Effect of cigarette smoke on airway inflammation, mucus hypersecretion and endoplasmic reticulum stress in a three-dimensional co-culture model of airway epithelium and smooth muscle

Abstract

Introduction: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally, in which cigarette smoke (CS) is the major risk factor. To investigate CS-induced effects, two-dimensional (2D) submerged culture model was widely performed as the model for primary airway cells research, which could not reproduce the characteristics of respiratory tract in vivo. In addition, the interaction between airway epithelium and smooth muscle in response to CS is still unclear. Therefore, there is a need for setting up a robust and reliable three-dimensional (3D) model to study the interaction between airway epithelium and smooth muscle recapitulating comparable characteristic of respiratory tract in vivo for CS exposure. We aimed to investigate CS-induced airway inflammation, mucus hypersecretion and endoplasmic reticulum (ER) stress in the well-differentiated co-culture model. Methods: Primary normal human bronchial epithelial cells (HBECs) differentiated at the air-liquid interface (ALI) for 28 days were co-cultured with normal human airway smooth muscle cells (HASMCs). Cigarette smoke medium (CSM) was directly applied to the apical side of well-differentiated HBECs for 24 hours (n=4). The inflammatory, mucin and ER stress markers were assessed by qPCR, ELISA and Western blot assays. Results: A functional multilayer epithelium consisting of basal cells, goblet cells and ciliated cells was successfully developed under ALI culture. Co-culture of well-differentiated airway epithelium and smooth muscle showed higher sensitivity in response to CS, with significantly increase in airway inflammation and mucus hypersecretion in comparison with single cell culture model. CS could also cause induction of ER stress via activation of the unfolded protein response (UPR) pathway activating transcription factor 6 (ATF6) in the co-culture model. Conclusion: The current findings provide evidence of interaction between airway epithelium and smooth muscle, leading to greater responses in CS-induced airway inflammation and mucus hypersecretion via regulation of UPR-ATF6 pathway. Acknowledgement: This work was supported by YC Chan Scientist Award.