Cigarette smoke impairs autophagic flux in bronchial epithelial cells

Background : Autophagy describes the protective mechanism cells use to keep their interior “clean” and function optimal. Chronic obstructive pulmonary disease (COPD) is a progressive and life-threatening condition caused mainly by cigarette smoking. The role of autophagy in COPD remains controversial due to a lack of adequate measurements. Aim and objectives : We hypothesize that cigarette smoke exposure can impair autophagy in an in vitro model that mimics COPD. Methods : BEAS2B, a human lung epithelial cell line, were treated with cigarette smoke extracts (CSE) for 24 hours, media was replaced and the cells were incubated for another 24 hours. The formation of autophagosomes (AP) was assessed by the conversion of LC3B-I to LCE3B-II using Western blot and fluorescent microscopy. The flux of autophagy was investigated by measuring LC3B-II by CSE in the presence and absence of lysosomal inhibitor ammonium chloride and by the turnover of autophagy substrate protein p62. Autophagy initiation was measured by looking at phosphorylation of AMPK by Western blot. Lysosomal proteins LAMP1 and LAMP2 and the endosomal marker Rab7 were measured by Western blot. Results : CSE concentration-dependently increased the formation of autophagosomes as shown by increased LC3B-II and AP. Only high concentration of CSE (40%) impaired the flux of autophagy as shown by accumulation of p62. CSE (40%) decreased AMPK activity (>50%, p 75%) and LAMP1 protein levels but not Rab7. Conclusions : High levels of CSE can impair the autophagic flux in a bronchial epithelial cell line by possibly reducing AMPK activity and LAMP proteins. We propose that defective autophagy could play an important role in COPD pathogenesis.