Neutrophil extracellular traps-induced pyroptosis of liver sinusoidal endothelial cells exacerbates intrahepatic coagulation in cholestatic mice

BACKGROUND: Neutrophil extracellular traps (NETs) and NOD-like receptor protein 3 (NLRP3) inflammation are key contributors to cholestatic liver disease (CLD). However, the relationship between NETs release and inflammasome activation, as well as its contribution to intrahepatic coagulation in CLD, remains unexplored. This study explores NETs-induced liver sinusoidal endothelial cells (LSECs) pyroptosis on intrahepatic coagulation in CLD. METHODS: mice underwent bile duct ligation (BDL) or sham surgery for 7 or 14 days. The liver analysis assessed intrahepatic coagulation, inflammation, fibrosis, NETs release, and NLRP3 activation. Primary LSECs were exposed to NETs with or without MCC950. Pyroptosis and LSECs procoagulant activity were quantified. RESULTS: BDL mice exhibited significantly increased inflammation, tissue factor (TF), and fibrin deposition compared with controls. NETs release in the liver was increased significantly in WT BDL mice and was responsible for intrahepatic coagulation. PAD4 deficiency reduced TF and fibrin expression, improving hepatic sinusoid function. RNA-seq revealed BDL-induced enrichment of coagulation, neutrophil activation, and pyroptosis pathways. In vivo, NETs increased intrahepatic NLRP3 and IL-1β expression in BDL mice. However, NLRP3 inhibition (MCC950) or activation (BMS-986299) did not alter NETs release. Furthermore, NETs-induced NLRP3 activation increased intrahepatic coagulation, inflammation, and fibrosis. Finally, we demonstrated that NETs triggered LSECs dysfunction and pyroptosis, upregulating TF and phosphatidylserine production and enhancing procoagulant activity. CONCLUSIONS: NETs-induced LSECs pyroptosis exacerbates intrahepatic coagulation in cholestasis. Targeting NETs and LSECs pyroptosis holds promise for treating chronic liver injury in CLD.