Volume 141, Issue 5, Pages 1842-1851.e10 (November 2011) Chronic Intestinal Inflammation Induces Stress-Response Genes in Commensal Escherichia coli  Laura G. Patwa, Ting–Jia Fan, Sandrine Tchaptchet, Yang Liu, Yves A. Lussier, R. Balfour Sartor, Jonathan J. Hansen  Gastroenterology  Volume 141, Issue 5, Pages 1842-1851.e10 (November 2011) DOI: 10.1053/j.gastro.2011.06.064 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Spontaneous intestinal inflammation develops in E coli NC101-monoassociated IL-10−/− mice. WT and IL-10−/− mice were monoassociated with E coli NC101 for 3 and 7 weeks. Histological inflammation in the cecum (A) and spontaneous secretion of IL-12/23p40 from proximal colon explant cultures (B) was measured. Data are mean + standard error of mean, n = 5 mice/group. *P < .05 relative to WT. Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Survival of NC101 is attenuated relative to NC101ΔibpAB in vivo, but not in vitro. (A) NC101 or NC101ΔibpAB were grown in Luria-Bertani broth under aerobic or anaerobic conditions and bacterial density was measured using OD600. Data are presented as the mean ± standard error of mean (SEM), n = 3 cultures/strain. (B) Luminal bacterial concentrations in monoassociated WT and IL-10−/− mice. Data are mean + SEM, n = 5–6 mice/group, *P < .05 relative to NC101-monoassociated mice. Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Expression of E coli ibpAB is associated with decreased inflammatory responses in NC101-monoassociated IL-10−/− and WT mice. (A) Composite blinded histological inflammation scores of 4 colonic segments from IL-10−/− or WT mice. (B) Representative photomicrographs of cecum and mid-colon from IL-10−/− mice monoassociated for 5 weeks, 20× magnification. (C) Western blots of NC101 lysate using sera from 13 IL-10−/− mice monoassociated with the indicated bacteria for 5 weeks. (D) Spontaneous secretion of IL-12/23p40 in culture supernatants from proximal colon explants. (E) IFN-gamma secretion by unseparated MLN cells stimulated ex vivo with NC101 lysate. Data are mean + standard error of mean, n = 5–6 mice/group, *P < .05 relative to NC101-monoassociated mice. Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Attenuated proinflammatory responses in IL-10−/− mice monoassociated with NC101 compared to NC101ΔibpAB are not proportional to differences in luminal bacterial concentrations at early time points. Luminal bacterial concentrations in the cecum (A) or mid-colon (B) from IL-10−/− mice monoassociated with NC101 or NC101ΔibpAB. (C) Spontaneous IL-12/23p40 secretion in proximal colon explant cultures. (D) IFN-gamma secretion by unseparated MLN cells stimulated ex vivo with NC101 lysate. (E) Blinded composite histological inflammation scores of 4 colonic segments. Data are mean ± standard error of mean, n = 5–6 mice/group, *P < .05 relative to NC101-monoassociated mice. Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Expression of ibpAB by E coli NC101 is associated with attenuated survival within macrophages and decreased TNF secretion. (A) Intracellular survival of NC101 and NC101ΔibpAB in WT and IL-10−/− bone marrow-derived macrophages. (B) Survival ratios (24 hours to 1 hour) of intracellular bacteria. (C) TNF secretion by NC101- or NC101ΔibpAB-infected macrophages. Data are mean + standard error of mean, 3 wells/condition, representative of results from 3 different mice, *P < .05 relative to NC101. Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Real-time PCR of the most highly up-regulated genes in E coli from IL-10−/− mice selected from the microarray analysis. Real-time PCR was performed using bacterial RNA isolated from cecal contents from WT and IL-10−/− mice monoassociated with NC101 for 7 weeks. Results are normalized to 16S ribosomal RNA expression and presented as fold-change IL-10−/− compared to WT (n = 4 samples per group). Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Real-time PCR of ibpA and ibpB in cecal bacteria from monoassociated IL-10−/− mice. Real-time PCR was performed using bacterial RNA isolated from cecal contents of IL-10−/− mice monoassociated with the indicated bacterial strains for 3 weeks. Results are normalized to 16S ribosomal RNA expression (n = 4 samples per group, *P < .05 relative to NC101-monoassociated mice). Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Complementation of NC101 ΔibpAB restores the E coli NC101 phenotype in monoassociated IL-10−/− mice. (A) Luminal bacterial concentrations in the cecum and mid-colon of IL-10−/− mice monoassociated with NC101, NC101 ΔibpAB, and NC101ΔibpAB(pGEN-MCSibpAB) for 3 weeks were measured by counting colonies on BHI agar plates that were inoculated with diluted luminal contents. (B) Composite blinded histological inflammation scores from colon fragments from the same monoassociated mice as in (A). (C) IFN-gamma production, measured using enzyme-linked immunosorbent assay, by unseparated MLN cells from the same monoassociated mice as in (A) stimulated ex vivo with E coli NC101 lysate for 72 hours. Data for all panels are presented as mean + standard error of mean, n = 5–6 mice per group, *P < .05 relative to NC101-monoassociated IL-10−/− mice. Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 NC101ΔibpAB-monoassociated and NC101+NC101ΔibpAB dual-associated IL-10−/− mice develop similar levels of colonic inflammation. (A) Luminal bacterial concentrations in the cecum and mid-colon of IL-10−/− mice monoassociated with NC101 or NC101ΔibpAB, or dual associated with NC101+NC101ΔibpAB for 5 weeks. (B) Composite blinded histological inflammation scores from colon fragments from the same mice as in (A). (C) Spontaneous secretion of IL-12/23p40 in colonic explant cultures from the same mice as in (A). (D) IFN-gamma production by unseparated MLN cells from the same mice as in (A) stimulated ex vivo with E coli NC101 lysate for 72 hours. Data for all panels are presented as mean + standard error of mean, n = 5 NC101-monoassociated mice, n = 8 NC101ΔibpAB-monoassociated mice, n = 7 dual-associated mice. For all data, P < .05 relative to NC101-monoassociated IL-10−/− mice. Other P values as shown on graphs. Gastroenterology 2011 141, 1842-1851.e10DOI: (10.1053/j.gastro.2011.06.064) Copyright © 2011 AGA Institute Terms and Conditions