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.

ILC2s are potent mucosal effector cells that participate in type 2 inflammatory responses. Stier et al. demonstrate that IL-33 negatively regulates CXCR4, mediating the egress of ILC2 lineage cells from the bone marrow for potential hematogenous trafficking.

Rationale: Group 2 innate lymphoid cells (ILC2s) robustly produce IL-5 and IL-13, cytokines central to the asthma phenotype; however, the effect of prostaglandin (PG) I 2 on ILC2 function is unknown.Objectives: To determine the effect of PGI 2 on mouse and human ILC2 cytokine expression in vitro and the effect of endogenous PGI 2 and the PGI 2 analog cicaprost on lung ILC2s in vivo.Methods: Flow-sorted bone marrow ILC2s of wild-type (WT) and PGI 2 receptor-deficient (IP 2/2 ) mice were cultured with IL-33 and treated with the PGI 2 analog cicaprost. WT and IP 2/2 mice were challenged intranasally with Alternaria alternata extract for 4 consecutive days to induce ILC2 responses, and these were quantified. Prior to A. alternata extract, challenged WT mice were treated with cicaprost. Human flow-sorted peripheral blood ILC2s were cultured with IL-33 and IL-2 and treated with the PGI 2 analog cicaprost. Measurement and Main Results:We demonstrate that PGI 2 inhibits IL-5 and IL-13 protein expression by IL-33-stimulated ILC2s purified from mouse bone marrow in a manner that was dependent on signaling through the PGI 2 receptor IP. In a mouse model of 4 consecutive days of airway challenge with an extract of A. alternata, a fungal aeroallergen associated with severe asthma exacerbations, endogenous PGI 2 signaling significantly inhibited lung IL-5 and IL-13 protein expression, and reduced the number of lung IL-5-and IL-13-expressing ILC2s, as well as the mean fluorescence intensity of IL-5 and IL-13 staining. In addition, exogenous administration of a PGI 2 analog inhibited Alternaria extract-induced lung IL-5 and IL-13 protein expression, and reduced the number of lung IL-5-and IL-13-expressing ILC2s and the mean fluorescence intensity of IL-5 and IL-13 staining. Finally, a PGI 2 analog inhibited IL-5 and IL-13 expression by human ILC2s that were stimulated with IL-2 and IL-33.Conclusions: These results suggest that PGI 2 may be a potential therapy to reduce the ILC2 response to protease-containing aeroallergens, such as Alternaria.

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 Women have an increased prevalence of severe asthma compared to men. IL-17A is associated with severe asthma and requires IL-23 receptor (IL-23R) signaling, which is negatively regulated by Let-7f miRNA. Objective Determine the mechanism by which 17β-estradiol (E2) and progesterone (P4) increase IL-17A production. Methods IL-17A production was determined by flow cytometry in Th17 cells from women (n=14) and men (n=15) with severe asthma. Cytokine levels were measured by ELISA and IL-23R and Let-7f expression by qPCR in Th17 differentiated cells from healthy women (n=13) and men (n=14). In sham-operated or ovariectomized female mice, 17β-E2, P4, 17β-E2+P4, or vehicle pellets were administered for 3 weeks prior to ex vivo Th17 cell differentiation. Airway neutrophil infiltration and KC expression was also determined in OVA-challenged WT female recipient mice with an adoptive transfer of OVA-specific Th17 cells from female and male mice. Results In severe asthma patients and healthy controls, IL-17A production was increased in Th17 cells from women compared to men. IL-23R expression was increased and Let-7f expression was decreased in Th17 differentiated cells from women compared to men. In ovariectomized mice, IL-17A and IL-23R expression was increased and Let-7f expression was decreased in the Th17 cells from mice administered 17β-E2+P4 compared to vehicle. Further, transfer of female OVA-specific Th17 cells increased acute neutrophil infiltration in the lungs of OVA-challenged recipient mice compared to transfer of male OVA-specific Th17 cells. Conclusions 17β-E2+P4 increased IL-17A production from Th17 cells, providing a potential mechanism for the increased prevalence of severe asthma in women compared to men.

The appropriate orchestration of different arms of the immune response is critical during viral infection to promote efficient viral clearance while limiting immunopathology. However, the signals and mechanisms that guide this coordination are not fully understood. Interferons are produced at high levels during viral infection and have convergent signaling through signal transducer and activator of transcription 1 (STAT1). We hypothesized that STAT1 signaling during viral infection would regulate the balance of innate lymphoid cells (ILC), a diverse class of lymphocytes that are poised to respond to environmental insults including viral infections with the potential for both anti-viral or immunopathologic functions. During infection with respiratory syncytial virus (RSV), STAT1-deficient mice had reduced numbers of anti-viral IFNγ+ ILC1 and increased numbers of immunopathologic IL-5+ and IL-13+ ILC2 and IL-17A+ ILC3 compared to RSV-infected WT mice. Using bone marrow chimeric mice, we found that both ILC-intrinsic and ILC-extrinsic factors were responsible for this ILC dysregulation during viral infection in STAT1-deficient mice. Regarding ILC-extrinsic mechanisms, we found that STAT1-deficient mice had significantly increased expression of IL-33 and IL-23, cytokines that promote ILC2 and ILC3 respectively, compared to WT mice during RSV infection. Moreover, disruption of IL-33 or IL-23 signaling attenuated cytokine-producing ILC2 and ILC3 responses in STAT1-deficient mice during RSV-infection. Collectively, these data demonstrate that STAT1 is a key orchestrator of cytokine-producing ILC responses during viral infection via ILC-extrinsic regulation of IL-33 and IL-23.

Allergic airway diseases are immune disorders associated with heightened type 2 immune responses and IL-5 and IL-13 production at the site of inflammation. We have previously reported that cyclooxygenase (COX) inhibition by indomethacin augmented allergic airway inflammation in a STAT6-independent manner. However, the key COX product(s) responsible for restraining indomethacin-mediated STAT6-independent allergic inflammation is unknown. In this study, using the mouse model of OVA-induced allergic airway inflammation, we identified that PGI2 receptor (IP) signaling was critical for indomethacin-induced, STAT6-independent proallergic effects. We demonstrated that IP deficiency increased inflammatory cell infiltration, eosinophilia, and IL-5 and IL-13 expression in the lung in a STAT6-independent manner. The augmented STAT6-independent allergic inflammation correlated with enhanced primary immune responses to allergic sensitization and elevated production of multiple inflammatory chemokines (CCL11, CCL17, CCL22, and CXCL12) in the lung after allergen challenge. We also showed that the PGI2 analogue cicaprost inhibited CD4 T cell proliferation and IL-5 and IL-13 expression in vitro, and IP deficiency diminished the stimulatory effect of indomethacin on STAT6-independent IL-5 and IL-13 responses in vivo. The inhibitory effects of PGI2 and the IP signaling pathway on CD4 T cell activation, inflammatory chemokine production, and allergic sensitization and airway inflammation suggest that PGI2 and its analogue iloprost, both Food and Drug Administration–approved drugs, may be useful in treating allergic diseases and asthma. In addition, inhibiting PGI2 signaling by drugs that either block PGI2 production or restrain IP signaling may augment STAT6-independent pathways of allergic inflammation.

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.