Transient inhibition of ROR-γt therapeutically limits intestinal inflammation by reducing TH17 cells and preserving group 3 innate lymphoid cells

Abstract

RAR-related orphan receptor-γt (ROR-γt) directs differentiation of proinflammatory T helper 17 (TH17) cells and is a potential therapeutic target in chronic autoimmune and inflammatory diseases. However, ROR-γt-dependent group 3 innate lymphoid cells ILC3s provide essential immunity and tissue protection in the intestine, suggesting that targeting ROR-γt could also result in impaired host defense after infection or enhanced tissue damage. Here, we demonstrate that transient chemical inhibition of ROR-γt in mice selectively reduces cytokine production from TH17 but not ILCs in the context of intestinal infection with Citrobacter rodentium, resulting in preserved innate immunity. Temporal deletion of Rorc (encoding ROR-γt) in mature ILCs also did not impair cytokine response in the steady state or during infection. Finally, pharmacologic inhibition of ROR-γt provided therapeutic benefit in mouse models of intestinal inflammation and reduced the frequency of TH17 cells but not ILCs isolated from primary intestinal samples of individuals with inflammatory bowel disease (IBD). Collectively, these results reveal differential requirements for ROR-γt in the maintenance of TH17 cell and ILC3 responses and suggest that transient inhibition of ROR-γt is a safe and effective therapeutic approach during intestinal inflammation.

Figure 1. Transient chemical inhibition of ROR-γt selectively reduces T_H17 responses but not ILC3s during infection

(a–i) C57BL/6 mice were infected with C. rodentium and treated daily with vehicle control or GSK805 (10 mg/kg) starting on day –1 and continued for the duration of the experiment (a) Frequency of IL-17A-producing T_H17 cells and (b) IL-17A- or (c) IL-22-producing ILC3 at day 10 post infection. (d) Total numbers of IL-17A⁺ and IL-22⁺ T_H17 cells, (e) IFN-γ⁺ T cells and (f) IL-17A⁺ and IL-22⁺ ILC3 in the colon at day 10 post infection. (g) Survival curve of infected mice. (h) Colon length and (i) Hematoxylin and eosin stained histologic sections of the distal colon at day 10 post infection, representative of n = 5. Scale bar, 200 μm. All data are representative of five individual mice (n = 5) and was replicated in two independent experiments. Data shown are mean ± SEM (error bars). Statistics compare treatment versus control groups using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Transient genetic deletion of ROR-γt impairs T_H17 cells but not ILC3s during homeostasis and intestinal infection

(a–g) Analyses of ILC3s in Rorc^floxed control and Id2^iΔROR-γt mice following daily treatment with tamoxifen for two weeks. (a) Gating for ILCs and subsequent analysis for ROR-γt. Lin^–CD127⁺ cells from the mLN were analyzed for (b) CCR6 and (c) CD4 protein, as well as sequential gating for MHCII protein. (d) Relative expression of ILC3-associated genes from mLN sort purified CCR6⁺ ILCs was assessed by genome-wide microarrays. (e–h) Mice were infected with C. rodentium and treated daily with tamoxifen starting on day –1. (e) Identification of IL-22-producing ILC3 and (f) IL-17-producing T_H17 cells at day 10 post infection. (i) Survival curve of infected mice. (g) Colon length and (h) Hematoxylin and eosin stained histologic sections of the distal colon at day 10 post infection, representative of n = 5. Scale bar, 200 μm. All data are representative of five individual mice (n = 5) and was replicated in two independent experiments. Data shown are mean ± SEM (error bars). Statistics compare treatment versus control groups using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 3. Transient inhibition of ROR-γt selectively limits T_H17 cell responses and reduces intestinal inflammation

(a–d) Ten-week-old Il10^−/− mice were treated with vehicle control or GSK805 (10 mg/kg) for two weeks. (a) Frequency of IL-17A-producing T_H17 cells (red) or ILC3 (blue). Amount of fecal lipocalin-2 (b), colon length (c), and Hematoxylin and eosin stained histologic sections of the distal colon (d). Scale bar, 200 μm, representative of n = 5. (e–h) Adoptive transfer of CBir1 TCR transgenic T cells into Rag1^−/− mice, followed 2–3 weeks later by daily treatment with either vehicle control or GSK805 (10 mg/kg) for 2 weeks. (e) Analyses of IL-17A-producing T_H17 cells (top) or ILC3 (bottom), (f) amount of fecal lipocalin-2, (g) colon length, and (h) Hematoxylin and eosin stained histologic sections of the distal colon. Scale bar, 200 μm, representative of n = 5. All data are representative of five individual mice (n = 5) and was replicated in two independent experiments. Data shown are mean ± SEM (error bars). Statistics compare treatment versus control groups using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 4. Transient inhibition of ROR-γt selectively reduces T_H17 cells in intestinal tissue from pediatric individuals with Crohn’s disease

(a–i) Intestinal resection tissue from pediatric individuals with Crohn’s disease was cultured with DMSO control or GSK805 (0.5 μM) for 12 hours. (a) Identification of Lin^–IL-7Rα⁺ ILCs. (b) Percentage of NKp44⁺c-kit⁺ ILC3s. (c) Production of IL-22 and (d) TNF by gated c-kit⁺ ILC3s. (e) Expression of IL-17A and (f) IL-22 by CD3⁺ T_H17 cells. (g) Fold change in the percentage of IL-17A⁺ or IL-22⁺ T_H17 cells, (h) IFN-γ⁺ T_H1 cells and (i) IL-17A⁺ or IL-22⁺ ILC3 following culture with GSK805 relative to DMSO. All data are representative or shown for n = 10 samples from pediatric individuals with Crohn’s disease.