Interleukin-23 drives innate and T cell-mediated intestinal inflammation

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract involving aberrant activation of innate and adaptive immune responses. We have used two complementary models of IBD to examine the roles of interleukin (IL)-12 family cytokines in bacterially induced intestinal inflammation. Our results clearly show that IL-23, but not IL-12, is essential for the induction of chronic intestinal inflammation mediated by innate or adaptive immune mechanisms. Depletion of IL-23 was associated with decreased proinflammatory responses in the intestine but had little impact on systemic T cell inflammatory responses. These results newly identify IL-23 as a driver of innate immune pathology in the intestine and suggest that selective targeting of IL-23 represents an attractive therapeutic approach in human IBD.

Figure 1.

Increased expression of IL-23 and IL-17 in inflamed intestine. (A and B) RNA was purified from tissue samples isolated from H. hepaticus–infected (≥8 wk after infection) and control uninfected 129SvEvRAG^−/− mice, and cytokine gene expression was assayed using Q-PCR. For each individual sample, cytokine gene expression was normalized relative to expression of HPRT (×10⁴). Data shown represent mean relative expression levels (±SEM) from two independent experiments (n = 8–9 total mice per group). (C) Freshly isolated colon explants from H. hepaticus–infected and control uninfected 129SvEvRAG^−/− mice were cultured overnight in complete RPMI, and cytokine release was measured using cytometric bead assay or ELISA. Results represent mean cytokine levels (±SEM; pg/100 mg colonic tissue) in samples pooled from three independent experiments (n = 9–12 total mice per group). (D and E) Spleen cells and LPLs (pooled from 6–12 mice) were isolated from H. hepaticus–infected 129SvEvRAG^−/− mice and separated into the indicated subpopulations using FACS sorting. IL-17 gene expression was assayed using Q-PCR and normalized relative to expression of HPRT (×10⁴). Data shown represent mean relative expression levels (±SEM) from two independent experiments.

Figure 2.

IL-23 drives innate immune typhlocolitis. (A) 129SvEvRAG^−/− mice were infected with H. hepaticus and treated i.p. with 1 mg/wk of anti–IL-23p19 or isotype control antibody throughout the course of the experiment. Mice were killed 6–8 wk later, and pathology in the cecum and colon was assessed histologically. Each symbol represents a single animal, and the data shown represent combined results from two independent experiments (n = 4 total controls; and n = 8–9 total mice for experimental groups). Horizontal lines represent means. (B) Representative photomicrographs of tissue sections isolated from the mice outlined in A. Numbers indicate inflammation scores for the cecal sections shown. Bar, 500 μm.

Figure 3.

IL-23 drives systemic innate immune activation in H. hepaticus–infected 129SvEvRAG^−/− mice. 129SvEvRAG^−/− mice were infected with H. hepaticus and treated i.p. with 1 mg/wk of anti–IL-23p19 or isotype control antibody throughout the course of the experiment (6–8 wk). Spleen cell populations were enumerated using FACS analysis (reference 58). Graphs represent means ± SEM of (A) spleen weights, (B) spleen cell numbers, (C) granulocytes (FSC^HiSSC^HiGr1^HiCD11b^HiCD11c⁻), and (D) monocytes/macrophages (FSC^HiSSC^LoGr1^IntCD11b^HiCD11c⁻). Data shown represent combined results from two independent experiments (n = 4 total controls; and n = 8–9 total mice for experimental groups).

Figure 4.

IL-23 blockade reduces proinflammatory cytokine production in the intestine. 129SvEvRAG^−/− mice were infected with H. hepaticus and treated i.p. with 1 mg/wk of anti–IL-23p19 or isotype control antibody throughout the course of the experiment (6–8 wk). (A and B) Cytokine concentrations in colon homogenates were measured and normalized to total protein content for each sample and are given as pg/mg total protein. Results represent mean cytokine levels (±SEM) from samples pooled from two similar experiments (n = 3–4 total samples per group). (C) Total amount of H. hepaticus DNA present in each cecal sample was determined using Q-PCR. Results represent mean H. hepaticus DNA levels (±SEM; n = 5 mice per group). H. hepaticus DNA was undetectable in cecal samples isolated from control uninfected mice.

Figure 5.

IL-23 drives T cell–mediated colitis. Cohorts of wild-type or IL-12/23–deficient RAG^−/− mice were reconstituted with 4 × 10⁵ CD4⁺CD45RB^high T cells by i.p. injection. Mice were killed 6–8 wk later, and pathology in the colon was assessed histologically. Each symbol represents a single animal, and data shown represent combined results from two independent experiments (n = 7–11 total mice per group). Horizontal lines represent means.

Figure 6.

T cell–mediated systemic inflammation does not require IL-12 or IL-23. Cohorts of wild-type or IL-12/23–deficient RAG^−/− mice were reconstituted with 4 × 10⁵ CD4⁺CD45RB^high T cells by i.p. injection and killed 6–8 wk later. (A–C) Splenomegaly was assessed by measuring spleen weights (A) and spleen cell number (B), and CD4⁺ T cell reconstitution (C) was assessed using FACS analysis. Data represent means ± SEM from two similar experiments (n = 7–11 total mice per group). (D) Representative photomicrographs of liver sections isolated from the mice outlined earlier in this legend. Bar, 500 μm.

Figure 7.

IL-23 deficiency results in decreased levels of proinflammatory cytokine production in the intestine. Cohorts of wild-type or IL-12/23–deficient RAG^−/− mice were reconstituted with 4 × 10⁵ CD4⁺CD45RB^high T cells by i.p. injection and killed 6–8 wk later. Cytokine concentrations in colon homogenates were measured and normalized to total protein content for each sample and are given as pg/mg total protein. Results represent mean cytokine levels (±SEM) from one of two similar experiments (n = 3–5 mice per group).

Figure 8.

IL-12 or IL-23 are not required for differentiation of IL-17–secreting CD4⁺ T cells in vivo. Cohorts of wild-type or IL-12/23–deficient RAG^−/− mice were reconstituted with 4 × 10⁵ CD4⁺CD45RB^high T cells by i.p. injection and killed 6–8 wk later. (A) IL-17 concentrations in colon homogenates were measured and normalized to total protein content for each sample and are given as pg/10 mg total protein. Results represent mean cytokine levels (±SEM) from one of two similar experiments (n = 3–5 mice per group). (B) Pooled MLN cells were restimulated for 4 h with PMA and ionomycin in the presence of Brefeldin A. IFN-γ– and IL-17–secreting cells were detected using intracellular FACS analysis. Results shown represent frequencies of cytokine-secreting cells among gated CD4⁺ cells and are representative of two similar experiments.