IL-33 dysregulates regulatory T cells and impairs established immunologic tolerance in the lungs Chien-Chang Chen, PhD, Takao Kobayashi, PhD, Koji Iijima,

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IL-33 dysregulates regulatory T cells and impairs established immunologic tolerance in the lungs Chien-Chang Chen, PhD, Takao Kobayashi, PhD, Koji Iijima, PhD, Fan-Chi Hsu, PhD, Hirohito Kita, MD Journal of Allergy and Clinical Immunology Volume 140, Issue 5, Pages 1351-1363.e7 (November 2017) DOI: 10.1016/j.jaci.2017.01.015 Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Journal of Allergy and Clinical Immunology 2017 140, 1351-1363 Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 CD4+Foxp3+ Treg cells in the lungs expressed IL-33 receptor ST2. A, ST2 expression on CD4+Foxp3+ Treg cells was examined in spleens and lungs of BALB/c-Foxp3EGFP reporter mice. Foxp3EGFPSt2-/- mice were used as a control. 7-AAD, 7-Aminoactinomycin D; FSC, forward scatter; SSC, side scatter. B, ST2 expression in CD4+Foxp3eGFP+ and CD4+Foxp3eGFP− cell populations is shown in histograms. In Fig 1, A and B, data are representative of 3 independent experiments. C, Frequencies of ST2+ cells within the CD4+Foxp3eGFP+ and CD4+Foxp3eGFP− populations in the spleen and lung are shown. Data are shown as means ± SEMs from 4 mice. **P < .01 compared with the spleen. D, ST2+CD4+Foxp3eGFP+ and ST2−CD4+Foxp3eGFP+ cells were isolated by means of sorting from the lungs of naive Foxp3EGFP mice and cultured with medium or IL-33 for 24 hours. St2 mRNA expression was examined by using real-time qRT-PCR and normalized to its expression in CD4+Foxp3eGFP− cells isolated from naive Foxp3EGFP reporter mice. Data are shown as means ± SEMs from 3 mice. *P < .05 and **P < .01 between the groups indicated by horizontal lines. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 IL-33 increased numbers of TH2 cell–like Treg cells in the lungs in vivo. A, Experimental protocol. Naive Foxp3eGFP+ reporter mice were treated with IL-33 (100 ng per dose) or PBS intranasally (i.n.) every other day for 4 days. Lungs were collected 24 hours after the last administration. B, Lung single cells were stained for Foxp3 and GATA3 and analyzed by gating on CD4+ T cells. Representative FACS scattergrams are shown. C, Quantitative analyses of total Foxp3+ Treg cells and Foxp3+/GATA3+ double-positive Treg cells are shown. Data are shown as means ± SEMs from 3 mice per group and are representative of 2 experiments. **P < .01 compared with PBS. D, CD4+Foxp3eGFP+ cells were isolated from the lungs of PBS- or IL-33–treated mice by means of sorting. mRNA expression levels were analyzed by using real-time qRT-PCR and normalized to levels of CD4+Foxp3eGFP+ cells from naive Foxp3EGFP reporter mice. Data are shown as means ± SEMs from 3 mice and are representative of 2 independent experiments. *P < .05 compared with PBS-treated mice. E, CD4+Foxp3eGFP+ cells were isolated by means of sorting and cultured with PMA plus ionomycin for 6 hours. Cytokine levels in cell-free supernatants were analyzed by means of ELISA. Date are shown as means ± SEMs of a pool of 2 independent experiments, each of which used 3 mice per group. F, Lung single-cell suspensions from IL-33–treated Foxp3EGFP mice (WT mice) and Foxp3EGFPSt2−/− mice (ST2−/− mice) were cultured with PMA plus ionomycin for 6 hours in the presence of GolgiPlug. Cells were stained for Foxp3 and GATA3, intracellular IL-5 and IL-13, and cell-surface ST2. Representative data from 3 mice per group are shown. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 IL-33 impaired the suppressive ability of Treg cells. A and B, CD4+Foxp3+ Treg cells and CD4+Foxp3− T-responder cells were isolated by means of sorting from spleens of naive Foxp3EGFP mice. T-responder cells were labeled with a tracer dye and stimulated with anti-CD3/anti-CD28 microbeads with or without Treg cells (at a 1:1 ratio) or IL-33 (40 ng/mL) for 4 days. Representative histograms are shown. C, Frequencies of nonproliferating T-responder cells are shown. Data (means ± SEMs) are a pool of 4 independent experiments. **P < .01 between the groups indicated by horizontal lines. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Culture with IL-33 induced TH2 cell–like Treg cells in vitro. CD4+Foxp3eGFP+ Treg cells were isolated from the lungs of naive Foxp3EGFP mice by means of sorting and were cultured with IL-2 (20 ng/mL) with or without IL-33 (40 ng/mL) for 96 hours. A, mRNA expression levels were analyzed by using real-time qRT-PCR and normalized to levels in CD4+Foxp3eGFP+ cells isolated from naive nontreated Foxp3EGFP mice. B, Cells were stimulated with PMA plus ionomycin for 6 hours. Cytokine levels in cell-free supernatants were analyzed by means of ELISA. Data (n = 6, means ± SEMs) are a pool of 2 independent experiments. *P < .05 and **P < .01 compared with IL-2 alone. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 IL-33 broke down established airway tolerance in vivo. A, Experimental protocol. To establish tolerance, naive BALB/c mice were treated intranasally with endotoxin-free OVA. The state of tolerance was verified by means of sensitization with intraperitoneal administration of OVA plus alum. Mice were then exposed intranasally to OVA with or without IL-33. Immune responses were examined by analyzing plasma levels of anti-OVA IgE on day 28 and then challenging mice by means of intranasal administration of OVA on days 29, 30, and 31. BAL fluid and lung tissue were collected 24 hours after the last challenge. B, Plasma levels of anti-OVA IgE are shown. C, Cell differential counts and cytokine levels in BAL fluid are shown. Data are shown as means ± SEMs of 3 to 8 mice per group. *P < .05 and **P < .01 between the groups indicated by horizontal lines. Eos, Eosinophils; i.n., intranasal; i.p., intraperitoneal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Airway exposure of antigen with IL-33 was sufficient to break down established airway immunologic tolerance without systemic administration of antigen with an adjuvant. A, Experimental protocol. Naive BALB/c mice were treated intranasally with endotoxin-free OVA to establish tolerance. Mice were then exposed intranasally to OVA with IL-33 or sensitized by means of intraperitoneal injection of OVA plus alum. Immune responses were examined by analyzing plasma levels of anti-OVA IgE on day 28 and then challenging mice with intranasal administration of OVA on days 29, 30, and 31. BAL fluid and lung tissue were collected 24 hours after the last challenge. B, Plasma levels of anti-OVA IgE are shown. C, Cell differential counts and cytokine levels in BAL fluid are shown. Data are shown as means ± SEMs of 3 to 6 mice per group and are representative of 2 independent experiments. *P < .05 and **P < .01 between the groups indicated by horizontal lines. Eos, Eosinophils; i.n., intranasal; i.p., intraperitoneal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 ST2+ Treg cells in the lungs produced TH2-type cytokines during allergic airway inflammation. A, Experimental protocol. Naive BALB/c Foxp3EGFP reporter mice were exposed to PBS or a cocktail of airborne allergens, including extracts of A alternata, A fumigatus, and HDM and OVA, every other day for 2 weeks. BAL fluid and lung tissue were collected 12 hours after the last exposure. B, Cell differential counts in BAL fluid are shown. C, Single-cell suspensions of lungs were stained for CD4 and ST2 and analyzed by using FACS. Representative FACS scattergrams are shown. D, Quantitative analyses of total Foxp3+ Treg cells and ST2+ and ST2− Treg cell subpopulations are shown. E, The Foxp3eGFP+ST2+ Treg cell population was isolated by means of sorting, and mRNA expression was analyzed by using real-time qRT-PCR. Data were normalized to levels of total Foxp3eGFP+ Treg cells from naive mice. In Fig 7, B, D, and E, data are shown as means ± SEMs of at least 3 mice per group and are representative of 2 independent experiments. *P < .05 and **P <.01 between the groups indicated by horizontal lines. Eos, Eosinophils; i.n., intranasal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 IL-33 signaling was required for the increase in numbers of GATA3+ Treg cells in the lungs. A, Experimental protocol. Naive WT BALB/c mice (BALB/c) or BALB/c St2−/− mice (ST2−/−) were exposed to PBS or a cocktail of airborne allergens, as described in Fig 7, every other day for 4 weeks. Lungs were collected 24 hours after the last exposure. B, Single-cell suspensions of lungs were stained for Foxp3 and GATA3 and analyzed by using FACS. Representative FACS scattergrams are shown. C, Quantitative analyses of total Foxp3+ Treg cells and the GATA3+ population of Treg cells are shown. Data are shown as means ± SEMs from a pool of 2 experiments, each of which used 3 mice per group. *P < .05 between the groups indicated by horizontal lines. i.n., Intranasal. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Lung Treg cells from WT mice that had been administered IL-33 produced type 2 cytokines. A, Experimental protocol. Naive WT BALB/c mice received intranasal (i.n.) administration of 100 ng of IL-33 or PBS per dose for 4 days. Twenty-four hours after the last administration, lung single-cell suspensions were collected and analyzed by using FACS. B, Cells were stained for intracellular expression of Foxp3, GATA3, and type 2 cytokines, as well as cell-surface expression of ST2. Representative histograms are shown by gating on CD4+Foxp3+GATA3+ cells or CD4+Foxp3+GATA3− cells. C, Representative histograms are shown by gating on CD4+Foxp3+GATA3+ cells. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Airway immunologic tolerance model. A, Experimental protocol. Naive BALB/c mice were treated intranasally with endotoxin-free OVA to establish tolerance. Mice were then sensitized by means of intraperitoneal injection of OVA plus alum to verify the state of tolerance. Immune responses were examined by analyzing plasma levels of anti-OVA antibodies on day 28 and then challenging mice by means of intranasal administration of OVA on days 29, 30, and 31. BAL fluid was collected 24 hours after the last challenge. B, Plasma levels of anti-OVA IgE, IgG1, and IgG2a are shown. C, Cell differential counts and cytokine levels in BAL fluid are shown. Data are shown as means ± SEMs of at least 3 mice per group and are representative of 2 independent experiments. *P < .05 and **P < .01 between the groups indicated by horizontal lines. Eos, Eosinophils; i.n., intranasal; i.p., intraperitoneal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 IL-33 alone without antigen did not break down immunologic tolerance. A, Experimental protocol. Naive BALB/c mice were treated intranasally with endotoxin-free OVA to establish tolerance. The state of tolerance was verified by means of sensitization with intraperitoneal administration of OVA plus alum. Mice were then exposed intranasally to OVA alone, IL-33 alone, or OVA plus IL-33. Immune responses were examined by challenging the mice by means of intranasal administration of OVA on days 29, 30, and 31. Plasma and BAL fluid were collected 24 hours after the last challenge. B, Plasma levels of anti-OVA IgE are shown. C, Cell differential counts and cytokine levels in BAL fluid are shown. Data are shown as means ± SEMs of 3 to 6 mice per group and are representative of 2 independent experiments. **P < .01 between the groups indicated by horizontal lines. Eos, Eosinophils; i.n., intranasal; i.p., intraperitoneal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Airway histology of the lungs. Lung specimens from the experiments shown in Fig 5 were stained with hematoxylin and eosin (H&E; left) and periodic acid-Schiff (PAS; right). Photomicrographs are representative of 3 to 8 mice per group. i.n., Intranasal; i.p., intraperitoneal. Panels A, B, C, and D represent the experimental groups as listed in the top of the Figure. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Airway exposure to a mixture of allergens breaks down established airway immunologic tolerance. A, Experimental protocol. Naive BALB/c mice were treated intranasally with endotoxin-free OVA to establish tolerance. Mice were then exposed intranasally to OVA with a mixture of allergens (50 μg each of A alternata, A fumigatus, and HDM) or sensitized by means of intraperitoneal injection of OVA plus alum. Immune responses were examined by analyzing plasma levels of anti-OVA IgE on day 28 and then challenging mice with intranasal administration of OVA on days 29, 30, and 31. BAL fluid and lung tissue were collected 24 hours after the last challenge. B, Plasma levels of anti-OVA IgE are shown. C, Cell differential counts and cytokine levels in BAL fluid are shown. Data are shown as means ± SEMs of 6 mice per group. *P < .05 and **P < .01 between the groups indicated by horizontal lines. Eos, Eosinophils; i.n., intranasal; i.p., intraperitoneal; Lym, lymphocytes; Mac, macrophages; Neu, neutrophils. Journal of Allergy and Clinical Immunology 2017 140, 1351-1363.e7DOI: (10.1016/j.jaci.2017.01.015) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions