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Oxidative stress is of importance in the pathophysiology of sickle cell disease (SCD). In this open label randomized pilot study the effects of oral N-acetylcysteine (NAC) on phosphatidylserine (PS) expression as marker of cellular oxidative damage (primary end point), and markers of hemolysis, coagulation and endothelial activation and NAC tolerability (secondary end points) were studied. Eleven consecutive patients (ten homozygous [HbSS] sickle cell patients, one HbSβ(0)-thalassemia patient) were randomly assigned to treatment with either 1,200 or 2,400 mg NAC daily during 6 weeks. The data indicate an increment in whole blood glutathione levels and a decrease in erythrocyte outer membrane phosphatidylserine exposure, plasma levels of advanced glycation end-products (AGEs) and cell-free hemoglobin after 6 weeks of NAC treatment in both dose groups. One patient did not tolerate the 2,400 mg dose and continued with the 1,200 mg dose. During the study period, none of the patients experienced painful crises or other significant SCD or NAC related complications. These data indicate that N-acetylcysteine treatment of sickle cell patients may reduce SCD related oxidative stress. [ABSTRACT FROM AUTHOR]

Accumulation of neurotoxic bilirubin due to a transient neonatal or persistent inherited deficiency of bilirubin glucuronidation activity can cause irreversible brain damage and death. Strategies to inhibit bilirubin production and prevent neurotoxicity in neonatal and adult settings seem promising. We evaluated the impact of Bvra deficiency in neonatal and aged mice, in a background of unconjugated hyperbilirubinemia, by abolishing bilirubin production. We also investigated the disposal of biliverdin during fetal development. In Ugt1 -/- mice, Bvra deficiency appeared sufficient to prevent lethality and to normalize bilirubin level in adults. Although biliverdin accumulated in Bvra -deficient fetuses, both Bvra -/- and Bvra -/- Ugt1 -/- pups were healthy and reached adulthood having normal liver, brain, and spleen histology, albeit with increased iron levels in the latter. During aging, both Bvra -/- and Bvra -/- Ugt1 -/- mice presented normal levels of relevant hematological and metabolic parameters. Interestingly, the oxidative status in erythrocytes from 9-months-old Bvra -/- and Bvra -/- Ugt1 -/- mice was significantly reduced. In addition, triglycerides levels in these 9-months-old Bvra -/- mice were significantly higher than WT controls, while Bvra -/- Ugt1 -/- tested normal. The normal parameters observed in Bvra -/- Ugt1 -/- mice fed chow diet indicate that Bvra inhibition to treat unconjugated hyperbilirubinemia seems safe and effective.

Progressive Familial Intrahepatic Cholestasis (PFIC) are inherited severe liver disorders presenting early in life, with high serum bile salt and bilirubin levels. Six types have been reported, two of these are caused by deficiency of an ABC transporter; ABCB11 (bile salt export pump) in type 2; ABCB4 (phosphatidylcholine floppase) in type 3. In addition, ABCB11 function is affected in 3 other types of PFIC. A lack of effective treatment makes a liver transplantation necessary in most patients. In view of long-term adverse effects, for instance due to life-long immune suppression needed to prevent organ rejection, gene therapy could be a preferable approach, as supported by proof of concept in animal models for PFIC3. This review discusses the feasibility of gene therapy as an alternative for liver transplantation for all forms of PFIC based on their pathological mechanism. Conclusion: Using presently available gene therapy vectors, major hurdles need to be overcome to make gene therapy for all types of PFIC a reality.

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (h UGT1A1 ) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-h UGT1A1 , and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs., Competing Interests: F.C., G.B., G.R., A.F.M., and F.M. are inventors of patents describing liver gene transfer approaches for metabolic diseases and/or treatment of hyperbilirubinemia. F.M. is an employee of Spark Therapeutics, a Roche company. The remaining authors declare no competing interests., (© 2020 The Author(s).)

The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD). We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation. These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Human liver cell line HepaRG is a well-known source of human hepatocyte-like cells which, however, displays limited biotransformation and a tendency to transform after 20 passages. The new HepaRG-CAR cell line overexpressing constitutive androstane receptor (CAR, NR1I3), a regulator of detoxification and energy metabolism outperforms the parental HepaRG cell line in various liver functions. To further characterize this cell line and assess its stability we compared HepaRG-CAR with HepaRG cells at different passages for their expression profile, ammonia and lactate metabolism, bile acid and reactive oxygen species (ROS) production. Transcriptomic profiling of HepaRG-CAR vs. HepaRG early-passage revealed downregulation of hypoxia, glycolysis and proliferation and upregulation of oxidative phosphorylation genesets. In addition CAR overexpression downregulated the mTORC1 signaling pathway, which, as mediator of proliferation and metabolic reprogramming, may play an important role in the establishment of the HepaRG-CAR phenotype. The ammonia and lactate metabolism and bile acid production of HepaRG-CAR cells was stable for 10 additional passages compared to HepaRG cells. Interestingly, bile acid production was 4.5-fold higher in HepaRG-CAR vs. HepaRG cells, whereas lactate and ROS production were 2.7- and 2.0-fold lower, respectively. Principal component analysis showed clustering of HepaRG-CAR (early- and late-passage) and HepaRG early-passage and not with HepaRG late-passage indicating that passaging exerted larger effect on the transcriptional profile of HepaRG than HepaRG-CAR cells. In conclusion, overexpression of CAR in HepaRG cells improves their bile acid production, mitochondrial energy metabolism, and stability, with the latter possibly due to reduced ROS production, resulting in an optimized source of human hepatocytes.

Background & Aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3), for which there are limited therapeutic options, often leads to end-stage liver disease before adulthood due to impaired ABCB4-dependent phospholipid transport to bile. Using adeno-associated virus serotype 8 (AAV8)-mediated gene therapy, we aimed to restore the phospholipid content in bile to levels that prevent liver damage, thereby enabling stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3., Methods: Ten-week-old Abcb4 -/- mice received a single dose of AAV8-hABCB4 (n = 10) or AAV8-GFP (n = 7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by immunohistochemistry., Results: Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration. AAV8-hABCB4 expression restored biliary phospholipid excretion, increasing the phospholipid and cholesterol content in bile to levels that ameliorate liver damage. This resulted in normalization of the plasma cholestatic markers, alkaline phosphatase and bilirubin. In addition, AAV8-hABCB4 prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study., Conclusion: Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Translational studies that verify the clinical feasibility of this approach are warranted., Lay Summary: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe genetic liver disease that results from impaired transport of lipids to bile, which makes the bile toxic to liver cells. Because therapeutic options are currently limited, this study aims to evaluate gene therapy to correct the underlying genetic defect in a mouse model of this disease. By introducing a functional copy of the missing gene in liver cells of mice, we were able to restore lipid transport to bile and strongly reduce damage to the liver. The proliferation of liver cells was also reduced, which contributes to long-term correction of the phenotype. Further studies are required to evaluate whether this approach can be applied to patients with PFIC3., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Bile acids play a major role in the regulation of lipid and energy metabolism. Here we propose the hepatic bile acid uptake transporter Na+ taurocholate co-transporting polypeptide (NTCP) as a target to prolong postprandial bile acid elevations in plasma. Reducing hepatic clearance of bile acids from plasma by genetic deletion of NTCP moderately increased plasma bile acid levels, reduced diet-induced obesity, attenuated hepatic steatosis, and lowered plasma cholesterol levels. NTCP-G protein-coupled bile acid receptor (TGR5) double knockout mice were equally protected against diet-induced-obesity as NTCP single knockout mice. NTCP knockout mice displayed decreased intestinal fat absorption and a trend towards higher fecal energy output. Furthermore, NTCP deficiency was associated with an increased uncoupled respiration in brown adipose tissue, leading to increased energy expenditure. We conclude that targeting NTCP-mediated bile acid uptake can be a novel approach to treat obesity and obesity-related hepatosteatosis by simultaneously dampening intestinal fat absorption and increasing energy expenditure.

Intrahepatic cholestasis of pregnancy (ICP) is frequently accompanied by pruritus, whose etiology has been associated with an enhanced production of lysophosphatidic acid (LPA) by the combined action of phospholipase A1/A2 (PLA1/PLA2) and autotaxin (ATX). Here, we have investigated whether the placenta is involved in LPA release to maternal circulation during ICP. Serum levels of ATX and LPA (determined by ELISA) were elevated in women with ICP, and a correlation between both parameters was found. No relationship between serum levels of ATX or LPA and bile acids was found. Expression levels of ATX and PLA2 were determined by RT-qPCR and Western blot. Placenta ATX but not PLA2 was significantly upregulated in ICP, and a tendency to increase was found at the protein level. A correlation between serum ATX and placental ATX mRNA levels was found. In human placenta at term, ATX was clearly detected (by immunofluorescence) in Hofbauer cells, but only faintly in trophoblast cells. In pregnant rats, the expression of Atx and Pla2 in placenta was lower than in liver. When obstructive cholestasis was imposed by bile duct ligation from day 14 of gestation until term, placenta Atx and Pla2 expression was markedly enhanced, and overexpression was confirmed at the protein level for Pla2, whereas Atx protein was not detected. In conclusion, the placenta substantially participates in LPA production during gestation. This contribution is markedly higher during maternal cholestasis and hence, may be involved in ICP-associated pruritus. NEW & NOTEWORTHY Fetal placental macrophages and, to a lesser extent, trophoblast cells express high levels of autotaxin at term. An increased expression of mRNA and protein autotaxin, the key secretory enzyme responsible for the production of lysophosphatidic acid in serum, has been observed in placentas of women with cholestasis of pregnancy, which supports that the placenta can contribute to an increased production of this pruritogenic compound in women suffering from this liver disease.

The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O 2 ), normoxia (21%O 2 ) or hyperoxia (40%O 2 ). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBPα, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1α. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1α was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBPα expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1α.

Objective: Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan immune-mediated disease that predominantly affects the biliary tract (IgG4-associated cholangitis, IAC) and pancreas (autoimmune pancreatitis, AIP). We recently identified highly expanded IgG4+ B-cell receptor clones in blood and affected tissues of patients with IAC/AIP suggestive of specific (auto)antigenic stimuli involved in initiating and/or maintaining the inflammatory response. This study aimed to identify (auto)antigen(s) that are responsible for the clonal expansion of IgG4+ B cells in IgG4-RD., Design: We screened sera of patients with IAC/AIP (n=50), in comparison to control sera of patients with primary sclerosing cholangitis (PSC) and pancreatobiliary malignancies (n=47), for reactivity against human H69 cholangiocyte lysates on immunoblot. Subsequently, target antigens were immunoprecipitated and analysed by mass spectrometry., Results: Prominent reactivity against a 56 kDa protein was detected in human H69 cholangiocyte lysates exposed to sera of nine patients with IAC/AIP. Affinity purification and mass spectrometry analysis identified annexin A11, a calcium-dependent phospholipid-binding protein. Annexin A11-specific IgG4 and IgG1 antibodies were only detected in serum of patients with IgG4-RD of the biliary tract/pancreas/salivary glands and not in disease mimickers with PSC and pancreatobiliary malignancies. Epitope analysis showed that two annexin A11 epitopes targeted by IgG1 and IgG4 autoantibodies were shared between patients with IAC/AIP and IgG4 antibodies blocked binding of IgG1 antibodies to the shared annexin A11 epitopes., Conclusion: Our data suggest that IgG1-mediated pro-inflammatory autoreactivity against annexin A11 in patients with IgG4-RD may be attenuated by formation of annexin A11-specific IgG4 antibodies supporting an anti-inflammatory role of IgG4 in IgG4-RD., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease associated with autoimmune phenomena targeting intrahepatic bile duct cells (cholangiocytes). Although its etiopathogenesis remains obscure, development of antimitochondrial autoantibodies against pyruvate dehydrogenase complex E2 is a common feature. MicroRNA (miR) dysregulation occurs in liver and immune cells of PBC patients, but its functional relevance is largely unknown. We previously reported that miR-506 is overexpressed in PBC cholangiocytes and directly targets both Cl - / HCO3- anion exchanger 2 and type III inositol 1,4,5-trisphosphate receptor, leading to cholestasis. Here, the regulation of miR-506 gene expression and its role in cholangiocyte pathophysiology and immune activation was studied. Several proinflammatory cytokines overexpressed in PBC livers (such as interleukin-8 [IL8], IL12, IL17, IL18, and tumor necrosis factor alpha) stimulated miR-506 promoter activity in human cholangiocytes, as revealed by luciferase reporter assays. Experimental overexpression of miR-506 in cholangiocytes dysregulated the cell proteomic profile (by mass spectrometry), affecting proteins involved in different biological processes including mitochondrial metabolism. In cholangiocytes, miR-506 (1) induced dedifferentiation with down-regulation of biliary and epithelial markers together with up-regulation of mesenchymal, proinflammatory, and profibrotic markers; (2) impaired cell proliferation and adhesion; (3) increased oxidative and endoplasmic reticulum stress; (4) caused DNA damage; and (5) sensitized to caspase-3-dependent apoptosis induced by cytotoxic bile acids. These events were also associated with impaired energy metabolism in mitochondria (proton leak and less adenosine triphosphate production) and pyruvate dehydrogenase complex E2 overexpression. Coculture of miR-506 overexpressing cholangiocytes with PBC immunocytes induced activation and proliferation of PBC immunocytes., Conclusion: Different proinflammatory cytokines enhance the expression of miR-506 in biliary epithelial cells; miR-506 induces PBC-like features in cholangiocytes and promotes immune activation, representing a potential therapeutic target for PBC patients. (Hepatology 2018;67:1420-1440)., (© 2017 by the American Association for the Study of Liver Diseases.)

Background: Human liver cell lines, like HepaRG and C3A, acquire higher functionality when cultured in the AMC-Bio-Artificial Liver (AMC-BAL). The three main differences between BAL and monolayer culture are the oxygenation (40% vs 20%O 2 ), dynamic vs absent medium perfusion and 3D vs 2D configuration. Here, we investigated the background of the differences between BAL-cultures and monolayers., Methods: We performed whole-genome microarray analysis on HepaRG monolayer and BAL-cultures. Next, mitochondrial biogenesis was studied in monolayer and BAL-cultures of HepaRG and C3A. The driving forces for mitochondrial biogenesis by BAL-culturing were investigated in representative culture models differing in oxygenation level, medium flow or 2D vs 3D configuration., Results: Gene-sets related to mitochondrial energy metabolism were most prominently up-regulated in HepaRG-BAL vs monolayer cultures. This was confirmed by a 2.4-fold higher mitochondrial abundance with increased expression of mitochondrial OxPhos complexes. Moreover, the transcript levels of mitochondria-encoded genes were up to 3.6-fold induced and mitochondrial membrane potential activity was 8.3-fold increased in BAL vs monolayers. Culturing with 40% O 2 , dynamic medium flow and/or in 3D increased the mitochondrial abundance and expression of mitochondrial complexes vs standard monolayer culturing. The stimulatory effect of the BAL culture on mitochondrial biogenesis was confirmed in C3A cells in which mitochondrial abundance increased 2.2-fold with induction of mitochondria-encoded genes., Conclusions and General Significance: The increased functionality of liver cell lines upon AMC-BAL culturing is associated with increased mitochondrial biogenesis. High oxygenation, medium perfusion and 3D configuration contribute to the up-regulation of the mitochondrial biogenesis., (Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Background: Up to 70% of patients with primary biliary cholangitis develop pruritus (itch) during the course of their disease. Treatment of pruritus in primary biliary cholangitis is challenging and novel therapies are needed. Ursodeoxycholic acid, the standard first-line treatment for primary biliary cholangitis, is largely ineffective for pruritus. We investigated the efficacy and safety of GSK2330672, a selective inhibitor of human ileal bile acid transporter (IBAT), in patients with primary biliary cholangitis with pruritus., Methods: We conducted this phase 2a, double-blind, randomised, placebo-controlled, crossover trial in two UK medical centres. Following 2 weeks of open placebo run-in, patients were randomly assigned in a 1:1 ratio with a block size of 4 to receive GSK2330672 or placebo twice daily during two consecutive 14-day treatment periods in a crossover sequence. The treatment periods were followed by a 14-day single-blinded placebo follow-up period. The primary endpoints were safety of GSK2330672, assessed using clinical and laboratory parameters, and tolerability as rated by the Gastrointestinal Symptom Rating Scale. The secondary endpoints were changes in pruritus scores measured using the 0 to 10 numerical rating scale (NRS), primary biliary cholangitis-40 (PBC-40) itch domain score and 5-D itch scale, changes in serum total bile acids and 7 alpha hydroxy-4-cholesten-3-one (C4), and changes in the pharmacokinetic parameters of ursodeoxycholic acid and its conjugates. The trial was registered with ClinicalTrials.gov, number NCT01899703., Findings: Between March 10, 2014, and Oct 7, 2015, we enrolled 22 patients. 11 patients were assigned to receive intervention followed by placebo (sequence 1), and 11 patients were assigned to receive placebo followed by intervention (sequence 2). One patient assigned to sequence 2 withdrew consent prior to receiving randomised therapy. One patient did not attend the placebo follow-up period, but was included in the final analysis. GSK2330672 treatment for 14 days was safe with no serious adverse events reported. Diarrhoea was the most frequent adverse event during treatment with GSK2330672 (seven with GSK2330672 vs one with placebo) and headache was the most frequent adverse event during treatment with placebo (seven with placebo vs six with GSK2330672). After GSK2330672 treatment, the percentage changes from baseline itch scores were -57% (95% CI -73 to -42, p<0·0001) in the NRS, -31% (-42 to -20, p<0·0001) in the PBC-40 itch domain and -35% (-45 to -25, p<0·0001) in the 5-D itch scale. GSK2330672 produced significantly greater reduction from baseline than the double-blind placebo in the NRS (-23%, 95% CI -45 to -1; p=0·037), PBC-40 itch domain, (-14%, -26 to -1; p=0·034), and 5-D itch scale (-20%, -34 to -7; p=0·0045). After GSK2330672 treatment, serum total bile acid concentrations declined by 50% (95% CI -37 to -61, p<0·0001) from 30 to 15 μM, with a significant 3·1-times increase (95% CI 2·4 to 4·0, p<0·0001) in serum C4 concentrations from 7·9 to 24·7ng/mL., Interpretation: In patients with primary biliary cholangitis with pruritus, 14 days of ileal bile acid transporter inhibition by GSK2330672 was generally well tolerated without serious adverse events, and demonstrated efficacy in reducing pruritus severity. GSK2330672 has the potential to be a significant and novel advance for the treatment of pruritus in primary biliary cholangitis. Diarrhoea, the most common adverse event associated with GSK2330672 treatment, might limit the long-term use of this drug., Funding: GlaxoSmithKline and National Institute for Health Research., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.

Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis) is a chronic fibrosing cholangiopathy with the signature of an autoimmune disease and features of intrahepatic cholestasis. Immunosuppressing treatments are largely unsuccessful. Responsiveness to ursodeoxycholic acid and reduced expression of anion exchanger 2 (AE2) on canalicular membranes and small bile ducts underline the importance of bicarbonate transportation in its disease mechanism. Soluble adenylyl cyclase (sAC; ADCY10) is an evolutionarily conserved bicarbonate sensor that regulates apoptosis, barrier function and TNF signaling. Key Messages: The biliary epithelium defends against the toxic bile by bicarbonate secretion and by maintaining a tight barrier. Passive diffusion of weak acid conjugates (e.g. bile salts and other toxins) across plasma membrane is pH-dependent. Reduced AE2 expression results in both reduced bicarbonate secretion and accumulation of bicarbonate in the cells. Increased intracellular bicarbonate leads to increased sAC activity, which regulates bile salt-induced apoptosis. Reduced bicarbonate secretion causes more bile salts to enter cells, which further increase sAC activity by releasing intracellular Ca2+ store. In vitro studies demonstrate that inhibition of sAC not only corrects sensitization to bile salt-induced apoptosis as a result of AE2 down-regulation but also prevents bile salt-induced apoptosis altogether. Targeting sAC is also likely to slow down disease progression by strengthening the barrier function of biliary epithelia and by reducing oxidative stress as a result of chronic inflammation., Conclusions: sAC is a potential therapeutic target for PBC. More in vitro and in vivo studies are needed to understand how sAC regulates bile salt-induced apoptosis and to establish its therapeutic value in PBC and other cholestatic cholangiopathies., (© 2017 S. Karger AG, Basel.)

Ethanol abuse is a serious public health problem that is associated with several stages of alcoholic liver disease (ALD) and a high incidence of morbidity and mortality. Alcoholic fatty liver disease (AFLD), the earliest stage of ALD, is a multifactorial injury that involves oxidative stress and disruptions of lipid metabolism. Although benign and reversible, no pharmacological treatments are available for this condition. In the present study, we induced AFLD in mice with 10% ethanol and a low-protein diet and then orally treated them with a hydroethanolic extract of Baccharis trimera (HEBT; 30 mg kg -1 ). HEBT reversed ethanol-induced oxidative stress in the liver, reduced lipoperoxidation, normalized GPx, GST, SOD and Cat activity, and GSH and total ROS levels. The reverser effect of HEBT was observed upon ethanol-induced increases in the levels of plasma and hepatic triglycerides, plasma cholesterol, plasma high-density lipoprotein, and plasma and hepatic low-density lipoprotein. Moreover, HEBT increased fecal triglycerides and reduced the histological ethanol-induced lesions in the liver. HEBT also altered the expression of genes that are involved in ethanol metabolism, antioxidant systems, and lipogenesis (i.e., CypE1, Nrf2, and Scd1, respectively). No signs of toxicity were observed in HEBT-treated mice. We propose that HEBT may be a promising pharmacological treatment for AFLD., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Menopause is often followed by obesity and, related to this, non-alcoholic fatty liver disease (NAFLD). Two bile acid (BA) receptors, farnesoid X receptor (FXR) and G-protein-coupled receptor TGR5, have emerged as putative therapeutic targets for obesity and NAFLD., Aim of This Study: to evaluate the efficacy of selective agonists INT747/obeticholic acid (FXR) and INT777 (TGR5) as novel treatments for the metabolic effects of oestrogen deficiency. Ovariectomized (OVX) or sham-operated (SHAM) mice were fed a high-fat diet (HFD) for 5weeks. During the last 4weeks two groups of OVX and SHAM mice received either INT747- or INT777-supplemented HFD. OVX mice had significantly higher bodyweight gain than SHAM mice, which was attenuated by INT747- or INT777-treatment. No significant changes in food intake or physical activity were found. OVX mice had significantly lower energy expenditure than SHAM mice; INT747- and INT777-treated OVX mice had intermediate energy expenditure. Liver triglyceride and cholesterol content was significantly increased in OVX compared to SHAM mice, which was normalized by INT747- or INT777-treatment. Significant changes in metabolic gene expression were found in liver (Cpt1, Acox1), muscle (Ucp3, Pdk4, Cpt1, Acox1, Fasn, Fgf21), brown adipocytes (Dio2) and white adipocytes (c/EBPα, Pparγ, Adipoq). For the first time, expression of FXR and induction of its target gene Pltp1 was shown in skeletal muscle. BA receptor agonists are suitable therapeutics to correct postmenopausal metabolic changes in an OVX mouse model. Potential mechanisms include increased energy expenditure and changes in expression patterns of key metabolic genes in liver, muscle and adipose tissues., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function. We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription. We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells. We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pruritus (itch) is an important symptom of primary biliary cirrhosis (PBC), an archetypal cholestatic liver disease. Cholestatic pruritus can be a debilitating symptom causing significant deterioration in patients' quality of life. Effective management of pruritus in PBC involves awareness among clinicians to adequately assess its severity, and treatment with specific drug therapies in line with current practice guidelines. In PBC, antipruritic drugs are not universally effective and/or have significant side effects, and despite best efforts with various combinations of drugs, some patients remain significantly symptomatic, eventually opting for invasive or experimental treatments. Therefore, there is a clear unmet need for better alternative treatments for patients with refractory or intractable cholestatic pruritus. Recent advances in the understanding of pathogenesis of cholestatic pruritus and bile acid physiology have raised hopes for novel therapies, some of which are currently under trial. In this review, we aim to provide a practical guide to the management of this important and complex problem, discussing current knowledge and recent advances in the pathogenesis, summarise the evidence base for available therapeutic approaches and update potential novel future therapies for the management of pruritus in PBC.

Unlabelled: ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a conjugated hyperbilirubinemia and an unconjugated hypercholanemia. Here, we have studied the hypothesis that ATP11C deficiency interferes with basolateral uptake of unconjugated bile salts, a process mediated by organic anion-transporting polypeptide (OATP) 1B2. ATP11C localized to the basolateral membrane of central hepatocytes in the liver lobule of control mice. In ATP11C-deficient mice, plasma total bilirubin levels were 6-fold increased, compared to control, of which ∼65% was conjugated and ∼35% unconjugated. Plasma total bile salts were 10-fold increased and were mostly present as unconjugated species. Functional studies in ATP11C-deficient mice indicated that hepatic uptake of unconjugated bile salts was strongly impaired whereas uptake of conjugated bile salts was unaffected. Western blotting and immunofluorescence analysis demonstrated near absence of basolateral bile salt uptake transporters OATP1B2, OATP1A1, OATP1A4, and Na(+) -taurocholate-cotransporting polypeptide only in central hepatocytes of ATP11C-deficient liver. In vivo application of the proteasome inhibitor, bortezomib, partially restored expression of these proteins, but not their localization. Furthermore, we observed post-translational down-regulation of ATP11C protein in livers from cholestatic mice, which coincided with reduced OATP1B2 levels., Conclusions: ATP11C is essential for basolateral membrane localization of multiple bile salt transport proteins in central hepatocytes and may act as a gatekeeper to prevent hepatic bile salt overload. Conjugated hyperbilirubinemia and unconjugated hypercholanemia and loss of OATP expression in ATP11C-deficient liver strongly resemble the characteristics of Rotor syndrome, suggesting that mutations in ATP11C can predispose to Rotor syndrome. (Hepatology 2016;64:161-174)., Competing Interests: Potential conflict of interest: Nothing to report., (© 2016 by the American Association for the Study of Liver Diseases.)

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

Objective: Pruritus is a common symptom of cholestatic liver disorders. The present study aimed at evaluating autotaxin (ATX), a lysophospholipase recently identified as potential cause for cholestatic pruritus, in pediatric cholestatic diseases presenting with or without itching., Methods: A cohort of 45 children consisting of 14 patients experiencing itching (Alagille syndrome [n = 10], complete extrahepatic biliary atresia [n = 2], neonatal sclerosing cholangitis (n = 1), progressive familial intrahepatic cholestasis type 2 [n = 1]), 9 patients with bile acid synthesis defects (3β-hydroxy-C27-steroid-oxidoreductase [n = 7] and Δ-3-oxosteroid-5β-reductase deficiency [n = 2]), and 22 healthy children were studied. Serum ATX activity and total serum bile salt were determined enzymatically, ATX protein content was semiquantified by Western blotting. Using real-time polymerase chain reaction, ATX mRNA expression was studied in HepG2 cells treated with farnesoid-X-receptor agonists or vehicle., Results: Serum ATX activity was increased in pruritic children with Alagille and other cholestatic syndromes (mean ± standard deviation: 16.1 ± 4.3 nmol · mL · min) compared with children with nonpruritic cholestatic diseases with bile acid synthesis defects (10.4 ± 4.7 nmol · mL · min; P < 0.01) and healthy controls (7.6 ± 2.3 nmol · mL · min; P < 0.001). ATX protein levels closely correlated with serum ATX activity. Serum ATX activity and total serum bile salt showed a linear correlation with itch intensity (r = 0.66, P < 0.001 and r = 0.80, P < 0.001, respectively). No correlation was observed between ATX activity and bilirubin. ATX mRNA expression in HepG2 cells was not induced by farnesoid-X-receptor ligands., Conclusions: Serum ATX activity correlated with itch intensity in children with cholestatic diseases. Bile salts did not increase ATX expression in vitro. ATX inhibitors may be useful antipruritic agents in pediatric cholestatic disorders.

Unlabelled: Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl-/HCO3- anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2a,b(-/-) mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2a,b(-/-) mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥ 10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8(+) T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2'-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8(+) T cells from aged knockouts., Conclusion: Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8+ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.

Unlabelled: The Na(+) -taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte-specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1(-/-) hepatocytes showed absence of sodium-dependent taurocholic acid uptake, whereas sodium-independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1(-/-) mice, explained by increased Asbt and Ostα/β expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down-regulation of OATP1A1 and up-regulation of OATP1A4. Furthermore, sodium-dependent taurocholic acid uptake was inhibited by Myrcludex B in wild-type hepatocytes, while Slc10a1(-/-) hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1(-/-) mice., Conclusion: The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation., (© 2015 by the American Association for the Study of Liver Diseases.)

IgG4-associated cholangitis (IAC) is a major manifestation of immunoglobulin G4-related disease (IgG4-RD), an inflammatory multiorgan disorder of unknown cause. IAC and autoimmune pancreatitis (AIP) may mimic sclerosing cholangitis, cholangiocarcinoma, or pancreatic carcinoma. Typically, elderly male patients present with abdominal discomfort, weight loss, jaundice, and itch. At present, no accurate diagnostic test for IAC and IgG4-RD is at hand, often causing significant diagnostic delay. Serum IgG4 is only diagnostic when markedly raised (>4× ULN). Imaging in IAC discloses mass-forming lesions and/or strictures in the biliary tract. Histology may show tissue infiltration of IgG4-expressing plasma cells. Diagnostic criteria for histologic and imaging findings, serum tests, organ manifestation pattern, and response to immunosuppressive therapy (HISORt) criteria are used for the diagnosis of IgG4-RD. Still, considering the difficulty in diagnosing IAC and AIP, unnecessary hepatic or pancreatic resections for presumed malignancies occur. The good response to corticosteroid therapy in IAC and other manifestations of IgG4-RD suggests an immune-mediated inflammatory disease. Maintenance immunosuppression after induction of remission is needed in the majority of patients to avoid relapse. The pathogenesis of IAC and IgG4-RD remains poorly understood. Unresolved questions include: (i) Does IgG4 have a pro- or anti-inflammatory role in IAC? (ii) Is IAC a B cell- and/or T cell-mediated disease? (iii) Which are the molecular targets attacked by the immune system in IgG4-RD? Here, we review the diagnostic and therapeutic management of the disease and discuss recent pathophysiological findings, which might help to better understand the molecular mechanisms contributing to IAC and other manifestations of IgG4-RD.

Fucosyltransferase 2 (FUT2) mediates the inclusion of fucose in sugar moieties of glycoproteins and glycolipids. ABO blood group antigens and host-microbe interactions are influenced by FUT2 activity. About 20 % of the population has a "non-secretor" status caused by inactivating variants of FUT2 on both alleles. The non-sense mutation G428A and the missense mutation A385T are responsible for the vast majority of the non-secretor status in Caucasians, Africans, and Asians, respectively. Non-secretor individuals do not secrete fucose-positive antigens and lack fucosylation in epithelia. They also appear to be protected against a number of infectious diseases, such as Norovirus and Rotavirus infections. In recent years, genome-wide association studies (GWAS) identified inactivating variants at the FUT2 locus to be associated with primary sclerosing cholangitis (PSC), Crohn's disease (CD), and biochemical markers of biliary damage. These associations are intriguing given the important roles of fucosylated glycans in host-microbe interactions and membrane stability. Non-secretors have a reduced fecal content of Bifidobacteria. The intestinal bacterial composition of CD patients resembles the one of non-secretors, with an increase in Firmicutes and decreases in Proteobacteria and Actinobacteria. Non-secretor individuals lack fucosylated glycans at the surface of biliary epithelium and display a different bacterial composition of bile compared to secretors. Notably, an intact biliary epithelial glycocalix is relevant for a stable 'biliary HCO3 (-) umbrella' to protect against toxic effects of hydrophobic bile salt monomers. Here, the biology of FUT2 will be discussed as well as hypotheses to explain the role of FUT2 in the pathophysiology of PSC and Crohn's disease.

Background & Aims: Intrahepatic cholestasis of pregnancy (ICP) is defined by pruritus, elevated total fasting serum bile salts (TBS) and transaminases, and an increased risk of adverse fetal outcome. An accurate diagnostic marker is needed. Increased serum autotaxin correlates with cholestasis-associated pruritus. We aimed at unraveling the diagnostic accuracy of autotaxin in ICP., Methods: Serum samples and placental tissue were collected from 44 women with uncomplicated pregnancies and 105 with pruritus and/or elevated serum transaminases. Autotaxin serum levels were quantified enzymatically and by Western blotting, autotaxin gene expression by quantitative PCR., Results: Serum autotaxin was increased in ICP (mean ± SD: 43.5 ± 18.2 nmol ml(-1)min(-1), n=55, p<0.0001) compared to other pruritic disorders of pregnancy (16.8 ± 6.7 nmol ml(-1)min(-1), n=33), pre-eclampsia complicated by HELLP-syndrome (16.8 ± 8.9 nmol ml(-1)min(-1), n=17), and pregnant controls (19.6 ± 5.7 nmol ml(-1)min(-1), n=44). Longitudinal analysis during pregnancy revealed a marked rise in serum autotaxin with onset of ICP-related pruritus. Serum autotaxin was increased in women taking oral contraceptives. Increased serum autotaxin during ICP was not associated with increased autotaxin mRNA in placenta. With a cut-off value of 27.0 nmol ml(-1)min(-1), autotaxin had an excellent sensitivity and specificity in distinguishing ICP from other pruritic disorders or pre-eclampsia/HELLP-syndrome. Serum autotaxin displayed no circadian rhythm and was not influenced by food intake., Conclusions: Increased serum autotaxin activity represents a highly sensitive, specific and robust diagnostic marker of ICP, distinguishing ICP from other pruritic disorders of pregnancy and pregnancy-related liver diseases. Pregnancy and oral contraception increase serum autotaxin to a much lesser extent than ICP., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

P4 ATPases are lipid flippases and transport phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. Lipid flipping is important for the biogenesis of transport vesicles. Recently it was shown that loss of the P4 ATPases ATP8B1 and ATP11C are associated with severe Cholestatic liver disease. Mutation of ATP8B1 cause progressive familial Intrahepatic Cholestasis type 1 (PFIC1)and benign recurrent intrahepatic cholestasis type 1 (BRIC 1). From our observations we hypothesized that ATP8B1 deficiency causes a phospholipids randomization at the canalicular membrane, which results in extraction of cholesterol due to increase sensitivity of the canalicular membrane. Deficiency of ATP11C causes conjugated hyperbilirubinemia. In our preliminary result we observed accumulation of unconjugated bile salts in Atp11c deficient mice probably because of regulation in the expression or function of OATP1B2. Similar to ATP8B1, ATP11C have regulation on membrane transporters., (2015 S. Karger AG, Basel.)

Pruritus is a preeminent symptom in patients with chronic cholestatic liver disorders such as primary biliary cirrhosis and primary sclerosing cholangitis. More than two-thirds of these patients experience itching during the course of their disease. This symptom is also frequently observed in patients with other causes of cholestasis such as cholangiocarcinoma, inherited forms of cholestasis and intrahepatic cholestasis of pregnancy, but may accompany almost any other liver disease. The pathogenesis of pruritus of cholestasis remains largely elusive. Increased concentrations of bile salts, histamine, serotonin, progesterone metabolites and endogenous opioids have been controversially discussed as potential pruritogens. However, for these molecules, neither a correlation with itch intensity nor a causative link could be established. The G protein-coupled receptor for bile salts, TGR5, has been shown to be expressed in dorsal root ganglia and give rise to itch in rodents, albeit upon stimuli with suprapathological concentrations of bile salts. The potent neuronal activator lysophosphatidic acid (LPA) and its forming enzyme, autotaxin (ATX), could be identified in the serum of patients with cholestatic pruritus. ATX activity correlated with itch severity and effectiveness of several anti-pruritic therapeutic interventions in cholestatic patients. Thus, the ATX-LPA-axis may represent a key element in the pathogenesis of this agonizing symptom. Treatment options for pruritus of cholestasis remain limited to a few evidence-based and several experimental medical and interventional therapies. The current guideline-based recommendations include the anion exchange resins colestyramine, the pregnane X receptor-agonist and enzyme inducer rifampicin, the μ-opioid antagonist naltrexone, and the selective serotonin reuptake inhibitors sertraline. Still, a considerable part of patients is unresponsive to these drugs and requires experimental approaches including phototherapy, plasmapheresis, albumin dialysis or nasobiliary drainage. This review outlines the current knowledge on pathogenesis of cholestatic pruritus and summarizes evidence-based and experimental therapeutic interventions for cholestatic patients with itch., (© 2015 S. Karger AG, Basel.)

Lysophosphatidic acid is a potent signaling lipid molecule that has initially been characterized as a growth factor. However, later studies have revealed many more functions such as modulation of cell shape, cell migration, prevention of apoptosis, platelet aggregation, wound healing, osteoclast differentiation, vasopressor activity, embryo implantation, angiogenesis, lung fibrosis, hair growth and more. The molecule mainly acts through the activation of a set of at least 6 G-protein-coupled receptors (LPA1-6), but intracellular LPA was also shown to signal through the activation of the nuclear receptor PPARγ. In this short review we discuss the recent observations which suggest that in pathological conditions LPA also modulates signaling in sensory neurons. Thus, LPA has been shown to play a role in the initiation of neuropathic pain and, more recently, a relation was observed between increased LPA levels in the circulation and cholestatic itch. The mechanism by which this occurs remains to be elucidated. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Destruction of cholangiocytes is the hallmark of chronic cholangiopathies such as primary biliary cirrhosis. Under physiologic conditions, cholangiocytes display a striking resistance to the high, millimolar concentrations of toxic bile salts present in bile. We recently showed that a 'biliary HCO3(-) umbrella', i.e. apical cholangiocellular HCO3(-) secretion, prevents cholangiotoxicity of bile acids, and speculated on a role for extracellular membrane-bound glycans in the stabilization of this protective layer. This paper summarizes published and thus far unpublished evidence supporting the role of the glycocalyx in stabilizing the 'biliary HCO3(-) umbrella' and thus preventing cholangiotoxicity of bile acids., Key Messages: The apical glycocalyx of a human cholangiocyte cell line and mouse liver sections were visualized by electron microscopy. FACS analysis was used to characterize the surface glycan profile of cultured human cholangiocytes. Using enzymatic digestion with neuraminidase the cholangiocyte glycocalyx was desialylated to test its protective function. Using lectin assays, we demonstrated that the main N-glycans in human and mouse cholangiocytes were sialylated biantennary structures, accompanied by high expression of the H-antigen (α1-2 fucose). Apical neuraminidase treatment induced desialylation without affecting cell viability, but lowered cholangiocellular resistance to bile acid-induced toxicity: both glycochenodeoxycholate and chenodeoxycholate (pKa ≥4), but not taurochenodeoxycholate (pKa <2), displayed cholangiotoxic effects after desialylation. A 24-hour reconstitution period allowed cholangiocytes to recover to a pretreatment bile salt susceptibility pattern., Conclusion: Experimental evidence indicates that an apical cholangiocyte glycocalyx with glycosylated mucins and other glycan-bearing membrane glycoproteins stabilizes the 'biliary HCO3(-) umbrella', thus aiding in the protection of human cholangiocytes against bile acid toxicity., (2015 S. Karger AG, Basel.)

Unlabelled: The enterohepatic circulation of bile salts is an important physiological route to recycle bile salts and ensure intestinal absorption of dietary lipids. The Na(+)-taurocholate cotransporting polypeptide SLC10A1 (NTCP) plays a key role in this process as the major transporter of conjugated bile salts from the plasma compartment into the hepatocyte. Here we present the first patient with NTCP deficiency, who was clinically characterized by mild hypotonia, growth retardation, and delayed motor milestones. Total bile salts in plasma were extremely elevated (up to 1,500 μM, ref. <16.3) but there were no clinical signs of cholestatic jaundice, pruritis, or liver dysfunction. Bile salt synthesis and intestinal bile salt signaling were not affected, as evidenced by normal plasma 7α-hydroxy-4-cholesten-3-one (C4) and FGF19 levels. Importantly, the presence of secondary bile salts in the circulation suggested residual enterohepatic cycling of bile salts. Sequencing of the SLC10A1 gene revealed a single homozygous nonsynonymous point mutation in the coding sequence of the gene, resulting in an arginine to histidine substitution at position 252. Functional studies showed that this mutation resulted in a markedly reduced uptake activity of taurocholic acid. Immunofluorescence studies and surface biotinylation experiments demonstrated that the mutant protein is virtually absent from the plasma membrane., Conclusion: We describe the identification of NTCP deficiency as a new inborn error of metabolism with a relatively mild clinical phenotype. The identification of NTCP deficiency confirms that this transporter is the main import system for conjugated bile salts into the liver but also indicates that auxiliary transporters are able to sustain the enterohepatic cycle in its absence., (© 2014 by the American Association for the Study of Liver Diseases.)

Deficiency of the phospholipid flippase ATPase, aminophospholipid transporter, class I, type 8B, member 1 (ATP8B1) causes progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1). Apart from cholestasis, many patients also suffer from diarrhea of yet unknown etiology. Here we have studied the hypothesis that intestinal ATP8B1 deficiency results in bile salt malabsorption as a possible cause of PFIC1/BRIC1 diarrhea. Bile salt transport was studied in ATP8B1-depleted intestinal Caco-2 cells. Apical membrane localization was studied by a biotinylation approach. Fecal bile salt and electrolyte contents were analyzed in stool samples of PFIC1 patients, of whom some had undergone biliary diversion or liver transplantation. Bile salt uptake by the apical sodium-dependent bile salt transporter solute carrier family 10 (sodium/bile acid cotransporter), member 2 (SLC10A2) was strongly impaired in ATP8B1-depleted Caco-2 cells. The reduced SLC10A2 activity coincided with strongly reduced apical membrane localization, which was caused by impaired apical membrane insertion of SLC10A2. Moreover, we show that endogenous ATP8B1 exists in a functional heterodimer with transmembrane protein 30A (CDC50A) in Caco-2 cells. Analyses of stool samples of post-transplant PFIC1 patients demonstrated that bile salt content was not changed, whereas sodium and chloride concentrations were elevated and potassium levels were decreased. The ATP8B1-CDC50A heterodimer is essential for the apical localization of SLC10A2 in Caco-2 cells. Diarrhea in PFIC1/BRIC1 patients has a secretory origin to which SLC10A2 deficiency may contribute. This results in elevated luminal bile salt concentrations and consequent enhanced electrolyte secretion and/or reduced electrolyte resorption., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Progressive familial intrahepatic cholestasis (PFIC) types 1 and 3 are severe cholestatic liver diseases caused by deficiency of ATB8B1 and ABCB4, respectively. Mouse models for PFIC display mild phenotypes compared with human patients, and this can be explained by the difference in bile salt pool composition. Mice, unlike humans, have the ability to detoxify hydrophobic bile salts by cytochrome P450-mediated (re)hydroxylation and thus have a less toxic bile salt pool. We have crossed mouse models for PFIC1 and PFIC3 with Hrn mice that have a reduced capacity to (re)hydroxylate bile salts. Double transgenes were obtained by backcrossing Atp8b1(G308V/G308V) and Abcb4(-/-) mice with Hrn mice that have a liver-specific disruption of the cytochrome P450 reductase gene and therefore have markedly reduced P450 activity. In these mice, a more hydrophobic bile salt pool was instilled by cholic acid supplementation of the diet, and bile formation and liver pathology was studied. As opposed to single transgenes, Atp8b1(G308V/G308V)/Hrn and Abcb4(-/-)/Hrn mice rapidly developed strong cholestasis that was evidenced by increased plasma bilirubin and bile salt levels. The bile salt pool was more toxic in both models; Atp8b1(G308V/G308V)/Hrn mice had a more hydrophobic plasma pool compared with the single transgene, whereas Abcb4(-/-)/Hrn mice had a more hydrophobic biliary pool compared with the single transgene. In line with these findings, liver damage was not aggravated in Atp8b1(G308V/G308V)/Hrn but was more severe in Abcb4(-/-)/Hrn mice. These data indicate that bile salt pool composition is a critical determinant in the initiation and progression of cholestasis and liver pathology in PFIC1 and PFIC3. Most importantly, our data suggest that the hydrophobicity of the plasma bile salt pool is an important determinant of the severity of cholestasis, whereas the hydrophobicity of the biliary bile salt pool is an important determinant of the severity of liver pathology.

Objective: Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations., Approach and Results: Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations., Conclusions: Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy., (© 2014 American Heart Association, Inc.)

Pruritus is a common symptom in patients with cholestatic liver diseases such as primary biliary cirrhosis, primary sclerosing cholangitis, intrahepatic cholestasis of pregnancy, or hereditary pediatric cholestatic disorders and may accompany, although less frequently, many other liver diseases. Recent findings indicate that lysophosphatidic acid (LPA), a potent neuronal activator, and autotaxin (ATX; ectonucleotide pyrophosphatase/phosphodiesterase 2), the enzyme which forms LPA, may form a key element of the long-sought pruritogenic signaling cascade in cholestatic patients suffering from itch. Serum ATX, but no other pruritogen candidate studied so far, correlates with pruritus intensity and responds to therapeutic interventions. In this comprehensive review, we provide a short update on actual insights in signal transmission related to pruritus and discuss pruritogen candidates in cholestasis. We also summarize evidence-based and guideline-approved as well as experimental therapeutic approaches for patients suffering from pruritus in cholestasis., (© 2014 by the American Association for the Study of Liver Diseases.)

Pruritus is a sensory phenomenon accompanying a broad range of systemic disorders including hematologic and lymphoproliferative disorders, metabolic and endocrine diseases, solid tumours, and infectious diseases. The molecular mechanisms involved in itch sensation remain enigmatic in most of these diseases. However, from studies in patients and animal models a large number of mediators and receptors responsible for scratching behaviour have been identified in recent years. New insights into the interplay between neuronal and non-neuronal cells in the initiation, modulation and sensitization of itch sensation have been acquired. This review highlights the current knowledge of the molecular mechanism involved in pruritus of systemic disorders and summarizes the signalling pathways of biogenic amines, neuropeptides, proteases, eicosanoids, cytokines, opioids, endocannabinoids, neurotrophins, phospholipids and other signalling molecules participating in pruritus., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background and Aim: Excessive ethanol consumption can lead to development of hepatic steatosis. Since the FXR receptor regulates adipose cell function and liver lipid metabolism, the aim of this work was to examine the effects of the FXR agonist 6ECDCA on alcoholic liver steatosis development and on oxidative stress induced by ethanol consumption., Methods: Swiss mice (n=24) received a low-protein diet (6%) and a liquid diet containing 10% ethanol or water for 6weeks. In the last 15days mice received oral treatment with 6ECDCA (3mgkg(-1)) or 1% tween (vehicle). The experimental groups (n=6) were: water+tween, water+6ECDCA, ethanol+tween and ethanol+6ECDCA. Moreover, as a diet control, we used a basal group (n=6), fed by a normal-proteic diet (23%) and water. After the treatment period, the animals were anesthetized for sample collection to perform plasma biochemistry assays, hepatic oxidative stress assays, hepatic cholesterol and triglycerides measurements, liver histology and hepatic gene expression., Results: Ethanol associated with low-protein diet induced hepatic oxidative stress, increased plasma transaminases and induced hepatic lipid accumulation. Many of these parameters were reversed by the administration of 6ECDCA, including amelioration of lipid accumulation and lipoperoxidation, and reduction of reactive oxygen species. These effects were possibly mediated by regulation of Srebpf1 and FAS gene expression, both reduced by the FXR agonist., Conclusions: Our data demonstrated that 6ECDCA reverses the accumulation of lipids in the liver and decreases the oxidative stress induced by ethanol and low-protein diet. This FXR agonist is promising as a potential therapy for alcoholic liver steatosis., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: The hydration of CO2 catalyzed by the ubiquitous carbonic anhydrase 2 (Ca2) is central for bicarbonate transport, bone metabolism and acid-base homeostasis in metazoans. There is evidence that in some tissues Ca2 expression can be acutely induced by cAMP, whereas in other cell types it is unresponsive to cAMP-mediated transcriptional activation., Methods: We isolated fibroblasts from wild type and mice lacking the ubiquitous chloride/bicarbonate exchanger (Ae2a,b(-/-) mice). In these cells the regulation of carbonic anhydrase 2 by cAMP was studied., Results: We show that Ca2 expression is strongly inhibited by chronic incubation with dibutyryl-cAMP, forskolin or alkaline pH in cultured mouse fibroblasts. Furthermore, fibroblasts obtained from anion exchanger 2 deficient (Ae2a,b(-/-)) mice, which display intracellular alkalosis and increased cAMP production, express less than 10% of control Ca2 mRNA and protein. Surprisingly, inhibition of the bicarbonate-sensitive soluble adenylyl cyclase (sAC) was found to reduce CA2 expression instead of increasing it., Conclusions: CA2 expression is strongly regulated by intracellular pH and by cAMP, suggesting a role for soluble adenylyl cyclase. Regulation occurs in opposite directions which may be explained by an incoherent feedforward loop consisting of activation by pCREB and repression by ICER., (Copyright © 2013 Elsevier B.V. All rights reserved.)

It has been established that bile salts play a role in the regulation of hepatic lipid metabolism. Accordingly, overt signs of steatosis have been observed in mice with reduced bile salt synthesis. The aim of this study was to identify the mechanism of hepatic steatosis in mice with bile salt deficiency due to a liver specific disruption of cytochrome P450 reductase. In this study mice lacking hepatic cytochrome P450 reductase (Hrn) or wild type (WT) mice were fed a diet supplemented with or without either 0.1% cholic acid (CA) or 0.025% obeticholic acid, a specific FXR-agonist. Feeding a CA-supplemented diet resulted in a significant decrease of plasma ALT in Hrn mice. Histologically, hepatic steatosis ameliorated after CA feeding and this was confirmed by reduced hepatic triglyceride content (115.5±7.3mg/g liver and 47.9±4.6mg/g liver in control- and CA-fed Hrn mice, respectively). The target genes of FXR-signaling were restored to normal levels in Hrn mice when fed cholic acid. VLDL secretion in both control and CA-fed Hrn mice was reduced by 25% compared to that in WT mice. In order to gain insight in the mechanism behind these bile salt effects, the FXR agonist also was administered for 3weeks. This resulted in a similar decrease in liver triglycerides, indicating that the effect seen in bile salt fed Hrn animals is FXR dependent. In conclusion, steatosis in Hrn mice is ameliorated when mice are fed bile salts. This effect is FXR dependent. Triglyceride accumulation in Hrn liver may partly involve impaired VLDL secretion., (Copyright © 2014 Elsevier B.V. All rights reserved.)