Abstract
Innate lymphoid cells (ILCs) are critical mediators of mucosal immunity, and group 1 ILCs (ILC1 cells) and group 3 ILCs (ILC3 cells) have been shown to be functionally plastic. Here we found that group 2 ILCs (ILC2 cells) also exhibited phenotypic plasticity in response to infectious or noxious agents, characterized by substantially lower expression of the transcription factor GATA-3 and a concomitant switch to being ILC1 cells that produced interferon-γ (IFN-γ). Interleukin 12 (IL-12) and IL-18 regulated this conversion, and during viral infection, ILC2 cells clustered within inflamed areas and acquired an ILC1-like phenotype. Mechanistically, these ILC1 cells augmented virus-induced inflammation in a manner dependent on the transcription factor T-bet. Notably, IL-12 converted human ILC2 cells into ILC1 cells, and the frequency of ILC1 cells in patients with chronic obstructive pulmonary disease (COPD) correlated with disease severity and susceptibility to exacerbations. Thus, functional plasticity of ILC2 cells exacerbates anti-viral immunity, which may have adverse consequences in respiratory diseases such as COPD.
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Change history
05 May 2016
In the version of this article initially published online, the units in the vertical axis for Figure 1l were incorrectly stated as '(fold)', and the figure lacked a key. The correct units are '(ng/ml; change from mock)'; the white bars are IL-5, the gray bars are IL-13, and the black bars are IFN-γ. Also, the lowest portions of the images in Figure 4i,j (including the inset in Figure 4j) were incorrectly cropped. The errors have been corrected for the print, PDF and HTML versions of this article.
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Acknowledgements
We thank the MedImmune Flow Cytometry core for all cell sorting; the MedImmune histology core for embedding tissues; the LAR staff for maintaining the experimental mice; M. Stämpfli for expertise and guidance in establishing a smoking system at MedImmune; A. Gonzales for running and maintaining the system for in-house exposure to cigarette smoke; the C. Lopez laboratory (University of Pennsylvania, School of Veterinary Medicine) for influenza virus strain PR8; M. Snaith for help with generating the ST2-GFP reporter mouse; M.E.P. Roberts for facilitating the collaboration with National Jewish Health; C. Schnell, T. Thorn and the rest of the team at NJH for commitment and contributions to this collaborative effort; and J. Jönsson and K. Jansner for histological work and image processing and analysis.
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J.S.S., J.K. and A.A.H. planned all experiments; J.S.S. and J.K. executed and analyzed all experiments; A.M.C., L.Y., G.H.P. and A.A.B. planned and executed specific experiments; C.S., M.M. and J.S.E. cut, stained and analyzed all histology sections and developed the algorithms for analysis of ILC location; L.Y. analyzed and generated the statistical data and graphs for the COPDGene study; G.H.P. and C.A.H. provided Tbx21−/− mice and reagents; R.B. provided blood samples from patients with COPD and control subjects; C.A.H., J.S.E., R.K. and A.A.H. provided feedback and edits; and J.S.S. and A.A.H. wrote the manuscript.
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J.S.S., J.K., A.M.C., L.Y., A.A.B., R.K. and A.A.H. are employed by and shareholders of MedImmune, and C.S., M.M., G.H.P., C.A.H., R.B. and J.S.E. have received funding from MedImmune.
Integrated supplementary information
Supplementary Figure 1 Generation of ST2-GFP reporter mice and characterization of ILC1 cells following infection with influenza virus.
(a) ST2-GFP reporter mice were generated as described in Methods. (b) Representative flow cytometric plots demonstrating Lineage vs. ST2-GFP staining in lung of littermate control or ST2-GFP reporter mice; and expression of CD90, IL-7Rα, CD44 and CD25 on Lin- ST2-GFP+ cells. Representative flow cytometric plots comparing ILC populations in naive lung, infected lung and draining lymph nodes at Day 7 post-infection (c). Representative flow cytometry plots comparing ILC populations in the bronchoalveolar lavage fluid from naive and infected mice (d). Live/dead fixable blue (e) or Annexin V (f) staining on ILCs in naïve and infected lungs at Day 7 post-infection. (g) Expression of GATA-3, T-bet, CD44, CD25, IL-7rα, cKit and ICOS on lung-resident ST2+ (red) or IL-18rα+ (blue) ILCs at Day 7 post-infection. (h) Percentage of lung ILCs positive for Ki67. (i) IL-5, IL-13 and IFN-γ levels in culture supernatants from lung ILCs isolated from the lung at day 7 post-infection and stimulated as indicated for 24 hours. Data are expressed as Mean±S.E.M., b-h are representative of 2 independent experiments with 3-5 mice per group; 1l is representative of 2 separate experiments with 9 mice/group. *P<.01 according to the Mann-whitney Wilcoxon test.
Supplementary Figure 2 Bacterial infection and/or exposure cigarette smoke trigger(s) phenotypic changes in lung-resident ILC2 cells.
(a-c) Quantification of MFI for IL-18Rα, Tbet and IL-12Rβ2 on lung-resident ILCs in naïve mice and mice infected with Staphylococcus aureus (S.aureus) at day 5 post-challenge. (d) Representative flow cytometric plots of GATA-3 expression in naive mice or mice infected with nontypeable Haemophilus influenzae (NTHi). at day 2 post-infection. (e-f) Representative flow cytometric plots and MFI of IL-12Rβ2 expression on lung-resident ILCs in naive mice and mice infected with NTHi at day 5 post-infection (g). Percentage of cells expressing GATA-3 in naïve mice, mice infected with influenza, or mice exposed to cigarette smoke prior to influenza infection. (h) Representative flow cytometric plots of ST2 and IL-18Rα expression on lung-resident ILCs in naive mice, influenza-infected mice or mice exposed to cigarette smoke prior to influenza infection. Correlation (i) and percentage (j) of ILCs expressing ST2 or IL-18Rα in naive mice, influenza-infected mice or mice exposed to cigarette smoke prior to influenza infection. (k-l) GATA-3 and T-bet expression in lung-resident ILCs from naive mice, influenza-infected mice or mice exposed to cigarette smoke prior to influenza infection. *p<.01, **p<.001. Data in a-f are representative of 2-3 independent experiments with ≥5 mice per group. Data in g are pooled from three independent experiments with 3-15 mice/group. Data in h-l are representative of 3-4 independent experiments with ≥5 mice per group. *p<.01, **p<.001.
Supplementary Figure 3 IL-12 and IL-18 induce IFN-γ production by ILC2 cells in vitro and in vivo.
(a) Representative flow cytometric plots of ILCs following double depletion of lineage positive cells. ILCs were defined as CD45+, viable, CD3-, CD49b-, Lin- CD44+ CD90+ IL-7rα+. Levels of IL-5 (b) and IL-13 (c) as measured in the culture supernatants after 4 days of culture with IL-12 and IL-18. MFI of GATA-3 (d), ST2 (e), T-bet (f) and IL-18Rα (g) on lung-resident ILCs from mice treated intranasally as indicated. (h-j) Representative flow cytometric plots showing IL-5 and IFN-g on ILCs were purified from lungs of mice treated as indicated. Data in a-c are representative of three independent experiments, where ILC2 cells were pooled from n≥8 mice. Data in d-g are representative of two independent experiments. For h-j, ILCs were pooled from n≥5 mice per treatment group. *p<.01, **p<.001.
Supplementary Figure 4 GFP+ ILC2 cells adopt an ILC1 phenotype following infection with influenza virus.
(a) ST2+IL-18Rα- ILC2 were sorted and transferred in RAG/γc-/- mice, which were subsequently infected with influenza A. Representative flow cytometric plots showing GATA-3 (b), IL-18Rα (c) and IL-12Rβ2 (d) expression on adoptively transferred ILC2 cells 10 days after virus infection. Representative flow cytometric plots showing IFNγ (d) or IL-13 (e) expression on adoptively transferred ILC2 cells purified from the lung and stimulated overnight with IL-12 and IL-18 (d) or IL-33 (e). MFI of GATA-3 (f), IL-18Rα (g) and IL-12Rβ2 (h) on adoptively transferred ILCs. (i) IFN-γ levels in the supernatants of ILCs cultured overnight in indicated conditions. Data are expressed as Mean and are representative of 2 independent experiments with 5-9 mice per group (g-i) or from 2 independent experiments where each group was pooled (j). ***p<0.0001. All data was analyzed at day 10 post-infection.
Supplementary Figure 5 Rate of influenza-virus infectivity and imaging of transferred GFP+ ILC2 cells in tissue.
(a) Quantification of influenza A staining in RAG/γc-/- deficient mice at day 10 post-infection. Data are expressed as Mean and are representative of 2 independent experiments with 8-11 mice/group, ***p<0.0001. (b) Positive control of GFP immunostaining with brown DAB chromogen in an infected RAG/γc-/- mouse that received GFP+ ILC2 cells. (c) Negative control with the same staining protocol in a virus-infected mouse that had not received any GFP cells. (d) Double in situ hybridization (ISH) with simultaneous visualization of IL-12 and IL18 mRNA by green and red chromogen, respectively. The image is from 6 d post infection and influenza virus particles are immunostained with brown DAB chromogen. (e) Positive ISH control with probe for the eukaryote house-keeping gene PPIB. (f) Negative control with the non-eukaryote bacterial gene DapB. (g,h) Positive staining control for IL-12 and IL-18 alone, respectively. Scale bars: b-h=20μm.
Supplementary Figure 6 T-bet promotes IFN-γ production by ILC2 cells and effects of CD90 depletion on lung-resident cells.
Representative flow cytometric plots (a) and quantification (b) of GATA-3 expression in lung-resident ILCs in C57BL/6 and Tbx21-/- mice after intranasal treatment with PBS or IL-12 and IL-18. Representative flow cytometric plots (c) of ST2 and IL-18Rα expression, quantified in (d), in lung-resident ILCs in C57BL/6 and Tbx21-/- mice treated intranasally with PBS or IL-12 and IL-18. Representative flow cytometric plots (e) and quantification (f) of IL-12Rβ2 expression in lung-resident ILCs in C57BL/6 and Tbx21-/- mice treated intranasally with PBS or IL-12 and IL-18. (g) Intracellular IFN-γ expression in purified ILCs from PBS- or IL-12/IL-18-treated C57BL/6 and Tbx21-/- mice that were stimulated overnight with IL-12 and IL-18. (h) IFN-γ levels in culture supernatants from purified ILCs stimulated overnight as indicated. Representative flow cytometric plots (i) showing Lin- CD90+ cells (gated on CD45+, viable, CD3- CD49b- cells) in mice treated with isotype control or anti-CD90.2 antibody. (j) Quantification of the percentage of ILCs in naive and infected mice treated with isotype control or anti-CD90.2 antibody. Representative flow cytometric plots (k) showing NK cell populations (defined as viable, CD45+, CD3- CD49b+ cells) in naive and infected SCID mice treated with isotype control or anti CD90.2 antibody. (l) Quantification of NK cells in the lungs of naïve and infected SCID mice treated with isotype control or anti CD90.2 antibody. Representative flow cytometric plots (m) showing mesenchymal stem cell populations (mSC, defined as viable, CD45-, CD90+ cells) in naive and infected mice treated with isotype control or anti CD90.2 antibody. (n) Quantification of mSC cells in the lungs of naïve and infected mice treated with isotype control or anti CD90.2 antibody. (o-p) CD166 and Sca-1 expression on CD90+ CD45- cells in the lung. (q) ILC2 and ILC1 cells were purified from mice treated with IL-33 or IL-33 +IL-12+IL-18, respectively, and transferred into RAG/γc deficient mice. Data are shown as Mean and are representative of 2 independent experiments with 4-9 mice/group (a-g) or 7-10 mice/group (j-n) or groups were pooled from 2 independent experiments (h). *p<.01, **p<.001, ***p<.0001.
Supplementary Figure 7 Flow cytometry gating strategy for ILC2 cells from healthy human donors.
Human PBMCs were pooled from 3-4 healthy donors and enriched for NK cells/ILCs using an NK cell enrichment kit. Purity of ILCs in post-NK-enrichment demonstrated by representative flow cytometric plots showing pre-sort (a) and post-sort (b) samples for in vitro culture of human ILC2 cells, defined here as CD45+ Viable CD3- CD19- Lin- IL-7Rα+ CRTH2+ CD161+. (c) Representative flow cytometric plots demonstrating post-culture purity human ILC2 cells cultured with IL-2 and IL-33.
Supplementary Figure 8 Flow cytometry gating strategy for the identification and quantification of ILC subsets in patients with COPD.
(a) Representative flow cytometric plots demonstrating ILC gating strategy for identifying ILCs in the peripheral blood of non-smokers (aged matched healthy controls), healthy smokers or COPD patients. Samples were depleted of T and B cells and ILCs were defined as CD45+ Viable CD3- CD19- Lin- IL-7Rα+ CD56-. (b) Correlative analysis between FEV1/FVC and % ILC1 cells. (c) Correlative analysis between FEV1 predicted and % ILC2 cells. (d) Correlative analysis between FEV1/FVC and % ILC2 cells in the peripheral blood of non-smokers, healthy smokers and COPD patients. (e) Quantification of the sum of % ILC1 and ILC2 cells in non-smokers, healthy smokers and COPD patients, dotted line indicates the mean ± SD across all groups; 42.3%±13.1%. Group sizes: b-d GOLD I=4; GOLD II=11; GOLD III=12; GOLD IV=14; e Non-smokers=11; Smokers=18; total COPD=42.
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Silver, J., Kearley, J., Copenhaver, A. et al. Inflammatory triggers associated with exacerbations of COPD orchestrate plasticity of group 2 innate lymphoid cells in the lungs. Nat Immunol 17, 626–635 (2016). https://doi.org/10.1038/ni.3443
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DOI: https://doi.org/10.1038/ni.3443
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