Bioactive Plasma Mitochondrial DNA is Associated with Disease Progression in Scleroderma Associated Interstitial Lung Disease.

OBJECTIVE SSc-ILD is characterized by variable clinical outcomes, activation of innate immune pattern recognition receptors (PRRs), and accumulation of αSMA expressing myofibroblasts. The association of these entities with mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA-sensing PRRs TLR9 and cGAS-STING, has yet to be determined. METHODS Human lung fibroblasts from normal donors (NHLFs) and SSc-ILD explants were treated with synthetic CpG DNA and assayed for αSMA expression and extracellular mtDNA using qPCR for the human MT-ATP6 gene. Plasma MT-ATP6 concentrations were evaluated in two independent SSc-ILD cohorts and demographically matched controls. The ability of control and SSc-ILD plasma to induce TLR9 and cGAS-STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons, IL-6, and oxidized DNA were measured with using electrochemiluminescence and ELISA-based methods, respectively. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT-ATP6 concentrations and proteomics via liquid chromatography-mass spectrometry. RESULTS NHLFs and SSc-ILD fibroblasts develop increased αSMA expression and MT-ATP6 release following CpG stimulation. Plasma mtDNA concentrations are increased in two SSc-ILD cohorts, reflective of ventilatory decline, and positively associated with both TLR9 and cGAS-STING activation as well as Type I interferon and IL-6 expression. Plasma mtDNA is not oxidized and is conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. CONCLUSION These findings demonstrate an unrecognized connection between EV-encapsulated mtDNA, clinical outcomes, and intracellular DNA-sensing PRR activation in SSc-ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc-ILD and related disorders.