The type I IFN induction pathway constrains Th17-mediated autoimmune inflammation in mice

Abstract

IFN-beta, a type I IFN, is widely used for the treatment of MS. However, the mechanisms behind its therapeutic efficacy are not well understood. Using a murine model of MS, EAE, we demonstrate that the Th17-mediated development of autoimmune disease is constrained by Toll-IL-1 receptor domain-containing adaptor inducing IFN-beta-dependent (TRIF-dependent) type I IFN production and its downstream signaling pathway. Mice with defects in TRIF or type I IFN receptor (IFNAR) developed more severe EAE. Notably, these mice exhibited marked CNS inflammation, as manifested by increased IL-17 production. In addition, IFNAR-dependent signaling events were essential for negatively regulating Th17 development. Finally, IFN-beta-mediated IL-27 production by innate immune cells was critical for the immunoregulatory role of IFN-beta in the CNS autoimmune disease. Together, our findings not only may provide a molecular mechanism for the clinical benefits of IFN-beta in MS but also demonstrate a regulatory role for type I IFN induction and its downstream signaling pathways in limiting Th17 development and autoimmune inflammation.

Figure 1. TRIF-deficient mice develop severe EAE.

(A) Mean EAE score and (B) disease incidence in WT mice (n = 12) and TRIF^Lps2/Lps2 mice (n = 12) at each time point. WT and TRIF-deficient mice were immunized with MOG peptide (MOG_35–55) emulsified in CFA. Mice were assigned a disease score of 0 to 5 based on the severity of EAE.

Figure 2. Increased CNS inflammation in TRIF-deficient mice during EAE.

Representative sections of lumbar spinal cord from WT and TRIF-deficient mice at day 12 after MOG/CFA immunization were stained with H&E to determine inflammation or immunostained with anti-CD4 or anti–IL-17 for infiltration of CD4⁺ and Th17 cells. Data are representative of 3 experiments with similar results.

Figure 3. Th17 development in TRIF-deficient mice.

(A) Flow cytometry analysis of CNS mononuclear cells from WT and TRIF-deficient mice at day 21 after immunization. CNS mononuclear cells isolated from WT and TRIF^–/– mice were stained for intracellular IL-17. Plots were gated on CD4⁺ T cells. Numbers indicate percentage of IL-17⁺CD4⁺ cells of total CD4⁺ cells. (B) T cells from TRIF-deficient mice immunized with antigen were hyperresponsive ex vivo. Total splenocytes were isolated from WT and TRIF-deficient mice 7 days after immunization and restimulated with MOG peptide ex vivo for 3 days. IL-17 production was measured by ELISA. (C and D) Ex vivo response of splenocytes from WT and TRIF-deficient mice 21 days after immunization. IL-17 or IFN-γ production was measured by ELISA. Results are reported as mean ± SD of duplicate samples from 1 representative experiment of 3 independent experiments.

Figure 4. Type I IFN induction pathway in innate immune system constrains the development of Th17 development and CNS autoimmune disease.

(A) Adoptive transfer experiments. Spleen cells and draining lymph node cells isolated from immunized WT mice were used as donor cells and restimulated with 20 μg/ml of MOG peptides in vitro. 3 × 10⁷ cells were transferred into WT or TRIF^–/– naive recipient mice via tail-vein injection (5 mice per group). The mice were monitored daily for clinical signs of disease. (B) Spleen cells and draining lymph node cells isolated from immunized TRIF^–/– mice were restimulated with MOG peptide in vitro for 72 hours. 3 × 10⁷ cells were transferred into WT or TRIF-deficient naive recipient mice via tail-vein injection (5 mice per group). (C) Flow cytometry analysis of Th17 development in CD4⁺ T cells cocultured with BMMs. BMMs from WT mice, TRIF-deficient, or IFNAR-deficient mice were stimulated with LPS (100 ng/ml) for 24 hours, then were cultured with WT naive CD4⁺ T cells in the presence of anti-CD3 (1 μg/ml) for 72 hours. Cells were stained for surface CD4 and intracellular IL-17. Plots were gated on CD4⁺ T cells. Numbers indicate percentage of IL-17⁺CD4⁺ cells of total CD4⁺ cells. (D) IL-17 production in the coculture of WT CD4⁺ T cells and BMMs in experiments described in C. (E) IL-17 production from TRIF-deficient CD4⁺ T cells cocultured with BMMs.

Figure 5. The TRIF pathway limits Th17 development through induction of antiinflammatory cytokine IL-27.

(A) WT and TRIF-deficient macrophages were stimulated with 100 ng/ml LPS. The amount of IL-27 protein in culture supernatants was measured after 24 hours of stimulation. (B) WT and TRIF-deficient DCs were stimulated with 100 ng/ml of LPS. The amount of IL-27 protein was measured after 24 hours of stimulation. (C) IL-27 inhibits IL-17 production induced by IL-6 and TGF-β. Naive CD4⁺ T cells isolated from WT mice were activated for 72 hours in Th17 culture in the presence of IL-27 as indicated. IL-17 production by CD4⁺ T cells was determined by ELISA. (D) IL-27 inhibits antigen-induced Th17 response. Total splenocytes isolated from WT mice 21 days after MOG/CFA immunization were restimulated with MOG peptide ex vivo in the presence of IL-27 for 3 days. IL-17 production was measured by ELISA. Data shown are representative of at least 3 experiments. (E and F) WT and IFNAR^–/– BMMs or DCs were stimulated with 100 ng/ml LPS. The amount of IL-27 protein was measured after 24 hours of stimulation.

Figure 6. Type I IFN–mediated IL-27 production in macrophages contributes to inhibition of IL-17 production.

(A) CM from IFN-β–treated macrophages suppresses Th17 development. WT, TRIF-deficient, and IFNAR-deficient BMMs were stimulated with IFN-β for 24 hours. Supernatants from IFN-β–stimulated macrophages were used as CM; they were added to Th17 culture and incubated for 72 hours. IL-17 production by CD4⁺ T cells was determined by ELISA. (B) WT and IFNAR-deficient BMMs were stimulated with IFN-β for 24 hours. The level of IL-27 protein was measured by ELISA. Data shown are representative of at least 3 experiments. (C) IFN-β–mediated inhibitory effects on Th17 development are reversed in the presence of anti–IL-27 antibody. CM from IFN-β–stimulated WT macrophages together with anti–IL-27 antibody or control IgG was added to Th17 cell culture. After 72 hours, IL-17 production by CD4⁺ T cells was determined by ELISA. (D) IL-27 contributes to IFN-β–mediated inhibition of encephalitogenic T cells. Lymphocytes isolated from immunized WT mice were restimulated with MOG peptide for 72 hours in the presence of CM from IFN-treated macrophages plus anti–IL-27 antibody or control IgG. IL-17 levels were measured by ELISA. (E) Lymphocytes isolated from immunized IFNAR^–/– mice were restimulated with MOG peptide in the presence of CM from IFN-treated macrophages plus anti–IL-27 antibody. IL-27 treatment was included as a positive control. IL-17 level was measured after 72 hours of culture.

Figure 7. IFNAR^–/– mice are sensitive to EAE.

(A) Mean disease score of WT mice and IFNAR^–/– mice during EAE induction. WT mice (n = 5) and IFNAR^–/– mice (n = 5) were immunized with MOG peptide (MOG_35–55) emulsified in CFA. Mice were assigned disease scores from 0 to 5 based on the severity of EAE. (B) Increased inflammation in IFNAR-deficient mice during EAE. Representative sections of spinal cord from WT and IFNAR-deficient mice at day 12 after MOG/CFA immunization were stained with H&E. (C) Total splenocytes isolated from WT and IFNAR^–/– mice 12 days after immunization were restimulated with MOG peptide ex vivo for 3 days. IL-17 production was measured by ELISA. (D) Spinal cord tissues isolated from WT (n = 3) and IFNAR^–/– (n = 3) mice 12 days after immunization were homogenized. The level of IL-27 protein in tissue samples was measured by ELISA. Data are representative of 3 experiments with similar results.

Figure 8. IL-27 reverses severe EAE phenotype in IFNAR^–/– mice in vivo.

(A) IL-27 inhibits adoptive transfer of EAE in IFNAR^–/– mice. Spleen and lymph node cells isolated from immunized IFNAR^–/– mice were restimulated in vitro with MOG peptide in the presence of IL-27 or PBS for 72 hours. 3 × 10⁷ cells were transferred into IFNAR^–/– naive recipient mice via tail-vein injection (5 mice per group). (B) IFN-β–mediated IL-27 production inhibits adoptive transfer EAE in IFNAR^–/– mice. Spleen and lymph node cells isolated from immunized IFNAR^–/– mice were restimulated in vitro with MOG peptide in the presence of CM from IFN-treated macrophages with or without anti–IL-27 antibody. After 72 hours, 3 × 10⁷ cells were transferred into naive IFNAR^–/–recipient mice via tail-vein injection (5 mice per group). (C) IL-27 inhibits EAE development in WT mice. WT mice (n = 5) were immunized with MOG peptide emulsified in CFA. Recombinant carrier-free mouse IL-27 (0.25 μg in 100 μl PBS) was administered by s.c. injection to immunized WT mice every other day from day 2 until day 20. (D) Splenocytes from IL-27–treated WT mice represented in C were restimulated in vitro with MOG peptides for 72 hours, and IL-17 production was measured. (E) IL-27 treatment reverses the phenotype of EAE in IFNAR^–/– mice. Recombinant mouse IL-27 was administered by s.c. injection to immunized IFNAR^–/– mice (n = 5) every other day from day 2 until day 20. (F) Splenocytes from IL-27–treated IFNAR^–/– mice represented in E were restimulated in vitro with MOG peptides for 72 hours, and IL-17 production was measured.