Epigenetic Regulation of Tollip by Cigarette Smoke Exposure Impairs Human Lung Defense: Unlocking the Mystery of Viral Infection

Abstract Rationale: Cigarette smoking is a primary cause of chronic obstructive pulmonary disease (COPD). Smokers have a higher risk of influenza-related mortality, but the underlying mechanisms remain unclear. Toll-interacting protein (TOLLIP) is an important immune regulator that plays a crucial role in host defense against pathogens. Studies have shown that TOLLIP effectively inhibits Influenza A virus (IAV) infection. So far, the role of TOLLIP in smoking-related pathologies, particularly during COPD exacerbations, remains largely unexplored. Additionally, epigenetic modifications, such as histone methylation, have been observed in COPD that may affect the expression of multitude of genes. In this study, we hypothesized that cigarette smoke (CS) exposure upregulates the histone methyltransferases EZH1 and EZH2, which in turn suppresses TOLLIP expression and weakens the host defense against IAV infection in human lungs. Methods: TOLLIP and histone-methylating enzymes EZH1 and EZH2 were measured in healthy and COPD human lungs, cultured human airway epithelial cells and precision-cut lung slices (PCLS) exposed to CS with or without IAV infection. EZH1 and EZH2 siRNA and inhibitors were utilized to investigate their effects on TOLLIP expression. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed to evaluate chromatin accessibility at the TOLLIP locus following CS exposure. Results: In COPD patients, TOLLIP levels decreased, while EZH1 and EZH2 expression increased. Repeated CS exposure decreased TOLLIP levels and increased EZH1, EZH2, H3K27me3 and IAV levels in cultured human airway epithelial cells and PCLS. EZH1/EZH2 siRNA or their pharmacological inhibitor valemetostat tosylate in part restored TOLLIP expression and reduced IAV levels in CS-exposed airway epithelial cells and PCLS. Chromatin accessibility (indicated by the height of peaks) at the TOLLIP locus was reduced after CS exposure. Conclusion: The present study reveals, for the first time, that repeated CS exposure reduces TOLLIP expression in part through EZH1/EZH2-H3K27me3-mediated epigenetic mechanisms. Targeting EZH1 and EZH2 may serve as a promising therapeutic strategy to restore TOLLIP expression and host defense against viral infections in COPD patients. Keywords: Cigarette smoke, COPD, IAV, TOLLIP, Histone methylation Acknowledgments: The authors acknowledge BioRender for providing the figure template. During the preparation of this work the authors used ChatGPT to improve language and readability. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.