Macrophage-derived DLL4 promotes liver fibrosis by activating the Notch pathway in hepatic stellate cells

Liver fibrosis, a common pathological endpoint of chronic liver diseases, is critically shaped by Notch signaling in establishing a pro-fibrotic microenvironment. However, the mechanisms by which Notch signaling orchestrates the transition from pro-inflammatory to pro-fibrotic microenvironments, particularly through crosstalk between inflammatory macrophages and fibrogenic hepatic stellate cells (HSCs), remain poorly defined. Here, using mouse models of methionine-choline-deficiency (MCD) diet-induced metabolic dysfunction-associated steatohepatitis (MASH) and CCl₄-induced liver fibrosis, we demonstrate that macrophage-derived Delta-like ligand 4 (DLL4) exacerbates liver fibrosis by activating HSCs. Macrophage-specific knockout Dll4 markedly attenuated the hepatitis and liver fibrosis, whereas Dll4 overexpression aggravated these pathologies. Mechanistically, macrophage-derived DLL4 activates the Notch2 receptor on HSCs, driving their activation and proliferation, as evidenced by upregulated α-SMA and COL1A1 and increased EdU⁺ cells. Critically, Notch2 knockdown in HSCs reversed DLL4-induced HSCs activation and proliferation. Notably, macrophage DLL4 expression is regulated by the TLR4-NF-κB pathway, as LPS stimulation increases DLL4 expression, while NF-κB inhibition inhibits its expression. Collectively, our findings identify the DLL4-Notch2 axis as a key mediator of the interaction between macrophages and HSCs during fibrosis, highlighting its therapeutic potential to block the progression of chronic liver diseases.