title: Crohn's disease-associated adherent invasive Escherichia coli modulate levels of microRNAs in intestinal epithelial cells to reduce autophagy.
abstract: OBJECTIVE
  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.
authors.affiliation: M2iSH, UMR 1071 Inserm/University of Auvergne, Clermont-Ferrand, France; INRA USC 2018, Clermont-Ferrand, France. Electronic address: hang.nguyen@udamail.fr.
authors.forename: Hang Thi Thu
authors.initials: HT
authors.lastname: Nguyen
authors.affiliation: M2iSH, UMR 1071 Inserm/University of Auvergne, Clermont-Ferrand, France; INRA USC 2018, Clermont-Ferrand, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Dalmasso
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Müller
authors.affiliation: M2iSH, UMR 1071 Inserm/University of Auvergne, Clermont-Ferrand, France; INRA USC 2018, Clermont-Ferrand, France.
authors.forename: Jessica
authors.initials: J
authors.lastname: Carrière
authors.affiliation: Department of Gastroenterology, Spitalnetz, Bern, Switzerland.
authors.forename: Frank
authors.initials: F
authors.lastname: Seibold
authors.affiliation: M2iSH, UMR 1071 Inserm/University of Auvergne, Clermont-Ferrand, France; INRA USC 2018, Clermont-Ferrand, France; Centre Hospitalier Universitaire, Clermont-Ferrand, France. Electronic address: arlette.darfeuille-michaud@udamail.fr.
authors.forename: Arlette
authors.initials: A
authors.lastname: Darfeuille-Michaud
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D000071187:Autophagy-Related Protein 5; D000071183:Autophagy-Related Proteins; D015415:Biomarkers / Q000378:metabolism; D001706:Biopsy; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003093:Colitis, Ulcerative / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D003424:Crohn Disease / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D004927:Escherichia coli Infections / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007079:Ileitis / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D007082:Ileum / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
chemical_list: C518959:ATG16L1 protein, human; C502778:ATG5 protein, human; C532820:Atg16l1 protein, mouse; C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; D000071183:Autophagy-Related Proteins; D015415:Biomarkers; D002352:Carrier Proteins; C554019:MIRN130 microRNA, human; C582652:MIRN130 microRNA, mouse; C546086:MIRN30b microRNA, human; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins; C534715:Mirn30d microRNA, mouse; D016328:NF-kappa B
keywords: AIEC; Bacterial Infection; CD; CEABAC10; Carcinoembryonic antigen bacterial artificial chromosome, 10 copies; Crohn's disease; IBD; IEC; IL; Immune Regulation; MODE-K; Mouse Model; NC; NF-κB; PDTC; Tg; UTR; adherent-invasive Escherichia coli; homo sapiens; hsa; inflammatory bowel diseases; interleukin; intestinal epithelial cell; m; mRNA; messenger RNA; miR; miRNA; microRNA; mmu; mouse; mouse duodenal epithelial cell line; mus musculus; negative control; nuclear factor-κB; pyrrolidine dithiocarbamate; qRT-PCR; quantitative reverse-transcription polymerase chain reaction; transgenic; untranslated region