CRTh2 (encoded by PTGDR2) is a G-protein coupled receptor expressed by Th2 cells as well as eosinophils, basophils and innate lymphoid cells (ILC)2s. Activation of CRTh2, by its ligand prostaglandin (PG)D2, mediates production of type 2 cytokines (IL-4, IL-5 and IL-13), chemotaxis and inhibition of apoptosis. As such, the PGD2-CRTh2 pathway is considered important to the development and maintenance of allergic inflammation. Expression of CRTh2 is mediated by the transcription factor GATA3 during Th2 cell differentiation and within ILC2s. Other than this, relatively little is known regarding the cellular and molecular mechanisms regulating expression of CRTh2. Here, we show using primary human Th2 cells that activation (24hrs) through TCR crosslinking (αCD3/αCD28) reduced expression of both mRNA and surface levels of CRTh2 assessed by flow cytometry and qRT-PCR. This effect took more than 4 hours and expression was recovered following removal of activation. EMSA analysis revealed that GATA3 and NFAT1 can bind independently to overlapping sites within a CRTh2 promoter probe. NFAT1 over-expression resulted in loss of GATA3-mediated CRTh2 promoter activity, while inhibition of NFAT using a peptide inhibitor (VIVIT) coincided with recovery of CRTh2 expression. Collectively these data indicate that expression of CRTh2 is regulated through the competitive action of GATA3 and NFAT1. Though prolonged activation led to NFAT1-mediated downregulation, CRTh2 was re-expressed when stimulus was removed suggesting this is a dynamic mechanism and may play a role in PGD2-CRTh2 mediated allergic inflammation.

These findings show an association between CRTh2 rs533116 and allergic asthma and suggest this may be mediated by elevated expression of CRTh2, leading to higher numbers of circulating eosinophils and Th2 cytokine production.

Human CRTh2+ Th2 cells express IL‐25 receptor (IL‐25R) and IL‐25 has been shown to potentiate production of Th2 cytokines. However, regulation of IL‐25R and whether it participates in Th2 differentiation of human cells have not been examined. We sought to characterize IL‐25R expression on CD4+ T cells and determine whether IL‐25 plays a role in Th2 differentiation. Naïve human CD4+ T cells were activated in the presence of IL‐25, IL‐4 (Th2 conditions) or both cytokines to assess their relative influence on Th2 differentiation. For experiments with differentiated Th2 cells, CRTh2‐expressing cells were isolated from differentiating cultures. IL‐25R, GATA3, CRTh2 and Th2 cytokine expression were assessed by flow cytometry, qRT‐PCR and ELISA. Expression of surface IL‐25R was induced early during Th2 differentiation (2 days). Addition of IL‐25 to naïve CD4+ T cells revealed that it induces expression of its own receptor, more strongly than IL‐4. IL‐25 also increased the proportions of IL‐4‐, GATA3‐ and CRTh2‐expressing cells and expression of IL‐5 and IL‐13. Activation of differentiated CRTh2+ Th2 cells through the TCR or by CRTh2 agonist increased surface expression of IL‐25R, though re‐expression of CRTh2 following TCR downregulation was impeded by IL‐25. These data suggest that IL‐25 may play various roles in Th2 mediated immunity. We establish here it regulates expression of its own receptor and can initiate Th2 differentiation, though not as strongly as IL‐4.