Guanylate cyclase C limits systemic dissemination of a murine enteric pathogen

BACKGROUND: Guanylate Cyclase C (GC-C) is an apically-oriented transmembrane receptor that is expressed on epithelial cells of the intestine. Activation of GC-C by the endogenous ligands guanylin or uroguanylin elevates intracellular cGMP and is implicated in intestinal ion secretion, cell proliferation, apoptosis, intestinal barrier function, as well as the susceptibility of the intestine to inflammation. Our aim was to determine if GC-C is required for host defense during infection by the murine enteric pathogen Citrobacter rodentium of the family Enterobacteriacea. METHODS: GC-C(+/+) control mice or those having GC-C genetically ablated (GC-C(−/−)) were administered C. rodentium by orogastric gavage and analyzed at multiple time points up to post-infection day 20. Commensal bacteria were characterized in uninfected GC-C(+/+) and GC-C(−/−) mice using 16S rRNA PCR analysis. RESULTS: GC-C(−/−) mice had an increase in C. rodentium bacterial load in stool relative to GC-C(+/+). C. rodentium infection strongly decreased guanylin expression in GC-C(+/+) mice and, to an even greater degree, in GC-C(−/−) animals. Fluorescent tracer studies indicated that mice lacking GC-C, unlike GC-C(+/+) animals, had a substantial loss of intestinal barrier function early in the course of infection. Epithelial cell apoptosis was significantly increased in GC-C(−/−) mice following 10 days of infection and this was associated with increased frequency and numbers of C. rodentium translocation out of the intestine. Infection led to significant liver histopathology in GC-C(−/−) mice as well as lymphocyte infiltration and elevated cytokine and chemokine expression. Relative to naïve GC-C(+/+) mice, the commensal microflora load in uninfected GC-C(−/−) mice was decreased and bacterial composition was imbalanced and included outgrowth of the Enterobacteriacea family. CONCLUSIONS: This work demonstrates the novel finding that GC-C signaling is an essential component of host defense during murine enteric infection by reducing bacterial load and preventing systemic dissemination of attaching/effacing-lesion forming bacterial pathogens such as C. rodentium

Deletion of GC-C accelerates the development of severe colitis in IL-10 deficient 6 week old mice.

<p>(A) Histological analysis of H&E stained colon clearly demonstrates that mice lacking both GC-C and IL-10 have severe colitis characterized by inflammatory cell infiltrate and epithelial hyperplasia. (B) Scoring of disease parameters indicates that GC-C^−/−IL-10^−/− mice have more severe colonic inflammation. n = 3–4 mice per group; *p = 0.05.</p

Analysis of pro-inflammatory gene expression using realtime RT-PCR demonstrates enhanced cytokine and chemokine production in GC-C^−/−IL-10^−/− tissue.

<p>Gene expression in colon of IL-10^−/− and GC-C^−/−IL-10^−/− mice are shown relative to wildtype colon which is arbitrarily set at 1. n = 4–12 mice per group; *p<0.05.</p

Infiltration of F4/80+ cells into the colon is greatly increased in the absence of GC-C and IL-10.

<p>Representative images show that while there are increased F4/80+ cells in IL-10^−/− colon, most of which are likely macrophages, there is far greater staining in the severely diseased colon of GC-C^−/−IL-10^−/− mice. F4/80 is depicted in red while nuclei are stained blue using DAPI. Magnification = 200X.</p