Dataset Information

Functional Transcriptomics in Diverse Intestinal Epithelial Cell Types Reveals Robust MicroRNA Sensitivity in Intestinal Stem Cells to Microbial Status.

ABSTRACT: Gut microbiota play an important role in regulating the development of the host immune system, metabolic rate, and at times, disease pathogenesis. The factors and mechanisms that mediate interactions between microbiota and the intestinal epithelium are not fully understood. We provide novel evidence that microbiota may control intestinal epithelial stem cell (IESC) proliferation in part through microRNAs (miRNAs). We demonstrate that miRNA profiles differ dramatically across functionally distinct cell types of the mouse jejunal intestinal epithelium and that miRNAs respond to microbiota in a highly cell type-specific manner. Importantly, we also show that miRNAs in IESCs are more prominently regulated by microbiota compared with miRNAs in any other intestinal epithelial cell subtype. We identify miR-375 as one miRNA that is significantly suppressed by the presence of microbiota in IESCs. Using a novel method to knockdown gene and miRNA expression ex vivo enteroids, we demonstrate that we can knock down gene expression in Lgr5⁺ IESCs. Furthermore, when we knock down miR-375 in IESCs, we observe significantly increased proliferative capacity. Understanding the mechanisms by which microbiota regulate miRNA expression in IESCs and other intestinal epithelial cell subtypes will elucidate a critical molecular network that controls intestinal homeostasis and, given the heightened interest in miRNA-based therapies, may offer novel therapeutic strategies in the treatment of gastrointestinal diseases associated with altered IESC function.

SUBMITTER: Peck BC

PROVIDER: S-EPMC5314158 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Functional Transcriptomics in Diverse Intestinal Epithelial Cell Types Reveals Robust MicroRNA Sensitivity in Intestinal Stem Cells to Microbial Status.

Peck Bailey C E BC Mah Amanda T AT Pitman Wendy A WA Ding Shengli S Lund P Kay PK Sethupathy Praveen P Sethupathy Praveen P

The Journal of biological chemistry 20170104 7

Gut microbiota play an important role in regulating the development of the host immune system, metabolic rate, and at times, disease pathogenesis. The factors and mechanisms that mediate interactions between microbiota and the intestinal epithelium are not fully understood. We provide novel evidence that microbiota may control intestinal epithelial stem cell (IESC) proliferation in part through microRNAs (miRNAs). We demonstrate that miRNA profiles differ dramatically across functionally distinc ...[more]

PMID: 28053090

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Project description:The Interleukin-22 (IL22) pathway plays a key role in maintaining homeostasis at the mucosal surfaces and is frequently dysregulated during infectious and inflammatory diseases. Human genome-wide association studies performed at the WTSI have identified SNPs in and around the human IL-22 gene that are associated with IBD susceptibility. The IL-22 cytokine is produced by cells of the innate and adaptive immune systems within the intestinal mucosa and acts on the IL-22 receptor (IL-22RA1) that is present specifically on intestinal epithelial cells. Studies to date have shown the that IL-22 pathway is linked to proliferative and anti-apoptotic pathways, as well as anti-microbial molecule production that help control bacterial invasion and prevent tissue damage. We are characterizing a novel IL-22RA1 KO mouse and have shown that these mice are highly susceptible to enteric bacterial pathogens, such as Citrobacter rodentium and Clostridium difficile. However, the mechanism of this protective immunity is poorly understood. The aim of this project is to identify novel molecules or pathways downstream of IL-22 signaling that provide the mechanistic link between IL-22RA1 and innate immunity. To maintain mouse colonic epithelial cells in vitro, we used an organoid culture system that faithfully recapitulates the crypt architecture of the gastrointestinal epithelium. Primary colonic epithelial crypts were isolated from age-matched wildtype and IL-22ra1 knock-out mice (n = 4 per genotype), and subsequently cultured in the presence of defined growth factors and Matrigel for 5 days. The resulting mature organoids were then either treated with Control medium (Advanced DMEM/F12) or mouse recombinant IL-22 (50ng/ml) for 16 hours. Total RNA were extracted using the QIAgen RNeasy Micro Kit, including an on-column DNAse digestion step, according to manufacturer's protocol. We are submit total RNA samples of good quality (RIN > 7) and quantity.The culture and stimulation conditions have been optimised to achieve significant and specific induction of known target genes such as the antimicrobial peptides RegIIIb, RegIIIg, S100a8 and S100a9. Two technical replicates will be available for each genotype x condition. We aim to sequence 5GBp per replicate, and 5 replicates per lane on an Illumina platform. Through this dataset, we hope to discover epithelial cell-specific pathways downstream of IL-22 signaling that are ablated in the absence of its receptor.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

Dataset Information

Functional Transcriptomics in Diverse Intestinal Epithelial Cell Types Reveals Robust MicroRNA Sensitivity in Intestinal Stem Cells to Microbial Status.

Publications

Functional Transcriptomics in Diverse Intestinal Epithelial Cell Types Reveals Robust MicroRNA Sensitivity in Intestinal Stem Cells to Microbial Status.

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