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Interferon and IL-27 antagonize the function of group 2 innate lymphoid cells and type 2 innate immune responses

Abstract

Group 2 innate lymphoid cells (ILC2 cells) are type 2 cytokine–producing cells of the innate immune system with important roles in helminth infection and allergic inflammation. Here we found that tissue-resident ILC2 cells proliferated in situ without migrating during inflammatory responses. Both type I and type II interferons and interleukin 27 (IL-27) suppressed ILC2 function in a manner dependent on the transcription factor STAT1. ILC2-mediated lung inflammation was enhanced in the absence of the interferon-γ (IFN-γ) receptor on ILC2 cells in vivo. IFN-γ effectively suppressed the function of tissue-resident ILC2 cells but not that of inflammatory ILC2 cells, and IL-27 suppressed tissue-resident ILC2 cells but not tissue-resident TH2 cells during lung inflammation induced by Alternaria alternata. Our results demonstrate that suppression mediated by interferon and IL-27 is a negative feedback mechanism for ILC2 function in vivo.

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Figure 1: Characteristics of tissue-resident ILC2 cells.
Figure 2: Cytokine receptors on ILC2 cells.
Figure 3: Interferons suppress the proliferation and cytokine production of ILC2 cells.
Figure 4: IFN-γ suppresses type 2 immune responses mediated by ILC2 cells.
Figure 5: A defect in the action of IFN-γ exacerbates type 2 immune responses.
Figure 6: STAT1-mediated suppression of ILC2 function by IFN-γ and IL-27.
Figure 7: IL-27 suppresses ILC2 cells in vitro and in vivo.

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Acknowledgements

We thank M. Kubo (Tokyo University of Science) for IfngVenus/+ mice; M. Miyasaka (Osaka University) for antibody to mouse IL-2Rβ (TM-β1); S. Wada, T. Yamamoto, T. Shitamichi, U. Tran and S. Tada for animal care; M. Yamamoto, S. Kagawa, T. Fukushima and J. Furusawa for help in some experiments; Kafi N. Ealey for critical reading of this manuscript; and members of the Laboratory for Immune Cell Systems at RIKEN Integrative Medical Sciences for discussion. Supported by Precursory Research for Embryonic Science and Technology from Japan Science and Technology Agency Japan Society for the Promotion of Science (Grant-in Aid for Scientific Research (B) 26293110; Grant-in-Aid for Challenging Exploratory Research 24659373 to K.M.; Grant-in-Aid for Scientific Research (S) 22229004 to S.K.) and the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research on Innovative Areas 15H01166 to K.M.).

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Authors and Affiliations

Authors

Contributions

K.M. designed and interpreted the experiments and wrote the manuscript; H.K. designed and performed IL-27 experiments and wrote the manuscript; M.T. maintained N. brasiliensis and supervised helminth infection experiments; Sa.K. assessed chemokine receptor expression and performed helminth infection experiments; N.T. and M.M. performed experiments under the supervision of K.M.; K.F., K.A. and T.B. discussed research with H.K. and provided insight into the study design; and Sh.K. interpreted the experiments and wrote the manuscript.

Corresponding author

Correspondence to Kazuyo Moro.

Ethics declarations

Competing interests

S.K. is a consultant for Medical and Biological Laboratories.

Integrated supplementary information

Supplementary Figure 1 Chemokine receptors on ILC2 cells.

The expression of chemokine receptors on ILC2s was examined by flow cytometry. Freshly sorted naive ILC2 cells from the mesentery were cultured for 7 days with the indicated cytokines. Gray lines indicate histogram s for isotype controls for those of specific mAbs (blue lines). Experiments were performed at least twice and similar results were obtained.

Supplementary Figure 2 Comparison of ILC2 cells from various tissues.

ILC2 cells were isolated from N. brasiliensis infected Rag2−/– mice 6 days after infection and stained with anti- IL-1RL1, anti-Thy-1.2, anti-KLRG1, anti-Sca-1 mAbs and mixture of antibodies for lineage markers. FALC ILC2 cells, BALF ILC2 cells and lung ILC2 cells are shown in purple, pink and blue, respectively, in merged plots. Cells were gated through an FCSloSSClo lymphocyte gate before analyzing in the indicated gates.

Supplementary Figure 3 Apoptosis of ILC2 cells is not induced by interferons.

ILC2 cells were isolated from mesentery and cultured (17,000 cells/well) with indicated cytokines for 36 hrs. Each sample was assessed by flow cytometry for PI and Annexin V staining.

Supplementary Figure 4 Dose-dependent suppression of ILC2 function by IFN-γ and IL-27 in vitro.

ILC2 cells were isolated from mesentery, cultured with 10 ng/ml IL-2 and then seeded (5,000 cells/well) with indicated doses of IFN-γ (10 ng/ml) and IL-27 (10 ng/ml) in the presence of 10 ng/ml IL-33. ILC2 cells were counted by flow cytometry and amounts of IL-5 and IL-13 in the supernatants were measured by ELISA on day 6.

Supplementary Figure 5 Gene expression in IFN-γ- and IL-27-treated ILC2 cells.

Freshly sorted ILC2 cells from the mesentery were stimulated with IFN-γ (10 ng/ml) or IL-27 (10 ng/ml) under IL-33 stimulation (10 ng/ml). RNA was extracted 48 hrs after stimulation and the indicated genes were measured by RNA sequencing. Heatmaps indicate results of 3 independent experiments.

Supplementary Figure 6 IFN-γ and IL-27 similarly suppress FALC ILC2 cells and lung ILC2 cells.

ILC2 cells were isolated from mesentery (FALC) or lung and cultured (5,000 cells/well) with 10 ng/ml IL-33 with or without IFN-γ and IL-27 (10 ng/ml for each cytokine). ILC2 cells were counted by flow cytometry and amounts of IL-5 and IL-13 in the supernatants were measured by ELISA on day 5. All statistics are compared to white dots, n.d.: none detected, **p<0.01 (Dunnett’s test after one-way ANOVA).

Supplementary Figure 7 IL-27 suppresses eotaxin production by ILC2 cells.

ILC2 cells were isolated from mesentery and cultured (10,000 cells/well) with 10 ng/ml IL-33 with or without IL-27 (10 ng/ml for each cytokine). Amounts of eotaxin in the supernatant were measured by Bio-plex on day 5 (n=5). n.d.: none detected, **p<0.01 (Mann-Whitney U test).

Supplementary Figure 8 Gating strategy for ILC2 cells in flow cytometry.

BALF cells and lung cells were isolated from C57BL/6 mouse 5 days after intratracheal injection of 0.5 μg/mouse IL-33 on days 0 and 2. Mesentery cells (FALC) were isolated from untreated C57BL/6 mouse. Cells were stained with indicated antibodies and examined by flow cytometry. FSC-Alo SSC-Alo gates were used for the detection of ILC2 cells, and FCS-AloSSC-Ahi gates were used for the detection of eosinophils. FSC-H, FSC-W, SSC-H and SSC-W were used to gate out doublet cells. Note that FSCloSSChiSiglec-Fhi cells are all eosinophils.

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Moro, K., Kabata, H., Tanabe, M. et al. Interferon and IL-27 antagonize the function of group 2 innate lymphoid cells and type 2 innate immune responses. Nat Immunol 17, 76–86 (2016). https://doi.org/10.1038/ni.3309

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