GPR35-positive mast cells are enriched in primary sclerosing cholangitis and show an enhanced secretory phenotype.

Abstract

Objective: Gene polymorphism in G protein-coupled receptor 35 (GPR35) is associated with primary sclerosing cholangitis (PSC). How GPR35 contributes to PSC pathogenesis remains poorly understood. We have observed infiltration of GPR35-positive mast cells in PSC explant liver tissue. Here, we aim to characterize GPR35 dependent mast cell biology relevant for cholangitis and biliary fibrosis.

Methods: GPR35 expressing cells were quantified by immunohistochemistry in liver tissue from PSC patients (n = 8) and disease controls (primary biliary cholangitis n = 3, alcohol-related liver disease n = 4). GPR35 -/- HMC-1 and LUVA mast cell lines were generated with CRISPR, stimulated with ionomycin and PMA, and phenotyped by RNA sequencing. Genes and pathways affected by GPR35 knockdown were determined by DESeq2 and gene set enrichment analysis. Selected cytokines, TNFα and IL13, were measured by qRT-PCR and ELISA. Mast cell-cholangiocyte interaction was studied in transwell co-culture with H69 cell line.

Results: GPR35-positive mast cells were increased around bile ducts in PSC liver explants compared to disease controls. PSC mast cells showed higher relative expression of GPR35 than macrophages. In vitro, GPR35-positive mast cells showed increased transcription of cytokine signaling and Golgi complex genes upon activation. These transcriptional changes translated to increased IL13 secretion. Finally, mast cells induced upregulation of TGFB1, FN1, and ACTA2 in H69 cholangiocytes in a GPR35 dependent manner.

Conclusion: GPR35 supports a pro-secretory mast cell phenotype with potential relevance to biliary fibrosis. The increased infiltration of GPR35 positive mast cells in PSC near bile ducts could be a target for biliary fibrogenesis.