Neutrophil extracellular traps-mediated thrombosis drive pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome

Hepatic sinusoidal obstruction syndrome (HSOS) caused by pyrrolizidine alkaloid (PA) is a life-threatening disease with limited treatments, yet the initiating vascular event remains undefined. Neutrophil extracellular traps (NETs), as key driver of immunothrombosis in various thromboinflammatory diseases, may represent a critical yet uncharacterized component in the pathogenesis of PA-HSOS. Rats and mice were separately administered monocrotaline (MCT) or senecionine (SEN) via gavage to induce HSOS. Human hepatic sinusoidal endothelial cell (HHSEC) and primary human neutrophil treated with MCT, SEN, or corresponding metabolites were used as in vitro models. In separate experiments, animals received a neutrophil elastase (NE) inhibitor or neutrophil-depleting antibody to evaluate the contribution of NETs. We detected NETs within the sinusoids of necrotic liver lobes, as evidenced by robust immunostaining for NETs markers, accompanied with elevated level of circulating cell-free DNA, and hepatic citrullinated histone H3. Sinusoidal microthrombi containing VWF, CD41, and fibrin(ogen) were also stained positive for neutrophils in PA-HSOS models. Treatment with NE inhibitor or neutrophil depletion markedly attenuated PA-induced liver injury by concomitantly suppressing NETs formation and intrahepatic thrombogenesis. In vitro, conditioned medium from HHSEC exposed to MCT or SEN metabolites potently induced NETs formation. HHSEC injured by PA metabolites released CXCL8. Furthermore, hepatic mRNA and protein levels of CXCL1 and CXCL2 (murine homologous to human CXCL8) were up-regulated in PA-HSOS models, accompanied by a parallel elevation of their high-affinity receptors, CXCR1 and CXCR2. Our study identifies NETs-mediated immunothrombosis as a key driver in HSOS, which provides a promising target for PA-HSOS treatment.