Chronic e-cigarette aerosol exposure dysregulates NF-kB signaling and alters IRF-dependent innate immune signaling in human lung epithelial cells

Background: Although electronic cigarettes (e-cigarettes) are marketed as safer alternatives to combustible tobacco, accumulating evidence indicates that chronic use may impair respiratory health. E-cigarette aerosols contain harmful constituents, including nicotine, carbonyl compounds, metals, carcinogens, and reactive oxygen species (ROS). Excess ROS can overwhelm antioxidant defenses, driving oxidative stress and chronic inflammation. NF-κB is a central regulator of inflammation and antiviral immunity, yet its modulation by chronic exposure to e-cigarette aerosol remains poorly defined. Objective: To determine how chronic exposure to e-cigarette aerosols alters NF-κB signaling and downstream inflammatory and antiviral pathways in lung epithelial cells. Methods: Normal human bronchial epithelial cells (NuLi-1) were exposed to aerosol extracts generated from two commercial tobacco-flavored e-cigarette products every other day for 2 weeks. Exposure delivered approximately 30 ng/ml of nicotine to mimic the median levels observed in the plasma of e-cigarette users. NF-κB mRNA levels were measured by real-time RT-qPCR. Protein expressions of NF-κB, TLR3, TLR4, IRF1, IRF3 and IRF7 were quantified by Western blotting. Statistical significance was evaluated by Student's t -test. Results: Chronic e-cigarette aerosol exposure significantly upregulated NF-κB mRNA and protein levels. TLR3, TLR4, and IRF7 proteins were also elevated after chronic exposure to e-cigarette aerosols, indicating activation of stress- and pathogen-sensing pathways. In contrast, IRF1, and IRF3 protein expression were significantly decreased, consistent with suppression of key antiviral transcription factors. This pattern reflects an inflammatory shift characterized by NF-κB activation with concurrent impairment of IRF-dependent innate immune signaling. Conclusion: Chronic e-cigarette aerosol exposure induces a dysregulated inflammatory phenotype in human lung epithelial cells, marked by NF-κB activation and suppression of IRF-mediated antiviral pathways. These findings suggest that chronic e-cigarette use may compromise epithelial innate immunity and increase susceptibility to respiratory infections and inflammatory disease. Grant support: NIH/NCI (R01CA242168, Queimado); TSET HPRC (Ganapathy); NHI/NIGMS (U54GM104938-140).