Epithelial effects of exposure to e-cigarettes alone or in combination with cigarette smoke using multicellular lung mucosa models

Pulmonary molecular toxicity data on electronic (e)-cigarettes, alone or in combination with cigarette smoke, remain limited. We developed air-liquid interface bronchial and alveolar lung mucosa models using primary human bronchial epithelial cells and human type II alveolar epithelial cell lines, respectively. Models were exposed to air (sham) or e-cigarette aerosol (15 min ×3 at one-hour intervals) alone and in combination (dual exposure) with 0.5% cigarette smoke condensate (CSC). Transcript levels of epithelial junction (CLDN1), ciliogenesis (FOXJ1), cilia function (DNAI1), ion channels (CFTR, KCNMA1), cytokines (IL6, IL8, IL10, IL13, IL1B), oxidative stress (HMOX1, GSTA1), and injury/repair markers (MMP9, TIMP1) were assessed by qRT-PCR at 6 h. IL6 and TIMP1 proteins were measured by ELISA at 24 h, and ROS and TLR expression by flow cytometry at 2 and 24 h, respectively. In alveolar models, CLDN1 expression decreased after e-cigarette exposure, while OCLN, JAMA, and CDH1 expression increased after CSC exposure. In bronchial models, mucociliary and ion channel genes (FOXJ1, DNAI1, CFTR, KCNMA1) increased after e-cigarette or dual exposures. IL6, IL13, and HMOX1 were increased in bronchial models, whereas alveolar models showed reduced IL8, IL10, HMOX1, MMP9, and TIMP1 expression after e-cigarette exposure, with MMP9 and TIMP1 also decreased after dual exposures. IL6 and TIMP1 protein levels increased in bronchial models. Both models showed higher ROS and TLR expression after CSC and dual exposures. These findings demonstrate lung-region-specific epithelial responses to e-cigarette and CSC, highlighting the potential long-term respiratory risks and need for further research. IMPACT STATEMENT: This study shows that e-cigarette and CSC elicit distinct, region-specific epithelial injuries in human multicellular bronchial and alveolar ALI models, altering barrier, mucociliary function, inflammatory, and oxidative pathways. These mechanistic insights strengthen respiratory risk assessment and can guide regulatory evaluation of emerging inhaled products.