This study reveals the central role of the IL-8-CXCR2 signaling pathway in neutrophil extracellular trap (NET) formation in children with biliary atresia and, for the first time, confirms that NETs can directly activate hepatic stellate cells, thereby advancing liver fibrosis, offering new insights for future targeted therapies.
Literature Overview
The article titled 'IL-8-CXCR2 pathway mediates neutrophil extracellular trap (NET) formation and activates hepatic stellate cells in children with biliary atresia,' published in the journal Hepatology, reviews and summarizes the mechanisms of NET formation in biliary atresia (BA) patients and their association with liver fibrosis progression. By analyzing plasma and liver tissue samples collected at diagnosis and liver transplantation, the research team found that NET formation levels were significantly elevated in BA patients and positively correlated with bilirubin and fibrosis markers (e.g., APRI score). Further co-culture experiments verified that BA plasma could activate NET formation in neutrophils from healthy donors, and this process was dependent on IL-8 and its receptor CXCR2. Additionally, the study identified fibrosis-promoting factors (e.g., tissue factor TF, IL-17) within NETs and confirmed their direct activation of hepatic stellate cells (LX2) using co-culture models, evidenced by upregulated ACTA2 and COL1A1 expression and increased collagen secretion. These findings provide novel insights into the immunopathological mechanisms of biliary atresia and suggest that targeting NET formation or its downstream effects may serve as a therapeutic strategy.
Background Knowledge
Biliary atresia is a rare yet severe inflammatory disorder of the bile ducts in infants, ultimately leading to cholestasis and cirrhosis. Current research has focused on the roles of immune cells (e.g., T cells, B cells, NK cells, macrophages), but the involvement of NETs remains unclear. IL-8 is a well-known neutrophil chemoattractant and has been linked to poor prognosis in BA patients. Based on their previous findings that both NETs and IL-8 levels were elevated prior to liver transplantation, the research team hypothesized that NETs may play a critical role in bile duct injury and liver fibrosis. Moreover, NETs have been confirmed to participate in various autoimmune and chronic inflammatory diseases (e.g., systemic lupus erythematosus, juvenile dermatomyositis, rheumatoid arthritis, cholelithiasis, alcoholic liver disease), and in mouse models, NET inhibition has been shown to alleviate bile duct injury. Therefore, this study further investigates the mechanisms underlying NET involvement in BA, particularly whether they directly activate hepatic stellate cells and contribute to fibrosis.
Research Methods and Experiments
The study included plasma and liver tissue samples from BA patients and cholestatic control groups. Plasma levels of neutrophil elastase, NETs (MPO-DNA complexes), and IL-8 were measured by ELISA. Multiplex immunohistochemistry (IHC) was used to assess NET, IL-8, TF, and IL-17 expression in liver tissues. Flow cytometry (FACS) was employed to evaluate neutrophil NET formation capacity and surface CXCR2 expression. Additionally, an in vitro co-culture system was established by incubating purified NETs with the LX2 hepatic stellate cell line to assess changes in ACTA2 and COL1A1 expression, collagen secretion, and cell surface markers. The study also tested the inhibitory effects of DNAse1 and CXCR2 inhibitors (Reparixin) on NET formation to validate the role of the IL-8-CXCR2 pathway.
Key Conclusions and Perspectives
Research Significance and Prospects
This study, for the first time, reveals the persistence of NET formation in BA patients and its positive correlation with fibrosis markers. It also provides experimental validation that NETs can directly activate hepatic stellate cells in vitro. These findings suggest that NETs may serve as potential biomarkers and therapeutic targets in BA. Future studies may focus on regulatory factors of NET formation (e.g., STAT3) and their roles in fibrosis progression, while exploring therapeutic strategies such as DNAse1 and CXCR2 inhibitors in the context of BA.
Conclusion
This study demonstrates that persistent NET formation occurs in children with biliary atresia (BA), primarily mediated by the IL-8-CXCR2 pathway. NET formation is significantly associated with liver injury and fibrosis markers (e.g., bilirubin, APRI score) and can directly activate hepatic stellate cells (LX2) in vitro, as evidenced by increased fibrosis-related gene expression and collagen secretion. These findings provide new insights into the immunopathological mechanisms of BA and suggest that targeting NET formation or its downstream signaling might help slow liver fibrosis progression. Future research should focus on the dynamic changes of NETs in BA, their regulatory pathways (e.g., STAT3), and potential therapeutic interventions such as DNAse1 and CXCR2 inhibitors.
