Tetrahydroxylated bile acids improve cholestatic liver and bile duct injury in the Mdr2-/- mouse model of sclerosing cholangitis via immunomodulatory effects

Abstract

Bile salt export pump (Bsep) (Abcb11)^-/- mice are protected from acquired cholestatic injury due to metabolic preconditioning with a hydrophilic bile acid (BA) pool with formation of tetrahydroxylated bile acids (THBAs). We aimed to explore whether loss of Bsep and subsequent elevation of THBA levels may have immunomodulatory effects, thus improving liver injury in the multidrug resistance protein 2 (Mdr2) (Abcb4)^-/- mouse. Cholestatic liver injury in Mdr2^-/- Bsep^-/- double knockout (DKO), Mdr2^-/- , Bsep^-/- , and wild-type mice was studied for comparison. Mdr2^-/- mice were treated with a THBA (3α,6α,7α,12α-Tetrahydroxycholanoic acid). RNA/protein expression of inflammatory/fibrotic markers were investigated. Serum BA-profiling was assessed by ultra-performance liquid chromatography tandem mass spectrometry. Hepatic immune cell profile was quantified by flow cytometric analysis (FACS). In vitro, the THBA effect on chenodeoxycholic acid (CDCA)-induced inflammatory signaling in hepatocyte and cholangiocytes as well as lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced macrophage activation was analyzed. In contrast to Mdr2^-/- , DKO mice showed no features of sclerosing cholangitis. Sixty-seven percent of serum BAs in DKO mice were polyhydroxylated (mostly THBAs), whereas Mdr2^-/- mice did not have these BAs. Compared with Mdr2^-/- , DKO animals were protected from hepatic inflammation/fibrosis. THBA feeding in Mdr2^-/- mice improved liver injury. FACS analysis in DKO and Mdr2^-/- THBA-fed mice showed changes of the hepatic immune cell profile towards an anti-inflammatory pattern. Early growth response 1 (EGR1) protein expression was reduced in DKO and in Mdr2^-/- THBA-fed mice compared with Mdr2^-/- control mice. In vitro, THBA-reduced CDCA induced EGR1 protein and mRNA expression of inflammatory markers in hepatocytes and cholangiocytes. LPS/IFN-γ-induced macrophage activation was ameliorated by THBA. THBAs repress EGR1-related key pro-inflammatory pathways. Conclusion: THBA and their downstream targets may represent a potential treatment strategy for cholestatic liver diseases.