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Apoptosis in response to microbial infection induces autoreactive TH17 cells

Nature Immunology volume 17, pages 1084–1092 (2016)Cite this article

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Abstract

Microbial infections often precede the onset of autoimmunity. How infections trigger autoimmunity remains poorly understood. We investigated the possibility that infection might create conditions that allow the stimulatory presentation of self peptides themselves and that this might suffice to elicit autoreactive T cell responses that lead to autoimmunity. Self-reactive CD4+ T cells are major drivers of autoimmune disease, but their activation is normally prevented through regulatory mechanisms that limit the immunostimulatory presentation of self antigens. Here we found that the apoptosis of infected host cells enabled the presentation of self antigens by major histocompatibility complex class II molecules in an inflammatory context. This was sufficient for the generation of an autoreactive TH17 subset of helper T cells, prominently associated with autoimmune disease. Once induced, the self-reactive TH17 cells promoted auto-inflammation and autoantibody generation. Our findings have implications for how infections precipitate autoimmunity.

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Figure 1: Presentation of apoptotic-cell-derived antigens during infection.
Figure 2: A C. rodentium–specific TH17 response after orogastric infection with C. rodentium.
Figure 3: Detection of self-reactive CD4+ T cells without an effector phenotype in DTg mice.
Figure 4: Infection promotes the proliferation of self-reactive CD4+ T cells and their commitment to the TH17 lineage.
Figure 5: Apoptosis and self-antigen presentation are necessary for the activation of self-reactive T cells.
Figure 6: The generation of self-reactive TH17 cells is associated with a self-reactive IgA response and intestinal pathology.

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Acknowledgements

We thank S. Lira, G. Furtado, H. Xiong, G. Yeretssian, M.K. Jenkins and members of the Blander laboratory for discussions; D. Amsen and R.J. Cummings for critical reading of the manuscript; A. Rialdi for help with statistical analyses; J. Ochando and C. Bare for flow cytometry; A. Soto and M.J. Suarez (the NIH Tetramer Core Facility at Emory University) for I-Ab–OVA(328–337) and control tetramers; H. Xiong (The Icahn School of Medicine at Mount Sinai) for LM-OVA bacteria; A. Morelli (University of Pittsburgh) for 1H3.1 mice; B. Finlay and M. Croxen (University of British Columbia) for wild-type and ΔEspF C. rodentium and plasmid pMAC5; H.P. Schweizer (Colorado State University) for plasmid pTNS2; D. Mazel (Institut Pasteur) for MFDpir bacteria; and I. Marazzi, V. Verhasselt, S.E.F. Campisi, G.C. Chiesa, Jr., M.A. Blander, S.J. Blander and the late L. Mayer for advice and support. Supported by the National Institute of Diabetes and Digestive and Kidney Diseases (DK072201 to J.M.B.), the National Institute of Allergy and Infectious Diseases (AI073899, AI080959 and AI095245 to J.M.B.), the Arthritis Foundation (L.C.), the Crohn's and Colitis Foundation of America (G.B.), the Burroughs Wellcome Fund (J.M.B.), the Irma Hirschl and Monique Weill-Caulier Charitable Trust Funds (J.M.B.), the American Cancer Society (J.M.B.) and the Leukemia and Lymphoma Society (J.M.B.).

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  1. Laura Campisi

    Present address: Present addresses: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA, and Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.,

Authors and Affiliations

  1. Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Gaetan Barbet & J Magarian Blander

  2. Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Gaetan Barbet & J Magarian Blander

  3. Department of Pathology, New York University Langone Medical Center, New York, New York, USA

    Yi Ding

  4. Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA

    Enric Esplugues & Richard A Flavell

  5. Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    J Magarian Blander

  6. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    J Magarian Blander

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Contributions

L.C. and J.M.B designed and directed the study and wrote the manuscript; L.C. conducted all experiments; G.B. assisted with T cell–sorting experiments; Y.D. conducted the histological and pathological assessments of colonic tissues; E.E. and R.A.F. provided the IL-17–eGFP reporter mice; and J.M.B conceived of the study.

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Integrated supplementary information

Supplementary Figure 1 Presentation of cellular antigens from LPS–B cell blasts in vitro and Eα peptide in vivo.

(a) Proliferation of splenic OT-II CD4+ T cells co-cultured for 5 days with BMDCs previously incubated with Eα(52-68) or OVA(329-337) peptides, heat killed (HK) E. coli (EC) bacteria expressing OVA or Eα or apoptotic splenic B cells from Act-mOVA BALB/c mice cultured with or without LPS. Apoptosis was induced by UV irradiation. (b) Schematic showing experimental strategy for assessing proliferation of OT-II and 1H3.1 CD4+ T cells in response to different cargo stimulated bone marrow derived DCs (BMDCs). (c) Schematic showing experimental strategy used in Figure 1b. 5x105 each OT-II and 1H3.1 CD4+ T cells were adoptively transferred together into chimeric actin (Act)-mOVA or wild-type (WT) mice reconstituted with CD11c-DTR BM cells. Mice were infected with WT or ΔEspF Citrobacter and injected with BrdU daily starting at 24 hours post-infection. Some mice received diphtheria toxin (DT) daily starting at 24 hours before infection. (d) Proliferation of splenic Vβ6+CD45.2 CD4+ T cells from 1H3.1 transgenic mice transferred into CD45.1 congenic C57BL/6J mice at day 5 after injection of 109 HK EC-Eα. (a) Data represent 3 independent experiments. (d) n=4 individual mice.

Supplementary Figure 2 The C. rodentium–specific CD4+ T cell response.

Mice were orogastrically infected with C. rodentium (CR) or left untreated. (a-c) and (e) Gated on Aqua−B220−CD45.2+CD3+ CD4+ T cells following ex-vivo stimulation as indicated. (a) Cytokine production and Foxp3 expression in large intestinal lamina propria (LI LP) CD4+ T cells from uninfected and infected C57BL/6J mice. (b) T-bet expression in LI LP and MLN IL-17+ CD4 T cells from infected C57BL/6J mice. (c) Percentages of IL-10+ LI LP CD4+ T cells from uninfected and infected IL-10-eGFP mice after ex-vivo re-stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. (d) Left, Pictures of large intestines from uninfected or WT or ΔEspF CR infected mice. Right, Colony-forming units (CFU) in the stools and colons of infected mice (log10 of CFU normalized by gram of stools or colons). (e) Foxp3, RORγt and T-bet expression by LI LP CD4 T cells from uninfected or day 9 infected mice. Mean and standard deviations (s.d.) are shown; p-values calculated using a t-test. ns=not significant. Data in (a) n=9 mice represent 3 independent experiments, (b) n=4, (c) n=5, (e) n=6 and (d) n=4 (left) and n=9 (right) represent 2 experiments each. Mean and standard deviations (s.d.) are shown; **P=0.0052 (c) and ns=not significant (d) were calculated using a t-test.

Supplementary Figure 3 Characterization of DTg mice.

(a) OT-II were crossed to Act-mOVA mice to generate double transgenic mice (DTg). (b, c) Aqua−B220−CD45+CD3+ MLN and LI LP CD4+ T cells analyzed in uninfected OT-II and DTg mice as indicated. (b) Top, Percentages of Vα2hiCD3+CD4+ T cells. Bottom, Vβ5hi T cells within the Vα2hiCD3+ CD4+ T cells are encircled in blue. (c) Left, I-Ab- OVA(328-337) (HAAHAEINEA) tetramer+ or control unrelated CLIP tetramer+ CD4+ T cells. Tetramer staining MFIs shown below corresponding dot plots. Right, Percentages of self-reactive CD4+ T cells detected with antibodies to Vα2 and Vβ5 or I-Ab-OVA(328-337) tetramer staining. Data in (b, c) n= 6 mice represent 3 experiments. Mean and standard deviations (s.d.) are shown.

Supplementary Figure 4 Intact immune response to C. rodentium in DTg mice.

Indicated mouse strains were infected with WT Citrobacter (a, b) CFU in the stools and colons on days 9 (a) or 30 (b) post-infection (log10 of CFU/ g of stools or colon). (a) Right, Colitis scores in WT and DTg mice on day 9 post-infection. (c) Detection of intracellular cytokines IL-17 and IL-22 in polyclonal Vα2−Vβ5− LI LP CD4+ T cells from DTg (UI and day 9 Inf) and Act-mOVA (Inf) mice after ex vivo restimulation with PMA+I. Gated on Aqua−B220−CD45+CD3+ CD4+ T cells. Data in (a, left) n=9 (OT-II and DTg), n=6 (WT), n=4 (OT-II and DTg, CFU in the colon). Data in (a, right) n= 8 (WT), n=7 (DTg), (b) n=8 (WT and DTg), n=7 (OT-II), n=6 (Act-mOVA) and (c) n=4 individual mice represent 2 experiments each. Mean and standard deviations (s.d.) are shown; NS= not significant calculated using One-way ANOVA test and Dunnet post-test (a, left), t-test (a, right) and One-way ANOVA test and Tukey’s Multiple Comparison post-test (b).

Supplementary Figure 5 Differentiation of self-reactive CD4+ T cells during infection with C. rodentium.

(a) Left, IL-10-eGFP and Foxp3/IL-17 expression in IL-10-eGFP LI LP CD4+ T cells in DTg mice crossed to IL-10-eGFP reporter mice. Right, Percentages of Foxp3+Vα2hiVβ5+ CD4+ T cells in DTg mice. (b) Left, Schematic showing experimental strategy used in Figure 5a: CD45.1+CD45.2+ (CD45.1.2) Act-mOVA chimeric mice reconstituted with BM cells from CD45.1 C57BL/6J and CD45.2+ Tcrα−/− OT-II mice. Right, Percentages of thymic single positive (SP) CD4+ T cells in naïve chimeric mice. R1, R2, R3 represent WT, endogenous, and Tcrα−/− OT-II CD4 T cell populations, respectively. (c) Detection of intracellular IL-17 and Foxp3 at day 9 post-infection with WT Citrobacter in LI LP Vα2hiVβ5+ CD4+ T cells from DTg mice, treated or not with the pan-caspase inhibitor Q-VD-OPH. (a, c) Gated on Aqua−B220−CD45+CD3+ LI LP CD4+ T cells. Data in (a, left and c) n=4 and (b) n=6 represent 2 experiments each, and (a, right) n=8 (uninfected) and n=10 (infected) represent 3 experiments. ***P ≤0.001 calculated using the Mann-Whitney test. Mean and standard deviations (s.d.) are shown.

Supplementary Figure 6 Autoantibody response in DTg mice after infection with C. rodentium.

(a,c) Optical densities (OD) of anti-OVA IgM and IgG1 in the serum of indicated mice on day 40 post-infection. (b) CD45.1+CD45.2+ Act-mOVA chimeric mice reconstituted with BM cells from CD45.1 C57BL/6J and CD45.2 Tcrα−/− OT-II mice and left untreated or infected with WT Citrobacter. (d) Colitis scores on day 40 post-infection in Act-mOVA chimeric mice left untreated or injected with anti-Thy1.1 antibody 24 hours before C. rodentium infection. (a) n=11 (IgM in DTg) and n=8 represent 3 independent experiments; (c, d) n=8 (untreated) and n=7 (treated) represent 2 experiments. Mean and standard deviations (s.d.) are shown. NS=not statistically significant (with P=0.08 for d) and *** P≤ 0.001 calculated using t- test. (e) Model for self-reactive CD4+ T cell activation and differentiation into TH17 cells upon dendritic cell phagocytosis of infected apoptotic host cells. TLR signals from phagosomes carrying infected apoptotic cells lead to MHC class II presentation of both apoptotic cell-derived self and pathogen-derived non-self peptides present within the same phagosome. Activation within the resultant TGF-β and IL-6 rich inflammatory milieu instructs differentiation of both pathogen-specific and self-specific CD4+ T cells into TH17 cells. Self-reactive TH17 cells promote intestinal inflammation and autoantibody production (likely through autoreactive plasma cells).

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Campisi, L., Barbet, G., Ding, Y. et al. Apoptosis in response to microbial infection induces autoreactive TH17 cells. Nat Immunol 17, 1084–1092 (2016). https://doi.org/10.1038/ni.3512

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