Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokine response and lesions similar to those in murine inflammatory bowel disease

Abstract

Citrobacter rodentium is a classically noninvasive pathogen of mice that is similar to enteropathogenic Escherichia coli (EPEC) in man. Following oral infection of young mice, the organism colonizes the distal colon, and within 1 week the colonic mucosa doubles in thickness and there is massive epithelial cell hyperplasia. Since T-cell responses in mouse models of inflammatory bowel disease (IBD) also cause epithelial hyperplasia, we have investigated the possibility that C. rodentium promotes similar T-cell responses in the mucosa, thereby increasing epithelial shedding, transmission, and replication of the organism. Beginning 6 days after infection, bacteria were observed to be in close association with the epithelial surface and were also visible scattered throughout the lamina propria and in the submucosa. There was a CD3(+)-cell infiltrate into the colonic lamina propria and epithelium as well as mucosal thickening and crypt hyperplasia. The majority of CD3(+) cells were CD4(+) and were not gammadelta+. Reverse transcription-PCR analysis of cytokines also revealed a highly polarized Th1 response (interleukin-12, gamma interferon, and tumor necrosis factor alpha) in the mucosa and a large increase in the epithelial cell mitogen keratinocyte growth factor. None of the changes were seen in mice inoculated with bacteria lacking intimin (which is necessary for colonization), but they were seen in mice inoculated with C. rodentium complemented with intimin from EPEC. This is the first example of a classically noninvasive bacterial pathogen which elicits a strong mucosal Th1 response and which produces pathology similar to that seen in mouse models of IBD, which is also characterized by a strong Th1 response. These results also suggest that the colonic mucosa responds in a stereotypic way to Th1 responses.

FIG. 1

Swiss NIH mice infected with wild-type C. rodentium showed severe colonic hyperplasia compared to mice infected with an intimin mutant strain. Crypt lengths were measured on days 2, 6, and 12 postinfection. Mean crypt lengths in the colons of mice infected with wild-type C. rodentium (open bars) were significantly greater than those of mice infected with the control mutant strain (closed bars). Error bars represent standard errors. Each group contained five mice. ∗, P < 0.05).

FIG. 2

Bacteria were visualized on the epithelial surface (a) and in the mucosa and submucosa (b) of C. rodentium-infected Swiss NIH mice. The clear area between the muscularis mucosa and the external muscle layer is the submucosa, which becomes edematous during Citrobacter infection. The arrows indicate bacteria. Immunoperoxidase immunohistochemistry was performed with anti-intimin antibody. Magnification, ×400.

FIG. 3

Mean cell counts for peroxidase-containing cells in the mucosa and submucosa (A) and for CD3⁺ (B), CD4⁺ (C), and CD8⁺ (D) cells infiltrating the lamina propria of Swiss NIH mice were determined on days 2, 6, and 12 postinfection. In addition to the massive increase in numbers of CD3⁺ and CD4⁺ cells in the mucosa of C. rodentium-infected mice, there was also a slight increase in numbers of these cells in control mice. This probably reflected the increase which is seen in normal mice around this time (21). Closed bars represent mice infected with an intimin mutant strain; open bars represent mice infected with wild-type C. rodentium. Error bars indicate standard errors. Each group contained five mice. ∗, P < 0.05).

FIG. 4

Immunoperoxidase immunohistochemistry of colon tissue of infected Swiss NIH mice. (a and b) CD4⁺ cells in mice infected with an intimin mutant strain (a) or wild-type C. rodentium (b). In colon tissues of control mice, only a few CD4⁺ cells are visible in the lamina propria (arrows). In Citrobacter-infected mice, there is a massive accumulation of CD4⁺ cells at the crypt bases and in the lamina propria (arrows). (c and d) MHC class II-positive cells in the lamina propria of intimin-mutant-infected mice (c) and wild-type-C. rodentium-infected mice (d). In control mice, MHC class II-positive cells can be seen in the lamina propria, and the surface epithelium is also positive (arrows). In Citrobacter-infected mice, every surface and crypt epithelial cell is strongly class II positive, as are all cells in the lamina propria. Serial sections stained with control antibodies showed only a few cells expressing endogenous peroxidase in the mucosa, although these cells were more abundant in the submucosa (see Fig. 3A). Original magnification, ×100.

FIG. 5

Quantitation of intraepithelial cells. Counts are expressed as the number of positive intraepithelial lymphocytes per 100 epithelial cells. Each group contained five Swiss NIH mice. Closed bars represent mice infected with the intimin mutant strain; open bars represent mice infected with wild-type C. rodentium. Values are means ± standard errors. (A) CD3⁺ cells; (B) CD4⁺ cells; (C) CD8⁺ cells. ∗, P < 0.05.

FIG. 6

Cytokine and KGF mRNA transcripts in distal colonic tissue as measured by RT-PCR on days 6 and 12 postinfection. Closed bars represent mice infected with the intimin mutant strain; open bars represent mice infected with wild-type C. rodentium. Each group contained five Swiss NIH mice. Values are means ± standard errors. (A) IL-1; (B) TNF-α; (C) IL-12; (D) IFN-γ; (E) IL-4; (F) KGF. ∗, P < 0.05.

FIG. 7

(A) Cell counts on day 12 postinfection of C3H mice with wild-type intimin-β-expressing C. rodentium (open bars), intimin-α-expressing DBS255(pCVD438) (hatched bars), and intimin mutant (closed bars) strains. (B) Crypt lengths (day 12) in the colons of wild-type-C. rodentium-infected mice (open bars) or mice infected with C. rodentium expressing human intimin α were significantly greater than those of mice infected with the control mutant strain (closed bars). Each group contained five mice. Values are means ± standard errors. ∗, P < 0.05.