A cooperative release of mitochondrial DNA from platelets and neutrophils drives an interferon signature in systemic sclerosis

Objective Mitochondria are organelles with a hypomethylated circular genome. Mitochondrial DNA (mtDNA) in the systemic circulation has been implicated in inflammation. This study investigates the role of circulating DNA in systemic sclerosis (SSc) and the cellular mechanisms governing its release. Methods Total DNA was isolated from plasma of healthy individuals and SSc patients. Copy numbers were analyzed for mtDNA (ATP‐6) and GAPDH abundance by qPCR. mtDNA was isolated from HC and SSc patients. Neutrophils and platelets were incubated with SSc patients’ plasma and mtDNA, and NET formation was assessed by SytoxGreen and immunostainings. Platelets were tested for mtDNA release propensity. DNA oxidation was evaluated by MitoSOX Red staining in vitro and 8‐OHdG ELISA of patient plasma. Plasma IFN type 1 and CXCL4 were measured by ELISA. IFN signaling activation capacity was evaluated utilizing THP1 reporter cells and confirmed by a whole blood bulk RNA transcriptomic analysis. Results Median plasma mtDNA levels were 152‐fold higher in SSc patients compared to healthy controls (HC), while nDNA levels were similar. mtDNA from SSc plasma was highly oxidized. SSc‐derived mtDNA efficiently promoted its own release by NETosis, most potently in SSc patient neutrophils, and by platelet activation. Oxidized mtDNA from SSc platelets in complex with CXCL4 further stimulated mtDNA release in both neutrophils and platelets. mtDNA plasma concentrations correlated with type I IFN concentrations in SSc patient blood, and SSc blood exhibited elevated interferon‐stimulated gene (ISG) expression. SSc plasma‐derived mtDNA induced IFN signaling and NET formation via endosomal TLR, cGAS/STING and the JAK/STAT pathway. The type I IFN pathway further promoted NETosis and mtDNA release since IFN receptor (IFNAR) and Janus kinase (JAK) inhibition antagonized the proNETotic effects of IFN. Conclusion SSc plasma is characterized by highly abundant mtDNA, which drives feedback loops amplifying its own release from both neutrophils and platelets. Thus, mtDNA contributes to inflammation and tissue damage in SSc. image