Estrogen receptor-α signaling increases allergen-induced IL-33 release and airway inflammation

Abstract

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.

Keywords: IL-33 release; ILC2; allergic airway inflammation; estrogen receptor alpha; sex disparity.

Figure 1:. Esr1 deficiency decreased Alt Ext-induced IL-5 and IL-13 protein expression and infiltration of inflammatory cells into the lung

A. Experimental design of Alt Ext (7.5 μg) or vehicle intranasal challenge protocol. Endpoints were taken 24 hours following final Alt Ext challenge. B-C. IL-5 and IL-13 protein expression in lung homogenates. D-G. Inflammatory cells in BAL fluid. H-J. Representative sections at 10x magnification and quantification of H&E and PAS staining to detect airway inflammation in lung sections on 48 hours after final challenge. Black arrowheads denote PAS+ staining. Data are mean ± SEM, n=5-15 mice; * p<0.05, ANOVA with Tukey post-hoc analysis.

Figure 2:. Esr1 deficiency decreased Alt Ext-induced ILC2 in the lung.

Lungs were harvested 24 hours following last Alt Ext challenge and restimulated with PMA, ionomycin, and golgi-stop to detect IL-5 and IL-13+ ILC2. A. Representative dot plots of cytokine producing ILC2 in lungs of mice. Dot plots are pre-gated on Lin- CD45+ CD90+ CD127+ cells. B. Percentage of lung ILC2 from viable cells. C.Total number of lung ILC2. D-E. Percentage and total number of IL-5+ IL-13+ ILC2. F-G. Mean fluorescence intensity (MFI) of IL-5 and IL-13 in ILC2. Data are mean ± SEM, n=5-8 mice; * p<0.05, ANOVA with Tukey post-hoc analysis (B and C) and t-test (D-E).

Figure 3:. Estrogen signaling increased Alt Ext-induced ILC2 cytokine production and inflammatory cell infiltration in the lung.

WT female mice were administered subcutaneous Tamoxifen (5mg) or vehicle pellets for 21 days and were challenged with Alt Ext protocol. A-B. IL-5 and IL-13 protein expression in lung homogenates. C-F. Inflammatory cells in BAL fluid. Data are mean ± SEM, n=3-5 mice; * p<0.05, ANOVA with Tukey post-hoc analysis.

Figure 4:. Esr1 signaling had no intrinsic effect on Alt Ext-induced ILC2 cytokine production.

A. Experimental design of mixed BM chimera experiments with a 1:1 BM mixture from WT (CD90.1) and Esr1^−/− (CD90.2) female mice transferred into lethally irradiated WT (CD90.1/CD90.2) female recipient mice. After 6 weeks of reconstitution, recipient mice were challenged with Alt Ext protocol. Recipient mouse lungs were harvested 24 hours following last challenge and restimulated with PMA, ionomycin, and golgi-stop to detect IL-5 and IL-13+ ILC2. B. Representative dot plots showing gating strategy with ILC2 defined as viable, Lin−, CD45+, ICOS+, ST2+ cells. C-E. Percent of ILC2 and IL-5+, IL-13+, or IL-5+ IL-13+ ILC2 in lungs. F. ST2 MFI in lung ILC2. Data are mean ± SEM, n=9 mice; * p<0.05, ANOVA with Student t-test.

Figure 5:. Esr1 deficiency decreased IL-33 production in lung cells after Alt Ext challenge.

A. IL-33 protein expression in BAL fluid 1 hour after last Alt Ext or vehicle challenge. B-D. Lungs were harvested 24 hours following last challenge and IL33eGFP was measured by flow cytometry. Number of IL-33eGFP+ cells in total lung cells, epithelial (CD45-IL-33eGFP+EpCAM+) and endothelial (CD45-IL-33eGFP+CD146+) cell subsets. Data are mean ± SEM, n=6–9 mice; * p<0.05, ANOVA with Tukey post-hoc analysis.

Figure 6:. Esr1 signaling increased IL-33 release from Alt Ext challenged hBE33 cells.

A human bronchial epithelial cell line that expresses IL-33 (hBE33) was pre-treated for 6 hours with 17β-E2, ER agonists, or vehicle followed by an allergen challenge with Alt Ext or vehicle. A. IL-33 protein expression in hBE33 cells treated with 17β-E2 or vehicle (ethanol). B-C. IL-33 protein expression in hBE33 cells treated with PPT (ER-α agonist), DPN (ER-β agonist) or vehicle (DMSO). D. Analysis of cell viability measured by LDH assay. E. Extracellular ATP concentrations in cell supernatants at various time points after challenge. F. Relative intensity of DUOX1 protein levels 1 hour after challenge with β-actin serving as a loading control. Lanes shown are from lanes 2-4 and 6-8 of the same blot. Data are mean ± SEM, n=4 wells combined from 2 experiments; * p<0.05, ANOVA with Tukey post-hoc analysis.

Figure 7:. Esr1 signaling increased IL-33 expression from Alt Ext challenged NHBE cells.

Normal human bronchial epithelial (NHBE) cells were pre-treated for 6 hours with 17β-E2, ER agonists, or vehicle followed by an allergen challenge (Alt Ext, HDM or vehicle). A. Relative quantification of IL33 expression in NHBE cells pre-treated with 17β-E2 or vehicle (ethanol) followed by Alt Ext or vehicle challenge. B-C. Relative quantification of IL33 expression in NHBE cells pre-treated with PPT, DPN or vehicle followed by Alt Ext or vehicle challenge. D. Relative quantification of IL33 expression in NHBE cells treated with 17β-E2 or vehicle (ethanol) followed by HDM or vehicle challenge. E-F. Relative quantification of IL33 expression in NHBE cells treated with PPT, DPN or vehicle followed by HDM or vehicle challenge. All relative expressions were normalized to housekeeping gene, GAPDH. Data are mean ± SEM, n=4 wells combined from 2 independent experiments; * p<0.05, ANOVA with Tukey post-hoc analysis.