Method for depletion of mitochondria DNA in human bronchial epithelial cells.

Mitochondria are increasingly recognized to play a role in the airway inflammation of asthma. Model systems to study the role of mitochondrial gene expression in bronchial epithelium are lacking. Here, we create custom bronchial epithelial cell lines that are depleted of mitochondrial DNA. One week of ethidium bromide (EtBr) treatment led to ∼95 % reduction of mtDNA copy number (mtDNA-CN) in cells, which was further reduced by addition of 25 µM 2',3'-dideoxycytidin (ddC). Treatment for up to three weeks with EtBr and ddC led to near complete loss of mtDNA. The basal oxygen consumption rate (OCR) of mtDNA-depleted BET-1A and BEAS-2B cells dropped to near zero. Glycolysis measured by extracellular acidification rate (ECAR) increased ∼two-fold in cells when mtDNA was eliminated. BET-1A ρ0 and BEAS-2B ρ0 cells were cultured for two months, frozen and thawed, cultured for two more months, and maintained near zero mtDNA-CN. Mitochondrial DNA-depleted BET-1A ρ0 and BEAS-2B ρ0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis.•BET-1A and BEAS-2B cells were treated with ethidium bromide (EtBr) with or without 2',3'-dideoxycytidine (ddC) to create cells lacking mitochondrial DNA (mtDNA).•Cells' mtDNA copy number relative to nuclear DNA (nDNA) were verified by quantitative polymerase chain reaction (qPCR).•Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer.

Mitochondrial DNA Copy Number Variation in Asthma Risk, Severity, and Exacerbations.

Rationale: Although airway oxidative stress and inflammation are central to asthma pathogenesis, there is limited knowledge of the relationship of asthma risk, severity, or exacerbations to mitochondrial dysfunction, which is pivotal to oxidant generation and inflammation. Objectives: We investigated whether mitochondrial DNA copy number (mtDNA-CN) as a measure of mitochondrial function is associated with asthma diagnosis, severity, oxidative stress, and exacerbations. Methods: We measured mtDNA-CN in blood in two cohorts. In the UK Biobank (UKB), we compared mtDNA-CN in mild and moderate-severe asthmatics to non-asthmatics. In the Severe Asthma Research Program (SARP), we evaluated mtDNA-CN in relation to asthma severity, biomarkers of oxidative stress and inflammation, and exacerbations. Measures and Main Results: In UK Biobank, asthmatics (n = 29,768) have lower mtDNA-CN compared to non-asthmatics (n = 239,158) (beta, -0.026 [95% CI, -0.038 to -0.014], P = 2.46×10-5). While lower mtDNA-CN is associated with asthma, mtDNA-CN did not differ by asthma severity in either UKB or SARP. Biomarkers of inflammation show that asthmatics have higher white blood cells (WBC), neutrophils, eosinophils, fraction exhaled nitric oxide (FENO), and lower superoxide dismutase (SOD) than non-asthmatics, confirming greater oxidative stress in asthma. In one year follow-up in SARP, higher mtDNA-CN is associated with reduced risk of three or more exacerbations in the subsequent year (OR 0.352 [95% CI, 0.164 to 0.753], P = 0.007). Conclusions: Asthma is characterized by mitochondrial dysfunction. Higher mtDNA-CN identifies an exacerbation-resistant asthma phenotype, suggesting mitochondrial function is important in exacerbation risk.