Role of DNA methylation in Muc5AC hyperexpression in severe asthma

Abstract

Background: In asthma, mucus overproduction contributes to airway obstruction and poor control of the disease. Epigenetic modifications modulate gene expression and have been implicated in asthma pathogenesis. Aim and Objectives: We hypothesized that altered DNA methylation is involved in mucus hyperproduction in severe asthma (SA). We investigated the role of DNA methylation in mucociliary differentiation and Muc5AC expression in the reconstituted bronchial epithelium of controls and SA patients. Methods: Epithelial cells were isolated from bronchial biopsies from control (n=9) and SA (n=26) subjects and differentiated at the air-liquid interface. In a separate set of experiments, the DNA methylation inhibitor 5-aza-2’-deoxycytidine (AZA) was added for the first 7 days of culture. Real-time PCR was used to quantify the mRNA levels of the phenotype markers MUC5AC and FOXJ1, of the differentiation drivers, SPDEF, FOXA2 and FOXA3, and of DNA methylation regulators, DNMT1, DNMT 3A and B and TET1-3. Promoter methylation was assessed by bisulfite pyrosequencing. Results: Inhibition of DNA methylation with AZA led to the repression of both Muc5AC and FOXJ1 expression in regenerated epithelia from controls and SA subjects. In the absence of AZA, Muc5AC was hyperexpressed in the regenerated bronchial epithelia from a sub-group of SA patients, and this was associated with hypomethylation of its promoter within an NF-κB binding site. In SA subjects, methylation levels of Muc5AC promoter correlated positively with Muc5AC mRNA levels but negatively with those of the DNA methyltransferases Dnmt3a and Dnmt3b. Conclusions: Alterations in DNA methylation may affect bronchial epithelium phenotype and favor MUC5AC overproduction in SA.