Effect of cigarette smoke extract on cell-free DNA release from bronchial epithelial cells.

<bold>Background:</bold> Human bronchial epithelial cells (HBECs) are the first line of defence against inhaled insults including cigarette smoke and viruses. In response to insult, HBECs release alarmin proteins. Additionally, cell-free DNA (cfDNA) is released from virally infected HBECs. Cigarette smoke extract (CSE) can dampen down inflammatory responses to viruses, however, the effect of CSE on cfDNA release is unknown. This study examines the effect of CSE on viral mimic (Poly (I:C))-induced release of cfDNA and inflammatory cytokines. <bold>Methods:</bold> Immortalised HBECs (iHBECs) were treated with and without CSE (3%) and/or Poly(I:C) 10 μg/ml for 24 hours. Inflammatory mediator release was measured using Luminex® Multiplex assay. Quantitative real-time PCR for TERT and TapeStation were used to investigate the concentration and size of cfDNA released. <bold>Results:</bold> CSE inhibited the release of alarmins but increased CXCL8 release in response to Poly (I:C). Cell-free DNA was released in response to Poly (I:C) but not CSE. Co-stimulation with CSE and Poly (I:C) significantly increased the concentration of released cfDNA, relative to Poly (I:C) alone, (0.03901 and 0.03696 Cq respectively p<0.005, n=3). Released cfDNA was (336.55 bp, SD=10.64, n=3) bases in length which might indicate the DNA is released as part of extracellular vesicles (EVs). <bold>Conclusion:</bold> Cigarette smoke modulates the response of HBECs to viruses through altering inflammatory responses and releasing cfDNA. Future studies will establish the mechanism of cfDNA release and its functional effect on lung structural cells including airway smooth muscle.