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Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells

Nature Immunology volume 17, pages 65–75 (2016)Cite this article

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Abstract

Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.

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Figure 1: Deficiency in signaling via type I interferon receptor results in increased ILC2 cells and type 2 immunopathology.
Figure 2: Type I interferons restrain the proliferation and cytokine production of mouse ILC2 cells.
Figure 3: Human ILC2 cells are regulated by type I interferons.
Figure 4: Mechanisms of type I interferon–mediated regulation of ILC2 cells.
Figure 5: Stimulation of ILC2 cells with type I interferons reduces proliferation, viability and cytokine production.
Figure 6: Type I interferons restrict ILC2 cells and type 2 immunopathology in vivo.
Figure 7: IL-27 and IFN-γ regulate ILC2 function.
Figure 8: IFN-γ restricts ILC2 cells and type 2 immunopathology in vivo.

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Acknowledgements

We thank J.L. Gommerman (University of Toronto) for Ifnar1−/− (Ifnar1tm1Agt) mice; D. Xue and G.A. Kaufman for help with AHR measurements; and B. Charbonneau for technical support. Supported by the Canadian Institutes of Health Research (J.H.F. and C.U.D.; MOP-114972 for the J.H.F. laboratory; postdoctoral fellowship for C.U.D.; MOP-89821 operating funds for S.M.V.), the Canadian Foundation of Innovation (Leaders Opportunity Fund infrastructure grant for the J.H.F. laboratory), the German National Academy of Sciences Leopoldina (C.U.D.), The American Association of Immunologists Careers in Immunology Fellowship Program (J.H.F. and C.U.D.) and Canada Research Chair in Host Responses to Virus Infections (S.M.V.).

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, Complex Traits Group, McGill University, Montréal, Canada

    Claudia U Duerr, Barbara C Mindt & Jörg H Fritz

  2. Department of Physiology, Complex Traits Group, McGill University, Montréal, Canada

    Connor D A McCarthy & Jörg H Fritz

  3. Immunoregulation Laboratories, The Research Center of the Centre Hospitalier de L'Université de Montréal, Montréal, Québec, Canada

    Manuel Rubio & Marika Sarfati

  4. Department of Microbiology and Immunology, Microbiome and Disease Tolerance Center, McGill University, Lyman Duff Medical Building, Montréal, Canada

    Alexandre P Meli & Irah L King

  5. Diaccurate, Institut Pasteur, Paris, France

    Julien Pothlichet

  6. Department of Human Genetics, Complex Traits Group, McGill University, Montréal, Canada

    Megan M Eva, Danielle Malo & Silvia M Vidal

  7. Department of Pharmacology, Université de Montréal, Montréal, Canada

    Jean-François Gauchat

  8. McGill University and Department of Critical Care, Meakins Christie Laboratories, The Research Institute of the McGill University Health Center, Montréal, Canada

    Salman T Qureshi

  9. Department of Pediatrics, McGill University, Meakins Christie Laboratories, The Research Institute of the McGill University Health Center, Montréal, Canada

    Bruce D Mazer

  10. Department of Pathology and Molecular Medicine, McMaster Immunology Research Center, Institute for Infectious Disease Research, McMaster University, Hamilton, Canada

    Karen L Mossman

  11. Department of Medicine, Complex Traits Group, McGill University, Montréal, Canada

    Danielle Malo & Silvia M Vidal

  12. Department of Medical Genetics and Molecular Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

    Ana M Gamero

  13. Department of Medicine, Université de Montréal, Montréal, Canada

    Marika Sarfati

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Contributions

C.U.D. and J.H.F. designed the conceptual framework of the study, designed experiments and wrote the paper; C.U.D., C.D.A.M. and B.C.M. designed and performed experiments and analyzed data; M.R. and M.S. performed experiments with human ILC2 cells and helped to design and interpret experiments; A.P.M. and I.L.K. provided H. polygyrus, performed infections and helped to design and interpret experiments; J.P. and S.M.V. provided influenza virus and helped to design and interpret experiments; M.M.E. and D.M. provided STAT4-mutant mice; J.-F.G. provided reagents and expertise for experiments with IL-27; S.T.Q. provided STAT1-deficient BM; B.D.M. provided equipment and expertise for AHR measurements; K.L.M. provided IRF9-deficient BM; A.M.G. provided STAT1- and STAT2-deficient BM; and all authors provided input throughout the study and the writing of the manuscript.

Corresponding author

Correspondence to Jörg H Fritz.

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Integrated supplementary information

Supplementary Figure 1 Wild-type and Ifnar1−/− mice recover from infection with a low dose of IAV equally well.

(a,b) WT and Ifnar1-/- mice (five age- and sex-matched animals per group) were either mock treated with PBS as a control or infected with 20 PFU of influenza virus. (a) Survival and (b) body weight loss were monitored throughout the course of infection. (c) Pulmonary expression of the non-structural 1 (Ns1) gene of influenza was determined by qRT-PCR 5, 10 and 15 days post infection. The bars represent the mean ± standard deviation of each cohort. Data are representative of two independent experiments; n.d., not detectable. (d-g) WT and Ifnar1-/- mice (three to seven age- and sex-matched animals per group) were either mock treated with PBS or infected intranasally with 20 PFU of influenza virus. Five days post infection lungs were perfused with PBS and the pulmonary expression levels of (d) IL1b, (e) Tnf and (f) Ccl2 were determined by qRT-PCR, and (g) the numbers of inflammatory monocytes were determined by flow cytometry. Data are representative of three independent experiments. The bars represent the mean ± standard deviation of triplicates of each cohort. The asterisks indicate statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). (h) WT and Ifnar1-/- mice (three to seven age- and sex-matched animals per group) were either mock treated with PBS or infected intranasally with 20 PFU of influenza virus. At indicated time points post infection lungs were perfused with PBS, homogenized, and the IL-33 content was determined by ELISA. Data are representative of two independent experiments. The bars represent the mean ± standard deviation of triplicates of each cohort. One-way ANOVA test including the Bonferroni’s multiple comparison was used to analyze statistical significance.

Supplementary Figure 2 IFNAR1 deficiency results in elevated innate and adaptive type 2 immunity upon infection with H. polygyrus.

(a,b) WT and Ifnar1-/- mice (three to five age- and sex-matched animals per group) were either mock treated with PBS as control (co) or infected with H. polygyrus (H. poly., 200 larvae/mouse). Counts of (a) neutrophils, eosinophils and (b) group 2 innate lymphoid cells (ILC2) in the lamina propria of the small intestine and (c) IgG1 and (d) IgE levels in sera of mice were determined five days post-infection (p.i.). Data are representative of two independent experiments. The asterisks indicate statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.01).

Supplementary Figure 3 Isolation and ex vivo expansion of mouse BM-derived ILC2 cells.

(a) Schematic representation of isolation and ex vivo expansion of murine bone marrow-derived group 2 innate lymphoid cells. (b) Bone marrow cells were stained for Sca-1, CD117 (c-Kit), CD25, and for lineage markers (TCRβ, TCRγδ, CD3ɛ, Gr-1, CD11b, TER-119, B220, NK1.1, CD5, CD11c) and lineage-negative Sca-1+c-Kit-CD25+ group 2 innate lymphoid cells were enriched by flow cytometric cell sorting. (c) Purity and (d) expression of CD127, T1/ST2, ICOS, KLRG1, CD90.2 and Gata3 of sorted bone marrow-derived ILC2 was analyzed by flow cytometry. Cells were then cultured in the presence of IL-2, IL-7, IL-25, IL-33 (all at 50 ng/ml), and TSLP (20 ng/ml) for 15 days. Cells were given fresh medium and cytokines every two days and split into multiple wells when necessary to avoid excessive crowding, yielding an increase in ILC2 numbers of approximately 500-fold. The resulting cells were then seeded in fresh medium and allowed to rest in IL-2 and IL-7 (both at 10 ng/ml) for two days. Medium was removed from wells and rested cells were stimulated as necessary for up to five days.

Supplementary Figure 4 Type I interferon–mediated inhibition of the proliferation and cytokine production of ILC2 cells occurs independently of iNOS and STAT6.

Bone marrow cells from WT, inducible nitric oxide synthase (iNos)-deficient or Stat6-deficient mice were stimulated for five days in medium as a control or with IL‐7 and IL-33 (both at 10 ng/ml). IFN-β (25 or 500 U/ml) was added to some wells, as indicated. Proliferation of cells was measured by alamarBlue assay and cytokine production was assessed by ELISA of cell culture supernatants. The bars represent the mean ± standard deviation of triplicates of each cohort. Data are representative of three independent experiments. RFU, relative fluorescence units; n.d., not detectable; ns, not statistically significant (p > 0.05). The asterisks indicate statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Supplementary Figure 5 Type I interferon restrains mouse ILC2 cells.

(a,b) Sorted and expanded bone marrow-derived ILC2 from WT mice were stimulated for five days with IL‐7 and IL-33 (both at 10 ng/ml) only (black bars) or with IL‐7 and IL-33 (both at 10 ng/ml) in combination with IFN-β (500 U/ml; red bars). (a) IFN-β was added at different time points (0h, 24h, 48h and 72h) and left in the culture until supernatants were taken. (b) IFN-β was added at the beginning of the culture (0h) and at different time points (not removed, 24h, 48h and 72h) cells were washed and re-seeded with IL‐7 and IL-33 (both at 10 ng/ml) only until supernatants were taken. Five days after culture proliferation of cells was measured by alamarBlue assay and cytokine production was determined by ELISA. The percentages of reduction due to the addition of IFN-β are shown in brackets above the red bars. Data are representative of two independent experiments. (c) Sorted and expanded bone marrow-derived ILC2 from WT mice were stimulated for three days with medium only, or with IL‐7 and IL-33 (both at 10 ng/ml). IFN-β (25 or 500 U/ml) was added to some wells as indicated. GATA3 expression was determined by intracellular flow cytometry staining and histograms and the ratio of the GATA3 mean fluorescence intensities (MFI) to the MFI of the internal isotype control stainings are shown. Data are representative of three independent experiments. (d) Sorted and expanded bone marrow-derived ILC2 from WT mice were stimulated for five days with IL‐7 and IL-33 (both at 10 ng/ml) only (black bars) or with IL‐7 and IL-33 (both at 10 ng/ml) in combination with IFN-β (500 U/ml; red bars). IFN-β was added at the beginning of the culture (0h) and at different time points (24h, 48h and 72h) cells were washed and re-seeded with IL‐7 and IL-33 (both at 10 ng/ml) only until supernatants were taken. Five days after culture GATA3 expression was determined by intracellular flow cytometry staining and the ratio of the GATA3 MFI to the MFI of the internal isotype control stainings are shown. Data are representative of two independent experiments. The bars represent the mean ± standard deviation of triplicates of each cohort. The asterisks indicate statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). RFU, relative fluorescence units.

Supplementary Figure 6 Type I interferon restrains IL-33-mediated inflammation.

(a) Schematic model of cytokine administration protocol. WT mice (five age- and sex-matched animals per group) were challenged intranasally with PBS as control, IFNβ, IL-33, or IL-33 + IFN-β for three consecutive days. 24 hours after the last treatment pulmonary expression of (b) Il6, (c) Ccl17, (d) Ccl22, (e) Chi3l3, (f) Fn1 and (g) Ccl24 was determined by qRT-PCR. Data are representative of two independent experiments. (h,i) WT mice (five age- and sex-matched animals per group) were challenged intranasally with phosphate buffered saline (PBS) as a control, IFN-β, IL-33, or IL-33 + IFN-β for three consecutive days. 24 hours after the last treatment pulmonary cytokine expression and cellular composition of lungs were analyzed. (h) Expression of cell surface CD11c of pulmonary eosinophils was determined by flow cytometry. After setting singlet gate, viable eosinophils were further defined as SiglecF+CD11c-. The frequencies of eosinophils are highlighted in the red gates. (i) The mean fluorescence intensities (MFI) of cell surface CD11c expression by eosinophils are shown. The bars represent the mean ± standard deviation of each cohort. Data are representative of two independent experiments. The asterisks indicate statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Supplementary Figure 7 IFN-γ-mediated inhibition of the proliferation and cytokine production of ILC2 cells occurs independently of STAT2, IRF9, IFNAR, STAT4, T-bet, iNOS and STAT6.

Bone marrow cells from (a) WT and Stat2-/-, (b) WT and Irf9-/-, (c) WT and Ifnar1-/-, (d) WT and Stat4-mutant, (e) WT and Tbx21-/-, (f) WT, iNos-/- and Stat6-/- mice were stimulated for five days in medium as a control or with IL‐7 and IL-33 (both at 10 ng/ml). IFN-γ (5 or 20 ng/ml) was added to some wells, as indicated. Proliferation of cells was measured by alamarBlue assay, cytokine production was determined by ELISA of cell culture supernatants. The bars represent the mean ± standard deviation of triplicates of each cohort. Data are representative of three independent experiments. RFU, relative fluorescence units; n.d., not detectable; ns, not statistically significant (p > 0.05). The asterisks indicate statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

Supplementary Figure 8 Type II interferon restrains IL-33-mediated inflammation.

Wild type (WT) C57BL/6 mice (five age- and sex-matched animals per group) were challenged intranasally with PBS as control, IFN-γ, IL-33, or IL-33 + IFN-γ for three consecutive days. 24 hours after the last treatment pulmonary expression of (a) Il6, (b) Ccl17, (c) Ccl22, (d) Chi3l3, (e) Fn1 and (f) Ccl24 was determined by qRT-PCR. The bars represent the mean ± standard deviation of each cohort. Data are representative of two independent experiments. The asterisks indicate statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

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Duerr, C., McCarthy, C., Mindt, B. et al. Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells. Nat Immunol 17, 65–75 (2016). https://doi.org/10.1038/ni.3308

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