The Role Of SIRT6 In The Pathogenesis Of Ulcerative Colitis

Background and Specific Aims Ulcerative colitis (UC) is an inflammatory bowel disease characterized by relapsing inflammation leading to chronic injury of the colonic epithelium in a genetically susceptible host. The pathogenesis of UC is still unclear, while nowadays it is widely accepted that the development of UC is associated with alterations in the immune response, barrier function, and metabolic homeostasis of the intestinal epithelium. SIRT6, one member of NAD+-dependent class III deacetylase sirtuin family, participates in multiple physiological and pathophysiological processes such as glucose homeostasis, aging, and inflammation. In this study, thus, we examined (1) whether SIRT6 is localized in the intestinal epithelium, (2) whether ulcerative colitis or induction of experimental colitis affected colonic SIRT6 expression, and if so, (3) whether alteration of SIRT6 contributed to the development of inflammatory injury in the colon.Methods The murine model of dextran sulfate sodium (DSS)-induced colitis was used for in vivo experiments, whereas Young Adult Mouse Colon (YAMC) cell culture model was utilized for in vitro studies. Disruption of SIRT6 in mouse intestinal epithelium was achieved using a Cre-loxP mediated intestinal epithelium-specific gene knockout strategy. The mucosal biopsies were taken from the inflamed sigmoid area of 15 patients with ulcerative colitis, while normal sigmoid biopsies were take from 17 healthy subjects. The open reading frame of full-length mouse SIRT6 was inserted into a pIRES2-ZsGreen1 vector and this plasmid was transfected into YAMC cells to obtain a SIRT6 overexpression cell model. siRNA-mediated knockdown of targeted gene expression was used to silence SIRT6 expression in YAMC cells. Immunofluorescent staining was used for detecting the localization of SIRT6 protein expressed in mice intestines. qRT-PCR, Western blot, RNA-seq and MSD ELISA were used for determining gene expression in human biopsies, mice colons, YAMC cells or mice plasmas.Results SIRT6 gene was found to be constitutively expressing in the mouse colon. With immunofluorescent staining, we revealed nuclear localization of SIRT6 in the base of crypt colonic epithelial cells. Administration of DSS (3.5% in drinking water for 7 days) to wild-type adult C57BL/6J mice induced moderate experimental colitis is associated with down-regulation of SIRT6 protein in the colon. SIRT6 mRNA level was shown to be significantly down-regulated in the biopsies from UC patients compared to those from healthy subjects. Mice with intestinal epithelial-specific knockout of SIRT6 (i.e. SIRT6IEC-KO) are viable and have normal intestinal development. However, SIRT6IEC-KO mice were found to be more susceptible to DSS-induced colitis than their littermates. In in vitro experiments, we found that interferon-y (60 ng/ml) and H2O2 (400μM) directly inhibited SIRT6 protein expression in YAMC cells, and the effect of interferon-γ and H2O2 to inhibit SIRT6 expression are in a dose- and time-dependent manner. Moreover, knockdown of SIRT6 rendered YAMC cells more susceptible to injury induced by various pathological insults, whereas YAMC cells with overexpression of SIRT6 were more resistant to the injury. Using RNA-seq and Western blot analysis, we revealed that TNFa (100 ng/ml) significantly inhibited expression of R-spondin-1 (Rspol) in Sirt6 knockdown but not wild-type YAMC cells. Because Rspol is a critical growth factor for intestinal epithelial cells, we hypothesize that down-regulation of Rspo1 may contribute to the enhanced susceptibility to inflammatory injury in SIRT6 knockdown epithelial cells.Conclusions Our results suggest that SIRT6 plays an important role in protecting intestinal epithelial cells against ulcerative colitis and it may be a potential therapeutic target for inflammatory bowel disease.