Figure Legends
Figure 1 Th2 inflammation is higher in women than men with severe asthma Proportion of circulating Th2 cells (CD4+CRTh2+ T cells/pWBC) in men and women with mild/moderate and severe asthma was assessed by flow cytometry (A). Proportion of circulating Th2 cells in severe asthmatic men and women not taking OCS or biologics (anti-IgE or IL-5; B). Correlation analyses between the proportion of circulating Th2 cells and total daily dose of ICS in women (C) and men (D), whole blood CRTh2 mRNA (relative to GAPDH) and ICS in women (E) and men (F) and CRTh2 mRNA and FEV1 (% predicted) in women (G) and men (H).pWBC , peripheral white blood cells; ICS , inhaled corticosteroid oral corticosteroid; OCS , oral corticosteroid,FEV1 , forced expiratory volume in 1 second.*P < 0.01 determined by One Way ANOVA (A), *P = 0.016 two-tailed Student’s t test (B)
Figure 2 Estrogen receptor signaling decreases Th2 cell sensitivity to GC‐induced apoptosis In vitro differentiated Th2 cells treated with DEX and either the ERα selective agonist PPT (1 µM, A) or DEX and β-estradiol (0.1 µM, E2; B) were assessed for apoptosis by flow cytometry using Annexin V positivity in the absence of 7-AAD staining (Annexin V+/7-AAD-). Level of BCL-2 mRNA (relative to GAPDH) was elevated by qRT-PCR in Th2 cells treated with DEX and PPT (C). Constitutive levels of mRNA for IL-5 and IL-13 from Th2 cells treated with Vehicle, DEX and/or PPT (D; 24 hours).GC , glucocorticoid; DEX , dexamethasone; PPT , propyl pyrazole triol (ERα selective agonist); ERα , estrogen receptor alpha; IL-5 , Interleukin 5; IL-13 , Interleukin 13.*P < 0.05 vs vehicle;#P < 0.05 vs DEX, determined by One Way RM ANOVA
Figure 3 Combined GC and estrogen receptor signaling upregulates CRTh2 expression. Effect of treating in vitrodifferentiated Th2 cells with DEX (0.1 µM) and/or PPT (10 µM) was assessed at the level of CRTh2 mRNA by qRT-PCR (A) and total CRTh2 protein by western blot and densitometry analysis (B). CRTh2 surface levels were determined by flow cytometry following DEX and/or PPT treatment (C) or DEX and/or E2 treatment (0.1µM, D). The effect of DEX and PPT on CRTh2 transcriptional activity was assessed using a CRTh2-pro/Luc reporter construct (E).GC , glucocorticoid;DEX , dexamethasone; PPT , propyl pyrazole triol (ERα selective agonist); ERα , estrogen receptor alpha; E2 , β-estradiol. *P < 0.05 vs vehicle;#P < 0.05 vs DEX, determined by One Way RM ANOVA
Figure 4 GC and estrogen receptor signaling enhance type 2 cytokine release following treatment with PGD2. In vitro differentiated Th2 cells pre-treated (24 hours) with DEX (0.1 µM) and/or PPT (10µM) were assessed by ELISA for IL-5 (A) and IL-13 (B) release following stimulation with the CRTh2 ligand PGD2(24 hours). GC , Glucocorticoid; DEX , dexamethasone; PPT , propyl pyrazole triol (ERα selective agonist);PGD2 , Prostaglandin D2;ERα , estrogen receptor alpha. *P< 0.05 vs vehicle; #P < 0.05 vs DEX, determined by One Way RM ANOVA