Volume 146, Issue 2, Pages 508-519 (February 2014) Crohn's Disease–Associated Adherent Invasive Escherichia coli Modulate Levels of microRNAs in Intestinal Epithelial Cells to Reduce Autophagy  Hang Thi Thu Nguyen, Guillaume Dalmasso, Stefan Müller, Jessica Carrière, Frank Seibold, Arlette Darfeuille–Michaud  Gastroenterology  Volume 146, Issue 2, Pages 508-519 (February 2014) DOI: 10.1053/j.gastro.2013.10.021 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 AIEC infection increases hsa-miR-30c and hsa-miR-130a levels, inhibiting ATG5 and ATG16L1 expression in T84 cells. (A–C) T84 cells were infected with AIEC LF82, K12, or the E coli HS strain. qRT-PCR analysis for (A) mature miRNA or (B) ATG5 and ATG16L1 mRNA levels. (C) ATG5 and ATG16L1 expression analyzed by Western blot. (D) The 3′-UTRs of ATG5 and ATG16L1 mRNAs were cloned downstream of a luciferase reporter gene (ATG-3′-UTR-luc). T84 cells were transfected with the ATG5 3′-UTR-luc or ATG16L1 3′-UTR-luc construct together with vehicle or 50 nmol/L of precursor of hsa-miR-30c, hsa-miR-130a, or a miRNA-negative control (miR-NC). Luciferase activity was normalized to lysate protein concentration. (E and F) T84 cells were transfected with vehicle or 50 nmol/L of a precursor of hsa-miR-30c, hsa-miR-130a, or miR-NC. ATG5 and ATG16L1 expression was assessed by (E) qRT-PCR and (F) Western blot. Data are means ± SEM of 6 replicates and are representative of 3 independent experiments. *P < .05; **P < .005; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Gastroenterology 2014 146, 508-519DOI: (10.1053/j.gastro.2013.10.021) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 Hsa-miR-30c and hsa-miR-130a are involved in the regulation of ATG5 and ATG16L1 expression in CD. Total RNAs were extracted from the terminal ileal biopsy specimens from normal controls or patients with ulcerative colitis (UC) and CD (with normal appearance [noninflamed CD] or with macroscopic inflammation [inflamed CD]). Expression of (A and B) mature miRNAs and (C and D) ATG5 and ATG16L1 mRNA in human biopsy specimens was quantified by qRT-PCR. Statistical analysis was performed using the nonparametric Mann–Whitney test. *P < .05; **P < .005; ***P < .001. Gastroenterology 2014 146, 508-519DOI: (10.1053/j.gastro.2013.10.021) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 AIEC infection induces hsa-miR-30c and hsa-miR-130a up-regulation by activating the NF-κB pathway. (A–C) T84 cells were treated or not with PDTC at 100 μmol/L for 30 minutes before infection and during infection. (A) LF82-induced NF-κB activation was assessed by Western blot analysis for degradation of inhibitor of κB-α (IκB-α). Expression of (B) hsa-miR-30c and hsa-miR-130a, and (C) ATG5 and ATG16L1 mRNAs were analyzed by qRT-PCR. Data are means ± SEM of 6 replicates and are representative of 3 independent experiments. *P < .05. (D and E) Chromatin immunoprecipitation analysis for the binding of the NF-κB p65 subunit to hsa-miR-30c and hsa-miR-130a gene promoters. The protein-DNA complexes were immunoprecipitated with anti-p65 or a negative control IgG. (D) Representative agarose gels for the p65-binding regions in the hsa-miR-30c and hsa-miR-130a gene promoters (i) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA in the input (ii) amplified by semiquantitative PCR. M, DNA SmartLadder (MW-1800-02; Eurogentec, San Diego, CA). (E) Increase in the binding of p65 to miRNA gene promoters, normalized to GAPDH DNA in the inputs, upon LF82 infection analyzed by qPCR. Data are means ± SEM of 4 replicates and are representative of 2 independent experiments. **P < .005; ***P < .001. Gastroenterology 2014 146, 508-519DOI: (10.1053/j.gastro.2013.10.021) Copyright © 2014 AGA Institute Terms and Conditions

Figure 4 Overexpression of hsa-miR-30c and hsa-miR-130a blocks autophagy response, increasing AIEC intracellular replication and AIEC-induced IL8 production in T84 cells. T84 cells were transfected with 50 nmol/L of precursor of a miRNA-negative control (miR-NC), or hsa-miR-30c, hsa-miR-130a, or a combination of both miRNA precursors before being infected with (A and C–E) LF82 or (B) LF82-GFP. (A) Western blot analysis for the shift of LC3-I toward LC3-II. (B) Representative confocal micrographs of cells infected with LF82-GFP (green) and immunolabeled for LC3 (red). Nuclei were stained with Hoechst (blue). Scale bars: 5 μm. The percentage of LF82-GFP in LC3-positive vacuole was determined by confocal microscopy from 3 independent experiments, counting 20 cells/experiment. (C) Intracellular LF82 number counted on Luria Broth agar plates. (D) qRT-PCR analysis of IL8 mRNA expression. (E) Enzyme-linked immunosorbent assay quantification of secreted IL8 amounts in cell culture supernatant. Data are means ± SEM of 6 replicates and are representative of 3 independent experiments. *P < .05; **P < .005; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein. Gastroenterology 2014 146, 508-519DOI: (10.1053/j.gastro.2013.10.021) Copyright © 2014 AGA Institute Terms and Conditions

Figure 5 Inhibition of hsa-miR-30c and hsa-miR-130a blocks AIEC-induced suppression of ATG5 and ATG16L1 expression and enhances autophagic activity, decreasing AIEC intracellular persistence and proinflammatory response. T84 cells were transfected with vehicle or 50 nmol/L of antisense of a miRNA-negative control (anti-miR-NC), antisense of hsa-miR-30c (anti-miR-30c), or antisense of hsa-miR-130a (anti-miR-130a), or a combination of anti-miR-30c and anti-miR-130a (anti-miRNAs) before being infected with LF82. ATG5 and ATG16L1 expression was assessed by (A) qRT-PCR and (B) Western blot. Autophagic activity was assessed by (C) Western blot analysis for the shift of LC3-I toward LC3-II or by (D) confocal microscopic analysis of cells infected with LF82-GFP (green) and immunolabeled for LC3 (red). Nuclei were stained with Hoechst (blue). Scale bars: 5 μm. (D) The percentage of LF82-GFP in LC3-positive vacuole was determined by confocal microscopy from 3 independent experiments, counting 20 cells/experiment. (E) Intracellular LF82 number counted on Luria Broth agar plates. (F) Secreted IL8 amounts in cell culture supernatant quantified by enzyme-linked immunosorbent assay. Data are means ± SEM of 6 replicates and are representative of 3 independent experiments. *P < .05; **P < .005; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein. Gastroenterology 2014 146, 508-519DOI: (10.1053/j.gastro.2013.10.021) Copyright © 2014 AGA Institute Terms and Conditions

Figure 6 AIEC infection of mouse enterocytes increases mmu-miR-30c and mmu-miR-130a levels, inhibiting ATG5 and ATG16L1 expression. (A–C) MODE-K cells were infected with AIEC LF82, K12, or the E coli HS strain. qRT-PCR analysis for (A) mmu-miR-30c, mmu-miR-130a and (B) ATG5 and ATG16L1 mRNA levels. (B) ATG5 and ATG16L1 expression at protein level was analyzed by Western blot. (C) MODE-K cells were transfected with vehicle or 50 nmol/L of a precursor of mmu-miR-30c, mmu-miR-130a, or miR-NC. ATG5 and ATG16L1 expression was assessed by qRT-PCR and Western blot. Data are means ± SEM of 6 replicates and are representative of 3 independent experiments. (D and E) CEABAC10 transgenic mice were infected with AIEC LF82, K12, or the E coli HS strain. (D) Mmu-miR-30c and mmu-miR-130a expression in ileal enterocytes was quantified by qRT-PCR. (E) ATG5 and ATG16L1 expression in ileal enterocytes was assessed by qRT-PCR and Western blot. Data are means ± SEM of 10 mice/group. *P < .05; **P < .005; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Gastroenterology 2014 146, 508-519DOI: (10.1053/j.gastro.2013.10.021) Copyright © 2014 AGA Institute Terms and Conditions

Figure 7 Inhibition of mmu-miR-30c and mmu-miR-130a in mouse enterocytes enhances autophagic activity, decreasing AIEC intracellular replication and proinflammatory response. Mouse enterocytes were transfected with vehicle or with antisenses of mmu-miR-30c and mmu-miR-130a (anti-miRNAs) using the ileal loop model. (A) Representative confocal micrographs of mouse ileum transfected with cyanine dye 3 (Cy3)-labeled anti-miR negative control (red). Actin cytoskeleton was stained with fluorescein isothiocyanate (FITC)-phalloidin (green). Nuclei were stained with Hoechst (blue). Scale bars: 20 μm. (B–E) Transfected mouse ileum was infected or not with LF82 using ileal loops. (B) Expression of mmu-miR-30c and mmu-miR-130a, and of mATG5 and mATG16L1 mRNAs was quantified by qRT-PCR. (C) Western blot analysis of mATG5, mATG16L1, and LC3 protein levels, and confocal microscopic analysis of infected ileal sections immunolabeled for LC3 (red) and E coli lipopolysaccharide (green). Nuclei were stained with Hoechst (blue). Scale bars: 20 μm. (D) Intracellular LF82 number counted on ampicillin/erythromycin-containing Luria Broth agar plates. (E) Secreted keratinocyte-derived chemokine (KC) amounts in tissue culture supernatant quantified by enzyme-linked immunosorbent assay. Data are means ± SEM of 8 mice/group and are representative of 2 independent experiments. (B) $$$P < .001; (**P < .005; ***P < .001) vs vehicle-uninfected. (D–F) ***P < .001. (F) CEABAC10 transgenic mice were transfected with vehicle or antisenses of mmu-miR-30c and mmu-miR-130a (anti-miRNAs) using ileal loops and infected with LF82 by gavage. Secreted keratinocyte-derived chemokine, IL6, and tumor necrosis factor (TNF)-α amounts in ileal tissue culture supernatant were quantified by enzyme-linked immunosorbent assay. Data are means ± SEM of 10 mice/group. ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Gastroenterology 2014 146, 508-519DOI: (10.1053/j.gastro.2013.10.021) Copyright © 2014 AGA Institute Terms and Conditions