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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type Mice and Toll-like Receptor 4 Intestianl Epithelial Conditional Knockout Mice Ileal Transcriptomes

ABSTRACT: Toll-like receptor 4 (TLR4), the receptor of gram-negative bacterial endotoxin, is present on the intestinal epithelium and plays pivotal roles in intestinal epithelial differentiation and host defense. To better understand TLR4 on the intestinal epithelium, the ileum samples from wild type mice and mice lacking TLR4 in the intestinal epithelium (TLR4ΔIEC) were subjected to RNA-seq analysis to interrogate the transcriptome. We mapped about 20 million sequence reads per sample to the mouse genome and identified 86477 genes in the ileum of wild type and TLR4ΔIEC mice. 455 genes were differentially expressed between wild type and TLR4ΔIEC mice and 1037 genes were absent either from wild type or TLR4ΔIEC mice with G-factor value 0.015 and Δ cutoff value 0.54. Pathway analysis via IPA indicated some impacted canonical KEGG pathways such as DNA replication, PPAR signaling, Toll-like receptor signaling, cytokine-cytokine interaction, adherens junction, Jak-STAT signaling, and tight junction.

ORGANISM(S): Mus musculus

PROVIDER: GSE89966 | GEO | 2019/10/20

REPOSITORIES: GEO

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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.

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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type Mice and Toll-like Receptor 4 Intestianl Epithelial Conditional Knockout Mice Ileal Transcriptomes

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