Abstract Body: Objective: Post-cardiac arrest syndrome (PCAS) is characterized by a rapid, dysregulated inflammatory response that exacerbates organ injury and undermines recovery. Mitochondrial DNA (mtDNA) has recently been identified as a potent stimulus of post-resuscitation immune cell activation via engagement of pattern recognition receptors (PRRs; TLR9 and cGAS). However, the extent to which mtDNA drives transcriptional changes that underlie leukocyte activation in PCAS is unclear. Accordingly, we performed transcriptomic profiling of porcine peripheral blood mononuclear cells (PBMCs) activated by mtDNA ex vivo and compared these profiles to PBMCs endogenously activated in vivo during PCAS.

Methods: Swine (n=5) underwent 10min of cardiac arrest and subsequent resuscitation. PBMCs were isolated at baseline and 4h after return of spontaneous circulation (ROSC; in vivo) or cultured ex vivo for 4h with either transfection reagent (TR; control) or 2µg/mL mtDNA+TR. Total RNA was isolated and subjected to bulk RNA-sequencing.

Results: PBMCs collected 4h post-ROSC showed 1,557 upregulated and 960 downregulated genes vs. baseline. CXCL10 (log2FC 2.9), TNFα (2.2), IL1α (4.7), and IL10 (2.4) were significantly upregulated, with TLR9-MyD88 and IRF3 pathway activation. Ex vivo mtDNA stimulation resulted in 948 upregulated and 1,343 downregulated genes, with enrichment of antiviral and cytosolic DNA-sensing pathways (ZBP1, TREX1, IRF7, CXCL10, CCL4), including increased cGAS expression (2.7-fold). Although the 26 downregulated genes shared between PCAS and mtDNA exposure lacked significant functional enrichment, the 98 shared upregulated genes included key pro-inflammatory cytokines (TNFα, IL6, IL10, CXCL10) and additional immune regulators (e.g., IL27, CXCL9/10/11, CCL3L1, SOCS1, IRF1/7, STAT1/2), suggesting convergence on NF-κB and JAK-STAT signaling pathways.

Conclusion: PBMCs activated in vivo following resuscitation from cardiac arrest and ex vivo with mtDNA exhibit distinct upstream PRR transcriptional profiles yet consistently engage cytosolic DNA sensing and antiviral response pathways. Key mediators, including ZBP1, TREX1, IRF7, STAT2, and CXCL10, were upregulated across both conditions, underscoring mtDNA as a key driver of innate immune activation in PCAS. These findings highlight a shared inflammatory axis governed by cytosolic DNA sensing, which may be mechanistically and therapeutically relevant in modulating immune responses following cardiac arrest.