Hepatocyte-Derived IL-25 Promotes Macrophage Extracellular Trap Formation and Drives Liver Fibrosis Progression

Background: Liver Fibrosis represents a significant global health burden as a chronic progressive disease. Emerging evidence has established a critical link between macrophage extracellular traps (METs) and liver fibrosis; however, the precise triggers of MET formationand their mechanistic contributions to fibrosis remain poorly understood. Substantial evidence indicates that interleukin-25 (IL-25) potently regulates macrophage metabolism and fibrotic progression. Methods: A carbon tetrachloride (CCl₄)-induced mouse model of liver fibrosis spanning distinct stages (2 to 8 weeks) was established. In vitro studies utilized co-culture systems and conditioned medium treatment to assess MET-mediated activation of hepatic stellate cells (HSCs). To elucidate the specific role of IL-25, hepatocyte-specific IL-25 knockout (IL-25CKO) mice were generated using CRISPR/Cas9 technology and subjected to the fibrosis model. Mechanistic investigations involved stimulating RAW 264.7 macrophages with IL-25 and specific inhibitors. Techniques such as scanning/transmission electron microscopy, immunofluorescence, Western blot and ELISA were employed to analyze MET formation, ROS production, lysosomal activation, mitophagy, and related signaling pathways. Results: Fibrotic livers exhibited strong early co-localization of IL-25 with hepatocytes, which preceded MET formation. This demonstrates that early production of IL-25 by hepatocytes acts as a triggering factor for MET formation. In vitro co-culture systems revealed MET-mediated activation of HSCs, while mechanistic studies showed that IL-25 promotes IL-17RB receptor activation, triggering downstream reactive oxygen species (ROS) bursts, lysosomal activation, and MET formation. Conclusions: This work identifies a pathogenic hepatocyte–macrophage–HSC axis and supports IL-25 blockade as a promising clinical strategy for liver fibrosis.