IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6

Abstract

Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel disease (IBD), such as Crohn disease and ulcerative colitis. In early clinical trials as well as in animal models, IL-12 has been implicated as a major mediator of these diseases based on the ability of anti-p40 mAb treatment to reverse intestinal inflammation. The cytokine IL-23 shares the same p40 subunit with IL-12, and the anti-p40 mAbs used in human and mouse IBD studies neutralized the activities of both IL-12 and IL-23. IL-10-deficient mice spontaneously develop enterocolitis. To determine how IL-23 contributes to intestinal inflammation, we studied the disease susceptibility in the absence of either IL-23 or IL-12 in this model, as well as the ability of recombinant IL-23 to exacerbate IBD induced by T cell transfer. Our study shows that in these models, IL-23 is essential for manifestation of chronic intestinal inflammation, whereas IL-12 is not. A critical target of IL-23 is a unique subset of tissue-homing memory T cells, which are specifically activated by IL-23 to produce the proinflammatory mediators IL-17 and IL-6. This pathway may be responsible for chronic intestinal inflammation as well as other chronic autoimmune inflammatory diseases.

Figure 1. IL-23, but not IL-12, is essential for spontaneous colitis induced by IL-10 deficiency.

(A) Histologic changes were evaluated at 3 time points in the colons of mice that were IL-10 deficient and/or IL-23 (p19) deficient. (B) Histologic changes were evaluated at 3 months in the colons of mice that were IL-10 deficient and/or IL-12 (p35) deficient. The disease scores for each group (n = 5–8) were obtained as previously described (41). (C) Photomicrographs of the descending colons from 12-month-old IL-10 × p19–KO mice (upper panel) and IL-10 –KO mice (lower panel). The lower panel shows marked mucosal thickening and epithelial hyperplasia. *Inflammation extended into the submucosa and tunica muscularis. Scale bar: 50 μm.

Figure 2. Normal production of IFN-γ in the absence of IL-23, but not IL-12.

CD4⁺ splenocytes from IL-10 –deficient mice and mice doubly deficient for IL-10 and IL-23p19 (A) or IL-12p35 (B) were stimulated with anti-CD3 and anti-CD28 for 3 days. Supernatants were assessed for IFN-γ as described in Methods. Bars represent 3 mice per group; error bars indicate SD. Data shown are representative of 2 experiments.

Figure 3. IL-23 treatment accelerates the onset of colitis in T cell–reconstituted immunodeficient recipients.

(A) Disease scores of Rag-KO mice reconstituted with naive or memory CD4⁺ T cells from IL-10 –KO donors followed by daily infusions with IL-23 (1 μg/dose) or saline for 4 weeks (n = 8). (B) T cell and accessory cell numbers in mesenteric lymph nodes of reconstituted Rag-KO mice treated with IL-23 or saline for 4 weeks.

Figure 4. Gene expression induced by IL-23 treatment of Rag-KO mice reconstituted with memory CD4⁺ T cells from IL-10 –KO mice.

(A) Increased mRNA expression in colons of Rag-KO mice reconstituted with memory CD4⁺ T cells from IL-10 –KO mice and treated with IL-23 for 4 weeks. mRNA from the colons of 4 mice were pooled, and gene expression levels were determined by real-time quantitative PCR. Data are presented as values normalized to ubiquitin. (B) Gene expression by memory CD4⁺ T cells from IL-10 –KO mice following in vitro stimulation with anti-CD3 mAb plus IL-2, IL-12, or IL-23 relative to stimulation with anti-CD3 mAb alone.

Figure 5. IL-23, but not IL-12, specifically stimulates a subset of memory CD4⁺ T cells that produce IL-17.

FACS-purified CD4⁺CD45RB^low splenic T cells (2 × 10⁵/ml; >95% purity) were isolated and cultured on CD3-coated plates. (A) Cytokine production by memory CD4⁺ T cells. Supernatants from cells stimulated with IL-23 (10 ng/ml) or IL-12 (1 ng/ml) were analyzed by ELISA. (B) IL-23–dependent proliferation of memory CD4⁺ T cells was assessed by[³H]-thymidine incorporation after 4 days of culture on anti-CD3 mAb–coated plates in the presence of anti–IL-2 mAb. (C) FACS analysis of memory CD4⁺ T cells from IL-10 –KO and IL-10 × p19–KO mice after cells were stained for intracellular IL-4, IL-17, and IFN-γ proteins.

Figure 6. Blocking IL-6 and IL-17 significantly reduced the intestinal inflammation, by 50%.

Recipient mice were dosed i.p. with isotype, anti–IL-6, anti–IL-17, or anti–IL-6 plus anti–IL-17 Abs (2 mg/mouse) a day prior to T cell reconstitution. Rag-KO mice were reconstituted with sorted splenic CD4⁺CD45RB^hi (naive) T cells (5 × 10⁵ cells/mouse) from diseased IL-10 –KO mice and treated daily with 1 μg/mouse IL-23 protein. Subsequent rounds of Ab were administered weekly for 6 weeks. The graph shows the path scores from 2 independent but identical experiments. The disease scores for each group were obtained as previously described (41). Horizontal bars represent the median value for each group. **P < 0.05, compared with isotype Ab (unpaired Student’s t test). Histologic examination was performed and scored using formalin-fixed tissue sections stained with H&E, as previously described (40).