Volume 21, Issue 9, Pages 2487-2499 (November 2017) Testosterone Attenuates Group 2 Innate Lymphoid Cell-Mediated Airway Inflammation  Jacqueline-Yvonne Cephus, Matthew T. Stier, Hubaida Fuseini, Jeffrey A. Yung, Shinji Toki, Melissa H. Bloodworth, Weisong Zhou, Kasia Goleniewska, Jian Zhang, Sarah L. Garon, Robert G. Hamilton, Vasiliy V. Poloshukin, Kelli L. Boyd, R. Stokes Peebles, Dawn C. Newcomb  Cell Reports  Volume 21, Issue 9, Pages 2487-2499 (November 2017) DOI: 10.1016/j.celrep.2017.10.110 Copyright © 2017 The Authors Terms and Conditions

Figure 1 The Number of Circulating ILC2 Is Increased in Women with Asthma Compared to Men with Asthma (A–C) Blood was collected from men and women with moderate to severe asthma or from healthy controls. Representative dot plots from a woman and a man with asthma showing the flow cytometry gating strategy for circulating ILC2, defined as Lin−, CD45+, CD127+, CD161+, CD25+, and CRTH2+ cells (A). The number of ILC2 as a percentage of PBMCs per milliliter of blood (B). Representative histograms of Gata3+ ILC2 from a woman with asthma (red outline) compared to a man with asthma (blue outline); gray peak represents isotype control (C). (D and E) The number of Gata3+ ILC2 as a percentage of PBMCs per milliliter of blood (D) and Gata3 MFI (E). (F–H) ILC2 were restimulated with PMA, ionomycin, and GolgiStop to determine IL-5 production. Representative dot plots from a woman and a man with asthma (F). Representative histograms of IL-5+ ILC2; gray peak represents isotype control (G). The number of Gata3+ ILC2 as a percentage of PBMCs per milliliter of blood and GATA3 MFI (H). Data are mean ± SEM, n = 4 healthy women or men, n = 6 women with asthma and n = 7 men with asthma; ∗p < 0.05, ns, not significant, Kruskal-Wallis test. Cell Reports 2017 21, 2487-2499DOI: (10.1016/j.celrep.2017.10.110) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Lung ILC Numbers and CD25 Expression Are Decreased in Male Mice Compared to Female Mice ILCs were sorted from the lungs of naive adult and pre-pubescent male and female mice. (A and B) Representative dot plot and quantification of sorted ILCs. (C–E) Representative histograms and quantification of CD25+ or CD127+ staining and MFI on lung ILCs. (F–I) Sorted ILCs were stimulated with IL-2 and IL-33 for 6 days. IL-5 and IL-13 protein expression and Gata3 and Rorα mRNA relative expression normalized to GAPDH was determined. Data are mean ± SEM n = 6–9 mice per group, ∗p < 0.05, ANOVA (B, D, and E) or t test (F–I). Cell Reports 2017 21, 2487-2499DOI: (10.1016/j.celrep.2017.10.110) Copyright © 2017 The Authors Terms and Conditions

Figure 3 IL-2-Mediated ILC2 Proliferation Is Decreased in Male Mice Compared to Female Mice (A) Representative histogram and quantitation of ILC2 proliferation over 3 days from IL-33- and IL-2-stimulated ILC2 from adult male and female mice. Gray peak represents baseline of cell tracer dye added immediately before flow cytometry analysis. (B) Ki-67+ ILC2 from adult male and female mice measured after 3 days of stimulation with IL-2 plus IL-33 or IL-7 plus IL-33. (C) Phospho-STAT5 in ILC2 24 hr after stimulation. (D and E) Representative dot plots (D) and quantification of ILC2 (E) cultured for 6 days with IL-2 plus IL-33 or IL-7 plus IL-33 and restimulated to measure IL-5. Data are mean ± SEM, n = 6 wells from 2 combined experiments; ∗p < 0.05, t test (A and B) or ANOVA with Tukey’s post hoc analysis (C–F). Cell Reports 2017 21, 2487-2499DOI: (10.1016/j.celrep.2017.10.110) Copyright © 2017 The Authors Terms and Conditions

Figure 4 5α-DHT Inhibited IL-5 and IL-13 Protein Expression by ILC2 (A–I) ILCs sorted from the lungs of sham-operated or gonadectomized male and sham-operated female mice were administered hormone or vehicle pellets for 21 days. (A) Total number of sorted ILCs. (B–D) Representative histograms (B) and quantification of CD25+ or CD127+ staining (C) and MFI on lung ILCs. (E–H) ILC2 were stimulated with IL-33 and IL-2 for 6 days. IL-5 (E) and IL-13 (F) protein expression as well as Gata3 (G) and Rorα (H) mRNA relative expression normalized to GAPDH was determined. (I) AR mRNA expression normalized to GAPDH on ILC2. (J–M) 5α-DHT or vehicle was added concurrently with IL-33 and IL-2 stimulation of ILCs for 6 days. IL-5 and IL-13 protein expression and Gata3 and Rorα mRNA relative expression normalized to GAPDH was determined. Data are mean ± SEM, n = 4–5 wells from 3 combined experiments; ∗ p < 0.05, ns, not significant, ANOVA with Tukey’s post hoc analysis. Cell Reports 2017 21, 2487-2499DOI: (10.1016/j.celrep.2017.10.110) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Testosterone Decreased and Ovarian Hormones Increased Alt-Ext-Induced Airway Inflammation (A) Time line for Alt Ext protocol on sham-operated and gonadectomized male and female mice. (B and C) IL-5 (B) and IL-13 (C) protein expression in lung homogenates of male and female mice. Data are mean ± SEM, n = 5 mice; ∗p < 0.05, ANOVA with Tukey’s post hoc analysis. (D) Total number of ILC2 in the lungs 24 hr after last Alt Ext challenge. (E) Representative histogram of CD25 staining in ILC2, pregated on viable, Lin− CD45+ CD127+ ST2+ cells. Gray peak represents isotype control. (F) Quantification of CD25+ ILC2 and CD25 MFI. (G) Representative dot plots of IL-5- and IL-13-producing ILC2. (H) Quantification of ST2+ ILC2. (I) Quantification of IL-5+ ILC2 (top quadrants) and IL-13+ ILC2 (right quadrants). (J) Eosinophils in BAL fluid. (K) Representative sections at 20× magnification and quantification of PAS staining to detect mucus in lung sections on 48 hr after final challenge. Bar represents 100 μm. (L) IL-33 was measured in BAL fluid of mice 1 hr following the last Alt Ext challenge, and TSLP protein expression was measured in whole-lung homogenates 6 hr following the last Alt Ext challenge. Data are mean ± SEM, n = 5–9 mice per group; ∗p < 0.05, ANOVA with Tukey’s post hoc analysis (D–L). Cell Reports 2017 21, 2487-2499DOI: (10.1016/j.celrep.2017.10.110) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Testosterone Intrinsically Attenuated Alt-Ext-Induced Lung ILC2 (A) Experimental design of mixed bone marrow chimera experiment with a 1:1 bone marrow mixture from WT (CD90.1) or ARtfm (CD90.2) adult male mice being transferred into lethally irradiated heterozygous CD90.1+ CD90.2+ WT adult male recipient mice. After 6 weeks of reconstitution, recipient mice underwent the Alt Ext protocol. (B and C) Representative gating strategy (B) and quantification of the total number of IL-5 and IL-13+ ILC2 from WT (CD90.1) and ARtfm (CD90.2) mice (C). (D–F) Histogram (D) and quantification of CD25+ ILC2 (E) and CD25 MFI on WT and ARtfm ILC2. Gray peak represents isotype control. Data are mean ± SEM, n = 6–10 mice per group; ∗p < 0.05, ns, not significant, ANOVA with Tukey’s post hoc analysis. Cell Reports 2017 21, 2487-2499DOI: (10.1016/j.celrep.2017.10.110) Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 2487-2499DOI: (10.1016/j.celrep.2017.10.110) Copyright © 2017 The Authors Terms and Conditions