PPL + Cholangiocytes Define a Pro‐Regenerative Subset Associated With NRG1 – ERBB3 – PI3K / AKT Signalling During Liver Fibrosis

ABSTRACT Background & Aims Chronic biliary injury drives liver fibrosis, yet mechanisms governing injury resolution and hepatocyte regeneration remain unclear. Periplakin (PPL), a cholangiocyte‐enriched cytoskeletal linker, is upregulated during biliary injury, but its functional contribution to fibrosis and repair has not been defined. Methods Single‐nucleus RNA‐seq (snRNA‐seq) datasets from wild‐type and DDC‐treated mouse livers were analysed to map PPL expression. Hepatic PPL‐overexpression and knockdown models were generated using adenoviral (Ad) and adeno‐associated viral (AAV) vectors, respectively. Fluorescence‐activated cell sorting (FACS)‐sorted PPL + and PPL − cholangiocytes were isolated for morphological characterisation, adoptive transfer, and hepatocyte co‐culture assays. Liver fibrosis, proliferation, extracellular matrix (ECM) remodelling, cytokines, and growth factors were assessed by histology, qRT‐PCR, Western blot, ELISA, and IHC. CellChat was used to infer intercellular signalling, and NRG1‐ERBB3‐PI3K/AKT signalling was validated in vitro. Results SnRNA‐seq identified PPL as a cholangiocyte‐enriched marker defining a morphologically distinct subset. PPL overexpression reduced, whereas PPL knockdown aggravated, DDC‐induced fibrosis. Adoptive transfer of PPL + cholangiocytes improved liver function, limited ductular reaction and collagen deposition, increased MMP2/MMP12/MMP13/MMP14, elevated IL‐10, EGF, and HGF, and reduced TGF‐β1 and TIMP1. PPL + cholangiocytes enhanced hepatocyte proliferation in vivo and in vitro. CellChat and co‐culture assays demonstrated that PPL + cells activate hepatocytes via an NRG1‐ERBB3‐PI3K/AKT axis. Conclusions PPL + cholangiocytes represent a pro‐regenerative epithelial subset that is associated with attenuated fibrosis and enhanced hepatocyte proliferation, potentially involving NRG1‐ERBB3‐PI3K/AKT signalling.