B75-10 Electronic Cigarette Extract Exaggerates Influenza A Virus-induced Cell Death and Disrupts Barrier Function in Alveolar Epithelial Cells

Abstract Background Electronic-cigarette (EC) usage in the United States is a growing public health concern. EC particles are deposited in the distal respiratory tract, in contrast to traditional cigarette smoke aerosols which primarily affect the proximal respiratory epithelium. Viral infections can be potentially life threatening, with studies estimating over 500,000 annual deaths globally because of seasonal influenza. Influenza A virus (IAV) can affect the entire bronchial tree; when contained to the upper respiratory tract, symptoms are mild. However, infection of the alveolar epithelium (AE) can have more serious consequences, including disruption of the alveolar epithelial barrier, gas exchange abnormalities, and pneumonia. Tight junctions, which are essential to maintaining barrier integrity in the alveolar epithelium, are primarily formed by claudins. Claudin-18.1 is the only lung-specific claudin, and deficiency in claudin-18.1 is associated with barrier dysfunction. Little is known about the interactions of ECE, IAV, and barrier function especially in the alveolar epithelium where EC deposits and IAV infects. Methods We used immortalized human alveolar epithelial (hAELVI) cells for all experiments, assessing multiple doses of e-Cigarette Extract (ECE). Barrier function was assessed by measuring transepithelial electrical resistance (TEER) in hAELVI cells grown at air-liquid interface (ALI) for 14 days. Infection was assessed using GFP-expressing IAV using CellProfiler, using a bespoke analytic pipeline to analyze the images, calculating IAV-positive cells, the infection percentage, and mean cellular fluorescence intensity. Cell death was assayed using CellTiterGlo 2.0, and programmed cell death via the RealTimeGlo Annexin V assay. Results hAELVI cells grown at ALI formed tight junctions as demonstrated by TEERs 1000. ECE treatment decreased TEER values of hAELVI cells grown at ALI by 51.8% (mean = 1098 vs 678, SD = 286 vs 297, p = 0.040 by Student’s T-test) after 24h treatment as compared to control. Concomitantly, we observed reduction in claudin-18.1 assessed by immunoblot with ECE treatment. In hAELVI grown in submerged culture, ECE pre-treatment (72h) followed by IAV infection (0.2 MOI) resulted in an ECE dose-dependent increase in programmed cell death at both 2h and 24h compared to IAV alone. Furthermore, IAV infection (0.2 MOI) had no effect on claudin levels, either with- or without-ECE, as assessed by immunoblot. Conclusion ECE exposure disrupts alveolar epithelial barrier integrity by reducing claudin-18.1 protein levels. Pre-exposure to ECE enhances IAV-induced programmed cell death in a dose-dependent manner. These findings suggest that ECE compromises alveolar defenses, potentially worsening viral respiratory infections. This abstract is funded by: 1TfR01ActfHL174649Projectf01A1Yearf