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T cell–intrinsic ASC critically promotes TH17-mediated experimental autoimmune encephalomyelitis

Nature Immunology volume 17, pages 583–592 (2016)Cite this article

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Abstract

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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Figure 1: Genetic deletion of the inflammasome adaptor ASC in T cells protects from EAE.
Figure 2: MOG35-55-reactive T effector cell priming in the secondary lymphoid organs is unaffected by ASC deficiency in T cells.
Figure 3: T cell–specific ASC deficiency is required for TH17 cell–mediated but not TH1 cell–mediated EAE.
Figure 4: TH17 cells process IL-1β in response to stimulation with extracellular ATP in an ASC–NLRP3–caspase-8–dependent manner.
Figure 5: TH17 cells express IL-1R and pro-IL-1β.
Figure 6: IL-1β is produced by CD4+ T cells in vivo.
Figure 7: T cell–intrinsic inflammasome activation is required for TH17 cell survival and proliferation in the CNS.

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Acknowledgements

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.).

Author information

Author notes

  1. Richard M Ransohoff

    Present address: Present address: Biogen Idec, Cambridge, Massachusetts, USA.,

  2. Bradley N Martin and Chenhui Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA

    Bradley N Martin, Chenhui Wang, Cun-jin Zhang, Muhammet Fatih Gulen, Jarod A Zepp, Junjie Zhao, Guanglin Bian, Jeong-su Do, Booki Min, Paul G Pavicic Jr & Xiaoxia Li

  2. Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA

    Bradley N Martin

  3. Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

    Chenhui Wang

  4. Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China

    Cun-jin Zhang

  5. Department of Immunology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China

    Cun-jin Zhang

  6. Shanghai Institute of Immunology, Shanghai Jiaotong University School of Medicine, Shanghai, China

    Zizhen Kang

  7. Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA

    Caroline El-Sanadi & George R Dubyak

  8. Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA

    Paul L Fox

  9. Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki, Noda, Japan

    Aoi Akitsu & Yoichiro Iwakura

  10. Davis Heart and Lung Research Institute, Ohio State University College of Medicine, Columbus, Ohio, USA

    Anasuya Sarkar & Mark D Wewers

  11. Department of Microbiology and Immunology, Emory Vaccine Center, Atlanta, Georgia, USA

    William J Kaiser & Edward S Mocarski

  12. Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA

    Marc E Rothenberg

  13. Center for Global Health and Diseases, Case Western Reserve University, Cleveland, Ohio, USA

    Amy G Hise

  14. Department of Neurosciences, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA

    Richard M Ransohoff

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Contributions

B.N.M. and C.W. did the experiments and analyzed the data; C.Z., Z.K., M.F.G., J.A.Z., J.Z., C.E.-S. and G.B. contributed to the experiments; J.D. and P.G.P. helped to make the Cd3e−/− and Il1b−/− bone marrow chimera mice; A.A., Y.I., A.S., M.D.W., W.J.K., E.S.M., M.E.R., P.L.F. and A.G.H. provided reagents and participated in discussion; B.M. and G.R.D. contributed reagents, helped design experiments and helped edit the manuscript; B.N.M., C.W. and X.L. wrote the manuscript; R.M.R. and X.L. conceived the study, oversaw the experiments and analyzed the data.

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Correspondence to Richard M Ransohoff or Xiaoxia Li.

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Integrated supplementary information

Supplementary Figure 1 Generation of the Pycard conditional knockout allele.

(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).

Supplementary Figure 4 Comparison of T cell and macrophage secretion of IL-1β.

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.

Supplementary Figure 5 Model for T cell intrinsic ASC function during CNS inflammation.

Supplementary Figure 6 The EAE phenotype of Cd3e−/−Il1b−/− chimera mice.

(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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