Interleukin 8-CXCR2 mediated neutrophil extracellular trap (NET) formation in biliary atresia associated with NET-Induced stellate cell activation

Background & Aims: Biliary atresia (BA) entails an inflammatory sclerosing lesion of the biliary tree, with prominent fibrosis in infancy. Previous studies revealed neutrophil-activating IL-8 and neutrophil extracellular traps (NETs) positively correlated with bilirubin and risk of liver transplant. The aims of this study were to determine the mechanism of NET formation (NETosis) in BA and if NETs induce stellate cell activation. Approach: BA and other liver disease control plasma and tissue were obtained at diagnosis and transplant. Elastase, NETs, & IL-8 were quantified by ELISA for plasma and by immunohistochemistry (IHC) for liver tissue. FACS analysis of neutrophils co-cultured with BA or control plasma measured BA-specific NETosis. Stellate cell activation from co-culture studies of stellate cells with NETs was measured by RT-qPCR, ELISA and FACS. Results: Liver neutrophils, NETs and plasma elastase, NETs and IL-8 were significantly increased in BA at diagnosis and transplant. Normal neutrophils co-cultured with BA plasma had increased NETosis and activation of CXCR2, an IL-8 receptor; CXCR2 inhibition decreased NET production. IHC identified increased NET expression of pro-fibrogenic tissue factor and IL-17. NETs co-cultured with stellate cells resulted in stellate cell activation, based on increased ACTA2 & COL1A1 mRNA, collagen protein and cell surface expression of actin, collagen1A and PDGFRβ. Conclusions: BA patients have persistent IL-8-CXCR2-mediated NETosis that correlates with biomarkers of injury and fibrosis and NETs induce stellate cell activation, suggesting a role for NETs in the immunopathogenesis of disease. Future investigations should focus on therapeutic agents that inhibit NETs in BA.