Single-cell RNA Sequencing Analysis Reveals That Targeting PLG-PLGRKT Signaling-mediated Pro-fibrotic Scar-associated Macrophages Ameliorates Liver Fibrosis in Biliary Atresia.

BACKGROUND AND AIMS: Biliary atresia (BA) is a severe pediatric cholangiopathy characterized by rapidly progressive liver fibrosis. This study aimed to characterize scar-associated macrophages and investigate the role of plasminogen (PLG)-plasminogen receptor with a C-terminal lysine (PLGRKT) signaling in BA-associated fibrogenesis.

METHODS

Single-cell RNA sequencing and spatial transcriptomics were applied to liver tissues from patients with BA and non-BA controls. Key findings were validated in an independent cohort using quantitative polymerase chain reaction and multiplex immunohistochemistry. The functional role of the PLGRKT pathway was further examined using primary human peripheral blood mononuclear cell-derived macrophages with small interfering RNA (siRNA)-mediated PLGRKT knockdown, together with co-culture systems involving LX-2 hepatic stellate cells and human liver organoids. In vivo therapeutic potential was evaluated in a murine bile duct ligation model of cholestatic liver fibrosis using macrophage-targeted Plgrkt-Trem2 antibody-siRNA conjugates.

RESULTS

In BA, scar-associated macrophages (SAMs) increased with fibrosis progression and co-localized with hepatic stellate cells in fibrotic areas. PLGRKT was upregulated in BA liver tissue and increased during monocyte-to-macrophage differentiation. In primary human macrophages, PLG induced a PLGRKT-dependent pro-fibrotic phenotype, and conditioned medium from these cells increased COL1A1 deposition in hepatic stellate cells and human liver organoids. In vivo, macrophage-targeted Plgrkt-Trem2 antibody-siRNA conjugates reduced SAM accumulation and attenuated bile duct ligation-induced liver fibrosis.

CONCLUSIONS

These findings support a role for SAMs and the PLGRKT-SAM axis in BA-associated liver fibrosis and suggest that PLGRKT warrants further investigation as a potential macrophage-directed anti-fibrotic target.