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. Author manuscript; available in PMC: 2017 Dec 15.
Published in final edited form as: Cell Rep. 2017 Nov 28;21(9):2487–2499. doi: 10.1016/j.celrep.2017.10.110

Testosterone attenuates group 2 innate lymphoid cell-mediated airway inflammation

JY Cephus 1, MT Stier 2, H Fuseini 2, JA Yung 1, S Toki 1, MT Bloodworth 2, W Zhou 1, K Goleniewska 1, J Zhang 1, SL Garon 1, RG Hamilton 3, V Poloshukin 1, KL Boyd 2, RS Peebles Jr 1,2, DC Newcomb 1,2
PMCID: PMC5731254  NIHMSID: NIHMS922191  PMID: 29186686

Summary

Sex hormones regulate many autoimmune and inflammatory diseases, including asthma. As adults, asthma prevalence is 2-fold greater in women compared to men. Group 2 innate lymphoid cells (ILC2) are increased in asthma, and we investigated how testosterone attenuated ILC2 function. In patients with moderate to severe asthma, we determined that women had increased circulating ILC2 numbers compared to men. In mice, ILC2 from adult females had increased IL-2-mediated ILC2 proliferation versus ILC2 from adult males and pre-pubescent females and males. Further, 5α-dihydrotestosterone, a hormone downstream of testosterone, decreased lung ILC2 numbers and IL-5 and IL-13 expression from ILC2. In vivo, testosterone attenuated Alternaria extract-induced IL-5+ and IL-13+ ILC2 numbers and lung eosinophils by intrinsically decreasing lung ILC2 numbers and cytokine expression as well as decreasing expression of IL-33 and TSLP, ILC2 stimulating cytokines. Collectively, these findings provide a foundational understanding in the sexual dimorphism in ILC2 function.

Keywords: innate lymphoid cells, testosterone, sex hormones, asthma

eTOC

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Women have higher asthma prevalence compared than men, and ILC2 are increased in patients with asthma. Cephus et al show women with asthma had higher circulating ILC2 numbers compared to men with asthma. Testosterone negatively regulated ILC2 proliferation and cytokine expression as well as ILC2-mediated allergic airway inflammation.

Gender disparities in immune-mediated responses are driven by differences in sex hormone concentrations, anatomy, and/or X- or Y-linked genes (Fish, 2008). A female dominance exists in many autoimmune diseases, including lupus, multiple sclerosis, and rheumatoid arthritis, as well as in inflammatory diseases, such as asthma (Carey et al., 2007a; Fish, 2008). During childhood, asthma prevalence is greater in boys compared to girls (ALA, 2012; Carey et al., 2007a; Fitzpatrick et al., 2011). However, around puberty there is a shift in asthma prevalence and at mid-life, women are about twice as likely as men to have asthma (ALA, 2012; Carey et al., 2007a). Changes in asthma prevalence that coincide with changes in sex hormones strongly suggest a role for sex hormones in asthma pathogenesis. While sex hormone regulation of inflammation in asthma has been described (Fuseini and Newcomb, 2017), the role of sex hormones in modulating lung innate lymphoid cell (ILC)-mediated responses is less clear.

ILCs are a rare population of lymphocytes that are important for the initiation of inflammatory responses (Serafini et al., 2015) that express the surface markers CD25 and CD127, components of the IL-2 receptor (R) and the IL-7R, respectively. Once stimulated, ILCs rapidly produce cytokines in an antigen independent manner (Barlow et al., 2012; Halim et al., 2012a; Klein Wolterink et al., 2012), and ILCs are classified into groups, ILC1, 2, or 3, that broadly mimic the transcriptional programming, cytokine production, and effector function of adaptive CD4+ Th1, Th2, and Th17 cells, respectively (Serafini et al., 2015).

ILC2 play a central role in the propagation of allergic responses and asthma. ILC2 are activated by the cytokines IL-33, TSLP, and/or IL-25 leading to increased Gata3 and Rorα expression and the production of predominantly IL-5 and IL-13, as well as IL-4, IL-9, and amphiregulin (Hoyler et al., 2012; Mjosberg et al., 2012). IL-5 production is imperative for infiltration of eosinophils into the airway, a key pathologic feature of allergy, and IL-13 is important for increased airway hyperresponsiveness and mucus production (Halim et al., 2012a). ILC2 secrete more IL-5 and IL-13 compared to CD4+ T cells and are also important for establishing a memory Th2 allergic response in the lung and skin, underscoring the substantial pathophysiologic potential of ILC2 (Halim et al., 2014; Klein Wolterink et al., 2012; Zhou et al., 2016).

ILC2 were increased in the peripheral blood mononuclear cells (PBMCs) of patients with allergic asthma compared to healthy controls (Bartemes et al., 2014; Liu et al., 2015). Further, ILC2 were increased in the induced sputum and PBMCs of patients with severe, eosinophilic asthma compared to milder forms of asthma (Smith et al., 2016). Mouse in vivo experiments revealed that lung ILC2 have a critical role in rapid Th2-type inflammation in response to protease aeroallergens such as Alternaria alternata, which is associated with severe exacerbations of asthma (Agarwal, 2011; Dales et al., 2000; Neukirch et al., 1999; Zureik et al., 2002). Collectively, these data highlight the significance of ILC2 in allergic inflammation and emphasize the need to better understand the mechanisms that regulate ILC2, particularly in the context of asthma where a gender disparity is prominent.

Recently, androgen receptor (AR) signaling decreased ILC2 progenitor cell numbers in the bone marrow as well as IL-33-induced ILC2-mediated airway inflammation (Laffont et al., 2017). However, it remained unknown if women with asthma have increased circulating ILC2 compared to men with asthma. Further, the mechanisms by which androgen receptor signaling mediated ILC2 functions had not been fully elucidated. We hypothesized that testosterone and its derivative, 5α-dihydrotestosterone (DHT), signaling through AR negatively regulates ILC2 cytokine expression and ILC2-mediated airway inflammation. Our data shows that women with asthma have increased circulating ILC2 compared to men with asthma, while testosterone and 5α-DHT negatively regulates ILC2 proliferation and cytokine expression, as well as ILC2-mediated Alt-Ext induced allergic airway inflammation.

Results

Women with asthma have increased ILC2 in peripheral blood compared to men with asthma

ILC2 are important for allergic airway inflammation associated with asthma (Halim et al., 2012a; Neill et al., 2010), and patients with asthma have increased circulating ILC2 compared to healthy controls (Bartemes et al., 2014; Liu et al., 2015). To determine if there was a gender difference in circulating ILC2, we recruited women and men of reproductive age (18–45 years old) with moderate to severe asthma as well as healthy control participants. Participants were excluded for infections within 2 weeks, and women were excluded if pregnant, breastfeeding, or taking hormonal birth control medications. Demographic information on participants with asthma and healthy controls can be seen in Table S1. Clinical characteristics of participants with asthma can be seen in Table S2. No differences in inhaled corticosteroid use, %FEV1 predicted, or serum total IgE concentrations were determined in women and men with asthma. PBMCs and total blood volume were quantified from each patient, and the number of ILC2, defined as Lineage negative (Lin-) CD45+ CD127+ CD161+ CD25+ CRTH2+ cells (Figure 1A), was assessed by flow cytometry. Women and men with asthma had increased circulating ILC2, defined as a percentage of PBMCs/ml of blood, as well we increased % Gata3+ ILC2 compared to healthy women and men, respectively, (Figure 1B–C). In comparing participants with asthma based on gender, women with asthma had increased ILC2 percentage compared to men with asthma. No gender difference was seen in ILC2 of healthy women compared to healthy men (0.012 ± 0.006% vs 0.009 ± 0.001% ILC2, respectively). Women with asthma also had an increased percentage of GATA3+ ILC2 and increased GATA3 mean fluorescent intensity (MFI) compared to men with asthma (Figure 1C–E). To ensure the increased circulating ILC2 in women compared to men with asthma were not due to differential surface expression of CD161 or CRTH2, we quantified the number of Lin- CD45+ CD127+ GATA3+ cells. Using this gating strategy, we saw similar results with women with asthma having increased ILC2 compared to men with asthma (Figure S1A–B).

Figure 1. Circulating ILC2 are increased in women compared to men with asthma.

Figure 1

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Blood was collected from men and women with moderate to severe asthma or healthy controls. (A) Representative dot plots from a woman and man with asthma showing flow cytometry gating strategy for circulating ILC2, defined as Lin-, CD45+, CD127+, CD161+, CD25+, and CRTH2+ cells. (B) ILC2 as a percentage of PBMCs/ml of blood. (C) Representative histograms of Gata3+ ILC2 from woman with asthma (red outline) compared to man with asthma (blue outline). Gray peak represents isotype control. (D–E). Gata3+ ILC2 as a percentage of PBMCs/ml of blood and GATA3 MFI. (F–H). ILC2 were restimulated with PMA, ionomycin and golgi-stop to determine IL-5 production. (F) Representative dot plots from a woman and man with asthma. (G). Representative histograms of IL-5+ ILC2, gray peak represents isotype control. (H). IL-5+ ILC2 as a percentage of PBMCs/ml of blood. Data are mean ± SEM, n=4 healthy women or men, n=6 women with asthma and n=7 men with asthma; * p<0.05, n.s., not significant, Kruskal-Wallis test.

We also measured IL-5 production in ILC2 from healthy and asthmatic women and men. Enriched Lin-cells were restimulated with PMA, ionomycin, and golgi stop to induce cytokine production. After restimulation, IL-5+ ILC2 were increased in participants with asthma compared to healthy controls, and women with asthma had increased IL-5+ ILC2 compared to men with asthma (Figure 1F–H). Combined these data show that women with asthma have increased circulating ILC2 compared to men with asthma.

Sex hormones regulate IL-2-dependent ILC2 proliferation

To determine if sex hormones regulated ILC2, we isolated ILCs from the lungs of male and female adult mice or pre-pubescent mice (< 4 weeks of age). Total lung numbers were similar between all groups of mice (Figure S2A). Lung ILCs, defined as viable, Lin- CD45+ CD90+ CD25+ CD127+ cells (Figure S2B), were sorted for in vitro stimulation, but surprisingly, the number of sorted lung ILCs were increased in adult female mice compared to adult male mice and pre-pubescent female and male mice (Figure 2A–B). No differences in lung ILCs were detected in pre-pubescent female and male mice. To determine why decreased ILCs were sorted from the lungs of adult male mice compared to adult female mice, we quantitated the percentage of CD25+ and CD127+ cells as well as the MFI of CD25 and CD127. The percentage of CD25+ ILCs as well as the CD25 MFI was decreased on lung ILCs from naïve adult male mice compared to naïve adult female mice (Figure 2C–E). However, no differences in CD127+ ILCs or CD127 MFI were determined in adult male and female ILCs. Pre-pubescent male and female mice had similar percentages of CD25+ and CD127+ ILCs and no change in CD25 or CD127 MFI (Figure 2C–E). The IL-2R is comprised of CD25, IL-2RB, and the common gamma chain (common γ chain). The IL-7R is comprised of CD127 and the common γ chain. Since we detected decreased CD25 MFI in ILCs from adult male mice compared to female mice, we quantitated IL-2RB MFI and common γ chain MFI in ILCs. IL-2RB MFI was slightly, but significantly, decreased in ILCs from adult male compared to female mice, but no differences were detected in common gamma chain MFI (Figure S3). These results suggested that sex hormones were important for lung ILC numbers by regulating IL-2R signaling.

Figure 2. Lung ILC numbers and CD25 expression are decreased in male mice compared to female mice.

Figure 2

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ILCs were sorted from the lungs of naïve adult and pre-pubescent male and female mice. (A–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 as well as 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, E) or t-test (F–I).

To activate ILC2, sorted ILCs from adult male and female mice were stimulated with IL-2 and IL-33 for 6 days and IL-5 and IL-13 protein expression and transcription factor mRNA expression was determined. We did not include pre-pubescent mice, since no differences in CD25 and CD127 expression was determined in these mice. IL-5 and IL-13 protein expression was decreased in ILC2 from male mice compared to ILC2 from female mice (Figure 2F–G). Further, Gata3 and Rorα expression was significantly decreased in lung ILC2 from male mice compared to female mice (Figure 2H–I).

IL-2R signaling and RORα are important for ILC proliferation (Halim et al., 2012b; Serafini et al., 2015; Wong et al., 2012), and we next determined the proliferative capacity of lung ILCs from adult male and female mice following IL-2 plus IL-33 stimulation. ILC2 from male mice had decreased proliferation compared to ILC2 from female mice (Figure 3A). To determine if the gender difference in ILC2 proliferation was mediated by IL-2R signaling, and not IL-7R signaling, we stimulated ILC2 from adult female and male mice with IL-2, IL-7, IL-33, IL-2 plus IL-33, or IL-7 plus IL-33 for 3 days and quantified ILC2 proliferation with Ki67+ staining (Figure 3 and S4). Very few Ki67+ ILC2 were quantified when cells were stimulated with IL-2 alone or IL-7 alone. Stimulation with only IL-33 resulted in Ki67+ ILC2, with males having decreased Ki67+ ILC2 compared to females. IL-2 plus IL-33 stimulated ILC2 from female mice had increased Ki67+ staining compared to IL-33 alone or IL-7 plus IL-33 stimulation. IL-2 plus IL-33 stimulated ILC2 male mice had decreased Ki67+ ILC2 compared to ILC2 from female mice, but there were no gender differences in Ki67+ ILC2 in IL-7 plus IL-33 stimulated cells (Fig. 3B). Based on these data, we performed additional experiments using IL-2 plus IL-33 or IL-7 plus IL-33 stimulation conditions.

Figure 3. IL-2-mediated ILC2 proliferation is decreased in male mice compared to female mice.

Figure 3

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(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 hours after stimulation. (D–E) Representative dot plots and quantification of ILC2 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–B) or ANOVA with Tukey post-hoc analysis (C–F).

IL-2 signaling through the IL-2R or IL-7 signaling through the IL-7R increases STAT5 phosphorylation and regulates ILC2 proliferation. Therefore, we next measured phosphorylation of STAT5 by flow cytometry in IL-2 plus IL-33 or IL-7 plus IL-33 stimulated ILC2 from male and female mice. ILC2 from male mice stimulated with IL-2 plus IL-33 had decreased STAT5 phosphorylation compared to female ILC2, but no difference in STAT5 phosphorylation was determined in male and female ILC2 stimulated with IL-7 plus IL-33 (Figure 3C). Combined, these data showed gender differences in ILC2 proliferation with IL-2 plus IL-33, but not IL-7 plus IL-33, stimulation.

To determine cytokine expression from male and female ILC2, we cultured ILC2 in varying conditions for 6 days and then restimulated ILC2 with PMA, ionomycin, and golgi-stop to measure IL-5 and IL-13 production by flow cytometry. Male lung ILC2 stimulated with IL-2 plus IL-33 had decreased IL-5+ and IL-13+ ILC2 compared to female lung ILC2. No difference in IL-5+ and IL-13+ ILC2 was observed with IL-7 plus IL-33 stimulation (Figure 3D–E). However, the percentage of IL-5+ and IL-13+ ILC2 was similar in male and female ILC2 stimulated with either IL-2 plus IL-33 or IL-7 plus IL-33. Collectively, these data showed that male mice had decreased ILC2 proliferation that resulted in decreased IL-5+ and IL-13+ ILC2, compared to female mice. However, it remained unclear which sex hormone(s) were responsible for the observed changes.

5α-DHT inhibited IL-5 and IL-13 protein expression from lung ILC2

Since lung ILCs were decreased in male mice compared to female mice, we hypothesized that endogenous sex hormones were important in regulating ILC2 proliferation and cytokine expression. Previously we showed that prolonged administration of 17β-estradiol (17β-E2) and progesterone (P4) significantly increased Th17 cell differentiation and IL-17A protein expression (Newcomb et al., 2015). Therefore, we administered slow-release pellets containing 5α-DHT, 17β-E2, P4, the combination of 17β-E2 and P4, or vehicle for 21 days to gonadectomized male mice, which lack testosterone. Vehicle pellets were also administered to sham-operated male and female mice, with normal levels of testosterone and ovarian hormones, respectively. CD25+ CD127+ ILCs were then sorted from the lungs of sham-operated and gonadectomized mice. Sham-operated male mice administered vehicle pellets had decreased sorted lung ILCs compared to sham-operated female mice and gonadectomized male mice administered vehicle pellets (Figure 4A). Gonadectomized male mice administered 5α-DHT pellets also had decreased sorted lung ILC numbers compared to gonadectomized male mice administered vehicle pellets. No significant differences in sorted lung ILC numbers were detected in gonadectomized male mice administered 17β-E2, P4, or the combination of 17β-E2 and P4 pellets compared to gonadectomized male mice administered vehicle. These results showed that 5α-DHT, and not 17β-E2 or P4, modulated the number of sorted lung ILCs. Therefore, we focused on 5α-DHT regulation of ILC2. Sham-operated male mice administered vehicle and gonadectomized male mice administered 5α-DHT pellets had decreased % CD25+ cells and CD25 MFI compared to sham-operated female mice and gonadectomized male mice administered vehicle pellets (Figure 4B–D). No difference in CD127 MFI was detected between any groups (Figure 4B–D).

Figure 4. 5α-DHT inhibited IL-5 and IL-13 protein expression by ILC2 cells.

Figure 4

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(A–I) ILCs were sorted from the lungs of sham-operated or gonadectomized male and female mice were administered hormone or vehicle pellets for 21 days. (A) Total number of sorted ILCs. (B–D) Representative histograms and quantification of CD25+ or CD127+ staining and MFI on lung ILCs. (E–H) ILC2 were stimulated with IL-33 and IL-2 for 6 days. IL-5 and IL-13 protein expression as well as Gata3 and Rorα mRNA relative expression normalized to GAPDH was determined. (I). AR mRNA expression normalized to GAPDH on ILC2. (H–I) 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 as well as 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, n.s., not significant, ANOVA with Tukey post-hoc analysis.

We next determined if administration of 5α-DHT negatively regulated cytokine expression from ILC2. IL-5 and IL-13 protein expression as well as Rorα expression were significantly decreased in ILC2 from sham-operated male mice administered vehicle and gonadectomized male mice administered 5α-DHT compared to ILC2 from sham-operated female mice and gonadectomized male mice administered vehicle (Figure 4E–F, H). While ILC2 from sham-operated male mice had decreased Gata3 expression compared to ILC2 from sham-operated female mice, no differences in Gata3 expression were detected in ILC2 from sham-operated or gonadectomized male mice (Figure 4G). Similar to our results in Figure 4A, administration of 17β-E2, P4, or the combination of 17β-E2 and P4 to gonadectomized male mice had no effect on IL-5 and IL-13 protein expression nor Rorα expression from ILC2 compared to gonadectomized male mice administered vehicle (Fig. S5). However, ILC2 from mice administered 17β-E2 and P4 had increased Gata3 expression compared to ILC2 from mice administered vehicle.

While long-term administration of 5α-DHT to mice negatively regulated IL-5 and IL-13 production from ILC2 ex vivo, it was unknown whether 5α-DHT would rapidly inhibit ILC2 cytokine expression in culture. AR has been reported to be expressed on ILC2 progenitor cells (Laffont et al., 2017), and we determined that AR mRNA expression was similar in lung ILC2 from females and males (Figure 4I). Therefore, female ILC2 were stimulated with IL-2 and IL-33 in the presence of 5α-DHT (0.1–3nM) or vehicle (methanol). ILC2 from female mice were used because these cells developed in a low testosterone environment. IL-5 and IL-13 were significantly decreased in 5α-DHT-treated ILC2 compared to vehicle treated ILC2 (Figure 4J–K). 5α-DHT also decreased Rorα expression but had no effect on Gata3 expression (Fig. 4L–M). Combined, these data suggested that 5α-DHT negatively regulated Rorα expression and IL-5 and IL-13 protein expression in ILC2, but that Gata3 expression was regulated by 17β-E2 and P4.

Male mice have decreased Alt Ext-induced airway inflammation in mice compared to female mice

Our in vitro data demonstrated that 5α-DHT negatively regulated IL-5 and IL-13+ ILC2, but the role of sex hormones in an innate immune-mediated model of airway inflammation remained unknown. Previous studies had examined ILC2 function in models that have both ILC2 and CD4+ T cell mediated inflammation (Laffont et al., 2017; Warren et al., 2017). Alternaria alternata caused allergic airway inflammation and asthma exacerbations in persons sensitized to this fungus (Agarwal, 2011). We have previously shown that 4 day exposure to Alternaria alternata extract (Alt Ext) increased ILC2 cytokine expression and eosinophil infiltration (Toki et al., 2016; Zhou et al., 2016). Therefore, to determine if sex hormones regulated Alt Ext-induced airway inflammation, male and female WT mice were challenged with Alt Ext or PBS for 4 consecutive days and lungs were collected 24 hours after the final Alt Ext challenge and analyzed for IL-5 and IL-13 production (Figure 5A). Alt Ext-challenged male and female mice had increased lung IL-5 and IL-13 protein expression compared to PBS control mice (Figure 5B–C). However, Alt Ext-challenged male mice had a significantly less IL-5 and IL-13 production compared to Alt Ext-challenged female mice. To ensure the responses we were seeing were independent of adaptive lymphocytes, we repeated the experiment in Alt Ext-challenged male and female Rag1−/− mice, which lack T and B lymphocytes. Alt Ext-challenged male Rag1−/− mice had decreased lung IL-5 and IL-13 protein expression compared to Alt Ext-challenged female Rag1−/− mice (Fig. S6A–B). Combined, these data suggested that the innate immune-mediated response to Alt Ext is diminished in male mice compared to female mice.

Figure 5. Testosterone decreased and ovarian hormones increased Alt Ext-induced airway inflammation.

Figure 5

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(A) Timeline for Alt Ext protocol on sham-operated and gonadectomized male and female mice. (B–C) IL-5 and IL-13 protein expression in lung homogenates of male and female mice. Data are mean ± SEM, n=5 mice; * p<0.05, ANOVA with Tukey post-hoc analysis. (D) Total number of ILC2 in the lungs 24 hours 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 hours after final challenge. Bar represents 100µm. (L) IL-33 was measured in BAL fluid of mice 1 hour following the last Alt Ext challenge and TSLP protein expression was measured in whole lung homogenates 6 hours following the last Alt Ext challenge. (D–L) Data are mean ± SEM, n=5–9 mice per group; * p<0.05, ANOVA with Tukey post-hoc analysis.

Testosterone decreased Alt Ext-induced airway inflammation in mice

We next elucidated the hormone(s) responsible for the altered innate immune responses in male and female mice. Sham-operated male and female mice as well as gonadectomized male and female mice were challenged with Alt Ext. The number of lung ILC2, defined as Lin- CD45+ CD127+ ST2+ cells, were determined in Alt Ext-challenged mice, and lung ILC2 were significantly decreased in sham male mice compared to sham and gonadectomized female mice and gonadectomized male mice (Figure 5D). Surprisingly, no differences were determined in the number of lung ILC2 in sham-operated and gonadectomized female mice. CD25+ ILC2 were also decreased in sham-operated male mice compared to sham-operated female, gonadectomized female, and gonadectomized male mice, but no differences in MFI were detected (Figure 5F), but there was no change in CD25 MFI. Next, we determined the number of IL-5 and IL-13 producing ILC2. Cells were pre-gated on Lin-, CD45+, CD127+ and then gated on CD45+ ST2+ (left panel) followed by gating on IL-5 and IL-13 (right panel). Isotype controls for IL-5 and IL-13 staining are shown in Figure S7. The number of Alt Ext-induced ST2+ ILC2 cells was significantly decreased in sham-operated male compared to sham-operated female and gonadectomized male mice (Figure 5G–H). Further, the total number of Alt Ext-induced IL-5+ ILC2 cells was significantly decreased in gonadectomized female, sham-operated male, and gonadectomized male mice compared to sham-operated female mice (Figure 5G and I) but increased in gonadectomized male mice compared to sham-operated male mice. As shown previously, Alt Ext induced less IL-13 compared to IL-5 in ILC2 (Zhou et al., 2016). Nevertheless, IL-13+ ILC2 cells were also significantly decreased in sham-operated male mice compared to gonadectomized male mice and sham-operated female mice (Figure 5G and I). Taken together these data suggested that testosterone negatively regulated ILC2 in the lung, ST2+ ILC2, ILC2-derived IL-5 and IL-13 production whereas ovarian hormones enhanced IL-5 production from lung ILC2.

IL-5 is important for the development and maturation of eosinophils, and our Alt Ext challenge protocol increased eosinophil infiltration into the airway compared to vehicle (Zhou et al., 2016). Alt Ext-challenged sham-operated male mice had a significant decrease in BAL eosinophils compared to Alt Ext-challenged sham-operated female and gonadectomized male mice (Figure 5J). Alt Ext-challenged gonadectomized male and gonadectomized female mice had a significant decrease in BAL eosinophils compared to Alt Ext-challenged sham-operated female mice, suggesting that testosterone decreased and ovarian hormones increased eosinophil infiltration into the lung, consistent with IL-5 expression in the ILC2 compartment (Figure 5I). No significant changes were detected in the percentages or numbers of lymphocytes or neutrophils in the BAL fluid between groups after Alt Ext challenge (data not shown). Mucus production after Alt Ext challenge was also determined in the lung. Sham-operated and gonadectomized male mice as well as gonadectomized female mice had decreased mucus production compared to sham-operated female mice (Figure 5K). These results showed that testosterone decreased, and ovarian hormones increased eosinophilic inflammation after Alt Ext challenge, but it remained unclear if testosterone directly or indirectly affected ILC2 cytokine expression in vivo.

We also quantified IL-33 protein expression in the BAL fluid and TSLP protein expression in whole lung homogenates at one hour or six hours, respectively, following the last Alt Ext-challenge to determine whether testosterone affected the release of these cytokines. IL-33 and TSLP protein expression was significantly increased in the BAL fluid of Alt Ext-challenged mice compared to vehicle (PBS) challenged mice (data not shown). Sham-operated male mice had decreased IL-33 and TSLP protein expression compared to sham-operated female mice and gonadectomized male and female mice (Figure 5L). IL-33 and TSLP protein expression was similar in Alt Ext-challenged sham-operated and gonadectomized female. These data showed that testosterone negatively regulated Alt Ext-induced IL-33 and TSLP, but that ovarian hormones had no effect on IL-33 and TSLP secretion.

To determine the sufficiency of IL-33 alone to induce gender-mediated differences in ILC2 cytokine production, mice were intranasally challenged with 350ng of rmIL-33 daily for 4 consecutive days and then BAL fluid was collected and lungs were analyzed for IL-5 and IL-13+ ILC2 by flow cytometry. Similar to Alt Ext-induced airway inflammation, the number of rmIL-33-induced ST2+ ILC2 cells was significantly decreased in sham-operated male compared to sham-operated female and gonadectomized male mice (Figure S8A–B). IL-33-induced IL-5+ ILC2 were decreased in gonadectomized female, sham-operated male, and gonadectomized male mice compared to sham-operated female mice and increased in gonadectomized male mice compared to sham-operated male mice. IL-13+ ILC2 were minimal with rmIL-33 administration and were not quantified. We also determined IL-33 in whole lung homogenates. After rmIL-33 administration, sham-operated male mice and gonadectomized female mice had decreased whole lung IL-33 compared to sham-operated female mice and gonadectomized male mice (Figure S8C). These results suggest that sex hormones are important in regulating IL-33 production, degradation, and/or oxidation as well as cytokine producing ILC2.

Testosterone negatively regulated ILC2 via a cell-intrinsic mechanism

To determine if testosterone directly attenuated ILC2 cytokine expression, we utilized a mixed bone marrow chimera model whereby lethally irradiated WT CD90.1+ CD90.2+ male mice were reconstituted with a 1:1 mixture of CD90.1+ WT and CD90.2+ androgen receptortfm (ARtfm) bone marrow. ARtfm mice have a mutation in the AR and are unresponsive to androgens, including testosterone and testosterone derivatives. ARtfm mice are on a C57/BL6 background, and we determined that WT C57/BL6 male mice have significantly decreased lung ILC2 (3133 ± 1362 lung ILC2, n=4, p<0.05) compared to WT B57/BL6 female mice (12,140 ± 3181 lung ILC2, n=4). Six weeks after transplant, recipient mice were challenged with 4 intranasal daily doses of Alt Ext. Twenty-four hours after the final challenge, lungs from the recipient mice were harvested and the number of WT and ARtfm IL-5+ and/or IL-13+ ILC2 were determined. The number of IL-5+ and/or IL-13+ ILC2 from the ARtfm lineage was significantly increased in both the PBS and Alt Ext treated groups compared to the number of ILC2 from the WT lineage (Figure 6B–C). Further, CD25+ ILC2 were significantly increased in the Alt Ext-treated ARtfm ILC2 compared to Alt Ext-treated WT ILC2, but CD25 MFI was similar between groups (Figure 6D–F). Combined, these data show that androgen receptor signaling decreases ILC2 numbers in the lung at baseline and that upon allergen challenge with Alt Ext that androgen receptor signaling further represses ILC2 numbers and decreases CD25 expressing cells.

Figure 6. Testosterone intrinsically attenuated Alt Ext-induced lung ILC2.

Figure 6

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(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–C) Representative gating strategy and quantification of the total number of IL-5 and IL-13+ ILC2 from WT (CD90.1) and ARtfm (CD90.2) mice. (D–F). Histogram and quantification of CD25+ ILC2 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, n.s., not significant, ANOVA with Tukey post-hoc analysis.

Discussion

Sex hormones mediate cellular and humoral immune pathways and may partially explain the increased prevalence of autoimmune diseases and inflammation in women compared to men (Fish, 2008). Asthma prevalence increases in females at the time of puberty, and epidemiological studies have associated puberty, measured by increasing Tanner stages, with decreased asthma symptoms in boys and increased asthma symptoms in girls (Fu et al., 2014). Several studies in women showed changes in hormone levels, either during the menstrual cycle or in taking oral contraceptives, modulated asthma symptoms (Fuseini and Newcomb, 2017). However, the mechanisms by which sex hormones regulate asthma are not fully elucidated.

Mouse studies showed that ILC2 are important for establishing and maintaining allergic responses, including allergic airway inflammation associated with asthma. Previously, ex vivo IL-33 stimulated ILC2 from OVA-challenged male mice had decreased IL-5 and IL-13 production compared to ILC2 from OVA-challenged female mice (Warren et al., 2017). Further, androgen signaling has also shown to negatively regulate ILC2 cytokine production and ILC2 numbers in the lung after IL-33 administration (Laffont et al., 2017). In this study, we expand upon these findings and determined that ILC2 circulating cells in men with asthma are decreased compared to women with asthma, despite having similar baseline characteristics. Further, testosterone and androgen signaling attenuated IL-2R-mediated ILC proliferation, ILC2 numbers in the lung, and ILC2-mediated airway inflammation. Combined, these studies provide a foundational understanding in the sexual dimorphism in ILC2 function, and one potential explanation for increased prevalence of asthma in women compared to men.

IL-2 signaling through the IL-2R are important for ILC proliferation and T-cell activation and proliferation (Serafini et al., 2015). It has previously been reported that soluble CD25 and IL-2 are increased in the BAL fluid of patients with asthma compared to healthy controls (Park et al., 1994). A randomized, double-blinded, placebo controlled clinical trial was conducted on daclizumab, a CD25 monoclonal antibody, in patients with moderate to severe asthma that do not respond to inhaled corticosteroids. Patients administered daclizumab had increased %FEV1 and decreased daytime asthma symptoms with no significant differences in adverse side effect patients compared to placebo (Busse et al., 2008). Our data showed that testosterone and androgen signaling negatively regulated CD25 expression on ILC2 in vitro, and that Alt Ext challenge decreased the number of CD25+ ILC2, but had no effect on CD25 MFI, in lungs of mice with intact testosterone and androgen receptor signaling. CD25 MFI was decreased on sorted ILCs from male mice compared to female mice (Figure 2). The discordant findings between in vitro and in vivo CD25 MFI expression are likely due to culturing and/or kinetic differences. Nevertheless, testosterone and androgen signaling decreased CD25+ ILC2 both in vitro and after Alt Ext challenge as well as regulated IL-2R mediated proliferation, suggesting that targeting IL-2R signaling may particularly benefit women with moderate to severe allergic asthma (Busse et al., 2008; Laffont et al., 2017).

The roles of estrogen signaling and 5α -DHT in allergic airway inflammation have been conducted in mouse models with both CD4+ Th2-mediated and ILC2-mediated inflammation (Card et al., 2006; Carey et al., 2007b; Laffont et al., 2017). Our study is unique in using a 4 day allergic airway inflammation model to specifically study the role of innate immune cells on airway inflammation. Our data showed that 5α-DHT decreased ILC2 cytokine expression and Rorα mRNA expression in mice, but had no effect on Gata3 mRNA expression. It is currently unclear if androgen receptor signaling transcriptionally or post-transcriptionally regulates ILC2 proliferation and cytokine production. Since Rorα mRNA expression was decreased with the addition of 5α-DHT, these data suggest a transcriptional regulation of testosterone on ILC2 function. However, additional studies are needed to determine if AR signaling is transcriptionally or post-transcriptionally regulating ILC2function.

While 5α-DHT had no effect on Gata3 mRNA expression in ILC2 from mice, ILC2 from men with asthma as well as IL-2 plus IL-33 stimulated ILC2 from male mice had decreased Gata3 expression compared to ILC2 from women with asthma or female mice. Further, Gata3 expression was decreased in ILC2 from male mice compared to female mice. ILC2 from gonadectomized male mice administered 17β-E2 and P4 administration increased Gata3 expression compared to ILC2 from gonadectomized male mice administered vehicle, suggesting that estrogen and progesterone are important in upregulating Gata3 expression in ILC2. These results show that 5α-DHT is important in regulating proliferation and cytokine expression, but that estrogen and progesterone are important in regulating Gata3 expression.

We confirmed our in vitro findings in a physiologically relevant Alt Ext challenge in vivo model of innate-immune mediated allergic airway inflammation. Testosterone negatively regulated allergic airway inflammation by intrinsically decreasing ILC2 numbers and IL-5+ or IL-13+ ILC2 cells, as shown in the mixed bone marrow chimera experiments. Further, testosterone extrinsically regulated IL-5+ ILC2 and IL-13+ ILC2 by decreasing IL-33 and TSLP expression in the BAL fluid (Figure 5 and S6). IL-33 signaling through ST2 and TSLP signaling through the TSLP receptor stimulate Alt Ext-induced ILC2 airway inflammation. Therefore, combined these results show that testosterone has a dual mechanisms for regulating ILC2 production of IL-5 and IL-13, by acting directly on the ILC2 as well as reducing the release of IL-33 and TSLP.

Despite testosterone negatively regulating Alt Ext-induced IL-13+ ILC2, testosterone did not decrease mucus production. Surprisingly, ovarian hormones increased Alt Ext-induced mucus production even though ovarian hormones did not increase IL-13+ ILC2. Previous reports have shown that estrogen, signaling through estrogen receptor-β, increased MUC5AC expression and mucus production in cultured airway epithelial cells (Tam et al., 2014), suggesting that sex hormones may regulate mucus production through multiple pathways. Our studies did not determine the mechanisms by which ovarian hormones regulated ILC2-induced airway inflammation nor take into account other cell types, including T cells, macrophages, and dendritic cells that sex hormones are known to regulate (Fuseini and Newcomb, 2017). However, our results did show that ovarian hormones increased Alt Ext-induced IL-5+ ILC2, eosinophils in the BAL, and mucus production. Combined, these findings show that ovarian hormones and testosterone have differential pathways of regulation, and that further analysis of how ovarian hormones regulate cytokine producing ILC2 in vivo is warranted.

ILC2 produce approximately 10 fold more IL-5 and IL-13 compared to CD4+ Th2 cells, and ILCs are critical for Th2 memory responses in the lung (Halim et al., 2014). ILC2 therapeutic targets are actively being developed or ongoing in clinical trials (Gauvreau et al., 2014). Therefore, defining the role of sex hormones on ILC2-mediated airway inflammation is imperative to effectively design future clinical trials and develop new therapeutic strategies for asthma and other ILC-mediated diseases.

Experimental Procedures

Analysis of circulating ILC2 in men and women with asthma and healthy controls

Healthy women and men as well as patients with moderate to severe asthma (ages 18–45), as defined by the Severe Asthma Research Program and the NHLBI guidelines, were recruited from the Vanderbilt University Medical Center. Participants were excluded for any viral or bacterial symptoms 2 weeks prior to blood draw. Women were also excluded if pregnant, breastfeeding, taking hormonal contraceptive medications, taking estrogen replacement therapy, menopausal, or if they had undergone an oophorectomy or hysterectomy. All participants were consented in accordance with Vanderbilt University's Institutional Review Board policies. PBMCs were isolated from the blood. The total volume of blood and the total number of PBMCs were recorded for each patient. Lineage negative cells were enriched using StemCell Technologies Human ILC2 Enrichment Kit. Fifty percent of the Lin- cells were surface stained with FITC anti-human Lineage cocktail (Biolegend), FITC anti-CD123 (clone 7G3), FITC anti- FcεRI (clone AER-37(CRA1), redFluor 710 anti-CD45 (clone HI30), PE-Cy7 anti-CD127 (clone A019D5), PerCP-Cy5.5 anti-CD161 (clone HP-3G10) and BV421 anti-CD25 (clone M-A251) and Alexa Fluor 647 anti-CD294 (clone BM16) antibodies. Cells were fixed and permeabolized using Foxp3 / Transcription Factor Staining Buffer Kit (Tonbo), and then intracellularly stained with PE anti-Gata3 (clone TWAJ). The remaining 50% of cells were restimulated with 50ng/ml PMA (Sigma-Aldrich), 1µg/ml ionomycin (Sigma-Aldrich), and 0.07% golgi stop (BD Biosciences) for 4 hours at 37°C in RPMI + 10% FBS. Restimulated Lin- cells were then surface stained, fixed, and permeabolized as described above. Cells were then intracellularly stained for PE- anti-IL-5 (clone JES1-39D10). Flow cytometry was conducted and ILC2 were defined as Lin-, CD45+, CD127+, CD161+, CD25+, CRTH2+, GATA3+ or IL-5+ cells. All data was analyzed via FlowJo software.

Mice

WT BALB/c mice were purchased from Charles River Laboratories or bred in house. Veterinary staff at Charles River Laboratories performed sham or gonadectomy surgeries on 3–4 week mice. ARtfm (B6.Cg-Aw-J EdaTa-6J +/+ ArTfm/J), congenic CD90.1+ B6 Thy1.1 (B6.PL-Thy1a/CyJ), and WT C57BL/6 mice were obtained from the Jackson Laboratory. Heterozygous CD90.1+ CD90.2+ C57BL/6 mice were generated from breeding C57BL/6 mice with B6.PL-Thy1a/CyJ mice. For experiments, adult mice were greater than 8 weeks old and pre-pubescent mice were 4 weeks old. Experiments were approved by the Institutional Animal Care and Use Committee at Vanderbilt University and conducted according to the Care and Use of Laboratory Animals guidelines.

Sorting and stimulation of lung ILC2 from mice

Lungs were extracted, homogenized, and digested into a single cell suspension and counted. Lineage negative (Lin-) cells were enriched by negative selection as previously described (Zhou et al., 2016). Lin- cocktail contained biotinylated antibodies against CD3, CD5, CD11b, CD45R, 7-4, Gr-1, and Ter-119. Enriched Lin - cells were blocked with an anti-FcR antibody (clone 2.4G2) and surface stained with lineage cell detection cocktail (Miltenyi), biotin-labelled anti-CD3 (clone 17A2), APC anti-CD90 (clone 53-2.1), Alexa Fluor 700 anti-CD45 (clone 30-F11), APC anti-CD25 (clone PC61) and PE-Cy7 anti-CD127 (clone A7R34) antibodies. FITC conjugated to streptavidin (1:1500 dilution) was added following surface staining. ILCs, defined as Lin-, CD45+, CD90+, CD127+, CD25+ cells, were sorted by flow cytometry. All antibodies were purchased from BD Biosciences, Biolegend, or eBiosciences unless otherwise stated. Sorted lung ILCs from mice were plated at 2000 cells/well in 96 well round bottom plates and stimulated with rmIL-2 (10ng/ml) and rmIL-33 (10ng/ml) or rmIL-7 (10ng/ml) and rmIL-33 (10ng/ml) for 1–6 days. All recombinant proteins were purchased from Peprotech. In select experiments, vehicle (methanol) or testosterone (0.1–3nM, Sigma-Aldrich) was added at the time of stimulation.

Measuring ILC2 proliferation and STAT5 phosphorylation

CellTrace™ Violet Cell Proliferation Kit was added to ILC2 at the time of stimulation per manufacturer’s instructions (Life Technologies). After 3 days in culture, ILC2 were stained with a viability dye (Tonbo), blocked with an anti-FcR antibody (clone 2.4G2), and then surface stained with biotinylated lineage cell detection cocktail, biotinylated anti-CD3 (clone 17A2), Alexa Fluor 488 anti-CD25 (clone PC61), PE-Cy5 anti-CD127 (clone A7R34) antibodies, PerCp efluor 710 anti-ST2 (clone RMST2-2), and PE-Cy7 anti-ICOS (clone C98.4A) followed by APC-Cy7 strepavidin staining. CellTrace Violet was measured in ILC2, defined as Lin-, CD127+, CD25+, ST2+, ICOS+ cells. For Ki67 staining, ILC2 were harvested after 2 days of stimulation, surface stained as described above, fixed and permeabolized using Foxp3 / Transcription Factor Staining Buffer Kit (Tonbo), and intracellularly stained with PE anti-Gata3 (clone TWAJ), and eFluor 450 anti-Ki67 (clone SolA15). For STAT5 phosphorylation studies, ILC2 were stimulated for 1 day, harvested, and stained for surface markers as described above. ILC2 were then fixed with 4% paraformaldehyde for 10 minutes at room temperature, permeabolized with methanol overnight at 4°C, and intracellularly stained with PE anti-STAT5 (clone 47/Stat5(pY694)). STAT5 phosphorylation was then measured in ILC2 by flow cytometry. Just prior to flow cytometry analysis, count beads (Invitrogen) were added in per manufacturer’s protocol to determine total ILC2 number. Flow analysis was conducted on LSRII flow cytometer, and all data was processed using FlowJo software.

Quantification of IL-5+ and IL-13+ ILC2 by flow cytometry

After 6 days in culture, ILC2 were collected and restimulated with 50ng/ml PMA (Sigma-Aldrich), 1µg/ml ionomycin (Sigma-Aldrich), and 0.07% golgi stop (BD Biosciences) for 4 hours at 37°C. ILC2 were then surface stained, fixed and permeabolized using methods described in above section. ILC2 were then intracellularly stained with PE anti-gata3 (clone TWAJ), APC anti-IL-5 (clone TRFK5) and eFluor 450 anti-IL-13 (clone eBio13A). Just prior to flow cytometry analysis, count beads (Invitrogen) were added in per manufacturer’s protocol to determine total ILC2 number.

Cytokine Measurements

Cytokine levels were determined from BAL fluid or whole lung homogenates using the Duoset and Quantikine ELISA kits (R&D Systems) following the manufacturer’s instructions. Any value below the lower limit of detection was assigned half the value of the lowest detectable standard.

Quantitative PCR

Total RNA was extracted from ILC2 using RNeasy Micro Kit and cDNA was generated using 100ng of total RNA. qPCR was conducted using TaqMan primers purchased from Applied Biosystems. Data were reported as relative expression normalized to the housekeeping gene Gapdh.

Administration of hormone pellets to mice

At 6–7 weeks of age, 60-day slow-release pellets from Innovative Research of America containing 5α-DHT(15 mg), 17β-E2 (0.1mg), P4 (25mg) or a combination of 17β-E2 and P4 (25.1mg) were subcutaneously implanted into gonadectomized male BALB/c mice (Newcomb et al., 2015). As a control, vehicle pellets were surgically placed into sham-operated male, sham-operated female, or gonadectomized male mice. Three weeks (21 days) after pellets were implanted, ILCs were harvested from the lungs of mice, sorted by flow cytometry, and stimulated with IL-33 and IL-2 as described above.

In vivo Alternaria extract challenge

Mice were anesthetized with isoflurane and challenged intranasally with 5 µg in 100µl of Alt Ext or vehicle (PBS) for 4 consecutive days (Toki et al., 2016; Zhou et al., 2016). At various time points after the last challenge, mice were sacrificed.

Analysis of IL-5+ and IL-13+ ILC2 after Alt Ext challenge protocol

Lungs were harvested, digested, and single cell suspensions were restimulated with 50ng/ml PMA, 1µg/ml ionomycin, and 0.07% golgi stop for 6 hours at 37°C in RPMI + 10% FBS. Following restimulation, lung cells were stained with the viability dye (Tonbo), blocked with an anti-FcR antibody (clone 2.4G2), and surface stained with biotin-labelled lineage cell detection antibody cocktail (Miltenyi), biotin-labelled anti-CD3 (clone 17A2), BV786 anti-CD90.2 (clone 53-2.1), Alexa fluor700 CD45 (clone 30-F11), Alexa fluor 488 anti-CD25 (clone PC61), PE-Cy5 anti-CD127 (clone A7R34) antibodies, PerCp efluor 710 anti-ST2 (clone RMST2-2), and PE-Cy7 anti-ICOS (clone C98.4A) followed by APC-Cy7 strepavidin staining (1:1500). Cells were then fixed and permeabolized as described above and intracellularly stained with APC anti-IL-5 (clone TRFK5) and eFluor 450 anti-IL-13 (clone eBio13A). Flow analysis was conducted on LSRII flow cytometer, and all data was processed using FlowJo software.

Bronchoalveolar lavage (BAL)

BAL was performed by instilling 800 µL of saline through a tracheostomy tube and then withdrawing the fluid with gentle suction through a syringe. Cells in the BAL fluid were fixed to a slide, stained, and categorized as neutrophils, eosinophils, lymphocytes, or monocytes by using standard morphologic criteria, as previously described (Newcomb et al., 2012). Supernatants from BAL were used for analysis of cytokine production.

Histopathology of lungs

Lungs were perfused, inflated, and fixed in 10% buffered formalin overnight. The lungs were transferred to 70% ethanol and then embedded in paraffin blocks. Tissue sections (5 µm) were stained with periodic acid-Schiff (PAS) to assess mucous cell metaplasia as a measure of mucin expression. Slides were examined and scored by a pathologist who was blinded to the experimental groups according to the following scale: 0, no PAS-positive cells; 1, <5% PAS-positive cells; 2, 5 to 10% PAS-positive cells; 3, 10 to 25% PAS-positive cells; and 4, > 25% PAS-positive cells.

Bone marrow chimeras

Six- to 10-week old heterozygous CD90.1+ CD90.2+ C57BL/6 male recipient mice were lethally irradiated (11 Gy) using a cesium irradiator. Two to three hours following irradiation, a 1:1 mixture of whole bone marrow from age matched 6–10 week old WT (CD90.1+) C57BL/6 male mice or ARtfm male mice (CD90.2+) was transplanted into male CD90.1+ CD90.2+ C57BL/6 recipient mice via retro-orbital injection. After 6 weeks of bone marrow engraftment, recipient mice were challenged intranasally with 5 µg in 100µl of Alt Ext or vehicle (PBS) for 4 consecutive days. Twenty-four hours after last intranasal challenge, lungs were harvested and restimulated as described above section. Following restimulation, lung cells were stained with the viability dye (Tonbo), blocked with an anti-FcR antibody (clone 2.4G2), and surface stained with biotin-labelled lineage cell detection antibody cocktail (Miltenyi), biotin-labelled anti-CD3 (clone 17A2), BV786 anti-CD90.2, BV510 anti-CD90.1, Alexa Fluor 488 anti-CD25, PE-Cy5 anti-CD127, PerCp efluor 710 anti-ST2, and PE-Cy7 anti-ICOS, followed by APC-Cy7 strepavidin staining (1:1500). Cells were then fixed and permeabolized as described above and intracellularly stained with APC anti-IL-5 and eFluor 450 anti-IL-13. Flow analysis was conducted on LSRII flow cytometer and ARtfm (CD90.2) and WT (CD90.1) cytokine producing ILC2 were gated as Lin-, CD45+, CD127+, CD25+, ST2+, ICOS+, IL-5 and/or IL-13+ cells. All data was processed using FlowJo software. Residual cells remaining from the recipient mice (CD90.1+ CD90.2+) were excluded from analysis.

Statistical analysis

The p values were calculated by using unpaired Kruskall-Wallis test, Mann-Whitney U test, Student’s t-test, or one-way ANOVA with Tukey post hoc test. Values of p < 0.05 were considered significant.

Supplementary Material

supplement

Highlights.

  • Women with asthma have increased circulating ILC2 compared to men with asthma

  • Sex hormones regulated IL-2 dependent ILC2 proliferation and cytokine expression

  • Testosterone intrinsically and extrinsically attenuated ILC2 airway inflammation

Acknowledgments

This work was supported by National Institute of Health and the Veteran Affairs: R01 HL122554 (DCN), R21 AI121420 (DCN), R01 HL122554S1 (DCN), R01 U19AI095227 (RSP), Veteran Affairs (1I01BX000624) (RSP).

Abbreviations

5α-DHT

5-alpha dihydrotestosterone

17β-E2

17β-estradiol

Alt Ext

Alternaria extract

BAL

Bronchoalveolar lavage

CLPs

common lymphoid progenitor cells

E2

estradiol

Id2

inhibitor of DNA binding

ILC

innate lymphoid cell

Lin-

lineage negative

MFI

mean fluorescence intensity

n.s.

not significant

PAS

periodic acid-Schiff

PBS

phosphate-buffered saline

PBMCs

peripheral blood mononuclear cells

PMA

phorbol 12-myristate 13-acetate

P4

progesterone

R

receptor

Rag

recombination activating gene

veh

vehicle

WT

wild type.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Supplemental Information:

Supplemental information includes supplemental experimental procedures, one table, and eight figures.

Author contributions:

JYC and MTS designed and performed the experiments, analyzed and interpreted the data, and edited the manuscript; HF, ST, MTB, WZ and RSP contributed to the experimental design, interpretation of data, edited manuscript, and provided scientific discussion; SLG recruited and consented asthma patients, VP and KLB processed and scored histological slides; RGH conducted human IgE serum concentrations; RSP contributed to interpretation of data, edited manuscript, provided scientific discussion, and reagents; DCN conceived the study, designed and performed the experiments, analyzed and interpreted the data, and wrote the manuscript.

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