Chronic intestinal inflammation induces stress-response genes in commensal Escherichia coli

Abstract

Background & aims: Intestinal microbes induce homeostatic mucosal immune responses, but can also cause inappropriate immune activation in genetically susceptible hosts. Although immune responses to bacterial products have been studied extensively, little is known about how intestinal inflammation affects functions of commensal luminal microbes.

Methods: Microarrays and real-time polymerase chain reaction were used to profile transcriptional changes in luminal bacteria from wild-type and IL-10(-/-) mice monoassociated with a nonpathogenic, murine isolate of Escherichia coli (NC101, which causes colitis in gnotobiotic IL-10(-/-) mice). Colonic inflammation and innate and adaptive immune responses were measured in wild-type and IL-10(-/-) mice monoassociated with mutant NC101 that lack selected, up-regulated genes, and in IL-10(-/-) mice that were colonized with a combination of mutant and parental NC101. We measured intracellular survival of bacteria within primary macrophages from mice and resulting production of tumor necrosis factor.

Results: Bacteria from IL-10(-/-) mice with colitis had significant up-regulation of the stress-response regulon, including the small heat shock proteins IbpA and IbpB that protect E coli from oxidative stress, compared to healthy, wild-type controls. In IL-10(-/-) mice, expression of ibpAB reduced histologic signs of colon inflammation, secretion of interleukin-12/23p40 in colonic explant cultures, serologic reactivity to NC101 antigens, and secretion of interferon-gamma by stimulated mesenteric lymph node cells. Infection of primary macrophages by bacteria that express ibpAB was associated with decreased intracellular survival and reduced secretion of tumor necrosis factor.

Conclusions: Chronic intestinal inflammation causes functional alterations in gene expression in commensal gut bacterium (E coli NC101). Further studies of these expression patterns might identify therapeutic targets for patients with inflammatory bowel diseases.

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 means + SEM, n=5 mice/group,*p<0.05 relative to WT.

Figure 2

Survival of NC101 is attenuated relative to NC101ΔibpAB in vivo, but not in vitro. (A) NC101 or NC101ΔibpAB were grown in LB under aerobic or anaerobic conditions and bacterial density was measured using OD₆₀₀. Data are presented as the mean+/−SEM, n=3 cultures/strain. (B) Luminal bacterial concentrations in monoassociated WT and IL-10^−/− mice. Data are means + SEM, n=5–6 mice/group, *p<0.05 relative to NC101-monoassociated mice.

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, 20x 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-γ secretion by unseparated MLN cells stimulated ex vivo with NC101 lysate. Data are means + SEM, n=5–6 mice/group, *p<0.05 relative to NC101-monoassociated mice.

Figure 4

Attenuated pro-inflammatory 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-γ secretion by unseparated MLN cells stimulated ex vivo with NC101 lysate. (E) Blinded composite histological inflammation scores of 4 colonic segments. Data are means +/− SEM, n=5–6 mice/group, *p<0.05 relative to NC101-monoassociated mice.

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 (24h/1h) of intracellular bacteria. (C) TNF secretion by NC101- or NC101ΔibpAB-infected macrophages. Data are means + SEM, 3 wells/condition, representative of results from 3 different mice, *p<0.05 relative to NC101.