IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis - PubMed (original) (raw)

Comparative Study

. 2006 Oct 30;203(11):2485-94.

doi: 10.1084/jem.20061082. Epub 2006 Oct 9.

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Comparative Study

IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis

Marika C Kullberg et al. J Exp Med. 2006.

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract that is caused in part by a dysregulated immune response to the intestinal flora. The common interleukin (IL)-12/IL-23p40 subunit is thought to be critical for the pathogenesis of IBD. We have analyzed the role of IL-12 versus IL-23 in two models of Helicobacter hepaticus-triggered T cell-dependent colitis, one involving anti-IL-10R monoclonal antibody treatment of infected T cell-sufficient hosts, and the other involving CD4+ T cell transfer into infected Rag-/- recipients. Our data demonstrate that IL-23 and not IL-12 is essential for the development of maximal intestinal disease. Although IL-23 has been implicated in the differentiation of IL-17-producing CD4+ T cells that alone are sufficient to induce autoimmune tissue reactivity, our results instead support a model in which IL-23 drives both interferon gamma and IL-17 responses that together synergize to trigger severe intestinal inflammation.

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Figures

Figure 1.

Figure 1.

H. hepaticus infection plus anti–IL-10R mAb treatment leads to severe typhlocolitis in WT and p35−/− mice but reduced inflammation in the ascending colon of IFN-γ−/− animals. B6 WT, p40−/−, p35−/−, IFN-γ−/−, and B10 IL-10−/− mice were infected with H. hepaticus and treated with control or anti–IL-10R mAb once a week for 4 wk. Uninfected mice were included as controls. 1 wk after the last mAb administration, pathology was analyzed in the cecum (A) and ascending colon (B). MLN cells were cultured with 5 μg/ml SHelAg, and IFN-γ (C) and IL-17 (D) were measured in 72-h supernatants. No IFN-γ or IL-17 was detected from cells cultured in medium alone. Data in A and B are pooled from two separate experiments and show histology scores from individual mice, with the horizontal line indicating the average for each group. Bars in C and D represent means ± SEM of culture duplicates of three to five mice/group from one representative experiment of the two shown in A and B. (E) Single cell analysis of IL-17 and IFN-γ–producing CD4+ T cells in anti–IL-10R–treated Hh+ WT and Hh+ IL-10−/− mice. MLN cells from uninfected (top) or 6-wk-infected anti–IL-10R–treated WT (bottom left) or 6-wk-infected IL-10−/− mice (bottom right) were cultured with SHelAg for 72 h. After an additional incubation for 18 h in medium, cells were stimulated with PMA and ionomycin in the presence of brefeldin A, and three-color staining for CD4, IL-17, and IFN-γ was performed. The FACS dot plots shown are gated on CD4+ cells and are representative of more than three experiments performed.

Figure 2.

Figure 2.

Intestinal pathology in anti–IL-10R–treated Hh+ T cell–sufficient mice. B6 WT, p40−/−, p35−/−, and IFN-γ−/− mice were infected with H. hepaticus and treated with control or anti–IL-10R mAb once a week for 4 wk. 1 wk after the last mAb administration, pathology was analyzed in the cecum (A–E) and ascending colon (F–J). Shown is the histology of representative cecal and colonic sections from the mice in Fig. 1 (A and B). (A and F) Hh+ WT plus control mAb, (B and G) Hh+ WT plus anti–IL-10R, (C and H) Hh+ p40−/− plus anti–IL-10R, (D and I) Hh+ p35−/− plus anti–IL-10R, and (E and J) Hh+ IFN-γ−/− plus anti–IL-10R. The tissue photomicrographs shown were scored as follows: A, 0; B, 5; C, 0; D, 6; E, 7; F, 0; G, 3; H, 0; I, 2; J, 0. Note that the scores in Fig. 1 represent a total score for the whole section and include cells infiltrating the serosa as well as crypt abscesses and ulcers, all of which are absent in the above photomicrographs. Hematoxylin and eosin staining. Bars, 50 μm.

Figure 3.

Figure 3.

Cytokine mRNA levels in the colons of anti–IL-10R–treated Hh+ WT mice. Colonic tissues from uninfected or 4-wk-infected WT mice treated with control mAb or anti–IL-10R were collected and processed for real-time RT-PCR analysis of p19, p35, p40, IL-17A, IL-17F, and IFN-γ. Bars represent data from individual mice (two uninfected and four Hh+ mice per group) in one of the experiments shown in Fig. 1 (A and B). Error bars represent the range of fold difference in gene expression.

Figure 4.

Figure 4.

Anti-p40 mAb treatment prevents intestinal pathology in anti–IL-10R–treated Hh+ mice. B6 WT, p35−/−, and IFN-γ−/− mice were infected with H. hepaticus and treated with control, anti–IL-10R, or anti–IL-10R plus anti-p40 mAb once a week for 4 wk. 1 wk after the last mAb administration, pathology was analyzed in the cecum (A, black bars) and ascending colon (A, gray bars). Despite significant cecal pathology, anti–IL-10R–treated Hh+ IFN-γ−/− mice failed to develop inflammation in the colon. MLN cells were cultured with SHelAg, and IFN-γ (B) and IL-17 (C) were measured in 72-h supernatants. No IFN-γ or IL-17 was detected from cells cultured in medium alone. Bars in A represent mean histology scores ± SEM of four mice/group. Bars in B and C represent means ± SEM of culture duplicates of MLN cells pooled from the mice in A. nd, not detected.

Figure 5.

Figure 5.

Hh+ Rag−/− and p35Rag−/−, but not p40Rag−/−, mice develop intestinal inflammation after T cell reconstitution. Hh+ B6/B10 Rag−/−, p40Rag−/−, and p35Rag−/− mice were inoculated i.v. with 3 × 105 CD4+ CD45RBhi cells from uninfected WT or IFN-γ−/− animals as indicated. Separate groups of Hh+ Rag−/− and p35Rag−/− recipients of WT CD4+ CD45RBhi cells were also treated with anti-p40 or anti–IFN-γ on days 0, 7, 14, and 21 of cell transfer. Pathology in the cecum (A) and ascending colon (B) was analyzed 4 wk after cell transfer. Infected mice receiving no cells were included as controls (◯). Data are pooled from two separate experiments and show histology scores from individual mice, with the horizontal line indicating the average for each group.

Figure 6.

Figure 6.

IL-23 is essential for the development of maximal colitis and intestinal proinflammatory cytokine production. Hh+ B6 Rag−/−, p40Rag−/−, p35Rag−/−, and p19Rag−/− mice were inoculated i.p. with 4 × 105 CD4+ CD45RBhi cells from uninfected WT animals as indicated. Pathology in the cecum (A) and ascending colon (B) was analyzed 4 wk later. Data are pooled from two separate experiments and show histology scores from individual mice, with the horizontal line indicating the average for each group. (C) H. hepaticus colonization levels were quantified using real-time PCR. Bars represent means ± SEM of H. hepaticus DNA levels from the individual mice shown in A and B. (D–G) Cytokine levels in colon homogenates were determined by cytometric bead assay and normalized to the total amount of protein in each sample. Shown are TNF-α (D), IFN-γ (E), MCP-1 (F), and IL-6 (G). Data are pooled from the two experiments shown in A and B assayed as individual mice in experiment 1 and as two pools of three mice/group in experiment 2. The dotted line represents cytokine levels in unreconstituted Hh+ Rag−/− mice.

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