Gastroenterology

Gastroenterology

Volume 146, Issue 2, February 2014, Pages 508-519
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Crohn's Disease–Associated Adherent Invasive Escherichia coli Modulate Levels of microRNAs in Intestinal Epithelial Cells to Reduce Autophagy

https://doi.org/10.1053/j.gastro.2013.10.021Get rights and content

Background & Aims

Levels of microRNAs are altered in intestinal tissues of patients with Crohn's disease (CD). The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with CD, adhere to and invade intestinal epithelial cells. We investigated the mechanism by which AIEC infection alters the expression of microRNAs and the host immune response.

Methods

Levels of microRNAs in human intestinal epithelial T84 cells and in mouse enterocytes were measured using quantitative reverse-transcription polymerase chain reaction. Luciferase assays were used to measure binding of microRNAs to the 3′-untranslated region of messenger RNA targets. Binding of nuclear factor-κB to promoters of genes encoding microRNAs was assessed by chromatin immunoprecipitation assays. Autophagy was measured by immunoblot analyses and immunofluorescent labeling of LC3. Anti-microRNAs were transferred to mice using ileal loops. Biopsy specimens from the terminal ileum of patients with ulcerative colitis (n = 20), CD (n = 20), or individuals without inflammatory bowel disease undergoing surveillance colonoscopies (controls, n = 13) were collected during endoscopic examination.

Results

AIEC infection up-regulated levels of microRNA (MIR) 30C and MIR130A in T84 cells and in mouse enterocytes by activating nuclear factor-κB. Up-regulation of these microRNAs reduced the levels of ATG5 and ATG16L1 and inhibited autophagy, leading to increased numbers of intracellular AIEC and an increased inflammatory response. In ileal biopsy samples of patients with CD, there was an inverse correlation between levels of MIR30C and MIR130A and those of ATG5 and ATG16L1, supporting in vitro findings. Inhibition of MIR30C and MIR130A in cultured intestinal epithelial cells and in mouse enterocytes blocked AIEC-induced inhibition of ATG5 and ATG16L1 expression and restored functional autophagy. This resulted in more effective clearance of intracellular AIEC and reduced AIEC-induced inflammation.

Conclusions

Infection with AIEC up-regulates microRNAs to reduce expression of proteins required for autophagy and autophagy response in intestinal epithelial cells. Ileal samples from patients with CD have increased levels of these same microRNAs and reduced levels of ATG5 and ATG16L1.

Section snippets

Materials and Methods

Information on bacterial strains, human tissue samples, cell culture, invasion assays, miRNAs, plasmid construction, in vitro transfection and luciferase assay, protein extraction and Western blot analysis, RNA extraction and quantitative reverse-transcription polymerase chain reaction (qRT-PCR), miRNA target prediction, fluorescent microscopy, intestinal epithelial cell (IEC) isolation, chromatin immunoprecipitation assay, and enzyme-linked immunosorbent assay appears in the Supplementary

Identification of CD-Associated miRNAs That Are Dysregulated by AIEC Infection

To discover the CD-associated miRNAs involved in host response to AIEC infection, we analyzed miRNA expression in human intestinal epithelial T84 cells infected with the AIEC reference strain LF82 by qRT-PCR. Among the CD-associated miRNAs previously reported,19 expression of homo sapiens (hsa)-miR-30c and hsa-miR-130a was up-regulated significantly by infection with LF82 but not with the nonpathogenic E coli K12 MG1655 or the commensal E coli HS strain (Figure 1A). Infection with other

Discussion

The potential role of autophagy in CD onset has been highlighted with the identification of genetic risk polymorphisms in various autophagy-related genes and those that regulate autophagy. Exciting recent progress has been made in understanding the mechanistic links between defects in autophagy-mediated bacterial handling, innate immune activation, and CD pathogenesis. Among the infectious factors that have been shown to be involved in the dysregulated immune response observed in CD, much

Acknowledgments

The authors thank the Imagerie Confocale Clermont-Ferrand (ICCF) platform (IFR79 Santé Université d'Auvergne, Clermont-Ferrand, France) for confocal microscopy, Dr Pierre Lapaquette and Professor Richard Bonnet for valuable discussions, and Dr Emilie Vazeille and Dr Nicolas Barnich for help with human biopsy samples and the CEABAC10 transgenic mice.

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by the Ministère de la Recherche et de la Technologie, Inserm (UMR1071), INRA (USC-2018), and by grants from the Association François Aupetit (A.D.M.), the European Union FP7 People Marie Curie International Incoming Fellowship (H.N.) and Région Auvergne (Nouveau Chercheur to G.D.).

    Author names in bold designate shared co-first authorship.

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