Cigarette smoke extract induces barrier changes and senescence in primary bronchial epithelial cells

Abstract

Cigarette smoke (CS) induces pulmonary inflammation and is correlated with an increased risk of developing COPD. Our hypothesis is that CS drives a phenotypic shift in the bronchial epithelium resulting in altered function and inflammatory response. Differentiated primary human bronchial epithelial cells (HBECs) were cultured at air-liquid interface (ALI) and stimulated with cigarette smoke extract (CSE) for 48h or 15 days, followed by assessment of barrier permeability, gene expression and cell morphology. HBECs exposed to CSE for 48h demonstrated a dose-dependent alteration to the epithelial barrier seen as increased permeability. In addition, genes involved in antioxidant processes and senescence, were dose-dependently upregulated. Interestingly, after repeated exposure to the highest concentration of CSE, ALI cultures exhibited a remarkedly tight barrier with permeability lower than in control wells. Formalin-fixed and paraffin embedded cultures revealed signs of a phenotypic shift with a reduced number of ciliated cells and a thinner barrier. Further, genes linked to oxidative stress and senescence were significantly and dose-dependently upregulated in all donors. To conclude, CSE induces significant changes to the epithelial barrier. The dose and time are crucial in determining the type and severity of the response, where chronic treatment with a high concentration of CSE generates cultures with a different phenotype showing high similarity to some changes seen in patients with COPD. This model highlights the importance of the epithelial barrier in the response to external triggers and can be used to assess oxidative stress, senescence and changes in the epithelium