Mitochondrial DNA and Sex Differences in Scleroderma-Associated ILD

ABSTRACT Scleroderma (SSc) is a multisystem disease characterized by cutaneous and visceral fibrosis that most commonly affects the lung. Nearly 70% of patients with SSc experience clinically significant interstitial lung disease (SSc-ILD) that in 50% will lead to death within ten years of diagnosis. For unknown reasons, women are more commonly affected, yet men have worse clinical outcomes. Defining the molecular mechanisms of SSc- ILD sex differences may eradicate these disparities and improve patient care. SSc-ILD is believed to result from immune-driven accumulation of transforming growth factor beta (TGFβ)-1 responsive, extracellular matrix (ECM)-producing inflammatory fibroblasts. Innate immune pattern recognition receptors and their endogenous ligands are implicated in this process. The endosomal innate immune receptor Toll-Like Receptor 9 (TLR9), which recognizes and responds to danger associated molecular patterns such as mitochondrial DNA (mtDNA), has a recently discovered role in SSc-ILD. We found that SSc-ILD fibroblasts exhibit spontaneous mtDNA release and TLR9 activation. Extracellular mtDNA concentrations are increased in SSc-ILD bronchoalveolar lavage and plasma where they predict disease progression. Relative to their male counterparts, women with SSc-ILD show plasma mtDNA that is more bioactive and lung explants that are enriched for TLR9-responsive inflammatory genes. In chronic SSc-ILD mouse models, females show greater mtDNA bioactivity, TLR9 activation, and a stronger therapeutic response to pharmacologic TLR9 inhibition. The heightened inflammation- promoting consequences of mtDNA-TLR9 interaction in women could explain sex differences in clinical phenotypes experienced by SSc-ILD patients. Our world class team of experts will test the hypothesis that differential interactions between extracellular mtDNA and TLR9 drive SSc-ILD sexual dimorphism. Aim 1 will advance the study of extracellular mtDNA, TLR9, and SSc-ILD sex differences by a) characterizing extracellular mtDNA and TLR9 activation in SSc-ILD BAL samples from women and men; and b) using single nuclear sequencing and protein-based assays to compare TLR9 expression/activation in archived, explanted lung tissue specimens from women and men with SSc-ILD. Aim 2 will define mechanisms of sexual dimorphism in the extracellular mtDNA-TLR9 relationship in experimental mouse models of chronic lung fibrosis using a) pharmacologic and b) genomic methods in a tamoxifen-inducible model of fibroblast-specific Tlr9 deletion and two chronic models of SSc lung fibrosis. Aim 3 will probe sex differences in fibrogenic extracellular mtDNA-TLR9 interactions in human lung fibrosis using genetic and pharmacologic strategies in primary lung fibroblasts and a next generation model of lung fibrosis based on TGFβ1-stimulated human lung cores. If successful, these studies will frame extracellular mtDNA-TLR9 interactions as drivers of SSc-ILD sexual dimorphism and identify mechanisms that can be leveraged for the betterment of human health.