Extracellular Vesicle-Encapsulated Mitochondrial DNA Drives Immune Activation in Post-Cardiac Arrest Syndrome

Objective: Systemic inflammation is a pivotal component of post-cardiac arrest syndrome (PCAS) and contributes to poor outcomes among PCAS patients. Mitochondrial DNA (mtDNA) has recently emerged as a key trigger of immune activation following return of spontaneous circulation (ROSC) after cardiac arrest, but the mechanisms underlying mtDNA uptake and detection by innate immune cells remain unclear. Accordingly, we studied the immunomodulatory effects of post-ROSC plasma and extracellular vesicles (EVs) by selectively digesting EVs (lysis) and removing free-floating nucleotides (DNase I) to evaluate mechanisms of immune activation in porcine peripheral blood mononuclear cells (PBMCs). Methods: Blood plasma samples were collected from swine (n=3) before (baseline; BL) and 4 hours after resuscitation from 10 min of electrically-induced cardiac arrest. Plasma and EV depletion assays were conducted with naïve PBMCs (250k cells/well) in a 24-hour activation assay (biologic n=3, technical n=3) using 500µL of BL plasma, post-ROSC plasma, or post-ROSC plasma EVs with or without lysis buffer, DNase I, and/or mtDNA transfection (TR). Native mtDNA concentrations were verified via qPCR. Immune activation was assessed by flow cytometry (inflammatory cell quantification), ELISA (release of TNFα, IL-1β, IL-6, and IL-8), and qPCR (expression of NF-κB, TNFα, IL-1α, IL-1β, IL-6, IL-8, IFNα, and IFNβ). Results: Post-ROSC plasma increased PBMC immune activation compared to BL plasma, as shown by elevated inflammatory gene expression (NF-kB, TNFα, IL-1α, IL-1β, IL-6, IL-8, IFNα, IFNβ), cytokine secretion, and cell markers (CD14/CD163/CD172). Plasma lysis treatment alone reduced inflammatory gene expression of most targets, while DNase I affected NF-kB expression only. The addition of mtDNA+TR after lysis+DNase I restored inflammatory gene expression and cytokine secretion, highlighting the key role of mtDNA in post-ROSC plasma-induced PBMC activation. Flow cytometry confirmed that inflammatory cell enrichment was attenuated by plasma lysis but not DNase I, indicating that EV integrity, rather than free-floating DNA, drives mtDNA uptake and activation. Post-ROSC plasma EVs similarly activated PBMCs compared with BL EVs, as shown by increased inflammatory gene expression, cytokine secretion, and inflammatory cell markers. Plasma EV lysis alone significantly reduced IL-1α gene expression and tended to decrease expression of other genes, while lysis+DNase I treatment effects were reversed by addition of mtDNA+TR. ELISA showed that plasma EV lysis alone decreased TNFα and IL-8 secretion, with IL-1β reduced only by combined lysis+DNase I. Inflammatory responses, including cytokine release and cell marker enrichment, were restored by mtDNA+TR, further underscoring the importance of intact EV-encapsulated mtDNA in post-ROSC plasma-induced PBMC activation. Conclusion: This study demonstrates that EV-encapsulated mtDNA is essential for innate immune activation in PCAS, as intact EVs within post-ROSC plasma drive inflammatory gene expression, cytokine release, and immune cell activation. The dependence on EV uptake, rather than free-floating DNA, suggests that targeting EV uptake pathways could be a promising therapeutic strategy to mitigate mtDNA-mediated inflammation and improve outcomes in PCAS patients. Supported by funding from the NIH, AHA, and US Department of Veteran's Affairs. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.