A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis

Abstract

It was recently demonstrated that interleukin (IL)-23-driven IL-17-producing (ThIL-17) T cells mediate inflammatory pathology in certain autoimmune diseases. We show that the induction of antigen-specific ThIL-17 cells, but not T helper (Th)1 or Th2 cells, by immunization with antigens and adjuvants is abrogated in IL-1 receptor type I-deficient (IL-1RI(-/-)) mice. Furthermore, the incidence of experimental autoimmune encephalomyelitis (EAE) was significantly lower in IL-1RI(-/-) compared with wild-type mice, and this correlated with a failure to induce autoantigen-specific ThIL-17 cells, whereas induction of Th1 and Th2 responses was not substantially different. However, EAE was induced in IL-1RI(-/-) mice by adoptive transfer of autoantigen-specific cells from wild-type mice with EAE. IL-23 alone did not induce IL-17 production by T cells from IL-1RI(-/-) mice, and IL-23-induced IL-17 production was substantially enhanced by IL-1alpha or IL-1beta, even in the absence of T cell receptor stimulation. We demonstrate essential roles for phosphatidylinositol 3-kinase, nuclear factor kappaB, and novel protein kinase C isoforms in IL-1- and IL-23-mediated IL-17 production. Tumor necrosis factor alpha also synergized with IL-23 to enhance IL-17 production, and this was IL-1 dependent. Our findings demonstrate that IL-1 functions upstream of IL-17 to promote pathogenic ThIL-17 cells in EAE.

Figure 1.

IL-1RI is required for induction or activation of antigen-specific ThIL-17 T cells. (A) C57BL/6 or IL-1RI^−/− mice were immunized s.c. with 10 μg KLH either alone or with 10 μg LPS. Inguinal lymph nodes were harvested after 7 d and cells were restimulated with medium or 2, 10, or 50 μg/ml KLH. (B) Lymph node cells were restimulated with medium or 20 ng/ml PMA and 1 μg/ml anti-CD3. Supernatants were collected after 3 d, and IL-4, IL-10, IL-17, and IFN-γ concentrations were determined by ELISA. C57BL/6 versus IL-1RI^−/−: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

The induction of EAE and autoantigen-specific ThIL-17 cells is dependent on functional IL-1RI. C57BL/6 or IL-1RI^−/− mice were injected with 150 μg MOG in 200 μl CFA and 500 ng PT followed 2 d later with a second dose of PT. Mice were observed for clinical symptoms of EAE daily for 48 d. A separate group of mice were killed on d 21, and spleen cells were restimulated with MOG peptide. After 3 d, supernatants were collected and cytokine concentrations were determined by ELISA. Cell proliferation was determined on day 4 of culture by [³H]thymidine incorporation. (A) Disease scores (n = 15) ± SD. (B) Antigen-specific cytokine production and proliferation. C57BL/6 versus IL-1RI^−/−: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) IL-1RI^−/− mice (n = 7) were injected with MOG in CFA followed by PT as described above or with MOG-specific T cells (9 × 10⁶ cells/mouse) generated from the spleens of C57BL/6 mice with EAE. Disease scores (n = 5) ± SD.

Figure 3.

IL-1 synergizes with IL-23 to promote IL-17 production by spleen cells and purified CD4⁺ and CD8⁺ T cells. (A) Spleen cells (10⁶ cells/ml) from C57BL/6 or IL-1RI^−/− mice were cultured with 100 pg/ml IL-23 alone or together with 1 ng/ml IL-1α, and supernatants were collected after 3 d for analysis of IL-17 and IFN-γ by ELISA. C57BL/6 versus IL-1RI^−/−: *, P < 0.05; **, P < 0.01; ***, P < 0.001. IL-1α plus IL-23 versus IL-23; #, P < 0.001. (B) Spleen cells (2.25 × 10⁶ cells/ml) from C57BL/6 mice were cultured for 48 h with medium or cytokines and for the last 4 h in the presence of 10 μg/ml brefeldin A. Intracellular staining for IL-17 and IFN-γ was performed. Percentages refer to IL-17 production by gated CD3⁺ T cells. Splenic CD4⁺ (C) and CD8⁺ (D) T cells (5 × 10⁵cells/ml) from C57BL/6 mice were incubated with 10 U/ml IL-2 and 1 μg/ml anti-CD3 antibody alone or together with 0.1–1 ng/ml IL-23 and/or 1 ng/ml IL-1α or IL-1β. IL-17 concentrations were measured by ELISA in supernatants collected after 3 d. IL-23 plus IL-1 versus IL-23 only: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

IL-1 and IL-23 promote IL-17 production in the absence of TCR stimulation via stimulation of PI3K, NF-κB, and PKCθ. (A) Splenic CD3⁺ cells (5 × 10⁵ cells/ml) from C57BL/6 mice were incubated with 10 ng/ml IL-1β, 10 ng/ml IL-23, IL-1 and IL-23, or medium only in the presence or absence of anti-CD3, and supernatants were collected at 12, 24, 48, or 72 h for analysis by ELISA. (B) Splenic CD3⁺ cells from C57BL/6 mice were incubated with 1 μg/ml anti-CD3 for 2 h before stimulation with IL-1, IL-23, IL-1 and IL-23, or medium. IL-17 mRNA expression was evaluated by real-time PCR 48 h after stimulation, and the data represent the fold change for IL-17 mRNA in relation to 18S rRNA. IL-23 plus IL-1 versus IL-23 only: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) CD3⁺ cells (5 × 10⁵ cells/ml) were stimulated with medium (control) or with IL-23, IL-1, or IL-23 and IL-1 with or without anti-CD3 in the presence or absence of (30-min pretreatment) LY294002 (PI3K inhibitor at 20 and 40 μM), BAY 11-7082 (NF-κB inhibitor at 4 and 8 μM), rottlerin (novel PKC inhibitor at 10 and 100 μM), UO126 (ERK inhibitor at 5 μM), or SB203580 (p38 inhibitor at 5 μM). Supernatants were collected 3 d later for analysis of IL-17 production by ELISA. IL-23 plus IL-1 versus IL-23 plus IL-1 plus inhibitor: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) CD3⁺ cells were incubated with medium only or with IL-1, IL-23, or IL-1 and IL-23. Cell lysates were analyzed by Western blotting with antibodies specific for phosphorylated Akt and IκB-α or β-actin as a loading control.

Figure 5.

TNF-α promotes IL-23–induced IL-17 production in an IL-1–dependent manner. Spleen cells (10⁶ cells/ml) from C57BL/6 or IL-1RI^−/− were cultured with 1 ng/ml IL-23, 0.01–1 ng/ml TNF-α, 0.01–1 ng/ml IL-23 and TNF-α, medium, or PMA and anti-CD3. Supernatants were collected after 3 d for analysis of IL-17 and IFN-γ production by ELISA. C57BL/6 versus IL-1RI^−/−: *, P < 0.05; **, P < 0.01; ***, P < 0.001. TNF-α plus IL-23 versus IL-23: #, P < 0.05; ##, P < 0.01; ###, P < 0.001.