Mitochondrial DNA-Mediated Immune Activation After Resuscitation from Cardiac Arrest

BackgroundPost-cardiac arrest syndrome (PCAS) is characterized by a robust inflammatory response that contributes to significant morbidity and mortality among patients resuscitated from sudden cardiac arrest (SCA). Mitochondrial DNA (mtDNA), with its bacterial-like genomic motifs, has been implicated as a damage-associated molecular pattern in other inflammatory contexts, but its role as a pro-inflammatory stimulus in PCAS has not been studied. Accordingly, the present study was designed to determine if PCAS is characterized by a rise in circulating mtDNA and, if so, whether mtDNA is selectively released, how it activates immune cells, and if targeting mtDNA-sensing pathways attenuates leukocyte activation. MethodsPlasma mtDNA and nuclear DNA (nucDNA) levels were measured in peripheral blood samples collected [~]4-hours post-ROSC from swine with PCAS (n=8) and patients hospitalized after resuscitation from out-of-hospital cardiac arrest (OHCA; n= 57). Additionally, in vitro studies were performed where porcine peripheral blood mononuclear cells (PBMCs) were treated with mtDNA or extracellular vesicles (EVs) isolated from post-ROSC plasma. Pharmacological inhibitors were utilized to inhibit toll-like receptor 9 (TLR9)- and cyclic GMP-AMP synthase (cGAS)-mediated mtDNA sensing. ResultsA significant [~]250-fold elevation in circulating mtDNA was observed shortly after ROSC in swine despite negligible changes in circulating nucDNA, suggesting selective release of mtDNA in PCAS. This finding was corroborated in human OHCA survivors, in which circulating mtDNA was similarly elevated during the early post-ROSC period. Circulating mtDNA was largely encapsulated within EVs in swine and humans, suggesting a conserved mechanism of release across species. In vitro studies demonstrated that PBMC internalization of mtDNA-containing-EVs was required for immune activation and promoted development of a pro-inflammatory leukocyte phenotype characterized by altered surface marker expression and increased release of TNF, IL-1{beta}, and IL-6. Disrupting EVs or degrading enclosed DNA attenuated these responses, which were partially restored upon reintroduction of mtDNA. Pharmacological blockade of TLR9 or cGAS pathways significantly reduced mtDNA-induced inflammation, providing insight regarding signaling pathways that may be targeted to modulate mtDNA-mediated immune activation in PCAS. ConclusionsThese novel findings demonstrate that brief whole-body ischemia and reperfusion in the context of resuscitation from SCA triggers selective mtDNA release, primarily within EVs, that acts as a potent driver of immune activation in PCAS. By linking EV-encapsulated mtDNA to TLR9 and cGAS activation, this study provides a foundation for the development of novel therapeutic interventions aimed at limiting mtDNA release or disrupting its downstream sensing pathways to enhance survival and improve outcomes after SCA. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIOur study reveals that circulating mitochondrial DNA (mtDNA), primarily encapsulated in extracellular vesicles (EV), is selectively released into the bloodstream after resuscitation from sudden cardiac arrest. C_LIO_LIEV-encapsulated mtDNA triggers immune cell activation, evidenced by phenotypic shifts toward inflammatory dendritic cells and macrophages, as well as increased pro-inflammatory cytokine secretion. C_LIO_LIPharmacological inhibition of TLR9 and cGAS pathways significantly attenuates the mtDNA-induced inflammatory response, pointing to novel therapeutic avenues for modulating post-resuscitation immune activation in patients with post-cardiac arrest syndrome (PCAS). C_LI What are the clinical implications?O_LIIdentification of mtDNA as a key driver of sterile inflammation in PCAS highlights a potential target for interventions aimed at reducing multi-organ damage and improving neurological outcomes. C_LIO_LITherapeutic strategies to block mtDNA release or downstream signaling (e.g., TLR9/cGAS inhibition) may limit harmful pro-inflammatory cascades and bolster long-term survival following resuscitation from cardiac arrest. C_LIO_LIEarly clinical screening for elevated EV-encapsulated mtDNA could help refine prognostic evaluations, complement current biomarkers, and guide personalized therapy to lessen the inflammatory burden of PCAS. C_LI