Macrophage NEDD4L restrains liver fibrosis by preventing scar-associated macrophage expansion via ubiquitination of phospho-SMAD3

Background: Liver fibrosis is characterized by excessive extracellular matrix deposition and hepatic stellate cell (HSC) activation, driven by chronic liver injury and inflammation.Macrophages play dual roles in fibrogenesis; the dynamic balance between pro-fibrotic and anti-fibrotic subsets is critical in determining the progression or regression of the disease.NEDD4L, an E3 ubiquitin ligase, is well-known to be involved in cell biological processes by promoting protein degradation, yet its role in macrophages and liver fibrosis remains poorly understood.Methods: Myeloid cell-specific Nedd4l knockout (Nedd4l f/f Lyz-Cre+, Nedd4l Δ Mye ) were generated, and subjected to carbon tetrachloride (CCl4) and choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-induced experimental liver fibrosis models.Results: Single-cell RNA sequencing and transcriptomic analyses revealed significant upregulation of NEDD4L in macrophages from human and murine fibrotic livers.Strikingly, myeloid cell-specific Nedd4l deficiency exacerbated liver fibrosis in both mouse models, as evidenced by increased collagen deposition and elevated expression of fibrogenic genes in Nedd4l Δ Mye mice.Notably, mice with Nedd4l deficient in macrophages had more pro-fibrotic scar-associated macrophage (SAM) infiltration compared with Nedd4l f/f mice in two experimental models.More interestingly, coculture in vitro experiments further verified that TGF-β1-treated Nedd4l deficient macrophages promoted HSC activation due to greater activation of SMAD3 signaling.Mechanistically, NEDD4L directly ubiquitinated phosphorylated SMAD3 and led to its degradation, thus limiting TGF-β1/SMAD3 signaling in macrophages.Moreover, hepatic levels of NEDD4L were significantly elevated in patients with liver fibrosis, positively correlating with hepatic levels of several fibrogenic genes.Conclusions: NEDD4L serves as a critical negative regulator of liver fibrosis by restraining profibrotic SAM expansion through ubiquitination and degradation of p-SMAD3 in macrophages.These findings highlight that targeting the ubiquitin-proteasome system as a potential therapeutic strategy for the treatment of fibrotic disease.