RANKL blockade attenuates MASH by remodeling the hepatic immune microenvironment

Objective Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease worldwide and is largely driven by chronic inflammation and hepatic immune dysregulation. The receptor activator of nuclear factor-κB ligand (RANKL) is a master regulator of bone metabolism with emerging immunomodulatory functions. Although growing evidence links the RANK-RANKL-OPG axis to MASH pathophysiology, whether RANKL blockade attenuates MASH development remains unclear. This study therefore investigated the effects and underlying mechanisms of RANKL blockade in a mouse model of MASH. Methods Male mice were fed a choline-deficient high-fat diet (CDHFD) for 16 weeks to induce MASH and were treated with the RANKL-neutralizing antibody IK22/5 (1, 4, or 8 mg/kg) or an IgG control. Metabolic parameters, liver histology, and biochemical markers were assessed. Hepatic immune-cell infiltration was analyzed using CIBERSORT-based deconvolution, and immune populations were characterized by flow cytometry. Transcriptomic profiling identified RANKL-regulated genes, followed by cross-species analysis using human MASH datasets. Results RANKL blockade dose-responsively improved glucose tolerance, hepatic steatosis, inflammation, and fibrosis. Transcriptomic analysis identified 48 genes that were dysregulated in MASH but reversed by RANKL blockade; these genes were enriched in lipid metabolism, immune regulation, and fibrosis pathways. Immune profiling revealed broad remodeling of the hepatic immune microenvironment: IK22/5 reduced CD11b high F4/80 + monocyte-derived macrophages, CD86 + M1-like macrophages, CD3 - NK1.1 + NK cells, and CD3 + NK1.1 + CD4 - CD8 - NKT-like cells, while increasing γδ T cells and preserving CD11b int F4/80 + Tim4 + Kupffer cells. Immunofluorescence showed that RANKL blockade significantly reduced hepatic CD74 + F4/80 + macrophages, together with decreased expression of other lipid-associated macrophage markers. Conclusions Overall, these findings indicate that RANKL blockade attenuated MASH development and support RANKL as a potential immune–metabolic regulator, providing a preclinical rationale for further evaluation of RANKL-targeted strategies in clinically relevant models.