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A1201 - Cigarette Smoke Exposure of Airway Epithelial Cells Induces Specific NOTCH Signaling to Change the Balance of MUC5B and MUC5AC Expression
Author Block: N. Baumlin, J. S. Dennis, C. Aguiar, S. Paredes-Aller, A. Schmid, E. S. Mendes, M. Kim, M. Salathe; University of Miami, Miami, FL, United States.
Rational: Mucociliary clearance (MCC) is a major host defense mechanism in the airways. In patients with smoking-associated respiratory tract diseases such as chronic obstructive pulmonary disease (COPD) and chronic bronchitis, this mechanism is dysfunctional. Effective MCC requires an adequate airway surface liquid (ASL) volume for mucus hydration, and appropriate mucin composition (MUC5B vs. MUC5AC). Inadequate ASL volume increases mucus solids leading to mucociliary dysfunction. Here, we investigated the effect of cigarette smoke on NOTCH signaling and regulation of mucins in vitro and in vivo. We also investigated the effect of the anti-inflammatory drug losartan on smoke-mediated induction of MUC5AC in vitro and its benefits in vivo in smokers. Methods: Ex vivo: Human bronchial epithelial (HBE) cells were isolated from fresh tissue and processed for protein and mRNA. Tracheal rings were collected and embedded in paraffin. Sections were used for hematoxylin/eosin and immunofluorescence staining of MUC5B and MUC5AC. In vitro: HBE cells isolated from nonsmokers and smokers were re-differentiated at the air-liquid interface (ALI) for 3-4 weeks. Whole cigarette smoke (CS) exposure was executed with a smoking robot (Vitrocell). HBE cells were exposed to CS acutely (24 puffs once) and chronically (24 puffs, three times over 5 days) using 3R4F Kentucky research cigarettes. Mucus solids were determined using a microbalance. Losartan was administered throughout differentiation (10 µM). Protein and mRNA expression were performed as previously published. In vivo: nasal epithelial cells were collected using cytology brushes and processed for protein and mRNA isolation. Results: Ex vivo: Nonsmokers had more ciliated cells and less goblet cells compared to smokers. Histological expression of MUC5AC was more abundant in smokers, consistent with increased mRNA expression. On the other hand, MUC5B mRNA was decreased. NOTCH1 protein expression was upregulated in smokers. HES1, a downstream target of NOTCH signaling, was increased concomitantly. In vitro: Acute CS exposure increased mucus solids (rescued by losartan), protein expression of NOTCH1 and NOTCH3, and mRNA expression of MUC5B and MUC5AC. Chronic CS exposure downregulated MUC5B expression but upregulated MUC5AC. In vivo: three smokers were treated with 100 mg losartan for 2 months. Compared to the start of the study, their nasal cells showed reduced MUC5AC, TGF-β1 and MMP9 mRNA expression. Conclusion: Nonsmokers expressed different cells composition compared to smokers. CS upregulated NOTCH signaling thereby increasing MUC5AC predominantly. Long-term losartan use reduced mucus solids and MUC5AC mRNA expression. Thus, losartan could serve as a beneficial anti-inflammatory medication for smokers or ex-smokers.