GC and ERα cooperate to upregulate CRTh2 expression
Total daily dose of ICS correlated with whole blood levels ofCRTh2 mRNA in women, but not men (Fig. 1E, 1F). Therefore, we next examined whether GC and ERα signaling directly influences CRTh2 expression. Th2 cells treated with both DEX and PPT (24 hours) exhibited significantly more CRTh2 mRNA compared to vehicle or DEX alone (Fig. 3A). Western blot analysis showed higher abundance of total CRTh2 protein following co-treatment with PPT and DEX (Fig. 3B). CRTh2 is a plasma membrane receptor and so we assessed surface levels by flow cytometry and found CRTh2 was significantly increased following co-treatment with DEX and PPT (Fig. 3C) or DEX and E2 (Fig. 3D). Upregulation of CRTh2 was likely due to a transcriptional effect since the CRTh2 promoter construct contains putative GC response elements (GRE) and activity was increased by DEX treatment alone and further enhanced with both DEX and PPT (24 hours, Fig. 3E). Together, these experiments show that concomitant activation of GR and ERα increases CRTh2 expression.
GC and ERα enhance type 2 cytokine release following CRTh2 activation
Since concomitant DEX and PPT treatment increased CRTh2 expression (Fig. 3), we considered whether this exposure primed Th2 cells for heightened responsiveness to PGD2. Th2 cells were pre-treated with DEX alone or DEX and PPT (24 hours), washed and stimulated with PGD2 (24 hours) to activate the cells through CRTh2. We found that while pre-treatment with DEX alone reduced both cytokines, Th2 cells pre-treated with DEX and PPT released significantly more IL-5 (Fig. 4A) and IL-13 (Fig. 4B) in response to PGD2compared to DEX alone or no pre-treatment. These results suggest that the cooperative effect of GC and ERα on CRTh2 levels enhances the Th2 cell response to PGD2, increasing type 2 cytokine release.