The TGR5 protein amount is reduced in patients with primary sclerosing cholangitis (PSC)

Introduction: The localization of TGR5 in cholangiocytes, the choleretic, anti-inflammatory and anti-apoptotic functions of the receptor suggest that TGR5 is important for the pathogenesis of biliary diseases. Additionally, for the biliary tract disease PSC an association with the frequent TGR5 exon 1 SNP rs11554825 is described, which appears with a lower TGR5 mRNA expression (measured in lymphoblastic cell line) (Hov et al., 2010). Currently no data about TGR5 localization and expression in PSC patients are known. The milieu in PSC livers is insufficiently characterized, but elevated bile salt and cytokine levels are observed in chronic inflammatory bile duct disease.

Methods: TGR5 protein localization was analyzed using immunofluorescence staining in livers from PSC patients and controls as well as from MDR2-/- and WT mice. TGR5 protein amount in CK7 or CK19 positive cholangiocytes was determined by analysis of the mean fluorescence intensity per unit area (Axios Visio 4.8 software). TGR5 mRNA expression was quantified by qPCR in relation to an endogenous control (HPRT1), macrophage (CD14, CD163) or a cholangiocyte marker (CK19). To elucidate the TGR5 downregulation in livers macrophages isolated from blood mononuclear cells (PBMC) were used. Stimulation experiments with the most potent TGR5 bile acid ligand taurolithocholate (TLC), and the therapeutically used tauroursodeoxycholate (TUDC) and the cytokines TNFα and IL1β with subsequent TGR5 mRNA expression analysis were performed.

Results: The immunofluorescence analysis showed a significant reduction in TGR5 protein levels in the bile ducts of the PSC livers and in the bile ducts of MDR2-/- knockout animals compared to controls of human livers and mouse livers. In both studies, there was no change in the protein level of CK7 per bile duct. In contrast analysis of whole liver mRNA did not reveal a significant reduction in TGR5 mRNA in PSC or MDR2-/- livers as compared to the respective controls. However, mRNA for CD14 and CK19 also increased in livers from PSC patients or MDR2-/- mice. In chronic inflammatory biliary tract diseases bile salt and cytokine levels are increased. While the bile salts TLC and TUDC had no effects on TGR5 mRNA in isolated human macrophages, the inflammatory cytokines TNFα and IL1β significantly supressed TGR5 mRNA expression in these cells.

Discussion: Using immunofluorescence staining and quantification we can demonstrate a downregulation of TGR5 protein amounts in biliary epithelial cells of livers from PSC patients and MDR2-/- mice. Whether this decrease in TGR5 protein levels is caused by reduced TGR5 mRNA expression is unclear. However, stimulation of TGR5 expressing human macrophages with TNFα and IL1β significantly suppressed TGR5 mRNA levels, while various bile acids had no effect on the receptors mRNA expression. Since TGR5 exerts protective effects in liver and cholangiocytes the downregulation of the receptor in PSC livers may render cholangiocytes more susceptible to bile acid toxicity and may also explain why feeding of a TGR5 agonist (INT-767) failed to alleviate liver damage in MDR2-/- mice (Baghdasaryan et al., 2011).

Corresponding author: Spomer, Lina

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