Interleukin 1β (IL-1β) is critical for the in vivo survival, expansion and effector function of IL-17–producing helper T (TH17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1β during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell–intrinsic inflammasome that drives IL-1β production during TH17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1β, whereas ATP stimulation triggered T cell production of IL-1β via ASC-NLRP3–dependent caspase-8 activation. IL-1R was detected on TH17 cells but not on type 1 helper T (TH1) cells, and ATP-treated TH17 cells showed enhanced survival compared with ATP-treated TH1 cells, suggesting autocrine action of TH17-derived IL-1β. Together these data reveal a critical role for IL-1β produced by a TH17 cell–intrinsic ASC–NLRP3–caspase-8 inflammasome during inflammation of the central nervous system.
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This investigation was supported by the National Multiple Sclerosis Society (grant RG5130A2/1 to X.L.) and the US National Institutes of Health (grants 5R01NS071996-05, 1RO1AA023722 and MSTP-T32GM007250 to X.L.).
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Integrated supplementary information
(a) The Pycard gene is localized on chromosome 7 and contains three coding exons. For generation of the conditional knockout allele, the entire open reading frame including all three exons was targeted for conditional deletion. After generation of the targeting vector (using Pcr4.0 backbone), C57BL/6 embryonic stem cells were injected for gene targeting. Southern hybridization was used to identify progeny bearing the recombinant allele. (b) CD4+ T Cells were sorted from Ascf/+Lck-Cre and Ascf/-Lck-Cre mice by flow cytometry, and mRNA expression of three different exons of ASC was measured by Real-time PCR analysis. Unpaired t test was used to analyze the data. Data are representative of three independent experiments (b). n=3 mice per group in each experiment. Error bars represent s.e.m. *P < 0.05.
Supplementary Figure 2 Numbers of T cells and B cells were similar in the lymph nodes, spleens and thymus between Ascf/+Lck-Cre and Ascf/–Lck-Cre mice.
(a) The total cell number of CD4+, CD8+ and B220+CD19+ cells were counted from lymph nodes and spleen of Ascf/+Lck-Cre and Ascf/-Lck-Cre mice. (b) Total cell number of CD4-CD8-, CD4+CD8-, CD4+ and CD8+ were counted from thymus of Ascf/+Lck-Cre and Ascf/-Lck-Cre mice. Data are representative of two independent experiments (a-b). n=5 mice per group in each experiment. Error bars represent s.e.m.
Supplementary Figure 3 The EAE phenotype of adoptive transfer of sorted CD4+ T cells from Ascf/+Lck-Cre and Ascf/–Lck-Cre mice.
(a) Absolute numbers of CD4+ IL-17A+ cells (left panel) and concentrations of IL-17A in the supernatant of cells (right panel) from the draining lymph nodes and spleens of Ascf/+Lck-Cre and Ascf/-Lck-Cre mice immunized with MOG35-55 and harvested 10 days after immunization, followed by ex vivo re-stimulation in the presence of MOG35-55 and IL-23. (b-d) Cells from lymph nodes of Ascf/+Lck-Cre and Ascf/-Lck-Cre mice 10 days after immunization with MOG35-55 were re-stimulated with MOG 35-55 in vitro in the presence of recombinant IL-23 for 5 days, sorted for CD4+ T cells by flow cytometry, and then transferred into CD45.1 congenic mice. (b) Graph represents the average clinical score after T cell transfer. (c) Absolute numbers of CNS-infiltrating CD45.2+ CD4+ cells were determined at the peak of disease. Brains and spinal cords were harvested together and CD45.2+CD4+ cells were stained with anti-CD45.2 and anti-CD4 antibodies, followed by flow cytometric analysis. (d) Absolute numbers of CNS-infiltrating cells were determined at the peak of disease. Brains and spinal cords were harvested together and mononuclear infiltrating cells were stained with anti-CD45, anti-CD4, anti-CD8, anti-F4/80, anti-Ly6C, anti-CD19 and anti-Ly6G antibodies, followed by flow cytometric analysis. Two-way ANOVA (b) and Unpaired t test (a, c and d) were used to analyzed the data. Data are representative of three independent experiments (a-d). n=5 mice per group in each experiment. Error bars represent s.e.m. *P < 0.05 (a, c and d). Error bars represent s.d. *P < 0.05 (b).
Macrophages and TH17 cells were left untreated or treated with LPS for 4 hours (1ug/ul for macrophages) or LPS+ATP (5 mM, 30 mins) or ATP alone (5 mM, 8 hours). Cell pellet and supernatant were collected and subjected to western blotting for IL-1β and Actin. Data are representative of two independent experiments.
(a) Lethal irradiated WT mice were reconstituted with WT+Cd3e-/- bone marrow or Il1b-/-+Cd3e-/- bone marrow. 6 weeks after reconstitution, mice were immunized with MOG35-55 peptide and 200 ng pertussis toxin on days 1 and 4. Graph represents the average clinical score after active immunization. (b) Inflammatory gene expression in the lumbar spinal cord was assessed at the peak of disease. (c) Absolute numbers of CNS-infiltrating cells were determined at the peak of disease. Brains and spinal cords were harvested together and mononuclear infiltrating cells were stained with anti-CD45, anti-CD4, anti-CD8, anti-F4/80, anti-Ly6C and anti-Ly6G antibodies, followed by flow cytometric analysis. Two-way ANOVA (a) and Unpaired t test (b and c) were used to analyzed the data. Data are representative of three independent experiments (a-c). n=5 mice per group in each experiment. Error bars represent s.e.m. *P < 0.05.
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Martin, B., Wang, C., Zhang, Cj. et al. T cell–intrinsic ASC critically promotes TH17-mediated experimental autoimmune encephalomyelitis. Nat Immunol 17, 583–592 (2016). https://doi.org/10.1038/ni.3389
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