Mitochondrial DNA-Laden Extracellular Vesicles are Associated with Toll-Like Receptor 9 Activation and Disease Progression in Scleroderma Associated Interstitial Lung Disease

Background: The pathogenesis of scleroderma associated interstitial lung disease (SSc-ILD) involves the uncontrolled differentiation of normal human lung fibroblasts (NHLFs) to alpha-smooth muscle actin (αSMA) expressing myofibroblasts mediated by transforming growth factor-β1 (TGF-β1). SSc-ILD has a highly variable clinical trajectory as some patients experience rapid deterioration in their ventilatory function while others remain stable. The association between the toll-like receptor 9 (TLR9) ligand mitochondrial DNA (mtDNA) and fibroblast activation and clinical outcomes in SSc-ILD has yet to be explored. Methods: NHLFs grown in the absence and presence of TGFβ1, CpG DNA, and chloroquine were assayed for αSMA expression and extracellular mtDNA release using qPCR for the human MT-ATP6 gene. This approach was then used to detect plasma mtDNA concentrations in two SSc-ILD cohorts, Boston and Yale, and demographically matched controls. TLR9 activation was quantified using commercially available human TLR9-expressing HEK 293 cells. Isolation of extracellular vesicles (EVs) were performed in the plasma from controls and Yale SSc-ILD subjects, and EVs were analyzed for mtDNA content and proteomics via mass spectrometry. Findings: TLR9 activity modulates αSMA expression and extracellular mtDNA release. Plasma mtDNA concentrations are increased in two independent SSc-ILD cohorts and activates TLR9. Longitudinal analysis reveals that plasma mtDNA predicts ventilatory decline and is cargoed by EVs. Proteomic analysis reveals a multicellular origin of EVs that involves cellular pathways that could act as mediators of the SSc-ILD state. Interpretation: These findings demonstrate an unrecognized connection with TLR9 activation mediated by extravesicular mtDNA and myofibroblast differentiation and clinical outcomes in SSc-ILD. Further study into the interactions between TLR9 and mtDNA could lead to novel mechanistic and therapeutic insights in this difficult to manage disease. Funding Statement: Parker B. Francis Foundation, Foundation for Sarcoidosis Research, Scleroderma Foundation, American Thoracic Society, National Institutes of Health, Gabriel and Alma Elias Research Fund, and Greenfield Foundation. Declaration of Interests: The authors have no relevant conflicts of interest to disclose. Ethical Approval Statement: All human studies were performed with informed consent on protocols approved by the Institutional Review Board at each institution.