Critical role for tumor necrosis factor alpha in controlling the number of lumenal pathogenic bacteria and immunopathology in infectious colitis

Abstract

Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. In these latter models, and in patients with Crohn's disease, neutralization of tumor necrosis factor alpha (TNF-alpha) is of therapeutic benefit. Since there is no information on the role of TNF-alpha in either immunity to noninvasive bacterial pathogens or on the role of TNF-alpha in the immunopathology of infectious colitis, we investigated C. rodentium infection in TNFRp55(-/-) mice. In TNFRp55(-/-) mice, there were higher colonic bacterial burdens, but the organisms were cleared at the same rate as C57BL/6 mice, showing that TNF-alpha is not needed for protective antibacterial immunity. The most striking feature of infection in TNFRp55(-/-) mice, however, was the markedly enhanced pathology, with increased mucosal weight and thickness, increased T-cell infiltrate, and a markedly greater mucosal Th1 response. Interleukin-12 p40 transcripts were markedly elevated in C. rodentium-infected TNFRp55(-/-) mice, and this was associated with enhanced mucosal STAT4 phosphorylation. TNF-alpha is not obligatory for protective immunity to C. rodentium in mice; however, it appears to play some role in downregulating mucosal pathology and Th1 immune responses.

FIG. 1

Bacterial load in C. rodentium-infected C57BL/6 and TNFRp55^−/− mice. Bacterial counts were determined in the colon (A), spleen (B), and MLN (C) at the indicated time points. Each point reflects the means and SEM (error bars) of five mice per group (✽, P < 0.05). The data shown are from one experiment of three, which yielded identical results. The lower limit detection of the bacteria is 10 CFU per organ.

FIG. 2

Intimin staining of C. rodentium-infected C57BL/6 mice (magnifications: A, ×100; C, ×1,000; F, ×400) and TNFRp55^−/− mice (magnifications: B, ×100; D, ×1,000). (D) Bacteria can be seen along the surface epithelium in both groups of mice (arrows) and occasionally deep in the glands in the infected TNFRp55^−/− mice. (E) Rabbit isotype control IgG showed no staining (magnification, ×400). Another feature was that the anti-intimin antibody, in addition to staining the bacteria, also stained epithelial cells underlying areas of bacterial colonization (D, large arrows).

FIG. 3

Weight of the distal colon (A) and crypt length (B) in uninfected and C. rodentium-infected C57BL/6 and TNFRp55^−/− mice on days 7 and 14. Each group contained five mice (✽, P < 0.05).

FIG. 4

Cell counts for CD3⁺ (A), CD4⁺ (B), and CD8⁺ (C) cells infiltrating the lamina propria in uninfected and C. rodentium-infected C57BL/6 and TNFRp55^−/− mice on days 7 and 14. Each group represents five mice (the mean ± SEM) (✽, P < 0.05).

FIG. 5

Cytokine mRNA transcripts in gut tissue of uninfected and C. rodentium-infected C57BL/6 and TNFRp55^−/− mice on days 7 and 14. Each group represents five mice (the mean ± SEM; ✽, P < 0.05). (A) IFN-γ. (B) IL-12p40. (C) TNF-α. (D) IL-4.

FIG. 6

Western blot analysis of Stat4 and tyrosine-phosphorylated Stat4 in the gut tissue of uninfected and C. rodentium-infected C57BL/6 and TNFRp55^−/− mice on day 14. Three mice per group were analyzed in each of two independent experiments, and representative autoradiograph is shown in panel A. An increase in p-Stat4 expression was seen in the infected TNFRp55^−/− mice at day 14. (B) The density of the bands was also quantified. In total, we analyzed 5 to 6 mice per group. As positive control (⁺ve), we used proteins extracted from human peripheral blood mononuclear cells preactivated with PHA and then stimulated with IL-12, the major Stat4-inducing cytokine.