of MyD88 showed that protective Cologne, Joseph-Stelzmann-Str. exacerbated DSS-induced inflammatory responses that TLR signalling acts in intestinal epithelial cells 26, D50931 Cologne, ensued in the development of chronic colon (IEC), in B cells and in myeloid cells. Germany; inflammation. Antibiotic pretreatment abolished the [email protected] increased DSS susceptibility of these mice, showing that What are the new findings? ▸ fi KV and AP authors share first epithelial TRAF6 signalling pathways prevent the gut Mice with IEC-speci c MyD88/TRIF deletion http://gut.bmj.com/ authorship. microbiota from driving excessive colitis. However, in display unaltered DSS colitis severity, MP and AW authors share contrast to epithelial TRAF6 deletion, blocking epithelial unequivocally showing that the microbiota senior authorship. TLR signalling by simultaneous deletion of MyD88 and modulate colon inflammation by triggering fi TLRs residing on non-epithelial cells rather than Received 27 August 2014 TRIF speci cally in IECs did not affect DSS-induced colitis Revised 8 January 2015 severity. This in vivo functional comparison between on IECs. Accepted 17 February 2015 TRAF6 and MyD88/TRIF deletion in IECs shows that the ▸ Mice with IEC-specific TRAF6 deletion display Published Online First increased DSS colitis severity, showing that colitis-protecting effects of epithelial TRAF6 signalling on October 1, 2021 by guest. Protected copyright. 11 March 2015 are not triggered by TLRs. instead of TLR signalling, TLR-independent TRAF6 Conclusions Intestinal epithelial TRAF6-dependent but signalling in IECs limits acute DSS colitis. MyD88/TRIF-independent and, thus, TLR-independent ▸ Microbiota depletion abolished excessive colitis signalling pathways are critical for preventing in mice lacking intestinal epithelial TRAF6, propagation of DSS-induced colon inflammation by the demonstrating that epithelial TRAF6 signalling gut microbiota. Moreover, our experiments using mice limits DSS colitis by preventing the gut with dual MyD88/TRIF deletion in IECs unequivocally microbiota from exacerbating colon inflammation show that the gut microbiota trigger non-epithelial TLRs after DSS-induced epithelial damage. rather than epithelial TLRs to restrict DSS colitis severity. ▸ These findings establish a model of intestinal- immune homeostasis in which epithelial Open Access Scan to access more TLR-independent TRAF6 signalling prevents the free content microbiota from propagating colitis, while INTRODUCTION bacteria that invade the mucosa trigger TLRs on The maintenance of intestinal homeostasis depends non-epithelial cells to restrain colitis severity. on a tightly regulated cross-talk between the gut microbiota, intestinal epithelial cells (IEC) and mucosalimmuneandstromalcells.Eventhough in this model of colon inflammation. Consistent with deregulated immune responses to the gut microbiota this observation, mice lacking individual toll-like are thought to contribute to the development of receptors (TLR), or the critical TLR-signalling mol- To cite: Vlantis K, IBDs,1 germfree mice were shown to be more suscep- ecule, MyD88, showed increased sensitivity to DSS Polykratis A, Welz P-S, et al. tible to dextran sodium sulfate (DSS)-induced colitis, suggesting that microbiota-induced MyD88- – Gut 2016;65:935 943. colitis,2 suggesting a beneficial role for the microbiota dependent TLR responses protect from DSS-induced Vlantis K, et al. Gut 2016;65:935–943. doi:10.1136/gutjnl-2014-308323 935 Experimental colitis Villin promoter.18 These TRAF6IEC-KO mice showed efficient Significance of this study deletion of TRAF6 in IECs, but endoscopic and histological examination did not reveal any colon or small intestinal abnor- Gut: first published as 10.1136/gutjnl-2014-308323 on 11 March 2015. Downloaded from malities (see online supplementary figure S1), demonstrating How might it impact on clinical practice in the that epithelial TRAF6 does not control intestinal immune foreseeable future? homeostasis under basal conditions. ▸ The alterations in microbiota composition observed in To assess whether epithelial TRAF6 regulates colon homeosta- patients with IBD demonstrate the need for delineating the sis under conditions of inflammation, we treated TRAF6IEC-KO effects of different microbiota and the signalling pathways mice and their TRAF6FL control littermates for 7 days with 2% they induce in distinct intestinal cell types in order to DSS in the drinking water followed by 2 days recovery on normal understand the impact of such microbiota changes on IBD drinking water, after which we sacrificed the mice on day pathogenesis. Our results will direct future clinical research 9. Starting from day 5 of DSS treatment, TRAF6IEC-KO mice lost in this area towards identifying the intestinal bacteria that significantly more weight than control animals, indicating that trigger TLR-mediated beneficial effects in non-epithelial epithelial TRAF6 deficiency sensitised mice to DSS-induced intestinal cells. colitis (figure 1A). Consistent with the increased weight loss, TRAF6IEC-KO mice showed increased intestinal bleeding and suf- fered from more severe diarrhoea (figure 1B, C). Endoscopic – colon inflammation.3 7 Together with genetic mouse models analysis on day 6 revealed enhanced colon inflammation in showing that epithelial NF-κB activation prevents spontaneous TRAF6IEC-KO mice compared with their TRAF6FL littermates, as – and DSS-induced colon inflammation,8 11 the studies above col- indicated by a less translucent and more granular colonic wall lectively raised the possibility that bacteria might directly trigger and signs of diarrhoea (figure 1D). Moreover, TRAF6IEC-KO mice epithelial TLR/MyD88-dependent NF-κB activation for control- sacrificed on day 9 revealed considerably shorter colons com- ling intestinal inflammation.12 pared to their littermate controls (figure 1E). Histopathological This plausible hypothesis was questioned by an elegant study examination of colon sections showed more severe epithelial showing that mice lacking MyD88 specifically in B cells, but not erosion, loss of goblet cells and areas of mucosal ulceration, as mice lacking MyD88 in IECs, display increased lethality in well as increased numbers of infiltrating mucosal and submucosal response to DSS treatment.13 Additionally, restricted expression leukocytes in TRAF6IEC-KO mice compared with their control lit- of MyD88 in myeloid cells was shown to abolish the susceptibil- termates, resulting in higher histological scores for both tissue ity of MyD88-deficient mice to DSS colon injury.14 These obser- damage and inflammation (figure 1F, G). Immunostaining of vations indicated that MyD88-mediated protective effects in colon sections with specific antibodies revealed elevated DSS colitis originate from B cells and myeloid cells rather than numbers of infiltrating neutrophils as well as macrophages in epithelial cells. On the other hand, another study observed that TRAF6IEC-KO colons compared to their TRAF6FL littermates IEC-specific ablation of MyD88 compromised epithelial barrier (figure 1H, I), which was accompanied by enhanced expression function, and as such, did sensitise mice to DSS colitis,15 of several inflammatory cytokines and chemokines including arguing in favour of epithelial-intrinsic TLR/MyD88-driven pro- tumour necrosis factor (TNF), IL-1β, CCL3