Project description:Gut dysbiosis and host genetics are implicated as causative factors in inflammatory bowel disease, yet mechanistic insights are lacking. Longitudinal analysis of ulcerative colitis patients following total colectomy with ileal anal anastomosis (IPAA) where >50% develop pouchitis, offers a unique setting to examine cause vs. effect. To recapitulate human IPAA, we employed a mouse model of surgically-created blind self-filling (SFL) and self-emptying (SEL) ileal loops. SFL exhibit fecal stasis due to directional peristalsis motility oriented towards away from the loop end, whereas SEL remain empty. In wild type mice, SFL, but not SEL, develop pouch-like microbial communities without accompanying active inflammation. However, in genetically susceptible IL-10-/- deficient mice, SFL, but not SEL, exhibit severe inflammation and mucosal transcriptomes resembling human pouchitis. Germ-free IL10-/- mice conventionalized with wild type SFL, but not SEL, microbiota, develop severe colitis. These data demonstrate an essential role for fecal stasis, gut dysbiosis, and genetic susceptibility and offer insights into human pouchitis and ulcerative colitis. All animal protocols were approved by IACUC at the University of Chicago. All animals were C57Bl/6 mice that were bred and housed under standard 12:12 light/dark conditions at the University of Chicago. Female mice aged 6-8 weeks were fed ad libitum gel diet 76A (Cat# 72-07-5022, Clear H20, Portland, ME) for 5-days prior to surgery to prevent obstruction at the anastomosis. Animals were anesthetized with ketamine/xylazine. Aseptic surgery was performed to resect 2.5cm of ileum 3cm proximal to the ileal-cecal value with anastomosis to the ileum using 8-0 suture (Figure 1a). The abdominal wall was closed with interrupted 4-0 silk suture and skin was closed with staples. Analgesics (betanorphine mg/kg BW) were provided post-operatively. After 5 weeks, mice were humanely euthanized. Intestinal loops were collected for RNA, protein, and histology. Loop, sham ileum, and sham colon contents were collected and snap frozen at −80°C for microbiota analysis. Human biopsies and stool samples were obtained under IRB approval and privacy protocols were followed. Our initial work demonstrated up regulation of TLR4 signaling in the mucosa of self-filling ileal loops. We hypothesized that TLR4 may be in-part responsible for mediating the metaplasia and inflammatory responses observed. Therefore, TLR4 KO mice were used to test this hypothesis and subsequently demonstrated attenuated responses in these parameters.

Project description:Background: MicroRNAs (miRNAs) acting as negative regulators of gene expression are differentially expressed in intestinal tissues of patients with inflammatory bowel disease (IBD). Assessing the functional role of miRNAs in murine models of colitis facilitates elucidating the role of specific miRNAs in human IBD. The aim of this study was to determine the miRNA signature of murine models of colitis and to assess the influence of miR-21 on intestinal inflammation. Methods: miRNAs expression was accessed by microarray for acute and chronic murine model of colitis induced by DSS or TNBS. miR-21-deficient mouse and littermates controls were assessed in the standard DSS, TNBS and CD4+ T cell transfer models of colitis. RNAs of mouse colon and CD4+CD45RBHigh cells were analyzed by miRNA and mRNA microarray, and quantitative RT-PCR. Th1 polarization was accessed by flow-cytometry and ELISA. Results: Alterations of in miRNAs expression were identified for acute and chronic DSS colitis and TNBS colitis, receptively. The Expression of miRs-21, -142-3p and -223 was were distinct between DSS and TNBS models while overlap of numerous miRNAs was seen. Importantly, miRs-19b, -192 and -215, that are decreased in IBD, were significantly decreased in all 4 models of colitis. miR-21, which is increased in IBD, was increased in TNBS colitis but not the DSS colitis models. Further assessment of the miR-21-deficient 1-/- mice revealed that the deletion of miR-21 results in the exacerbation of both the TNBS and T cell-transfer models of colitis. Conclusions: miRNAs are differentially expressed in both human IBD and murine colitis, with overlap of several IBD-associated miRNAs. The demonstration that miR-21 deletion exacerbated CD4+ T cell-mediated models of colitis provides further evidence that miRNAs play significant roles in the pathogenesis of IBD. miRNAs expression was accesed for acute and chronic murine model of colitis induced by DSS or TNBS.Total of 20 samples with duplicates were analyed in this study.

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Dysbiosis MCJ colitis mice

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