Gut Microbiota-Derived Propionate Governs Hepatic N2 Neutrophils in Wilson's Disease

Neutrophil functions play a pivotal role in hepatic pathogenesis. Our previous work has established that N2-polarized neutrophils promote hepatic fibrogenesis in Wilson's disease depends on hepatic transforming growth factor-β1 (TGF-β1) production. However, the regulators governing TGF-β1 production in orchestrating disease-associated N2 neutrophils remain elusive. In this study, we investigated the immunomodulatory effects of gut microbiota-derived short-chain fatty acids (SCFAs) on neutrophil polarization.

Fecal metagenomic sequencing and short-chain fatty acid (SCFA) profiling were performed on ATP7B-knockout (ATP7B-KO) mice and their wild-type (WT) littermate controls. Fecal microbiota transplantation (FMT) experiments were conducted by transferring feces from WT mice or Akkermansia muciniphila into recipient mice. Additionally, propionate or trichostatin A (TSA) was administered to both ATP7B-KO and WT groups. Mice were assessed using histological analyses, Sirius Red staining, flow cytometry, biochemical assays, immunohistochemistry, measurement of TGF-β1 levels, immunofluorescence, and quantitative real-time polymerase chain reaction (qRT-PCR) for gene expression profiling. To elucidate the underlying molecular mechanisms, 4D label-free quantitative acetylated proteomics, site-directed mutagenesis, plasmid transfection, co-immunoprecipitation (IP), and luciferase reporter assays were employed.

We report that Akkermansia muciniphila was markedly reduced in the gut microbiota of mice with Wilson's disease, accompanied by decreased SCFA levels, especially propionate. Additionally, transplantation of fecal bacteria from wild-type mice or A muciniphila could promote an antifibrotic effect, elevate propionate levels, reduce TGF-β1 secretion, and decrease hepatic N2 neutrophils in mice with Wilson's disease. Moreover, administration of propionate also significantly enhanced antifibrotic immunity. Mechanistically, propionate reduced the production of TGF-β1 in hepatocytes by inhibiting histone deacetylase activity, increasing the acetylation of DNAJA3 at sites K134 and K385, thus decreasing expression of DNAJA3. Consistently, gut-derived propionate inversely correlated with hepatic injury severity in patients with Wilson's disease, which could be functionally mediated by TGF-β1.

Gut microbiota are pivotal for hepatic neutrophil polarization and liver fibrosis in Wilson's disease. Our findings suggest that therapeutic modulation of gut microbiota, SCFA profiles, and TGF-β1 production, particularly when combined with histone deacetylase inhibitors, may represent promising therapeutic approaches for Wilson's disease.