Effect of cigarette smoke on airway inflammation and mucus hypersecretion in submerged and well-differentiated airway epithelial cells

Abstract

Introduction: Cigarette smoke (CS) is one of the major risk factors for the development of chronic obstructive pulmonary disease. To understand the pathogenesis and mechanism of CS-induced airway injury, submerged culture is widely implemented as a model for primary human airway epithelial cell research. However, a more effective and reliable model should be developed to replicate the characteristics of in vivo airway epithelia. We aimed to compare CS-induced inflammation and mucus hypersecretion in submerged and well-differentiated airway epithelial cells. Methods: Primary normal human bronchial epithelial cells were cultured in either submerged condition in growth factor–supplemented medium to sub-confluence or differentiated at the air-liquid interface (ALI) for 28 days. The CS medium was directly applied to submerged culture or at the apical side of well-differentiated normal human bronchial epithelial cells for 24 hours (n=4). The expression of interleukin (IL)-6, IL-8, and MUC5AC were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Results: The basal secretions of both IL-6 and IL-8 were significantly greater in the supernatant of the basolateral side of the ALI culture than in the submerged culture (IL6: 812.5 ± 105.0 pg/mL vs 100.7 ± 7.5 pg/mL, P<0.001; IL-8: 28 955.0 ± 672.0 pg/mL vs 111.8 ± 10.2 pg/mL, P<0.001). Both IL-6 and IL-8 mRNA and protein were upregulated after CS medium exposure in both submerged and ALI culture. The CS medium also caused upregulation of MUC5AC mRNA in a dose-dependent manner with higher expression in ALI culture compared with submerged culture, in line with the observation of MUC5AC mucin elevation in ALI culture only. Conclusion: Although CS induces airway inflammation and mucus hypersecretion in both submerged and ALI culture, ALI culture more closely resembles the in vivo environment with more serious inflammation and mucus secretion.