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 ΔMye) 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 ΔMye 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.