Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and gamma interferon-dependent mechanism

Abstract

Mice rendered deficient in interleukin-10 (IL-10) by gene targeting (IL-10(-/-) mice) develop chronic enterocolitis resembling human inflammatory bowel disease (IBD) when maintained in conventional animal facilities. However, they display a minimal and delayed intestinal inflammatory response when reared under specific-pathogen-free (SPF) conditions, suggesting the involvement of a microbial component in pathogenesis. We show here that experimental infection with a single bacterial agent, Helicobacter hepaticus, induces chronic colitis in SPF-reared IL-10(-/-) mice and that the disease is accompanied by a type 1 cytokine response (gamma interferon [IFN-gamma], tumor necrosis factor alpha, and nitric oxide) detected by restimulation of spleen and mesenteric lymph node cells with a soluble H. hepaticus antigen (Ag) preparation. In contrast, wild-type (WT) animals infected with the same bacteria did not develop disease and produced IL-10 as the dominant cytokine in response to Helicobacter Ag. Strong H. hepaticus-reactive antibody responses as measured by Ag-specific total immunoglobulin G (IgG), IgG1, IgG2a, IgG2b, IgG3, and IgA were observed in both WT and IL-10(-/-) mice. In vivo neutralization of IFN-gamma or IL-12 resulted in a significant reduction of intestinal inflammation in H. hepaticus-infected IL-10(-/-) mice, suggesting an important role for these cytokines in the development of colitis in the model. Taken together, these microbial reconstitution experiments formally establish that a defined bacterial agent can serve as the immunological target in the development of large bowel inflammation in IL-10(-/-) mice and argue that in nonimmunocompromised hosts IL-10 stimulated in response to intestinal flora is important in preventing IBD.

FIG. 1

Immunostaining for CD3⁺ cells of Bouin’s-fixed cecum and colon of IL-10^−/− and WT mice, all with hematoxylin counterstain. (A to C) Ceca of representative uninfected WT (A), infected WT (B), and uninfected IL-10^−/− (C) mice at 4 weeks, showing few CD3⁺ cells. (D) Cecum of infected IL-10^−/− mouse at 4 weeks, with many CD3⁺ cells and marked thickening (hyperplasia) of the epithelium. (E) Colon of uninfected IL-10^−/− mouse at 4 weeks, with few CD3⁺ cells in the lamina propria. (F) Colon of infected IL-10^−/− mouse at 4 weeks, with many CD3⁺ cells in lamina propria and marked diffuse epithelial hyperplasia. Magnifications: (A to D) ×75, (E and F) ×150.

FIG. 2

Gross and microscopic intestinal pathology of H. hepaticus-infected mice. (A to C) Gross pathology of portions of proximal large bowels of 4-week-infected WT (A), uninfected IL-10^−/− (B), and 4-week-infected IL-10^−/− (C) mice. Note the smaller, paler cecum of the infected IL-10^−/− mouse; histologically, the mouse had typhlitis and diffuse hyperplasia. (D) Diffuse cecal hyperplasia in infected IL-10^−/− mouse at 9 weeks, showing marked inflammation in lamina propria and submucosa. H&E stain. (E) Severe focal atypical hyperplasia in cecum of infected IL-10^−/− mouse at 16 weeks. The muscularis mucosa is disrupted by atypical hyperplastic cells. H&E stain. (F) Steiner stain of cecum from infected IL-10^−/− mouse at 4 weeks, showing numerous bacteria within epithelial crypts. Magnifications, ×75 (D) and ×150 (E and F).

FIG. 3

SHelAg-induced proliferative responses of cells from H. hepaticus-infected WT and IL-10^−/− mice. Spleen cells (2.5 × 10⁶/ml) (A) and MLN cells (1.5 × 10⁶/ml) (B) from 4-week-infected (solid lines) and uninfected (dotted lines) WT (○) and IL-10^−/− (•) mice were stimulated in vitro with various doses of SHelAg. Cultures were pulsed with [³H]thymidine after 48 h and harvested 18 h later. Spleen cell results represent the mean values for three individual infected and a pool of two uninfected mice per group. MLN results represent means ± SD of duplicate cultures of pools of the same mice shown in A. Data for one of six experiments (using 4-, 6-, 10-, or 11-week-infected mice) with similar results are shown. An asterisk indicates a significant difference (P < 0.05) in proliferative response between infected WT and IL-10^−/− mice.

FIG. 4

SHelAg-specific Ab in sera of H. hepaticus-infected WT and IL-10^−/− mice. Sera from uninfected (▵, ▴) and 16-week-infected (○, •) WT (open symbols) and IL-10^−/− (closed symbols) mice were analyzed for levels of Ab (total IgG, IgG1, IgG2a, IgG2b, IgG3, and IgA) reactive to SHelAg by ELISA as described in Materials and Methods. Each dot represents the mean optical density (OD) value obtained from two separate pools of sera per group tested in duplicate (three females and three males in each pool).

FIG. 5

SHelAg-induced cytokine responses of spleen cells from H. hepaticus-infected WT and IL-10^−/− mice. Spleen cells (5 × 10⁶/ml) from uninfected (Uninf.) and 9-week-infected (Inf.) WT and IL-10^−/− mice were stimulated with medium alone (□) or 1 μg of SHelAg per ml (■), and IL-10 (A), IFN-γ (B), TNF-α (C), and NO (D) were measured in 72-h supernatants. Bars represent means ± SD of values obtained from two separate pools of mice per group tested in duplicate (three females and three males in each pool). Data for one of seven experiments (using 4-, 6-, 9-, 10-, or 11-week-infected mice) with similar results are shown.

FIG. 6

SHelAg-induced cytokine responses of MLN cells from H. hepaticus-infected WT and IL-10^−/− mice. MLN cells (3 × 10⁶/ml) from uninfected and 2-, 4-, 6-, and 10-week-infected WT and IL-10^−/− mice were stimulated with medium alone (□) or 1 μg of SHelAg per ml (■), and IL-10 (A), IFN-γ (B), and TNF-α (C) were measured in 72-h supernatants. Bars represent means ± SD of duplicate ELISA values from a pool of two to three infected WT and three to four infected IL-10^−/− mice per time point. Cytokine levels for the 0-week postinfection time point have been calculated after pooling data from three separate determinations for uninfected mice assayed in parallel with the infected mice at 4, 6, and 10 weeks postinfection.

FIG. 7

T-cell depletion (T-depl.) of MLN populations from H. hepaticus-infected mice abolishes SHelAg-induced cytokine secretion. Total (left) and anti-Thy1.2-plus-complement-treated (right) MLN cells from 4-week-infected WT and IL-10^−/− mice were cultured with medium alone (□) or 0.3 μg of SHelAg per ml (■), and IL-10 (A), IFN-γ (B), and TNF-α (C) were measured in 72-h supernatants. Bars represent means ± SD of duplicate ELISA values from a pool of three mice per group.

FIG. 8

Treatment with anti-IFN-γ or anti-IL-12 inhibits development of H. hepaticus-induced colitis in IL-10^−/− mice. IL-10^−/− mice were treated every 3 to 4 days with 1 mg of neutralizing MAb to IFN-γ or IL-12 or with a control MAb starting 1 day prior to H. hepaticus inoculation, and tissues were examined 4 weeks later. Shown are gross pathology (A to C) and histology (D to F) of ceca from 4-week-infected IL-10^−/− mice treated with control (A and D), anti-IFN-γ (B and E), or anti-IL-12 (C and F) MAb. Note the small, pale cecum from the mouse treated with control MAb (A) and much less inflammation and epithelial hyperplasia in mice receiving anticytokine MAb (E to F). (D to F) Formalin-fixed tissues stained with H&E; magnification, ×75.