Extracellular release of mitochondrial DNA is triggered by cigarette smoke and is detected in COPD

Chronic obstructive pulmonary disease (COPD) is characterized by continuous and irreversible inflammation frequently caused by persistent exposure to toxic inhalants such as cigarette smoke (CS). CS may trigger mitochondrial DNA (mtDNA) extrusion into the cytosol, extracellular space, or foster its transfer by extracellular vesicles (EVs). The present study aimed to elucidate whether mtDNA is released upon CS exposure and in COPD. We measured cell-free mtDNA (cf-mtDNA) in the plasma of former smokers affected by COPD, in the serum of mice that developed CS-induced emphysema, and in the extracellular milieu of human bronchial epithelial cells exposed to cigarette smoke extract (CSE). Further, we characterized cells exposed to sublethal and lethal doses of CSE by measuring mitochondrial membrane potential and dynamics, superoxide production and oxidative stress, cell cycle progression, and cytokine expression. Patients with COPD and mice that developed emphysema showed increased levels of cf-mtDNA. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased superoxide production and oxidative damage, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles. The release of mtDNA into the extracellular milieu occurred concomitantly with increased expression of DNase III, DNA-sensing receptors (cGAS, NLRP3), proinflammatory cytokines (IL-1β, IL-6, IL-8, IL-18, CXCL2), and markers of senescence (p16, p21). Exposure to a lethal dose of CSE preferentially induced mtDNA and nuclear DNA release in cell debris. Our findings demonstrate that CS-induced stress triggers mtDNA release and is associated with COPD, supporting cf-mtDNA as a novel signaling response to CS exposure. ### Competing Interest Statement The authors have declared no competing interest.