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.

Sex hormones are important in regulating asthma pathogenesis. However, additional studies need to be conducted to further elucidate how sex hormones are initiating and driving the inflammatory response(s) in asthma. Determining these pathways will provide the foundation necessary for the development of treatment strategies and potentially new therapeutics for patients, in particular females, with asthma.

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 AR 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.

Background: Group 2 innate lymphoid cells (ILC2) are stimulated by IL-33 to increase IL-5 and IL-13 production and airway inflammation. While sex hormones regulate airway inflammation, it remained unclear whether estrogen signaling through estrogen receptor-α (ER-α, Esr1) or ER-β (Esr2) increased ILC2-mediated airway inflammation. We hypothesize that estrogen signaling increases allergen-induced IL-33 release, ILC2 cytokine production, and airway inflammation. Methods: Female Esr1-/-, Esr2-/-, wild-type (WT), and IL33 fl/fl eGFP mice were challenged with Alternaria extract (Alt Ext) or vehicle for 4 days. In select experiments, mice were administered tamoxifen or vehicle pellets for 21 days prior to challenge. Lung ILC2, IL-5 and IL-13 production, and BAL inflammatory cells were measured on day 5 of Alt Ext challenge model. Bone marrow from WT and Esr1-/female mice was transferred (1:1 ratio) into WT female recipients for 6 weeks followed by Alt Ext challenge. hBE33 cells and normal human bronchial epithelial cells (NHBE) were pretreated with 17β-estradiol (E2), propyl-pyrazole-triol (PPT, ER-α agonist), or diarylpropionitrile (DPN, ER-β agonist) before allergen challenge to determine IL-33 gene expression and release, extracellular ATP release, DUOX-1 production, and necrosis. Results: Alt Ext challenged Esr1-/-, but not Esr2-/-, mice had decreased IL-5 and IL-13 production, BAL eosinophils, and IL-33 release compared to WT mice. Tamoxifen decreased IL-5 and IL-13 production and BAL eosinophils. IL-33eGFP + epithelial cells were decreased in Alt Ext challenged Esr1-/mice compared to WT mice. 17β-E2 or PPT, but not DPN, increased IL-33 gene expression, release, and DUOX-1 production in hBE33 or NHBE cells. Conclusion: Estrogen receptor-α signaling increased IL-33 release and ILC2-mediated airway inflammation.

RATIONALE: The purpose of this study was to determine whether conventionally used ILC2 markers, ST2 and CD127, are sufficient to include all Th2-cytokine producing ILCs. We hypothesized that ST2-and CD127-negative ILC2 populations exist in mouse lung and that these nontraditional populations might be activated by the clinically relevant fungal allergen Alternaria, a known potent inducer of IL-33. METHODS: Mice were challenged intranasally with Alternaria alternata for three consecutive days, and 24 hours after the last challenge brochoalveolar lavage fluid and lungs were harvested for flow cytometric analysis, ELISA, and lung section staining. ILCs were identified by flow cytometry as CD45+Lineage-Thy1.2+ lymphocyte-sized cells and were separated into four subsets based on ST2 and CD127 expression. Intracellular cytokine and transcription factor staining were performed on the ILC populations. RESULTS: Th2 cytokine-producing ILCs were identified in all four subpopulations, regardless of ST2 and CD127 expression, as evidenced by high GATA-3, IL-5, IL-13 and ILC2 surface marker expression. Further, ILC2 cytokines and surface markers were upregulated following Alternaria challenge, irrespective of ST2 and CD127 expression. The four ILC subpopulations were also found in RAG2 knockout mice, which is supportive of their presence being T-cell independent. Some of the unconventional ILC2s also produced IFNg and IL-17A suggesting the existence of ILC2 plasticity in these populations. CONCLUSIONS: Unconventional ST2-and CD127-negative ILC2 populations exist in mouse lung and are further induced by Alternaria. Thus, future mouse ILC2 investigations should be aware of ILC2 populations that do not express ST2 and CD127.

No abstract

RATIONALE: Airborne allergens stimulate the production of IL-25, IL-33 and TSLP from airway epithelial cells. However, the mechanism of allergen-induced cytokines production is not fully understood. The purpose of this study is to elucidate the role of oxidative stress, NADPH oxidase, on the allergen-induced production of epithelial-derived cytokines. METHODS: We examined the in vivo effects of NADPH oxidase inhibitor (DPI and VAS2870) in the nasal epithelium of mice exposed to multiple airborne allergens (MAA: House dust mite, Alternaria and Protease from S.aureus) were examined and the in vitro effects of NADPH oxidase on the secretion of the epithelial-derived cytokines in normal human bronchial epithelial (NHBE) cells. RESULTS: Intranasal instillations of NADPH oxidase inhibitor significantly inhibited MAA-induced production of IL-25, IL-33 and TSLP in mouse nasal epithelium. Eosinophil infiltration, epithelial disruption and production of Th2 cytokines were also significantly attenuated. NADPH oxidase was rapidly produced when NHBE cells were exposed to airborne allergens (house dust mite, alternaria, protease from S.aureus) and ATP. The airborne allergen induced epithelial-derived cytokines were partially blocked by infecting NHBE cells with small interfering RNA for one of NADPH family, DUOX1. CONCLUSIONS: Airborne allergen-induced oxidative stress may be involved in the production of IL-25, IL-33 and TSLP from airway epithelial cells.