Farnesoid X Receptor Critically Determines the Fibrotic Response in Mice but Is Expressed to a Low Extent in Human Hepatic Stellate Cells and Periductal Myofibroblasts

The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR^−/−) by CCl₄ intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl₄-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands.The farnesoid X receptor (FXR;NR1H4) is a key regulator of hepatic bile acid homeostasis, lipoprotein and glucose metabolism, bacterial colonization of the small intestine, the inflammatory response, and liver regeneration.^1,2,3 Hereditary and acquired FXR defects may contribute to cholestasis and gallstone formation in humans.^4,5,6,7 Defects in its target genes (eg, bile salt export pump/ABCB11; multidrug resistance gene 3/ABCB4 (a phosphatidylcholine floppase); multidrug related protein 2/ABCC2) cause well-characterized clinical syndromes.^8,9,10,11 Moreover, FXR knockout mice (FXR^−/−) have impaired resistance to bile acid feeding,^12,13 and show substantial differences in the cholestatic phenotype in response to common bile duct ligation,^14,15,16 have increased susceptibility for diet-induced gallstone disease,^17,18 and impaired liver regeneration following partial hepatectomy.¹⁹ FXR may also directly or indirectly (eg, by the interaction with other members of the nuclear receptor family such as PXR/NR1I2 and VDR/NR1I1) regulate the metabolism and hepatic clearance of xenobiotics.^20,21,22Recent studies also reported mRNA expression of FXR in hepatic stellate cells and FXR protein in renal proximal tubules^23,24,25 suggesting that FXR could represent a therapeutic target for the treatment of liver fibrosis and diabetic nephropathy.^23,24,25,26 Moreover, FXR ligands were claimed to repress collagen expression in HSCs in vitro via a postulated FXR/SHP-dependent mechanism.²³ It is also attractive to hypothesis that genetic FXR variants may predispose patients suffering from various forms of liver diseases to liver fibrosis as a kind of genetic disease modifier.^7,27 Taken together its pleiotrophic functions (eg, central regulator of bile acid homoeostasis, glucose and lipid metabolism, inflammation) make FXR an extremely attractive candidate for therapeutic targeting in cholestatic liver diseases and nonalcoholic fatty liver disease including their major sequel liver fibrosis.^28,30 However, little is known on hepatic cell-type FXR expression in human liver fibrosis.The aims of this study were threefold. First, we aimed to determine the impact of genetic FXR ablation on the degree of liver fibrosis in untreated mice and four different well established mouse models including CCl₄-intoxicated mice, 3,5 -diethoxycarbonyl-1,4-dihydrocollidine (DDC)-intoxicated mice and common bile duct-ligated (CBDL) mice for biliary fibrosis, and infection with Schistosoma mansoni (S.m.), which has been shown to induce “pipe-stem” fibrosis and granuloma formation.^31,32 Comparison of cholestatic (DDC, CBDL) and non-cholestatic (CCl₄, S.m.) mouse models for liver fibrosis should provide differentiated knowledge on the role of FXR in various types and etiologies of liver fibrosis. Based on previous studies reporting that pharmacological activation of FXR is antifibrotic in liver but also kidney^23,25 we hypothesized that FXR^−/− mice spontaneously develop liver fibrosis and are more susceptible to experimentally induced liver fibrosis due to the lack of a postulated FXR/SHP-dependent down-regulation of collagen mRNA expression in profibrotic states.^23,24 We therefore compared the extent of fibrosis in FXR^−/− mice and wild-type controls in a longitudinal study under baseline conditions and in response to cholestatic and non-cholestatic fibrogenic injury. Second, we aimed to determine the expression of genes involved in bile acid transport/metabolism and their regulatory nuclear receptors (including FXR, PXR, CAR/NR1I3, VDR, and SHP/NR0B2) in isolated profibrogenic rodent cells [ie, periductal myofibroblasts (MFBs), and quiescent as well as activated hepatic stellate cells (HSCs)] and to test the effects of FXR ligands on FXR target genes in vitro. Cell type-specific FXR protein expression was determined in five different in vivo models for liver fibrosis. Finally, we cross-validated these findings in isolated human HSCs and histological sections from human prototypic fibrotic liver diseases [eg, primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and alcoholic steatohepatitis (ASH)].