Mitochondrial DNA Released by Biliary Epithelial Cells Enhances Inflammation in Biliary Atresia Through the Cyclic GMP-AMP Synthase–Stimulator of Interferon Genes–Nuclear Factor-κB Pathway

BACKGROUND & AIMS: Biliary atresia, the leading cause of infant cholangiopathy, features progressive inflammation and fibrosis with undefined pathogenesis. In biliary atresia, biliary epithelial cells act as both targets and drivers of hepatic inflammation. The cyclic GMP-AMP synthase-stimulator of interferon genes pathway, a cytosolic DNA-sensing system linking cellular stress to inflammation, may contribute to biliary atresia pathogenesis. This study investigated whether cyclic GMP-AMP synthase-stimulator of interferon genes activation in biliary epithelial cells promotes disease progression. METHODS: Cyclic GMP-AMP synthase-stimulator of interferon genes expression and localization were assessed in biliary atresia and control livers by quantitative reverse transcription polymerase chain reaction, Western blotting, immunohistochemistry, and immunofluorescence. Oxidative injury in human intrahepatic biliary epithelial cells was modeled with hydrogen peroxide. Mitochondrial DNA depletion with ethidium bromide, cyclic GMP-AMP synthase and stimulator of interferon genes inhibitors (RU.521, H151), and the stimulator of interferon genes agonist (2'3'-c-di-AM(PS)2 (Rp,Rp)) were used to investigate mechanisms. Coenzyme Q10 was evaluated in vitro using biliary organoids and in vivo in a Rhesus rotavirus-induced neonatal mouse model of biliary atresia. RESULTS: biliary epithelial cells, accompanied by nuclear factor-κB activation without detectable interferon regulatory factor 3 phosphorylation. Human intrahepatic biliary epithelial cells exposed to hydrogen peroxide showed mitochondrial DNA release, cyclic GMP-AMP accumulation, stimulator of interferon genes activation, and p65 phosphorylation, selectively increasing C-X-C motif chemokine ligand 10 and interleukin32, which were markedly attenuated by mitochondrial DNA depletion or cyclic GMP-AMP synthase and stimulator of interferon genes inhibition. Coenzyme Q10 suppressed the mitochondrial DNA-stimulator of interferon genes-nuclear factor-κB axis in vitro and ameliorated liver injury while improving survival in vivo. CONCLUSIONS: Mitochondrial DNA leakage activates the cyclic GMP-AMP synthase-stimulator of interferon genes-nuclear factor-κB pathway in biliary epithelial cells, driving a C-X-C motif chemokine ligand 10- and interleukin32-dominant inflammatory response. Coenzyme Q10 mitigates this process and injury, supporting cyclic GMP-AMP synthase-stimulator of interferon genes modulation and mitochondrial stabilization as promising therapeutic strategies for biliary atresia.