Mitochondrial DNA-Mediated Immune Activation After Resuscitation from Cardiac Arrest

Background Post-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.

Plasma 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.

A 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 TNFα, IL-1β, 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.

These 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. What is new? Our 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. EV-encapsulated mtDNA triggers immune cell activation, evidenced by phenotypic shifts toward inflammatory dendritic cells and macrophages, as well as increased pro-inflammatory cytokine secretion. Pharmacological 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). What are the clinical implications? Identification 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. Therapeutic 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. Early 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. Competing Interest Statement The authors have declared no competing interest. Funding Statement The National Heart Lung and Blood Institute (1R01HL160538), the National Center for Advancing Translational Sciences (UL1TR001412), and the American Heart Association (24PRE1193924). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Statement Investigators were blinded to each patient and animal's experimental group (e.g., baseline vs. 1-hr post-ROSC vs. 4-hr post-ROSC) while performing blood sampling, in vitro experimentation, and data analysis. All experimental procedures and protocols conformed to institutional guidelines for the care and use of animals in research and were approved by the State University of New York at Buffalo Institutional Animal Care and Use Committee (ID: MED15083Y) and Institutional Review Board (ID: STUDY00000284). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability The data supporting the study's findings are available from the corresponding author upon reasonable request. Please see Supplemental Materials for complete methodological details. - Non-Standard Abbreviations and Acronyms - AMI - Acute Myocardial Infarction - ANOVA - analysis of variance - BI - brief ischemia - CM - conditioned media - CPR - cardiopulmonary resuscitation - dsDNA - double stranded DNA - DAMP - danger associated molecular pattern - EPI - epinephrine - EV - extracellular vesicle - FBS - fetal bovine serum - IQR - interquartile range - LAD - left anterior descending - LPS - lipopolysaccharide - MDV - mitochondria-derived vesicle - tDNA - mitochondrial - DNA nucDNA - nuclear DNA - OHCA - out-of-hospital cardiac arrest - PBMCs - peripheral blood mononuclear cells - PBS - phosphate buffered saline - PCAS - post cardiac arrest syndrome - ROS/RNS - reactive oxygen species/reactive nitrogen species - ROSC - return of spontaneous circulation - SBP - systolic blood pressur - SCA - sudden cardiac arrest - SEM - standard error of the mean - sR-OHCA - successfully resuscitated out-of-hospital cardiac arrest - TR - Transfection Reagent - TTM - targeted temperature management - VF - ventricular fibrillation - WBC - white blood cell