Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology

Abstract

The key role of interleukin (IL)-23 in the pathogenesis of autoimmune and chronic inflammatory disorders is supported by the identification of IL-23 receptor (IL-23R) susceptibility alleles associated with inflammatory bowel disease, psoriasis and ankylosing spondylitis. IL-23-driven inflammation has primarily been linked to the actions of T-helper type 17 (TH17) cells. Somewhat overlooked, IL-23 also has inflammatory effects on innate immune cells and can drive T-cell-independent colitis. However, the downstream cellular and molecular pathways involved in this innate intestinal inflammatory response are poorly characterized. Here we show that bacteria-driven innate colitis is associated with an increased production of IL-17 and interferon-gamma in the colon. Stimulation of colonic leukocytes with IL-23 induced the production of IL-17 and interferon-gamma exclusively by innate lymphoid cells expressing Thy1, stem cell antigen 1 (SCA-1), retinoic-acid-related orphan receptor (ROR)-gammat and IL-23R, and these cells markedly accumulated in the inflamed colon. IL-23-responsive innate intestinal cells are also a feature of T-cell-dependent models of colitis. The transcription factor ROR-gammat, which controls IL-23R expression, has a functional role, because Rag-/-Rorc-/- mice failed to develop innate colitis. Last, depletion of Thy1+ innate lymphoid cells completely abrogated acute and chronic innate colitis. These results identify a previously unrecognized IL-23-responsive innate lymphoid population that mediates intestinal immune pathology and may therefore represent a target in inflammatory bowel disease.

Figure 1

IL-23 induced IL-17 and IFN-γ are required for H. hepaticus-mediated innate colitis. a, Cytokine secretion following overnight culture of splenocytes or cLP cells from control or H. hepaticus–infected 129SvEvRag^−/− mice (n=6 per group). b, Cytokine secretion by cLP cells from control 129SvEvRag^−/− mice following overnight culture with IL-12 or IL-23 (n=6). Data represents mean ± s.e.m. Colitis scores (c), splenomegaly (d), and representative colon photomicrographs (×50) (e) from H. hepaticus infected 129SvEvRag^−/− mice treated with blocking α-IL-17 and/or α-IFNγ or isotype (Iso) control mAbs. Data represents two pooled experiments (n=5-12 per group). *P<0.05; **P<0.01; ***P<0.001.

Figure 2

IL-23-responsive innate lymphoid cells in inflamed colon are Thy1^hiSCA-1⁺ RORγt⁺. a,b IL-17, IFN-γ and Thy1 expression in Lin⁻ cLP cells from H. hepaticus–infected 129SvEvRag^−/− mice following overnight culture with or without IL-23 c, Phenotypic analysis of Lin⁻IL-17⁺ cLP cells from H. hepaticus-infected 129SvEvRag^−/− mice, using specific antibodies (black line) and isotype controls (grey line). d, Cytokine secretion and e, IL-23R, RORγt,AHR and Tbx21 mRNA expression, by sorted Thy1^hiSCA-1⁺ or the remaining cLP cells (Rest) from H. hepaticus–infected 129SvEvRag^−/− mice following overnight culture with or without IL-23. Results are representative of ≥ 2 independent experiments.

Figure 3

Thy1^hi innate lymphoid cells drive H. hepaticus-induced innate intestinal inflammation. a, Frequency of Thy1^hiSCA-1⁺ cells (n=3-6) and b, IL-17 expression among Thy1^hi cells following culture with or without IL-23 in cLP cells from control or H. hepaticus–infected 129SvEvRag^−/− mice. Data are representative of 2 independent experiments. Colitis and typhlitis scores (c), splenomegaly (d), and representative photomicrographs (×50; scale bars 200μm) (e) from H. hepaticus infected mice treated with α-Thy1 or isotype (Iso) control mAbs (n=6 per group). f, IL-17 and IFN-γ secretion by cLP cells from the mice described above, following stimulation with or without IL-23. Data represents mean ± s.e.m. (n=6). *P<0.05; ** P<0.01.

Figure 4

RORγt-expressing Thy1^hi innate lymphoid cells are required for α-CD40-induced innate intestinal inflammation. Colitis scores (a), colon photomicrographs (×50) (b) weight loss (c) and cLP cytokine secretion following overnight culture (d) in α-CD40 treated C57BL/6 Rag^−/− mice injected with or without α-Thy1 or isotype control mAb. e, RORγt mRNA expression by sorted Thy1^hi SCA-1⁺ or the remaining cLP cells (Rest) from α-CD40 treated mice. Results are representative of 2 independent experiments. Colitis scores (f) and photomicrographs (×50) (g) from α-CD40 treated C57BL/6 Rag^−/− or C57BL/6 Rag^−/−Rorc^−/− mice. (a-c) Data represent pooled results from two experiments (n=6-10 ) and (f,g) (n=11-13). (d) Data represents mean ± s.e.m. (n=5).**P<0.01; ***P<0.001.