The receptor SIGIRR suppresses Th17 cell proliferation via inhibition of the interleukin-1 receptor pathway and mTOR kinase activation

Abstract

Interleukin-1 (IL-1)-mediated signaling in T cells is essential for T helper 17 (Th17) cell differentiation. We showed here that SIGIRR, a negative regulator of IL-1 receptor and Toll-like receptor signaling, was induced during Th17 cell lineage commitment and governed Th17 cell differentiation and expansion through its inhibitory effects on IL-1 signaling. The absence of SIGIRR in T cells resulted in increased Th17 cell polarization in vivo upon myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide immunization. Recombinant IL-1 promoted a marked increase in the proliferation of SIGIRR-deficient T cells under an in vitro Th17 cell-polarization condition. Importantly, we detected increased IL-1-induced phosphorylation of JNK and mTOR kinase in SIGIRR-deficient Th17 cells compared to wild-type Th17 cells. IL-1-induced proliferation was abolished in mTOR-deficient Th17 cells, indicating the essential role of mTOR activation. Our results demonstrate an important mechanism by which SIGIRR controls Th17 cell expansion and effector function through the IL-1-induced mTOR signaling pathway.

Figure 1. SIGIRR expression is induced during Th17 cell differentiation

(A–C) Naïve CD4⁺ T cells (CD4⁺CD44^lo) were sorted by flow cytometry and polarized under Th1 (IL-12 and anti-IL-4) and Th17 (TGF-β, IL-6, anti-IFN-γ and anti-IL-4) cell-inducing conditions (on anti-CD3 and anti-CD28-coated plates) for 3 days, followed by (A) immunoblot analysis for the expression of SIGIRR, (B) real-time PCR analysis for the expression of IL-1R1 and IL-23R, and (C) ELISA assay for the production of IL-17 and IFN-γ. (D) Real-time PCR analysis for the expression of IL-17, SIGIRR, IL-1R1 and IL-23R over the time course of Th17 cell polarization. The presented data represent three independent experiments. Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test).

Figure 2. SIGIRR deficiency leads to increased susceptibility of Th17-dependent EAE and hyper activation of MOG-specific Th17 cells

(A) EAE was induced by MOG_35–55 immunization. Mean clinical scores were calculated each day for WT (n = 13) and Sigirr^−/− mice (n = 10). (B) Hematoxylin and eosin and anti-CD3 staining of spinal cord of WT and Sigirr^−/−mice 15 days after immunization with MOG_35–55. (C) Immune cell infiltration in the brain of MOG_35–55 immunized WT and Sigirr^−/− mice (n=3, 7 days after disease onset) was analyzed by flow cytometry. (D) Real-time PCR analysis of relative expression of IFN-γ, IL-17, TNF-α and IL-6 in spinal cords of MOG_35–55 immunized WT and Sigirr^−/− mice (n=3, 7 days after disease onset) as compared to CFA treated WT control mice. Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). Data are representative of three independent experiments. (E–F) Draining lymph node cells from wild-type mice and Sigirr^−/− mice were collected 10 days after immunization with either MOG_35–55 emulsified in complete Freund's adjuvant or complete Freund's adjuvant alone and were re-stimulated with MOG_35–55 in vitro for 4 days, followed by ELISPOT analysis (E) and ELISA (F) of IL-17 and IFN-γ. Error bars, s.d.; n = 10 mice per group. p<0.05; **.p<0.01 (two tailed t-test). (G) Primed MOG_35–55 specific T cells (10 days) were re-stimulated with MOG_35–55 in vitro in the presence of recombinant IL-23 for 4 days, and then transferred to naïve wild-type and Sigirr^−/− mice. Graph represents the average clinical score after T-cell transfer. n=5. *, p<0.05; (ANOVA). Data are representative of three independent experiments.

Figure 3. T-cell-derived SIGIRR regulates Th17 cell development in vivo

Wild-type and Sigirr^−/− naïve T cells were intravenousely transferred into Rag1^−/− mice and EAE was induced by MOG_35–55 immunization. (A) Mean clinical scores were calculated each day for Rag1^−/− mice transferred with wild-type and Sigirr^−/− T cells. n=5 *, p<0.05; (ANOVA). (B) Hematoxylin and eosin staining of spinal cord of Rag1^−/− mice transferred with wild-type and Sigirr^−/− T cells 15 days after immunization with MOG_35–55. (C) Real-time PCR analysis of relative expression of IFN-γ, IL-17, TNF-α and IL-6 in spinal cords of MOG_35–55 immunized Rag1^−/− mice transferred with wild-type and Sigirr^−/− T cells (n=3, 10 days after disease onset). (D–E) Draining lymph node cells from SIGIRR-TG-KO (TG-KO) and littermate Sigirr^−/− mice were collected 10 days after immunization with MOG_35–55 emulsified in complete Freund's adjuvant and were re-stimulated with MOG_35–55 in vitro for 4 days, followed by ELISA (D) and ELISPOT analysis (E) (See also Figure S1), Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). Data are representative of three independent experiments. (F–H) Naïve T cells (CD4⁺CD44^low) from wild-type and Sigirr^−/− mice were co-cultured with wild-type splenic antigen-presenting cells (APCs) in the presence of anti-CD3 and polarized them under Th1 (IL-12 and anti-IL-4), or Th17 (TGF-β, IL-6, anti-IFN-γ and anti-IL-4) conditions, followed by intracellular cytokine staining for IL-17 and IFN-γ (F) and ELISA for IL-17 (H). (G) Wild-type and Sigirr^−/− mice Th1 cells were treated with 5ng/ml IL-18 for 48 h, followed by ELISA for IFN-γ. (I) Real-time PCR analysis of relative expression of IL-17, IL-22, IL-23R, ROR-γt and IL-10 in wild-type and Sigirr^−/− Th17 cells as compared to naïve T cells. Data are representative of at least three (A–C) independent experiments. Error bars (A–C), s.d. *, p<0.05; **.p<0.01 (two tailed t-test).

Figure 4. SIGIRR suppresses Th17 differentiation and expansion through IL-1R signaling

(A) Naïve wild-type and Il1r1^−/− CD4⁺ T cells (CD4⁺CD44^low) were polarized to Th17 cells (TGFβ+IL-6 on anti-CD3 and anti-CD28 coated plates) in the presence and absence of IL-1β, followed by ELISA for IL-17 and IL-22. (B) Naïve wild-type and Sigirr^−/− CD4⁺ T cells (CD4⁺CD44^low) were polarized to Th17 cells (TGFβ+IL-6 on anti-CD3 and anti-CD28 coated plates) in the presence and absence of IL-1β, followed by intracellular cytokine staining for IL-17 and IFN-γ. While IL-1β promoted Th17 cell differentiation in both WT and Sigirr^−/− T cells, addition of IL-1β resulted in a significantly higher IL-17-positive population in Sigirr^−/− T cells than that in WT T cells. (C) Real-time PCR analysis of relative expression of IL-17, IL-22, IL-21, IL-23R, ROR-γt, IL-10 and IRF4 in wild-type and Sigirr^−/− Th17 cells as compared to the naïve T cells. Data are representative of at least three (A–C) separate experiments. Error bars (AC), s.e.m. *, p<0.05; **.p<0.01 (two tailed t-test).

Figure 5. SIGIRR suppresses Th17 expansion through IL-1-induced mTOR-mediated cell proliferation

(A) Cell lysates from wild-type and Sigirr^−/− Th17 cells untreated or treated with IL-1 (10ng/ml) for different time points were analyzed by western blot analysis using antibodies as indicated. (B) Wild-type and Sigirr^−/− Th17 cells were rested for two days, followed by incubation with different doses of IL-1 as indicated in the presence or absence of 50nM rapamycin, 2μM SB203580 or 10μM SP600125 for three days. The treated cells were analyzed for production of IL-17 by ELISA. (C) Cell lysates from Sigirr^−/− Th17 cells pre-treated with rapamycin for 2h and then treated IL-1 (10ng/ml) treatment for 15, 30 and 60 min were analyzed by western blot analysis using antibodies as indicated. (D) Wild-type and Sigirr^−/− Th17 cells were rested for two days, followed by incubation with different doses of IL-1. The treated cells were incubated one additional day with ³H for thymidine incorporation experiment. (E) Frap1^fl/fl naïve CD4⁺ T cells were differentiated into Th17 cells and infected with retrovirus expressing GFP or Cre/GFP. Infected CD4⁺GFP⁺ cells were isolated, analyzed for mTOR expression (left), and cultured in the presence and absence of IL-1. GFP⁺ cells were analyzed by thymidine incorporation assay for proliferation (right). Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). Data are representative of three independent experiments.

Figure 6. IL-1 activates mTOR pathway through IRAK proteins

(A) Cell lysates from wild-type, Irak4^−/− and Irak1^−/− Th17 cells untreat ed or treated with IL-1 (10ng/ml) for different time points were analyzed by western blot analysis using antibodies as indicated. (B) Naïve wild-type, Irak4^−/− and Irak1^−/− CD4⁺ T cells (CD4⁺CD44^low) were polarized to Th17 cells (TGFβ+IL-6 on anti-CD3 and anti-CD28 coated plates) in the presence and absence of IL-1β, followed by intracellular cytokine staining for IL-17 and IFN-γ, Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). (C) 293 cells transfected with IL-1R (293-IL-1R) cells, IRAK1-deficient cells and 293-IL-1R transfected with SIGIRR were treated or untreated with IL-1 for 15 min. TSC1/TSC2 protein complex was immunoprecipitated and analyzed by western blot analysis using antibodies as indicated. Data are representative of at least three independent experiments.