Fibroblast- and Macrophage-Derived Thrombospondin-1 Orchestrates the Fibroinflammatory Niche in Metabolic Dysfunction-Associated Steatohepatitis-Induced Fibrosis

Background: Thrombospondin-1 (THBS1) activates latent transforming growth factor-β1 (TGF-β1) and contributes to stromal- immune communication. However, how THBS1 derived from fibroblasts and macrophages collectively contributes to fibroinflammatory remodeling in metabolic dysfunction-associated steatohepatitis (MASH) has not been clearly defined. Methods: THBS1 expression was examined in liver tissue and serum from patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and in two dietary murine MASH models. We integrated single-cell RNA sequencing, immunohistochemistry, confocal microscopy, and flow cytometry with targeted perturbation of fibroblasts and macrophages using nintedanib, the THBS1-blocking peptide LSKL, and siRNA-mediated THBS1 silencing. Results: THBS1 and the fibroblast activation marker fibroblast activation protein (FAP) increased in association with fibrosis severity and interleukin-6 (IL-6) levels. Single-cell RNA sequencing and spatial imaging demonstrated THBS1 expression in both FAP+ fibroblasts and monocyte-derived macrophages, indicating a coordinated fibroinflammatory niche. Nintedanib reduced THBS1 expression in both compartments, decreased CD11b+MERTK+ macrophages and FAP+PD-L1+ fibroblasts, and improved histologic fibrosis. Direct THBS1 inhibition produced similar antifibrotic effects. In vitro, THBS1 knockdown or blockade suppressed TGF-β1-induced fibroblast activation and reduced lipopolysaccharide-stimulated IL-1β and IL-6 production in macrophages, supporting reciprocal THBS1-dependent stromal-immune signaling. Conclusion: THBS1 functions as a shared signaling mediator between fibroblasts and macrophages in MASH. Therapeutic suppression of THBS1, either through nintedanib or direct inhibition, disrupts this fibroinflammatory circuit and may represent a viable strategy to limit fibrosis progression in MASLD/MASH.