Stefanie Kluge

TGR5 is essential for bile acid-dependent cholangiocyte proliferation in vivo and in vitro

Objective Cholestatic liver diseases in humans as well as bile acid (BA)-feeding and common bile duct ligation (CBDL) in rodents trigger hyperplasia of cholangiocytes within the portal fields. Furthermore, elevation of BA levels enhances proliferation and invasiveness of cholangiocarcinoma (CCA) cells in animal models, thus promoting tumour progression. TGR5 is a G-protein coupled BA receptor, which is highly expressed in cholangiocytes and postulated to mediate the proliferative effects of BA. Design BA-dependent cholangiocyte proliferation was examined in TGR5-knockout and wild type mice following cholic acid (CA)-feeding and CBDL. TGR5-dependent proliferation and protection from apoptosis was studied in isolated cholangiocytes and CCA cell lines following stimulation with TGR5 ligands and kinase inhibitors. TGR5 expression was analysed in human CCA tissue. Results Cholangiocyte proliferation was significantly reduced in TGR5-knockout mice in response to CA-feeding and CBDL. Taurolithocholic acid and TGR5-selective agonists induced cholangiocyte proliferation through elevation of reactive oxygen species and cSrc mediated epidermal growth factor receptor transactivation and subsequent Erk1/2 phosphorylation only in wild type but not in TGR5-knockout-derived cells. In human CCA tissue TGR5 was overexpressed and the pathway of TGR5-dependent proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase (ERK)1/2 activation also translated to CCA cell lines. Furthermore, apoptosis was inhibited by TGR5-dependent CD95 receptor serine phosphorylation. Conclusions TGR5 is an important mediator of BA-induced cholangiocyte proliferation in vivo and in vitro. Furthermore, TGR5 protects cholangiocytes from death receptor-mediated apoptosis. These mechanisms may protect cholangiocytes from BA toxicity under cholestatic conditions, however, they may trigger proliferation and apoptosis resistance in malignantly transformed cholangiocytes, thus promoting CCA progression.

A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp).

The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ~30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.

Bile salt export pump‐reactive antibodies form a polyclonal, multi‐inhibitory response in antibody‐induced bile salt export pump deficiency

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC‐2 who developed PFIC‐like symptoms after orthotopic liver transplantation (OLT). BSEP‐reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody‐induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC‐2 patients who suffered from phenotypic disease recurrence post‐OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G‐class BSEP‐reactive antibodies in these patients. In all cases, the N‐terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C‐terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle‐based functional assay, transport inhibition by anti‐BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC‐2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post‐OLT. The antibody response is polyclonal, targeting both extra‐ and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis. (Hepatology 2016;63:524–537)

Bile Salt Export Pump‐reactive Antibodies Form a Polyclonal, Multi‐inhibitory Response in Antibody‐induced BSEP Deficiency

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC‐2 who developed PFIC‐like symptoms after orthotopic liver transplantation (OLT). BSEP‐reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody‐induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC‐2 patients who suffered from phenotypic disease recurrence post‐OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G‐class BSEP‐reactive antibodies in these patients. In all cases, the N‐terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C‐terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle‐based functional assay, transport inhibition by anti‐BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC‐2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post‐OLT. The antibody response is polyclonal, targeting both extra‐ and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis. (Hepatology 2016;63:524–537)

Genetic variations of bile salt transporters

Bile salt transporters directly or indirectly influence biological processes through physicochemical or signalling properties of bile salts. The coordinated action of uptake and efflux transporters in polarized epithelial cells of the liver, biliary tree, small intestine and kidney determine bile salt concentrations in different compartments of the body. Genetic variations of bile salt transporters lead to clinical relevant phenotypes of varying severity ranging from a predisposition for drug-induced liver injury to rapidly progressing end-stage liver disease. This review focuses on the impact of genetic variations of bile salt transporters including BSEP, NTCP, ASBT and OSTα/β and discusses approaches for transporter analysis.

MAPKAP kinase 2 regulates IL-10 expression and prevents formation of intrahepatic myeloid cell aggregates during cytomegalovirus infections

BACKGROUND & AIMS The kinase p38(MAPK) and its downstream target MAPKAP kinase (MK) 2 are critical regulators of inflammatory responses towards pathogens. To date, the relevance of MK2 for regulating IL-10 expression and other cytokine responses towards cytomegalovirus (CMV) infection and the impact of this pathway on viral replication in vitro and in vivo is unknown and the subject of this study. METHODS The effect of MK2, interferon-α receptor (IFNAR)1, tristetraprolin (TTP) and IL-10 on mouse (M)CMV virus titres, cytokine expression, signal transduction, transcript stability, liver enzymes release, immune cell recruitment and aggregation in response to MCMV infection were studied ex vivo in hepatocytes and macrophages, as well as in vivo. RESULTS MK2 is critical for MCMV-induced production of IL-10, IFN-α2 and 4, IFN-β, IL-6, and TNF-α but not for IFN-γ. The MCMV-induced IL-10 production requires activation of IFNAR1 and is further regulated by MK2 and TTP-dependent stabilization of IL-10 transcripts. MK2(-/-) mice are able to control acute MCMV replication, despite deregulated cytokine production. This may be related to the observation that MCMV-infected MK2(-/-) mice show enhanced formation of focal intrahepatic lymphocyte infiltrates resembling intrahepatic myeloid cell aggregates of T cell expansion (iMATEs), which were also observed in MCMV-infected IL-10(-/-) mice but are almost absent in MCMV-infected wild-type controls. CONCLUSIONS The data suggest that MK2 is critical for regulating cytokine responses towards acute MCMV infection, including that of IL-10 via IFNARI-mediated circuits. MCMV stimulates expression of MK2-dependent cytokines, in particular IL-10 and thereby prevents enhanced formation of intrahepatic iMATE-like cellular aggregates.

Two Case Reports of Successful Treatment of Cholestasis With Steroids in Patients With PFIC-2

Mutations in the gene encoding the canalicular bile salt export pump (BSEP) can result in progressive familial intrahepatic cholestasis type 2 (PFIC-2). Treatment options are limited, and PFIC-2 often necessitates liver transplantation. We report on a young woman and a boy who clinically presented with PFIC-2 phenotypes and dramatically improved with steroid treatment. Gene sequencing of ABCB11 encoding for BSEP revealed 2 relevant mutations in both patients. The young woman was compound heterozygous for p.T919del and p.R1235X. At the age of 5 years, partial biliary diversion was performed and rescued liver function but left serum bile salt levels elevated. At age 23 she developed systemic lupus erythematosus. Unexpectedly, steroid therapy normalized serum bile salt levels, with a strong correlation with the steroid dose. She is currently in clinical remission. The boy was compound heterozygous for the ABCB11 mutations c.150+3A>C and p.R832C and presented with intractable pruritus. When he developed colitis, he was treated with steroids. The pruritus completely disappeared and relapsed when steroids were withdrawn. To date, with low-dose budesonide, the boy has been symptom-free for >3 years. In conclusion, the clinical courses suggest that patients with BSEP deficiency and residual BSEP activity may benefit from steroid-based therapy, which represents a new treatment option.

High affinity anti‐BSEP antibodies after liver transplantation for PFIC‐2 – Successful treatment with immunoadsorption and B‐cell depletion

PFIC due to BSEP mutations (PFIC type 2) often necessitates OLT. It has recently been recognized that some PFIC‐2 patients develop phenotypic disease recurrence post‐OLT due to the appearance of anti‐BSEP antibodies. Here, we describe a boy who became cholestatic four yr after OLT during modification of immunosuppression. Canalicular antibody deposits were detected in biopsies of the transplant and antibodies specifically reacting with BSEP were identified at high titers in his serum. These antibodies bound extracellular epitopes of BSEP and inhibited BS transport and were assumed to cause disease recurrence. Consequently, anti‐BSEP antibody depletion was pursued by IA and B‐cell depletion by anti‐CD20 antibodies (rituximab) along with a switch of immunosuppression. This treatment resulted in prolonged relief of symptoms. Depletion of pathogenic anti‐BSEP antibodies causing AIBD after OLT in PFIC‐2 patients should be considered as a central therapeutic goal.

Role of primary bile salts in the regulation of sinusoidal substrate uptake in rat liver

Background Bile salts undergo enterohepatic circulation which is essential for bile salt homeostasis. From the biliary tract, they are excreted into the small intestine, absorbed into the blood and transported back to the liver. The sinusoidal uptake systems of the liver efficiently extract bile salts and other substrates from portal blood. In rat liver, these systems include Ntcp (sodium taurocholate cotransporting polypeptide) and the organic anion transporting polypeptides Oatp1a1, Oatp1a4 and Oatp1b2. While Ntcp represents the major basolateral uptake transporter for conjugated bile salts, the Oatps have broad substrate spectra including estrogenor leukotriene-conjugates. Their transport function is regulated by long-term and short-term mechanisms. Long-term adaptation of bile salt transporters involve changes at the level of gene expression and transporter degradation, while short-term regulation includes covalent transporter modifications, substrate availability and rapid endoand exocytosis of transporter-containing vesicles (reviewed in [1]). Ntcp as well as Oatp1a1 and Oatp1a4 underlie short-term control which has been demonstrated in several in vitro models. Hypoosmolarity or cAMP for instance increase the Ntcp-dependent bile salt uptake (increase of Vmax) within minutes by translocation of intracellular Ntcp to the plasma membrane [2,3]. In contrast, rapid clathrin-dependent endocytosis of Ntcp was demonstrated only recently by activation of PKC with phorbolesters [4,5]. However, to date in vivo-studies on short-term regulation of basolateral transporters are rare. Bile salt inflow to the liver may increase considerably under pathophysiological conditions or postprandial under physiological conditions [6]. To date, the interrelation between substrate load and transport activity is largely unknown. Therefore, the aim of this study was to investigate the role of primary bile salts in short-term feedback regulation of sinusoidal substrate uptake in perfused rat liver.