TLR-independent anti-inflammatory function of intestinal epithelial TRAF6 signalling prevents DSS-induced colitis in mice

Abstract

Objective: The gut microbiota modulates host susceptibility to intestinal inflammation, but the cell types and the signalling pathways orchestrating this bacterial regulation of intestinal homeostasis remain poorly understood. Here, we investigated the function of intestinal epithelial toll-like receptor (TLR) responses in the dextran sodium sulfate (DSS)-induced mouse model of colitis.

Design: We applied an in vivo genetic approach allowing intestinal epithelial cell (IEC)-specific deletion of the critical TLR signalling adaptors, MyD88 and/or TIR-domain-containing adapter-inducing interferon-β (TRIF), as well as the downstream ubiquitin ligase TRAF6 in order to reveal the IEC-intrinsic function of these TLR signalling molecules during DSS colitis.

Results: Mice lacking TRAF6 in IECs showed exacerbated DSS-induced inflammatory responses that ensued in the development of chronic colon inflammation. Antibiotic pretreatment abolished the increased DSS susceptibility of these mice, showing that epithelial TRAF6 signalling pathways prevent the gut microbiota from driving excessive colitis. However, in contrast to epithelial TRAF6 deletion, blocking epithelial TLR signalling by simultaneous deletion of MyD88 and TRIF specifically in IECs did not affect DSS-induced colitis severity. This in vivo functional comparison between TRAF6 and MyD88/TRIF deletion in IECs shows that the colitis-protecting effects of epithelial TRAF6 signalling are not triggered by TLRs.

Conclusions: Intestinal epithelial TRAF6-dependent but MyD88/TRIF-independent and, thus, TLR-independent signalling pathways are critical for preventing propagation of DSS-induced colon inflammation by the gut microbiota. Moreover, our experiments using mice with dual MyD88/TRIF deletion in IECs unequivocally show that the gut microbiota trigger non-epithelial TLRs rather than epithelial TLRs to restrict DSS colitis severity.

Keywords: EXPERIMENTAL COLITIS; GUT INFLAMMATION.

Figure 1

TRAF6^IEC-KO mice are more susceptible to dextran sodium sulfate (DSS)-induced acute colitis. (A) Body weight change, (B) rectal bleeding score, and (C) diarrhoea score of TRAF6^FL (n=9) and TRAF6^IEC-KO (n=10) mice administered 2% DSS for 7 days followed by 2 days of normal drinking water. (D) Representative endoscopic pictures and mean endoscopic index of colitis severity of TRAF6^FL and TRAF6^IEC-KO mice after 6 days of DSS treatment. (E) Colon length and (F) representative H&E stained colon cross-sections of TRAF6^FL and TRAF6^IEC-KO mice at day 9 of the DSS colitis protocol. Bars, 100 μm. (G) Histological tissue damage and inflammation scoring at day 9 of the DSS protocol. (H) Representative pictures and (I) mean number±SEM per 200× magnification field of myeloperoxidase (MPO)-stained neutrophils and F4/80-stained macrophages in TRAF6^FL and TRAF6^IEC-KO mice at day 9 of the DSS colitis protocol. Bars, 100 μm. (J) Cytokine and (K) chemokine mRNA induction±SEM in colon of TRAF6^FL and TRAF6^IEC-KO mice at day 9 of the DSS colitis protocol compared to mRNA levels of untreated TRAF6^FL and TRAF6^IEC-KO mice. All data shown are representative of at least 2 independent experiments. All statistical analyses were performed with unpaired two-sided Student's t tests with unequal variance.

Figure 2

TRAF6^IEC-KO mice develop chronic colon inflammation upon dextran sodium sulfate (DSS) treatment. (A) TRAF6^FL (n=8) and TRAF6^IEC-KO (n=9) mice were administered 2% DSS for 6 days followed by normal drinking water with endoscopies at the indicated time points until day 60 when they were sacrificed. (B) Body weight change and final survival at day 60. (C) Mean endoscopic index of colitis severity of TRAF6^FL and TRAF6^IEC-KO mice at indicated time points of the chronic DSS protocol. (D) Representative endoscopic pictures from TRAF6^FL and TRAF6^IEC-KO mice at day 54 of the chronic DSS colitis protocol. (E) Representative H&E stained colon cross-sections of TRAF6^FL and TRAF6^IEC-KO mice at day 60 of the chronic DSS colitis protocol. Bars, 100 μm. (F) Histological tissue damage and inflammation scoring at day 60 of the chronic DSS protocol. All data shown are representative of two independent experiments. All statistical analyses were performed with unpaired two-sided Student's t tests with unequal variance.

Figure 3

Excessive dextran sodium sulfate (DSS)-induced acute colitis in TRAF6^IEC-KO mice is driven by the gut microbiota. TRAF6^FL and TRAF6^IEC-KO mice were treated with antibiotics (+AB) or were left untreated (−AB) for 4 weeks before administration of DSS. (A) Body weight change, (B) rectal bleeding score, and (C) diarrhoea score of TRAF6^FL −AB (n=8), TRAF6^IEC-KO −AB (n=10), TRAF6^FL +AB (n=8), TRAF6^IEC-KO +AB (n=11) mice that were administered 2% DSS for 7 days followed by 2 days of normal drinking water. For A–C, asterisks above the data points indicate statistically significant differences between TRAF6^FL −AB and TRAF6^IEC-KO −AB mice, while asterisks below the data points indicate statistically significant differences between TRAF6^FL +AB and TRAF6^IEC-KO +AB mice. (D) Colon length, (E) mean endoscopic index of colitis severity and (F) representative endoscopic pictures of indicated mice at day 9 of the DSS colitis protocol. (G) Representative H&E stained colon cross-sections of indicated mice, and (H) histological tissue damage and inflammation scoring at day 9 of the DSS colitis protocol. Bars, 100 μm. All data shown are representative of two independent experiments. All statistical analyses were performed with unpaired two-sided Student's t tests with unequal variance.

Figure 4

MyD88^IEC-KO/TRIF^IEC-KO mice are not more susceptible to dextran sodium sulfate (DSS)-induced acute colitis. (A) Body weight change, (B) rectal bleeding score, and (C) diarrhoea score of TRAF6^FL (n=8), TRAF6^IEC-KO (n=8), MyD88^FL/TRIF^FL (n=7) and MyD88^IEC-KO/TRIF^IEC-KO (n=6) mice administered 1% DSS for 7 days followed by 2 days of normal drinking water. For A–C, asterisks indicate statistically significant differences between TRAF6^FL and TRAF6^IEC-KO mice. (D) Colon length, (E) histological tissue damage and inflammation scoring and (F) representative H&E stained colon cross-sections of indicated mice at day 9 of the DSS colitis protocol. Bars, 100 μm. All data shown are representative of two independent experiments. All statistical analyses were performed with unpaired two-sided Student's t tests with unequal variance.