LSC Abstract – Chronic cigarette smoke (CS) exposure alters differentiation of primary bronchial epithelial cells, cultured at the air-liquid interface (PBEC-ALI)

Abstract

During differentiation of PBEC at the ALI, basal cells differentiate into a pseudo stratified epithelial cell layer consisting of ciliated, mucus-producing goblet and Club cells. Recurrent exposure to CS, the main risk factor for COPD, may affect differentiation. The aim of this study was to investigate the effect of repeated CS exposure during the differentiation of PBEC-ALI, by assessing gene expression of mucins and various epithelial cell markers. PBEC (n = 4-5), isolated from tumour free bronchial tissue obtained during lung resection surgery, were cultured on transwell inserts until confluence was reached. Apical medium was removed and cells were exposed to volatile CS/air for 5 min for 21 days/5 days a week. After 1, 7, 14 and 21 days, trans-epithelial electrical resistance (TEER) was measured; cytotoxicity was assessed (by LDH measurement) in basal medium; mucin production (MUCAC and MUC5B) was measured by dotblot analysis in apical washes; and expression of mucins and markers of epithelial cell differentiation was assessed using both qPCR and confocal microscopy. TEER was reduced after 7 days of CS exposure compared to air-exposed controls, but restored after 14 and 21 days. Recurrent CS exposure decreased expression and release of MUC5B and expression of markers related to squamous-, neuroendocrine-, ciliated-, Club- and NGFR-positive basal cells, whereas P63-positive basal cells and mucin expressing goblet cells were unaffected. These results indicate that recurrent CS exposure decreases NGFR-positive basal cells and differentiation of bronchial epithelial cells.