Phase-dependent dynamics of circulating cell-free mitochondrial DNA reflect distinct immunometabolic states

Circulating cell-free mitochondrial DNA (ccf-mtDNA) has emerged as a potential biomarker of tissue injury and systemic inflammation, acting as a mitochondrial damage-associated molecular pattern (DAMP) capable of activating innate immune pathways. However, most studies have focused on acute inflammatory conditions, and the behavior of ccf-mtDNA across distinct immunological and metabolic disease states remains poorly characterized, particularly in pediatric populations. This study investigated whether circulating ccf-mtDNA levels vary according to the clinical and immunometabolic context rather than simply reflecting the presence of inflammation. Serum ccf-mtDNA copy number was quantified by quantitative polymerase chain reaction (qPCR) targeting the mitochondrial ND2 gene in 181 clinical samples obtained from five groups: adults with chronic-active and/or treatment-refractory pulmonary tuberculosis (n = 47); asymptomatic children with latent tuberculosis infection confirmed by interferon-gamma release assay (IGRA) without clinical, radiological or microbiological evidence of active disease (n = 11); children with severe chronic underlying diseases in clinically stable condition (n = 41); children undergoing cardiac surgery with cardiopulmonary bypass (CPB), with perioperative serial sampling (n = 52); and healthy young adult blood donors as controls (n = 30). Non-parametric statistical tests were applied due to non normal data distribution. Median ccf-mtDNA levels in controls were 649.3 copies/μL. Adults with chronic-active pulmonary and/or treatment-refractory tuberculosis and children undergoing cardiac surgery with CPB did not exhibit significantly elevated ccf-mtDNA levels compared with controls. In contrast, significantly higher levels were observed in IGRA-positive children with latent tuberculosis infection (median: 1,648.5 copies/μL; p = 0.0004) and in children with severe chronic underlying diseases despite the absence of overt infection or inflammation (median: 2,663.9 copies/μL; p = 0.0001). These findings suggest that circulating ccf-mtDNA does not behave as a simple linear marker of inflammatory intensity. Instead, its levels appear to reflect the interaction between mitochondrial injury, immune activation, metabolic competence, and clearance mechanisms. We propose a phase-dependent model in which mitochondrial DAMP signaling is amplified during sustained but metabolically competent immune engagement, and is attenuated during advanced immunometabolic exhaustion. These findings suggest that ccf-mtDNA levels are shaped by the host's underlying immunometabolic context, rather than simply mirroring the intensity of systemic inflammation.