As adults, women are twice as likely as men to have asthma; however, the mechanisms explaining this sexual dimorphism remain unclear. Increased type 2 cytokines and/or IL-17A, leading to increased airway eosinophils and neutrophils, respectively, are associated with asthma. Previous studies showed that testosterone, signaling through the androgen receptor (AR), decreased Th2-mediated allergic inflammation and type 2 innate immune responses during allergic inflammation. Therefore, we hypothesized that testosterone and AR signaling attenuate type 2 and IL-17A–mediated airway inflammation. To test our hypothesis, sham-operated and gonadectomized female and male mice were intranasally challenged with house dust mite (HDM) or vehicle (PBS) for 3 wk. Testosterone decreased and ovarian hormones increased HDM-induced eosinophilic and neutrophilic inflammation, IgE production, and airway hyperresponsiveness, as well as decreased the numbers of IL-13+ CD4 Th2 cells and IL-17A+ CD4 Th17 cells in the lung. Next, using wild-type male and female mice and ARtfm male mice that are unable to signal through the AR, we determined AR signaling intrinsically attenuated IL-17A+ Th17 cells but indirectly decreased IL-13+ CD4 Th2 cells in the lung by suppressing HDM-induced IL-4 production. In vitro Th2 and Th17 differentiation experiments showed AR signaling had no direct effect on Th2 cell differentiation but decreased IL-17A protein expression and IL-23R mRNA relative expression from Th17 cells. Combined, these findings show AR signaling attenuated type 2 and IL-17A inflammation through different mechanisms and provide a potential explanation for the increased prevalence of asthma in women compared with men.

A sexual dimorphism exits in asthma, and as adults, women have an increased prevalence of asthma compared with men. Epidemiological and clinical studies showed women with asthma had increased exacerbations, decreased forced expiratory volume in 1 s (FEV1) after bronchodilator use, and increased eosinophils and neutrophils in the bronchoalveolar lavage (BAL) fluid and sputum compared with men with asthma (13). Premenstrual worsening of asthma symptoms and changes in asthma control during pregnancy were also reported by women (49). Combined, these findings suggested that sex hormones are important in regulating mechanisms that drive airway inflammation in asthma.

Asthma is a heterogeneous disease that is regulated by different pathophysiologies. Increased eosinophils and neutrophils are found in the sputum of patients with more severe phenotypes of asthma (1013). Type 2 inflammation is mediated by increased secretion of IL-4, IL-5, and IL-13 from CD4+ Th2 cells, group 2 innate lymphoid cells (ILC2), and other cells (14, 15), resulting in increased airway eosinophils, mucus production, IgE production, and airway hyperresponsiveness (AHR) (14, 16, 17). Increased secretion of IL-17A, a cytokine secreted by CD4+ Th17 cells, γδ T cells, group 3 innate lymphoid cells (ILC3), and other cell types, leads to increased airway neutrophils and mucus production (18, 19). However, it remains unclear how sex hormones regulate inflammatory pathways that increase airway eosinophils and neutrophils.

Mouse models of allergic, type 2–mediated airway inflammation showed that testosterone decreased eosinophils and lymphocyte infiltration of the airways (20, 21), and ovarian hormones (estrogen and progesterone) increased IL-5 protein expression, airway eosinophils, AHR, and total serum IgE levels (22, 23). Recently, our group and others showed testosterone signaling through the androgen receptor (AR) attenuated both Alternaria alternata extract and house dust mite (HDM)–induced, ILC2–mediated airway inflammation (24, 25). Our group has also showed that ovarian hormones increased Th17 cell differentiation and IL-17A production through an IL-23R–dependent mechanism in mice and humans as well as increasing neutrophilic airway inflammation in mice (26). However, how testosterone regulates dual type 2– and IL-17A–mediated airway inflammation remains unknown. We hypothesized that testosterone signaling through AR attenuates type 2– and IL-17A–mediated airway inflammation. Our results showed that testosterone decreased HDM-induced total numbers of IL-13+ Th2 and IL-17A+ Th17 cells in the lung and that AR signaling directly attenuated total numbers of IL-17A+ Th17 cells in the lung and indirectly attenuated total numbers of IL-13+ Th2 cells by decreasing HDM-induced IL-4 production in the lung.

Wild-type (WT) BALB/cJ 6- to 8-wk-old female and male were purchased from Charles River Laboratories (Wilmington, MA). WT female, WT male, and AR testicular feminized (ARtfm) male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME), and breeding colonies were established at Vanderbilt University Medical Center. CD90.1/CD90.2 mice were bred in-house using homozygous CD90.1 and CD90.2 C57BL/6J mice from Jackson Laboratory. Gonadectomy or sham surgeries were conducted at 3–4 wk of age by Charles River laboratories veterinary staff, and experiments were started when gonadectomized or sham-operated mice were 6–8 wk old. All animal experiments were conducted in adherence to the rules and regulations of the Association for Assessment and Accreditation of Laboratory Animal Care and were approved by the International Animal Care and Use Committee at Vanderbilt University Medical Center.

Mice were intranasally administered 40 μg of HDM from Greer Laboratories (Lenoir, NC) or PBS as vehicle control in a 100-μl total volume for four times a week for 3 wk as shown in Fig. 1A.

BAL was performed by instilling 800 μl of saline solution through a tracheostomy tube and then withdrawing the fluid with gentle suction through a syringe, as previously described (27). The total cell count in the BAL fluid was counted using a hemocytometer and 0.04% trypan blue exclusion dye (Sigma-Aldrich). The cells from the BAL were then fixed to a slide and stained using the Three-Step Stain system (Richard-Allan Scientific, Waltham, MA). Two hundred BAL cells were classified as eosinophils, neutrophils, lymphocytes, or macrophages using standard morphologic criteria, and percentages of these inflammatory cells were determined. Total numbers of inflammatory cells were determined by multiplying the percentage of the inflammatory cells by the total numbers of viable cells in the BAL fluid.

Cytokine levels were measured from BAL fluid, lung homogenates, and/or Th0, Th2, and Th17 cell culture supernatants by ELISA using Quantikine and Duoset kits (R&D Systems). Total serum IgE levels were determined using an ELISA (BioLegend). All experiments were performed according to the manufacturer’s instructions. Any OD450 value less than the lower limit of detection was assigned half the value of the lowest detectable standard.

Mice were anesthetized with pentobarbital sodium (85 mg/kg), and an 18-gauge tracheostomy tube was placed in the trachea of the mice. Mice were then mechanically ventilated using the SCIREQ flexiVent machine with 150 breaths/min and a tidal volume of 10 ml/kg body weight. Airway resistance was determined at baseline and after administration of increasing doses of nebulized acetyl-β-methacholine (0–50 mg/ml) as previously described (28).

Lungs were harvested and digested, and lung single-cell suspensions were restimulated with 50 ng/ml of PMA (Sigma-Aldrich), 1 μg/ml of ionomcyin (Sigma-Aldrich), and 0.07% GolgiStop (BD Biosciences) for 5 h at 37°C in RPMI 1640 plus 10% FBS. Following restimulation, cells were stained with viability dye (Ghost Dye UV 450; Tonbo Biosciences), blocked with an anti-FcR Ab (clone 2.4G2), and surface stained with biotin-labeled anti-CD3 (clone 17A2), PE-Cy5 anti-CD4 (clone 129.19), BV786 anti-CD90.2 (clone 53-2.1), Alexa Fluor 700 anti-CD45 (clone 30-F11), Alexa Fluor 488 anti-CD25 (clone OC61), PE-Cy7 anti-CD127 (clone A7R34), and FITC anti-γδTCR (clone GL3) Abs, followed by allophycocyanin-Cy7 streptavidin staining (1:250). Cells were then fixed, permeabilized using the Foxp3/transcription factor staining kit (Tonbo Biosciences), and intracellularly stained with PE–IL-13 (clone eBio13A), PE-Cy7 anti–IL-17A (clone eBio17B7), and/or PE–IL-4 (clone 11B11). Flow cytometry analysis was conducted on LSR II flow cytometer, and all data were processed using FlowJo software version 10.

C57BL/6J heterozygous CD90.1+ CD90.2+ male recipient mice (6–10 wk old) were lethally irradiated (11 Gy) using a cesium irradiator. Three hours after irradiation, a 1:1 mixture of bone marrow (BM) from age-matched 6- to 10-wk-old WT (CD90.1+) and ARtfm male mice (CD90.2+) was transplanted in the male recipient CD90.1+ CD90.2+ mice via retro-orbital injection. Six weeks later, recipient mice were challenged intranasally with HDM or PBS. Twenty-four hours after the last challenge, lungs were harvested, digested, and restimulated as described in the above section. Following restimulation, cells were stained with viability dye (Ghost Dye UV 450; Tonbo Biosciences), blocked with an anti-FcR Ab (clone 2.4G2), and surface stained with biotin-labeled anti-CD3 (clone 17A2), PE-Cy5 anti-CD4 (clone 129.19), FITC anti-CD90.1 (clone HIS51), BV786 anti-CD90.2 (clone 53-2.1), Alexa Fluor 700 anti-CD45 (clone 30-F11), and Alexa Fluor 488 anti-CD25 (clone OC61) Abs, followed by allophycocyanin-Cy7 streptavidin staining (1:250). Cells were then fixed, permeabilized, and intracellularly stained with PE–IL-13 (clone eBio13A) and PE-Cy7 anti–IL-17A (clone eBio17B7). Flow cytometry analysis was conducted on LSR II flow cytometer, and ARtfm (CD90.2) and WT (CD90.1) cytokine-producing Th2 and Th17 cells were gated as CD3+CD4+ IL-13+ or CD3+ CD4+ IL-17A+ cells. All data were processed using FlowJo software version 10. Residual cells remaining from the heterozygous recipient mice (CD90.1+/CD90.2+) were excluded from the analysis.

Naive CD4 T cells were isolated from the spleens of WT C57BL/6J female, male, and ARtfm male mice using a commercially available naive CD4+ T cell isolation kit (Miltenyi Biotec). Following isolation, naive CD4+ T cells were activated with anti-CD3 (1 μg/ml; BD Biosciences) and anti-CD28 (0.5 μg/ml; BD Biosciences) and differentiated into Th2 cells by adding recombinant mouse (rm) IL-4 (10 ng/ml) and anti–IFN-γ (10 μg/ml). Naive CD4+ T cells were differentiated into Th17 cells by adding rmIL-23 (10 ng/ml), recombinant human TGF-β (1 ng/ml), rmIL-6 (20 ng/ml), anti–IFN-γ (10 μg/ml) and anti–IL-4 (10 μg/ml). Recombinant IL-23 protein, anti–IFN-γ, and anti–IL-4 Abs were purchased from R&D Systems. All other recombinant proteins were purchased from PeproTech (Rocky Hill, NJ).

Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was generated by using 100 ng of total RNA, and TaqMan quantitative PCR analysis of AR, Gata3, Rorc, Il4ra, Il23r, and Gapdh mRNA expression was conducted using commercially available primers and FAM/MGB probes (Applied Biosystems). Data were reported as relative expression normalized to the housekeeping gene Gapdh.

Unless otherwise indicated, data are represented as mean ± SEM where groups were compared by one-way ANOVA with Tukey post hoc analysis. For AHR experiments, ANOVA of repeated measures and Bonferroni post hoc analysis were used to calculate values. For all analysis, p < 0.05 was considered significant.

Type 2 and IL-17A inflammatory pathways are important in regulating airway eosinophils and neutrophils, respectively, in asthma. Because asthma prevalence is increased in women compared with men, we hypothesized that allergen-induced airway inflammation is increased in female mice compared with male mice. To test this hypothesis, male and female WT BALB/c mice were intranasally challenged four times per week for 3 wk with 40 μg of HDM or vehicle (PBS) (Fig. 1A). Twenty-four hours after the last challenge, on day 18, lung and BAL fluid were collected to measure cytokine expression and infiltration of inflammatory cells into the BAL fluid (26). In male and female mice, HDM significantly increased whole lung homogenate IL-13 and IL-17A protein expression as well as significantly increased total BAL inflammatory cells, eosinophils, neutrophils, macrophages, and lymphocytes compared with PBS-challenged mice (Fig. 1B–H). However, male mice had significantly decreased HDM-induced lung IL-13 and IL-17A protein expression and total BAL cell numbers, eosinophils, neutrophils, macrophages, and lymphocytes compared with HDM-challenged female mice (Fig. 1B–H). These data showed female mice had increased HDM-induced IL-13 and IL-17A protein expression and BAL inflammatory cells compared with male mice.

FIGURE 1.

HDM-induced BAL eosinophils and neutrophils were decreased in male compared with female mice. (A) Experimental design for HDM-induced airway inflammation. (B and C) IL-13 and IL-17A protein expression in the lung homogenates as measured by ELISA. (DH) Total BAL cells and total BAL eosinophils, neutrophils, macrophages, and lymphocytes. Data pooled from two independent experiments. *p < 0.05, one-way ANOVA with Tukey post hoc analysis (n = 6–9 mice per group).

FIGURE 1.

HDM-induced BAL eosinophils and neutrophils were decreased in male compared with female mice. (A) Experimental design for HDM-induced airway inflammation. (B and C) IL-13 and IL-17A protein expression in the lung homogenates as measured by ELISA. (DH) Total BAL cells and total BAL eosinophils, neutrophils, macrophages, and lymphocytes. Data pooled from two independent experiments. *p < 0.05, one-way ANOVA with Tukey post hoc analysis (n = 6–9 mice per group).

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Because there was a gender difference in HDM-induced airway eosinophils and neutrophils, we hypothesized that endogenous sex hormones were important in regulating eosinophilic and neutrophilic-mediated airway inflammation. To test our hypothesis, male and female WT BALB/c mice underwent gonadectomy or sham operation at 3–4 wk of age. This age is prior to sexual maturity and abundant production of sex hormones. When the mice were 6–8 wk old, the mice underwent the HDM or PBS challenge protocol. Twenty-four hours after the last challenge, BAL eosinophils and neutrophils were significantly increased in HDM-challenged sham-operated female mice and gonadectomized male mice compared with sham-operated male and gonadectomized female mice (Fig. 2A, 2B).

FIGURE 2.

Testosterone decreased and ovarian hormones increased HDM-induced airway eosinophilic and neutrophilic inflammation, IgE production, and AHR. WT gonadectomized and sham-operated male and female BALB/c mice were challenged with HDM or PBS. (A and B) Total BAL eosinophils and neutrophils. (C) Total serum IgE levels. (A–C) Representative sample of three independent experiments. *p < 0.05, one-way ANOVA with Tukey post hoc analysis (n = 3–7 mice per group). (D) AHR in response to increasing concentrations of methacholine 24 h after the last HDM challenge. Data pooled from two independent experiments. *p < 0.05 ANOVA of repeated measures with Bonferroni post hoc analysis. (n = 12–14 per group).

FIGURE 2.

Testosterone decreased and ovarian hormones increased HDM-induced airway eosinophilic and neutrophilic inflammation, IgE production, and AHR. WT gonadectomized and sham-operated male and female BALB/c mice were challenged with HDM or PBS. (A and B) Total BAL eosinophils and neutrophils. (C) Total serum IgE levels. (A–C) Representative sample of three independent experiments. *p < 0.05, one-way ANOVA with Tukey post hoc analysis (n = 3–7 mice per group). (D) AHR in response to increasing concentrations of methacholine 24 h after the last HDM challenge. Data pooled from two independent experiments. *p < 0.05 ANOVA of repeated measures with Bonferroni post hoc analysis. (n = 12–14 per group).

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HDM has been shown to increase IgE serum levels, AHR, and mucus production (29, 30). Thus, we wanted to determine how testosterone and ovarian hormones regulated these hallmarks of asthma. We measured total serum IgE levels by ELISA and AHR to increasing concentration of methacholine challenge using the flexiVent plethysmography system. Total serum IgE levels were increased in HDM-challenged sham-operated female mice and gonadectomized male mice compared with sham-operated male mice and gonadectomized female mice (Fig. 2C). HDM also significantly increased methacholine-induced AHR in the lungs of HDM-challenged mice from all groups (Fig. 2D), but no increase in AHR was seen with PBS-challenged mice (Supplemental Fig. 1A). Furthermore, HDM-induced AHR was increased in sham-operated female mice and gonadectomized male mice compared with sham-operated male mice and gonadectomized female mice (Fig. 2D). Forty-eight hours after the last challenge, when mucus production levels are upregulated (31), lungs were harvested, and mucus cell metaplasia was determined by periodic acid–Schiff staining. HDM-challenged mice in all groups had significantly increased mucus production compared with PBS-challenged mice, but no significant differences in periodic acid–Schiff staining were detected between HDM-challenged experimental groups (Supplemental Fig. 1B, 1C). Combined, these results showed that HDM-induced eosinophilic and neutrophilic inflammation, IgE production, and AHR were decreased by testosterone and increased by ovarian hormones.

IL-13 and IL-17A are increased with HDM exposure, and we next determined if sex hormones regulated IL-13– and IL-17A–producing CD45+ cells. On day 18, IL-13– and IL-17A–producing CD45+ cells were determined by flow cytometry using the gating strategy detailed in Supplemental Fig. 2. The total number, but not frequency, of IL-13+ CD45+ cells was increased in HDM-challenged sham-operated female mice and gonadectomized male mice compared with the HDM-challenged sham-operated male mice and gonadectomized female mice (Fig. 3B, 3C). Furthermore, the total number of IL-17A+ CD45+ cells was increased in HDM-challenged sham-operated female mice and gonadectomized male mice compared with the HDM-challenged sham-operated male mice and gonadectomized female mice (Fig. 3E). The frequency of IL-17A+ CD45+ was also increased in the HDM-challenged sham-operated female mice compared gonadectomized female mice, but the frequency of IL-17A+ CD45+ was similar in sham-operated and gonadectomized male mice (Fig. 3D). We did not detect a substantial population of dual IL-13– and IL-17A–producing cells in our model.

FIGURE 3.

Testosterone decreased and ovarian hormones increased HDM-induced total numbers of IL-13– and IL-17A–producing CD45+ cells. WT gonadectomized and sham-operated male and female BALB/c mice were challenged with HDM or PBS. Lungs were harvested 24 h after the last challenge, and lung cells were restimulated with PMA, ionomycin, and Golgi stop. (A) Representative dot plots of viable CD45+ IL-13+ or IL-17A+ cells. (B and C) Percentage and total numbers of IL-13+ CD45+ cells. (D and E) Percentage and total numbers of IL-17A+ CD45+ cells. Data pooled from two independent experiments. *p < 0.05, one-way ANOVA with Tukey post hoc analysis (n = 6–10 mice per group).

FIGURE 3.

Testosterone decreased and ovarian hormones increased HDM-induced total numbers of IL-13– and IL-17A–producing CD45+ cells. WT gonadectomized and sham-operated male and female BALB/c mice were challenged with HDM or PBS. Lungs were harvested 24 h after the last challenge, and lung cells were restimulated with PMA, ionomycin, and Golgi stop. (A) Representative dot plots of viable CD45+ IL-13+ or IL-17A+ cells. (B and C) Percentage and total numbers of IL-13+ CD45+ cells. (D and E) Percentage and total numbers of IL-17A+ CD45+ cells. Data pooled from two independent experiments. *p < 0.05, one-way ANOVA with Tukey post hoc analysis (n = 6–10 mice per group).

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Next, we determined the cell sources of CD45+ IL-13+ or IL-17A+ cells, and we found that the total number, but not the percentage, of IL-13+ Th2 cells was increased in HDM-challenged sham-operated female and gonadectomized male mice compared with sham-operated male and gonadectomized female mice (Fig. 4B, 4C). We found a significant increase in the percentage of IL-17A+ Th17 cells in the sham-operated female mice compared with the gonadectomized female mice (Fig. 4E). Furthermore, the total number of IL-17A+ Th17 cells was increased in HDM-challenged sham-operated female mice and gonadectomized male mice compared with sham-operated male mice and sham-operated female mice (Fig. 4F). Although the CD4 T cells accounted for ∼50–60% of the IL-13 and IL-17A produced in the lung, IL-13+ ILC2 and IL-17A+ γδ T cells were also increased in HDM-challenged sham-operated female mice and HDM-challenged gonadectomized male mice compared with HDM-challenged gonadectomized female and HDM-challenged sham-operated male mice (Fig. 4D, 4G). Combined, these studies showed that HDM-induced IL-13+ and IL-17A+ producing cells, predominantly Th2 and Th17 cells, were decreased by testosterone and increased by ovarian hormones and that the frequency of HDM-induced IL-17A+ Th17 cells was increased by ovarian hormones.

FIGURE 4.

Testosterone decreased and ovarian hormones increased HDM-induced total numbers of IL-13+ Th2 and IL-17A+ Th17 cells. WT gonadectomized and sham-operated male and female BALB/c mice were challenged with HDM or PBS. Lungs were harvested 24 h after the last challenge, and lung cells were restimulated with PMA, ionomycin, and Golgi stop. (A) Representative dot plots of viable, CD45+ CD3+ CD4+ IL-13+, or IL-17A+ cells. (BD) Percentage and total numbers of IL-13+ Th2 cells (defined as CD45+ CD3+ CD4+ IL-13+ cells) and IL-13+ ILC2 cells (defined as Lin CD45+ CD25+ CD90+ IL-13+ cells). (EG) Percentage and total numbers of IL-17A+ Th17 cells and IL-17A+ γδ T cells (defined as CD45+ CD3+ CD4-γδTCR+) cells. Data pooled from two independent experiments. *p < 0.05, one-way ANOVA with Tukey post hoc analysis (n = 6–10 mice per group).

FIGURE 4.

Testosterone decreased and ovarian hormones increased HDM-induced total numbers of IL-13+ Th2 and IL-17A+ Th17 cells. WT gonadectomized and sham-operated male and female BALB/c mice were challenged with HDM or PBS. Lungs were harvested 24 h after the last challenge, and lung cells were restimulated with PMA, ionomycin, and Golgi stop. (A) Representative dot plots of viable, CD45+ CD3+ CD4+ IL-13+, or IL-17A+ cells. (BD) Percentage and total numbers of IL-13+ Th2 cells (defined as CD45+ CD3+ CD4+ IL-13+ cells) and IL-13+ ILC2 cells (defined as Lin CD45+ CD25+ CD90+ IL-13+ cells). (EG) Percentage and total numbers of IL-17A+ Th17 cells and IL-17A+ γδ T cells (defined as CD45+ CD3+ CD4-γδTCR+) cells. Data pooled from two independent experiments. *p < 0.05, one-way ANOVA with Tukey post hoc analysis (n = 6–10 mice per group).

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Previous studies showed that testosterone and AR signaling negatively regulated allergen-induced ILC2-mediated airway inflammation (24, 25). Our in vivo results from the HDM-challenged gonadectomized and sham-operated male and female mice showed that total numbers of IL-13+ Th2 and IL-17A+ Th17 cells, IL-13+ ILC2, and IL-17A+ γδ T are decreased by testosterone and increased by ovarian hormones. Therefore, we hypothesized that AR signaling attenuates cell numbers of HDM-induced IL-13+ Th2 cells and IL-17A+ Th17 cells. To test this hypothesis, we intranasally challenged WT female, WT male, and ARtfm C57BL/6J with HDM or PBS as previously described in Fig. 1A. ARtfm male mice have a mutation in the AR mRNA transcript, and ARtfm male mice are unresponsive to androgens, including testosterone and testosterone derivatives (32). WT female and ARtfm male mice had increased airway eosinophils and neutrophils compared with WT male mice (Fig. 5A, 5B). Lung cells were restimulated with PMA, ionomycin, and Golgi stop and stained for IL-13– and IL-17A–producing Th2 and Th17 cells, respectively. Similar to Fig. 4, the percentage of IL-13+ Th2 cells and IL-17A+ Th17 were not different between HDM-treated groups, but IL-13+ Th2 and IL-17A+ Th17 cells were increased in WT female and ARtfm male mice compared with WT male mice (Fig. 5C–E). These results were similar to results in Figs. 14 from WT female and male BALB/c mice, suggesting that testosterone and AR signaling attenuating type 2 and IL-17A–mediated airway inflammation was independent of the mouse genetic background.

FIGURE 5.

AR signaling attenuated HDM-induced eosinophilic and neutrophilic inflammation. WT female, WT male, and ARtfm male C57BL/6 mice were challenged with HDM or PBS. (A and B) BAL fluid eosinophils and neutrophils. (C) Representative flow staining of IL-13– and IL-17A–expressing T cells. (D and E) Total numbers of IL-13+ Th2 cells and IL-17A+ Th17 cells. Data shown are from one representative experiment of three independent experiments. *p < 0.05, ANOVA with Tukey post hoc test. (n = 3–5 mice per group).

FIGURE 5.

AR signaling attenuated HDM-induced eosinophilic and neutrophilic inflammation. WT female, WT male, and ARtfm male C57BL/6 mice were challenged with HDM or PBS. (A and B) BAL fluid eosinophils and neutrophils. (C) Representative flow staining of IL-13– and IL-17A–expressing T cells. (D and E) Total numbers of IL-13+ Th2 cells and IL-17A+ Th17 cells. Data shown are from one representative experiment of three independent experiments. *p < 0.05, ANOVA with Tukey post hoc test. (n = 3–5 mice per group).

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To determine whether AR signaling decreased IL-13+ Th2 and IL-17A+ Th17 cells in the lung intrinsically, we used a mixed BM chimera model. Lethally irradiated WT CD90.1+/CD90.2+ male mice were reconstituted with a 1:1 mixture of BM cells from CD90.1+ WT male and CD90.2+ ARtfm male mice. Six weeks after reconstitution, the recipient mice were challenged with HDM intranasally four times a week for 3 wk (Fig. 6A). Twenty-four hours after the last challenge, we harvested the lungs and measured the total numbers of IL-13– and IL-17A–producing CD3+ CD4+ T cells from WT CD90.1+ or ARtfm CD90.2+ cells. Based on previous findings in ILC2-mediated airway inflammation (24, 25), we hypothesized that AR signaling directly attenuates IL-13– and IL-17A–producing Th2 and Th17 cells, respectively. Surprisingly, we found that the CD90.2+ IL-13+ Th2 cells from ARtfm mice were decreased compared with CD90.1+ IL-13+ Th2 cells from the WT mice (Fig. 6B, 6C). These findings were different from our results in Figs. 4 and 5, in which testosterone and AR signaling attenuated HDM-induced IL-13+ Th2 cells in the lung. However, it was possible that AR signaling was attenuating IL-13+ Th2 cells via an indirect, extrinsic mechanism. In contrast, CD90.2+ IL-17A+ Th17 cells from ARtfm mice were increased compared with CD90.1+ IL-17A+ Th17 cells from WT mice (Fig. 6D), suggesting that AR signaling decreased cell numbers of IL-17A+ Th17 cells present in the lung via a Th17-intrinisic mechanism.

FIGURE 6.

AR signaling intrinsically decreased IL-17+ Th17 cells and indirectly decreased IL-13+ Th2 cells. (A) Experimental design of mixed BM chimera experiments with 1:1 BM mixture from WT (CD90.1) or ARtfm (CD90.2) male C57BL/6J mice transferred into lethally irradiated heterozygous CD90.1+ CD90.2+ recipient C57BL/6J mice. After 6 wk of reconstitution, recipient mice underwent HDM or PBS challenge, and IL-17A+ and IL-13+ CD4+ T cells were determined for WT and ARtfm mice. (B) Representative dot plots showing gating strategy. (C and D) Total numbers of IL-13+ Th2 and IL-17A+ Th17 cells derived from WT CD90.1 or ARtfm CD90.2 lineages. Data pooled from two combined experiments. *p < 0.05, ANOVA with Tukey post hoc analysis (n = 5–10 mice per group).

FIGURE 6.

AR signaling intrinsically decreased IL-17+ Th17 cells and indirectly decreased IL-13+ Th2 cells. (A) Experimental design of mixed BM chimera experiments with 1:1 BM mixture from WT (CD90.1) or ARtfm (CD90.2) male C57BL/6J mice transferred into lethally irradiated heterozygous CD90.1+ CD90.2+ recipient C57BL/6J mice. After 6 wk of reconstitution, recipient mice underwent HDM or PBS challenge, and IL-17A+ and IL-13+ CD4+ T cells were determined for WT and ARtfm mice. (B) Representative dot plots showing gating strategy. (C and D) Total numbers of IL-13+ Th2 and IL-17A+ Th17 cells derived from WT CD90.1 or ARtfm CD90.2 lineages. Data pooled from two combined experiments. *p < 0.05, ANOVA with Tukey post hoc analysis (n = 5–10 mice per group).

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Our BM chimera experiment determined that AR signaling was differentially regulating cytokine production from Th2 and Th17 cells. Therefore, to determine how AR signaling regulated Th2 and Th17 differentiation, we conducted in vitro Th2 and Th17 cell differentiation experiments. Naive CD4+ T cells isolated from the spleens of WT female, WT male, and ARtfm male C57BL/6J mice were activated with anti-CD3 and anti-CD28 and differentiated into Th2 or Th17 cells for 4 d as previously described (26). After 4 d, IL-13 and IL-17A protein expression was measured in the cell culture supernatants. We also measured mRNA relative expression of AR, as well as transcription factors and receptors known to regulate Th2 and or Th17 differentiation, including Gata3, Il4ra, Rorc, and Il23r, by quantitative RT-PCR analysis. AR mRNA relative expression was significantly decreased in differentiated Th2 or Th17 cells from ARtfm male mice compared with WT female and male mice. Furthermore, AR relative mRNA expression in differentiated Th17 cells was significantly decreased in WT female mice compared with male mice (Fig. 7E), but no difference in AR mRNA expression was detected in Th2 differentiated cells between male and female mice (Fig. 7A). No significant differences in IL-13 protein expression, Gata3 mRNA relative expression, or Il4ra mRNA relative expression were found between Th2 cells from WT female, WT male, and/or ARtfm male mice (Fig. 7B–D). IL-17A protein expression and Il23r mRNA relative expression were increased in Th17 cells from WT female mice and ARtfm male mice compared with WT male mice, but no differences in Rorc expression were determined in Th17 cells from female, male, or ARtfm male mice (Fig. 7F–H). All together, these data suggest that in vitro, AR signaling attenuated IL-17A protein expression and Il23r mRNA relative expression in Th17 cells. However, AR signaling did not regulate IL-13 protein expression and/or transcription factors associated with Th2 cells.

FIGURE 7.

AR signaling attenuated Th17 but not Th2 differentiation in vitro. Naive CD4+ T cells from WT female, WT male, and ARtfm male mice were activated and differentiated into Th2 or Th17 cells. Cell culture supernatants and RNA were collected 4 d after differentiation. (A and E) AR mRNA expression in Th2 cells (left column) and Th17 cells (right column). (BD) IL-13 protein expression from Th2 cell culture supernatants and Gata3 and Il4rα mRNA relative expression in differentiated Th2 cells. (FH) IL-17A protein expression in Th17 cell culture supernatants and Rorc and Il23r mRNA relative expression in differentiated Th17 cells. IL-13 and IL-17A protein expression were measured by ELISA and mRNA relative expression measured by quantitative RT-PCR with relative expression in Th2 or Th17 cells were normalized to Gapdh. Data pooled from two independent experiments. *p < 0.05, ANOVA with Tukey post hoc analysis (n = 9–12 mice per group).

FIGURE 7.

AR signaling attenuated Th17 but not Th2 differentiation in vitro. Naive CD4+ T cells from WT female, WT male, and ARtfm male mice were activated and differentiated into Th2 or Th17 cells. Cell culture supernatants and RNA were collected 4 d after differentiation. (A and E) AR mRNA expression in Th2 cells (left column) and Th17 cells (right column). (BD) IL-13 protein expression from Th2 cell culture supernatants and Gata3 and Il4rα mRNA relative expression in differentiated Th2 cells. (FH) IL-17A protein expression in Th17 cell culture supernatants and Rorc and Il23r mRNA relative expression in differentiated Th17 cells. IL-13 and IL-17A protein expression were measured by ELISA and mRNA relative expression measured by quantitative RT-PCR with relative expression in Th2 or Th17 cells were normalized to Gapdh. Data pooled from two independent experiments. *p < 0.05, ANOVA with Tukey post hoc analysis (n = 9–12 mice per group).

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Our results from the HDM studies in ARtfm mice, the mixed BM chimeras, and in vitro Th2 differentiation experiments suggested that AR signaling decreased Th2 differentiation and cytokine production in the lung by attenuating cytokines and/or chemokines critical for Th2 cells. Therefore, we measured IL-4, IL-10, IL-25, CCL11, CCL24, CCL4, and TARC, cytokines and chemokines that are known to regulate Th2 differentiation and migration, 24 h after the last challenge in lung homogenates of WT female, WT male, and ARtfm male C57BL/6J mice (Supplemental Fig. 3). Only IL-4 protein expression was increased in the lung homogenates of WT female and ARtfm mice compared with WT male mice (Supplemental Fig. 3A).

IL-4 production is important for Th2 differentiation, and decreased levels of IL-4 in the cytokine milieu of the lung would decrease Th2 differentiation and cytokine production (33, 34). To determine the role of AR signaling on IL-4 production in the lung, we harvested the lungs of WT female, WT male, or ARtfm male mice and measured IL-4 production by flow cytometric analysis. IL-4+ Th2 cells, mast cells, and basophils were determined based on the gating strategy described in Fig. 8A and in Supplemental Fig. 4. IL-4+ Th2 cells, mast cells, and basophils were significantly increased in the lungs of WT female and ARtfm mice compared with WT male mice (Fig. 8C–F). No IL-4+ ILC2 were detected in PBS- or HDM-challenged WT or ARtfm mice (data not shown). These findings show that AR signaling decreased HDM-induced IL-4 production in vivo and provided a mechanism by which AR signaling decreased Th2 cell–mediated inflammation.

FIGURE 8.

AR signaling decreased total numbers of IL-4–producing cells. WT female, WT male, and ARtfm male mice were challenged with HDM or PBS. (A) Representative dot plots of IL-4–producing CD45+ cells. (B and C) Percentage and total numbers of viable IL-4+ CD45+ cells. (DF) Number of IL-4–producing Th2 cells, mast cells, and basophils. Th2 cells were defined as CD45+ CD3+ CD4+ cells, mast cells were defined as CD45+ FcεRI+ CD117+ DX5 cells, and basophils were defined as CD45+ FcεRI+ CD117 DX5+ cells. *p < 0.05, ANOVA with Tukey post hoc test (n = 3–9 mice per group) with data pooled from two independent experiments.

FIGURE 8.

AR signaling decreased total numbers of IL-4–producing cells. WT female, WT male, and ARtfm male mice were challenged with HDM or PBS. (A) Representative dot plots of IL-4–producing CD45+ cells. (B and C) Percentage and total numbers of viable IL-4+ CD45+ cells. (DF) Number of IL-4–producing Th2 cells, mast cells, and basophils. Th2 cells were defined as CD45+ CD3+ CD4+ cells, mast cells were defined as CD45+ FcεRI+ CD117+ DX5 cells, and basophils were defined as CD45+ FcεRI+ CD117 DX5+ cells. *p < 0.05, ANOVA with Tukey post hoc test (n = 3–9 mice per group) with data pooled from two independent experiments.

Close modal

Sex hormones modulate immune responses in many autoimmune and allergic diseases, including asthma (35, 36). Clinical and epidemiological studies showed that boys have a higher prevalence of asthma than girls before puberty. However, after puberty, women become twice as likely as men to have asthma (37, 38). The sex differences in asthma prevalence coincide with changes in sex hormones levels, suggesting that sex hormones are important in regulating asthma pathogenesis. However, the underlying molecular mechanisms by which sex hormones regulate airway inflammation associated with asthma remain unclear.

Dual type 2 and IL-17A–mediated airway inflammation increases eosinophil and neutrophil infiltration into the lung and is associated with more severe phenotypes of asthma (12, 18). Our study determined that testosterone decreased and ovarian hormones increased HDM-induced total numbers of IL-13+ Th2 cells and IL-17A+ Th17 cells, infiltration of eosinophils and neutrophils into the BAL fluid, total serum IgE, and AHR. Our 3 wk HDM protocol was sufficient to establish increased eosinophils and neutrophils but was not long enough to observe airway remodeling or smooth muscle hypertrophy that is associated with severe asthma [data not shown and (39)]. Therefore, our HDM challenge mouse model does not reflect all of the features of severe asthma in humans.

Increased AHR and mucus production are regulated by IL-13 and IL-17A, and previous studies showed female mice had increased allergen-induced AHR and/or mucus production compared with male mice (20, 21, 40). Additional studies showed that ovarian hormones increased OVA-induced AHR (20, 23), testosterone decreased contractility of guinea pig tracheal myocytes by negatively regulating IP3 signaling (41), estrogen increased mucus production compared with vehicle in the nasal mucosa of guinea pigs (42), and estrogen increased the production of the mucin proteins Muc5AC and Muc5B in human airway or nasal epithelial cells (43, 44). We expanded upon these findings and showed that testosterone decreased and ovarian hormones increased HDM-induced lung IL-13 and IL-17A production, AHR, and total serum IgE production in mice. However, to our surprise, no significant differences in mucus production were observed between HDM-challenged sham-operated and gonadectomized female and male mice. The conflicting results in our study compared with previous studies are potentially due to differential effects of IL-13 and IL-17A production on mucin protein expression in the airway epithelial cells. Taken together, these findings suggest that sex hormones differentially regulate allergen-induced AHR and mucus production through multiple pathways.

CD4+ Th2 and Th17 cells were the primary sources of HDM-induced IL-13 and IL-17A, respectively. AR signaling decreased IL-17A+ Th17 cells in the lung via Th17 cell intrinsic mechanisms and decreased IL-17A protein expression and Il23r mRNA expression in in vitro–differentiated Th17 cells. Other studies have also reported a critical role for IL-23R signaling in Th17 differentiation, in which inhibition of IL-23R expression decreases optimal IL-17A production in Th17 cells (19, 26, 45, 46). Surprisingly, AR signaling indirectly attenuated Th2 cells in the lung by attenuating lung IL-4 production. IL-4 signals through the IL-4R and is important for Th2 cell differentiation by increasing STAT6 activation and Gata3 expression (15). Our in vitro Th2 differentiation studies showed that Th2 cells from WT male and ARtfm male mice had similar IL-13 production, Gata3 expression, and mRNA expression of Il4ra, a component of the type 1 and 2 IL-4R. Although Th2 cells produce IL-4, our Th2 culture conditions included a large concentration of rmIL-4 (10 ng/ml). These data provide additional evidence to suggest that AR signaling attenuation of IL-4 cytokine expression in the lung limited the numbers of Th2 cells that differentiate or proliferate, leading to decreased type 2–mediated allergic inflammation in males compared with females.

Recent studies from our group and others reported that AR signaling intrinsically decreased ILC2-mediated allergic inflammation cell proliferation and cytokine production (24, 25). These findings differ from our study in that we determined that AR signaling negatively decreased Th2 cell–mediated inflammation by a Th2 cell indirect (extrinsic) mechanism. Previous reports have also shown that AR signaling extrinsically regulates T cell development and T cell function in the periphery (47). BM chimera models in which AR expression was attenuated in thymocytes or thymic epithelial cells revealed that suppression of AR expression on thymic epithelial cells inhibited the ability of the thymus to respond to androgens via indirect mechanisms (48). However, addition of 5α-dihydrotestosterone (DHT), a derivative of testosterone, to splenic, activated CD4+ T cells decreased IL-4, IL-6, IL-12, and IFN-γ levels (49). Therefore, AR signaling may intrinsically or extrinsically regulate immune responses depending on the cell type and/or tissue.

Understanding the foundational mechanisms underlying airway inflammation associated with type 2 and IL-17A inflammatory pathways is imperative for the development of novel asthma therapeutics that are not only effective but personalized specifically for women and men with asthma. Our studies showed that testosterone attenuated total numbers of IL-13– and IL-17A–producing CD4 T cells and suggest that increasing AR signaling may be important for decreasing airway inflammation associated with asthma. Recently, a phase IIa clinical trial has reported that nebulized dehydroepiandrosterone-3-sulfate (DHEAS), a nonvirilizing hormone derivative of testosterone that does not have unwanted side effects on females, improved asthma control in patients with moderate to severe asthma (50). These findings suggest a potential role for testosterone derivatives, including nebulized DHEA, in attenuating asthma symptoms and warrant additional investigation.

This work was supported by National Institutes of Health Grants R01 HL122554 (to D.C.N.), R21 AI121420 (to D.C.N.), and R01 HL122554S1 (to D.C.N.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

AHR

airway hyperresponsiveness

AR

androgen receptor

ARtfm

AR testicular feminized

BAL

bronchoalveolar lavage

BM

bone marrow

FEV1

forced expiratory volume in 1 s

HDM

house dust mite

ILC2

group 2 innate lymphoid cell

rm

recombinant mouse

WT

wild-type.

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The authors have no financial conflicts of interest.

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