Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction

• Type of Material: Article
• Authors: Viranuj Sueblinvong, Emory University; Xian Fan, Emory University; Craishun Hart, Emory University; Samuel Molina, FUJIFILM Irvine Scientific; Michael Koval, Emory University; David M Guidot, Emory University
• Language: English
• Date: 2023-08-29
• Publisher: John Wiley and Sons
• Publication Version: Preprint (Prior to Peer Review)
• Copyright Statement: © 2023 The Authors. Alcohol: Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): https://doi.org/10.1111/acer.15174
• Title of Journal or Parent Work: Alcohol, Clinical and Experimental Research
• Volume: 47
• Issue: 10
• Start Page: 1839
• End Page: 1849
• Grant/Funding Information: National Heart, Lung, and Blood Institute, Grant/Award Number: T32 HL116271; National Institute on Alcohol Abuse and Alcoholism, Grant/Award Number: R03 AA027662-01A1, R01 AA025854 and R01 AA025857
• Keywords: epithelial barrier function; TGFβ1; alcohol; GM-CSF; co-culture
• Research Categories: Health Sciences, General; Biology, Cell

Abstract

Background: Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGFβ1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGFβ1 and GM-CSF and thereby alter airway epithelial barrier function. Methods: Human or rat LF were cultured ± ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFβ1 receptor type 1 (TGFβR1) inhibitor (SB431542) or a TGFβ1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGFβ1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA. Results: AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and α-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGFβ1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFβ1 activity. Conclusions: Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFβ1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.