Inhibition of GATA3 and Th2 cytokines expression in type 2 high asthmatics by a small molecule targeting RNA-binding protein HuR

Abstract

Abstract RNA-binding protein human antigen R (HuR) is a master regulator of gene expression in human pathophysiology. Our animal studies in the past showed multiple critical roles for HuR in regulating proinflammatory markers in CD4+ T cells. We hypothesized HuR plays an important role in expression of GATA3 and Th2 cytokines in type 2 high asthma and its inhibition impairs these Th2 inflammatory responses. Peripheral CD4+ T cells were isolated from 26 healthy controls and 32 type 2 high asthmatics (blood eosinophil ≥300 cells/μl or FeNO levels ≥25ppb) and stimulated with anti-CD3/CD28 for 4 days, followed by determination of GATA3 and cytokines expression by flow cytometry and LEGENDplex. HuR levels were quantitated by Western blot, flow cytometry, and qPCR. Inhibition of inflammatory responses was evaluated using a cumarin-derived small molecule named CMLD-2 (a HuR-specific inhibitor). Actinomycin D treatment was used to measure mRNA decay rates of targeted HuR mRNAs. HuR protein levels in CD4+ T cells was significantly higher in type 2 high asthmatics compared to healthy controls. The expression and secretion of Th2 cytokines were significantly higher in asthmatics compared to controls. Our further mRNA and protein studies in type 2 high asthmatics showed reduced expression of GATA3 and Th2 cytokines after CMLD-2 treatment. There was also a significant reduction in gata3 mRNA stability with CMLD-2 treatment, compared with vehicle in the asthmatics. Our study is the first to demonstrate the role of RNA-binding protein HuR in type 2 high asthmatics and that interfering with HuR action using CMLD-2 ameliorates GATA3 and Th2 cytokines production. This data suggests HuR may serve as a novel and powerful therapeutic target to treat type 2 high asthmatics.