Soluble CD163 and mannose receptor as markers of liver disease severity and prognosis in patients with primary biliary cholangitis.

INTRODUCTION: In primary biliary cholangitis (PBC), macrophages are involved in liver inflammation and fibrosis. The macrophage activation markers, soluble (s)CD163 and mannose receptor (sMR) are associated with liver disease severity and prognosis in other chronic liver diseases. We aimed to investigate sCD163 and sMR in patients with PBC. METHODS: We investigated PBC patients from the Italian PBC Study Group cohort and measured macrophage activation markers in serum at study enrolment. Patients were followed from enrolment until they experienced an event or were censored at their last visit. Events were defined as follows: (a) death from a liver-related cause; or (b) liver transplantation (LT) for PBC. We used Cox regression to investigate the association between sCD163 and sMR and long-term prognosis. RESULTS: In total, 202 PBC patients were included. Median age was 62 years (interquartile range (IQR), 53-71) at enrolment and 93% were women. Median sCD163 was 3.43 mg/L (IQR 2.48-5.35) and median sMR was 0.35 mg/L (IQR 0.28-0.45). There was an increase in sCD163 and sMR with increasing alkaline phosphatase. Two hundred and one patients were followed for a median of 8.6 years, and sCD163 and sMR predicted long-term risk of liver-related death or LT in univariate analyses, while sCD163 was also associated with outcome after confounder adjusting (adjusted HR = 1.14, 95% CI 1.00-1.30). Finally, we showed an increase in the prediction accuracy of poor outcome by adding sCD163 to the UK-PBC risk score. CONCLUSION: The macrophage activation markers sCD163 and sMR represent a non-invasive measure of PBC disease severity that provides useful long-term prognostic information.

Correction to: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Secretin/secretin receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.

Primary biliary cholangitis (PBC) primarily targets cholangiocytes and is characterized by liver fibrosis and biliary proliferation. Activation of the secretin (Sct)/secretin receptor (SR) axis, expressed only by cholangiocytes, increases biliary proliferation, liver fibrosis, and bicarbonate secretion. We evaluated the effectiveness of SR antagonist treatment for early-stage PBC. Male and female dominant-negative TGF-ß receptor II (dnTGF-ßRII) (model of PBC) and wild-type mice at 12 wk of age were treated with saline or the SR antagonist, Sec 5-27, for 1 wk. dnTGF-ßRII mice expressed features of early-stage PBC along with enhanced Sct/SR axis activation and Sct secretion. dnTGF-ßRII mice had increased biliary proliferation or senescence, inflammation, and liver fibrosis. In dnTGF-ßRII mice, there was increased microRNA-125b/TGF-ß1/TGF-ß receptor 1/VEGF-A signaling. Human early-stage PBC patients had an increase in hepatobiliary Sct and SR expression and serum Sct levels. Increased biliary Sct/SR signaling promotes biliary and hepatic damage during early-stage PBC.-Kennedy, L., Francis, H., Invernizzi, P., Venter, J., Wu, N., Carbone, M., Gershwin, M. E., Bernuzzi, F., Franchitto, A., Alvaro, D., Marzioni, M., Onori, P., Gaudio, E., Sybenga, A., Fabris, L., Meng, F., Glaser, S., Alpini, G. Secretin/secretin receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.

Autoantibodies in patients with interleukin 12 receptor beta 1 deficiency.

OBJECTIVE: Interleukin 12 receptor beta 1 (IL-12Rß1) deficiency is a primary immunodeficiency that exposes affected individuals to an augmented risk of intracellular pathogen-mediated infections. The paradoxical presence of autoimmune manifestations in immune-deficient patients has been recognized, but the basis of this phenomenon is unclear, with the role of frequent infections being a possible trigger to break tolerance. Our study aimed to analyze extensively a profile of autoantibodies in a clinically well-defined case series of patients with IL-12Rß1 deficiency. METHODS: Eight patients with IL-12Rß1 deficiency referred to Children's Medical Center in Tunis, Tunisia, during 1995-2012 were enrolled in the study. Sixteen age- and gender-matched blood donors served as controls. Serum, liver-related autoantibodies immunoglobulin (Ig)G, IgM, IgA were tested by ELISA and by standard indirect immunofluorescence on Hep-2 cells. RESULTS: We found a significant prevalence of liver autoantibodies in the study group. Regarding primary biliary cholangitis (PBC), two of eight patients were positive for MIT3 autoantibodies, both confirmed by immunofluorescence, and one patient was positive for PBC-specific antinuclear antibodies, sp100. Moreover, two patients had significantly increased gamma-glutamyltransferase levels and one had IgM levels twice the upper limit of normal. Intriguingly two patients were positive for anti-actin antibodies; a typical feature of autoimmune hepatitis type 1, along with a significant increase in IgG levels. CONCLUSIONS: This is the first report of a serological analysis in patients with an IL-12Rß1 deficiency. Despite the difficulty in interpreting the role of the IL-12, the evidence of liver-specific autoantibodies confirms the importance its signal in liver autoimmunity.

Novel biomarkers for primary biliary cholangitis to improve diagnosis and understand underlying regulatory mechanisms.

BACKGROUND AND AIMS: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis. METHODS: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker. RESULTS: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients. CONCLUSIONS: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes.

Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA.

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell-derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)-/- mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2-/- mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2-/- mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.

Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP-/-) mouse model. α-CGRP-/- and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP-/- and BDL α-CGRP-/- mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP-/- mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

Ursodeoxycholate inhibits mast cell activation and reverses biliary injury and fibrosis in Mdr2-/- mice and human primary sclerosing cholangitis.

Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. MCs infiltrate Mdr2-/- mice liver (model of primary sclerosing cholangitis (PSC)). MC-derived histamine increases inflammation, hepatic stellate cell (HSC) activation and fibrosis. The objective was to determine the effects of UDCA treatment on MC infiltration, biliary damage, inflammation and fibrosis in Mdr2-/- mice and human PSC. Wild-type and Mdr2-/- mice were fed bile acid control diet or UDCA (0.5% wt/wt). Human samples were collected from control and PSC patients treated with placebo or UDCA (15 mg/kg/BW). MC infiltration was measured by immunhistochemistry and quantitative polymerase chain reaction (qPCR) for c-Kit, chymase, and tryptase. The HDC/histamine/histamine receptor (HR)-axis was evaluated by EIA and qPCR. Intrahepatic bile duct mass (IBDM) and biliary proliferation was evaluated by CK-19 and Ki-67 staining. Fibrosis was detected by immunostaining and qPCR for fibrotic markers. Inflammatory components were measured by qPCR. HSC activation was measured by SYP-9 staining. Inflammation was detected by qPCR for CD68. In vitro, MCs were treated with UDCA (40 µM) prior to HA secretion evaluation and coculturing with cholangiocytes or HSCs. BrDU incorporation and fibrosis by qPCR was performed. UDCA reduced MC number, the HDC/histamine/HR-axis, IBDM, HSC activation, inflammation, and fibrosis in Mdr2-/- mice and PSC patients. In vitro, UDCA decreases MC-histamine release, which was restored by blocking ASBT and FXRß. Proliferation and fibrosis decreased after treatment with UDCA-treated MCs. We conclude that UDCA acts on MCs reducing histamine levels and decreases the inflammatory/hyperplastic/fibrotic reaction seen in PSC. Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. Following liver injury like primary sclerosing cholangitis in mice and humans, MCs infiltrate. MC-derived histamine increases biliary damage, fibrosis, and inflammation. UDCA treatment decreases these parameters via reduced MC activation.

Pretreatment prediction of response to ursodeoxycholic acid in primary biliary cholangitis: development and validation of the UDCA Response Score.

BACKGROUND: Treatment guidelines recommend a stepwise approach to primary biliary cholangitis: all patients begin treatment with ursodeoxycholic acid (UDCA) monotherapy and those with an inadequate biochemical response after 12 months are subsequently considered for second-line therapies. However, as a result, patients at the highest risk can wait the longest for effective treatment. We determined whether UDCA response can be accurately predicted using pretreatment clinical parameters. METHODS: We did logistic regression analysis of pretreatment variables in a discovery cohort of patients in the UK with primary biliary cholangitis to derive the best-fitting model of UDCA response, defined as alkaline phosphatase less than 1·67 times the upper limit of normal (ULN), measured after 12 months of treatment with UDCA. We validated the model in an external cohort of patients with primary biliary cholangitis and treated with UDCA in Italy. Additionally, we assessed correlations between model predictions and key histological features, such as biliary injury and fibrosis, on liver biopsy samples. FINDINGS: 2703 participants diagnosed with primary biliary cholangitis between Jan 1, 1998, and May 31, 2015, were included in the UK-PBC cohort for derivation of the model. The following pretreatment parameters were associated with lower probability of UDCA response: higher alkaline phosphatase concentration (p<0·0001), higher total bilirubin concentration (p=0·0003), lower aminotransferase concentration (p=0·0012), younger age (p<0·0001), longer interval from diagnosis to the start of UDCA treatment (treatment time lag, p<0·0001), and worsening of alkaline phosphatase concentration from diagnosis (p<0·0001). Based on these variables, we derived a predictive score of UDCA response. In the external validation cohort, 460 patients diagnosed with primary biliary cholangitis were treated with UDCA, with follow-up data until May 31, 2016. In this validation cohort, the area under the receiver operating characteristic curve for the score was 0·83 (95% CI 0·79-0·87). In 20 liver biopsy samples from patients with primary biliary cholangitis, the UDCA response score was associated with ductular reaction (r=-0·556, p=0·0130) and intermediate hepatocytes (probability of response was 0·90 if intermediate hepatocytes were absent vs 0·51 if present). INTERPRETATION: We have derived and externally validated a model based on pretreatment variables that accurately predicts UDCA response. Association with histological features provides face validity. This model provides a basis to explore alternative approaches to treatment stratification in patients with primary biliary cholangitis. FUNDING: UK Medical Research Council and University of Milan-Bicocca.

Blocking H1/H2 histamine receptors inhibits damage/fibrosis in Mdr2-/- mice and human cholangiocarcinoma tumorigenesis.

Primary sclerosing cholangitis (PSC) patients are at risk of developing cholangiocarcinoma (CCA). We have shown that (1) histamine increases biliary hyperplasia through H1/H2 histamine receptors (HRs) and (2) histamine levels increase and mast cells (MCs) infiltrate during PSC and CCA. We examined the effects of chronic treatment with H1/H2HR antagonists on PSC and CCA. Wild-type and multidrug-resistant knockout (Mdr2-/- ) mice were treated by osmotic minipumps with saline, mepyramine, or ranitidine (10 mg/kg body weight/day) or a combination of mepyramine/ranitidine for 4 weeks. Liver damage was assessed by hematoxylin and eosin. We evaluated (1) H1/H2HR expression, (2) MC presence, (3) L-histidine decarboxylase/histamine axis, (4) cholangiocyte proliferation/bile duct mass, and (5) fibrosis/hepatic stellate cell activation. Nu/nu mice were implanted with Mz-ChA-1 cells into the hind flanks and treated with saline, mepyramine, or ranitidine. Tumor growth was measured, and (1) H1/H2HR expression, (2) proliferation, (3) MC activation, (4) angiogenesis, and (5) epithelial-mesenchymal transition (EMT) were evaluated. In vitro, human hepatic stellate cells were evaluated for H1HR and H2HR expression. Cultured cholangiocytes and CCA lines were treated with saline, mepyramine, or ranitidine (25 µM) before evaluating proliferation, angiogenesis, EMT, and potential signaling mechanisms. H1/H2HR and MC presence increased in human PSC and CCA. In H1/H2HR antagonist (alone or in combination)-treated Mdr2-/- mice, liver and biliary damage and fibrosis decreased compared to saline treatment. H1/H2HR antagonists decreased tumor growth, serum histamine, angiogenesis, and EMT. In vitro, H1/H2HR blockers reduced biliary proliferation, and CCA cells had decreased proliferation, angiogenesis, EMT, and migration. Conclusion: Inhibition of H1/H2HR reverses PSC-associated damage and decreases CCA growth, angiogenesis, and EMT; because PSC patients are at risk of developing CCA, using HR blockers may be therapeutic for these diseases. (Hepatology 2018).

Comprehensive review of autoantibodies in patients with hyper-IgM syndrome.

Hyper-immunoglobulin M syndrome is an X-linked primary immunodeficiency disease caused by mutations in the CD40 ligand gene. The CD40 ligand has been recently highlighted as playing a key role in the pathogenesis of primary biliary cholangitis. In the present study, we assessed an extensive set of serum autoantibodies in a series of well-defined patients with hyper-immunoglobulin M syndrome. Serum, liver-related and liver-not-related autoantibodies IgG, IgM and IgA were tested by ELISA and standard indirect immunofluorescence in HEp-2 cells in 13 Tunisian patients (8 males and 5 females, aged 1-12 years) with hyper-immunoglobulin M syndrome during 1995-2012 and, as controls, 21 age- and gender-matched blood donors. The level of IgM antibody against MIT3 was significantly higher in patients than in controls (35.8 vs 10.7, P=0.002). Half of the hyperimmunoglobulin M syndrome patients were found to be anti-MIT3 IgM positive vs none of the controls (P<0.0001). Twenty-three percent of patients were found to be anti-sp100 antibody positive vs only 0.05% of controls. By immunofluorescence, 92.3% of patients were MIT3 IgM positive vs none of the controls. In conclusion, the IgM class of anti-MIT3 antibodies was shown to be present by both ELISA and immunofluorescence in most of the patients with hyper-immunoglobulin M syndrome. The presence of the hallmark of primary biliary cholangitis, a disease where the CD40 ligand is a key player, in an immunodeficiency disease caused by mutations in the CD40 ligand gene is very intriguing and opens new scenarios in understanding the immune pathogenesis of primary biliary cholangitis.

From pathogenesis to novel therapies in the treatment of primary biliary cholangitis.

INTRODUCTION: Primary biliary cholangitis (PBC) is an immune-mediated liver disease characterized by chronic inflammation of the intrahepatic bile ducts, causing progressive ductopenia, cholestasis and fibrosis, and leading to liver failure. Ursodeoxycholic acid (UDCA) is the first-line therapy for the treatment of PBC patients. This is effective in majority of patients; however, up to 20 percent of patients have an incomplete response to UDCA therapy and have a reduced prognosis as compared to healthy individuals. Obeticholic acid (OCA) has been recently registered as second-line therapy for patients with incomplete response to UDCA, with plans to demonstrate the long-term clinical efficacy. Areas covered: Recent evolution in our understanding of disease mechanisms is leading to the advent of new and re-purposed therapeutic agents targeting key processes in the etiopathogenesis. Several therapeutic targets have been proposed which can be categorized into three compartments: immune, biliary and fibrosis. In this review we describe the main biological mechanisms underpinning disease development and progression in PBC and the new targeted therapies on the horizon. Expert commentary: Testing new drugs towards hard clinical endpoints is challenging in PBC due to its low prevalence and the slow progression of the disease. Novel promising biomarkers are under study and should be evaluated as surrogate endpoints in clinical trials.

Inhibition of microRNA-24 increases liver fibrosis by enhanced menin expression in Mdr2-/- mice.

BACKGROUND: Liver transplantation remains the primary treatment for primary sclerosing cholangitis (PSC). Mdr2-/- mice provide a reliable in vivo model of PSC and develop characteristic biliary inflammation and fibrosis. We tested the hypothesis that the tumor suppressor protein menin is implicated in the progression of liver fibrosis and that menin expression can be regulated in the liver via microRNA-24 (miR-24). MATERIALS AND METHODS: Menin expression was measured in human PSC and Mdr2-/- mice. Twelve-week-old FVB/NJ wild-type (WT) and Mdr2-/- mice were treated with miR-24 Vivo-Morpholino to knockdown miR-24 expression levels. Liver fibrosis was evaluated by Sirius Red staining and quantitative polymerase chain reaction (qPCR) for genes associated with liver fibrosis, such as fibronectin 1, collagen type 1 alpha 1, transforming growth factor-ß1 (TGF-ß1), and α-smooth muscle actin. Studies were also performed in vitro using immortalized murine cholangiocyte lines treated with miR-24 hairpin inhibitor and mimic. RESULTS: Menin gene expression was increased in Mdr2-/- mice and late-stage human PSC samples. Treatment of FVB/NJ WT and Mdr2-/- mice with miR-24 Vivo-Morpholino increased menin expression, which correlated with increased expression of fibrosis genes. In vitro, inhibition of miR-24 also significantly increased the expression of fibrosis genes. CONCLUSIONS: Inhibition of miR-24 increases menin and TGF-ß1 expression, subsequently increasing hepatic fibrosis in FVB/NJ WT and Mdr2-/- mice. Modulation of the menin/miR-24 axis may provide novel targeted therapies to slow the progression of hepatic fibrosis into cirrhosis in PSC patients by altering TGF-ß1 expression.

Prolonged darkness reduces liver fibrosis in a mouse model of primary sclerosing cholangitis by miR-200b down-regulation.

Melatonin therapy or prolonged exposure to complete darkness reduces biliary hyperplasia and liver fibrosis in bile-duct-ligated (BDL) rats; however, no information exists in primary sclerosing cholangitis (PSC). Thus, we aimed to determine the therapeutic effects of prolonged dark therapy or melatonin administration on hepatic fibrosis in the multidrug resistance gene 2-knockout (Mdr2-/-) mouse model of PSC. Melatonin levels, biliary mass, liver fibrosis, angiogenesis and miR-200b expression were evaluated in wild-type and Mdr2-/- mice exposed to darkness or melatonin treatment or in male patients with PSC and healthy controls. Mdr2-/- mice were also treated with miR-200b inhibitor or control before evaluating biliary mass, liver fibrosis, and angiogenesis. After overexpression of arylalkylamine N-acetyltransferase (AANAT; the enzyme regulating melatonin synthesis) or inhibition of miR-200b in cholangiocytes and hepatic stellate cells in vitro, we evaluated angiogenesis and fibrosis gene expression. After exposure to darkness or administration of melatonin, Mdr2-/- mice show elevated serum melatonin levels and inhibition of biliary mass, along with reduction of liver fibrosis and angiogenesis. MicroRNA PCR analysis demonstrated that miR-200b expression increased in Mdr2-/- mice and patients with PSC compared with controls and decreased in Mdr2-/- mice subjected to dark exposure or melatonin treatment. Inhibition of miR-200b in Mdr2-/- ablates biliary proliferation, liver fibrosis, and angiogenesis. In vitro, overexpression of AANAT or inhibition of miR-200b in cholangiocytes and hepatic stellate cells decreased the expression of miR-200b, angiogenesis, and fibrosis genes. Dark therapy or targeting melatonin/miR-200b axis may be important in the management of biliary damage and liver fibrosis in cholangiopathies including PSC.-Wu, N., Meng, F., Zhou, T., Han, Y., Kennedy, L., Venter, J., Francis, H., DeMorrow, S., Onori, P., Invernizzi, P., Bernuzzi, F., Mancinelli, R., Gaudio, E., Franchitto, A., Glaser, S., Alpini G. Prolonged darkness reduces liver fibrosis in a mouse model of primary sclerosing cholangitis by miR-200b down-regulation.

Knockdown of Hepatic Gonadotropin-Releasing Hormone by Vivo-Morpholino Decreases Liver Fibrosis in Multidrug Resistance Gene 2 Knockout Mice by Down-Regulation of miR-200b.

Hepatic fibrosis occurs during the progression of primary sclerosing cholangitis (PSC) and is characterized by accumulation of extracellular matrix proteins. Proliferating cholangiocytes and activated hepatic stellate cells (HSCs) participate in the promotion of liver fibrosis during cholestasis. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone synthesized by hypothalamic neurons and the biliary epithelium and exerts its biological effects on cholangiocytes by interaction with the receptor subtype (GnRHR1) expressed by cholangiocytes and HSCs. Previously, we demonstrated that administration of GnRH to normal rats increased intrahepatic biliary mass (IBDM) and hepatic fibrosis. Also, miR-200b is associated with the progression of hepatic fibrosis; however, the role of the GnRH/GnRHR1/miR-200b axis in the development of hepatic fibrosis in PSC is unknown. Herein, using the mouse model of PSC (multidrug resistance gene 2 knockout), the hepatic knockdown of GnRH decreased IBDM and liver fibrosis. In vivo and in vitro administration of GnRH increased the expression of miR-200b and fibrosis markers. The GnRH/GnRHR1 axis and miR-200b were up-regulated in human PSC samples. Cetrorelix, a GnRHR1 antagonist, inhibited the expression of fibrotic genes in vitro and decreased IBDM and hepatic fibrosis in vivo. Inhibition of miR-200b decreased the expression of fibrosis genes in vitro in cholangiocyte and HSC lines. Targeting the GnRH/GnRHR1/miR-200b axis may be key for the management of hepatic fibrosis during the progression of PSC.

Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells.

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct-ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R-/- ) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2-/- ) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2-/- mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2-/- mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-ß1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R-/- mice with BDL surgery or Mdr2-/- mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. CONCLUSION: Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528-541).

Nicotine Promotes Cholangiocarcinoma Growth in Xenograft Mice.

Nicotine, the main addictive substance in tobacco, is known to play a role in the development and/or progression of a number of malignant tumors. However, nicotine's involvement in the pathogenesis of cholangiocarcinoma is controversial. Therefore, we studied the effects of nicotine on the growth of cholangiocarcinoma cells in vitro and the progression of cholangiocarcinoma in a mouse xenograft model. The predominant subunit responsible for nicotine-mediated proliferation in normal and cancer cells, the α7 nicotinic acetylcholine receptor (α7-nAChR), was more highly expressed in human cholangiocarcinoma cell lines compared with normal human cholangiocytes. Nicotine also stimulated the proliferation of cholangiocarcinoma cell lines and promoted α7-nAChR-dependent activation of proliferation and phosphorylation of extracellular-regulated kinase in Mz-ChA-1 cells. In addition, nicotine and PNU282987 (α7-nAChR agonist) accelerated the growth of the cholangiocarcinoma tumors in our xenograft mouse model and increased fibrosis, proliferation of the tumor cells, and phosphorylation of extracellular-regulated kinase activation. Finally, α7-nAChR was expressed at significantly higher levels in human cholangiocarcinoma compared with normal human control liver samples. Taken together, results of this study suggest that nicotine acts through α7-nAChR and plays a novel role in the pathogenesis of cholangiocarcinoma. Furthermore, nicotine may act as a mitogen in cholestatic liver disease processes, thereby facilitating malignant transformation.

Enhanced liver fibrosis test predicts transplant-free survival in primary sclerosing cholangitis, a multi-centre study.

BACKGROUND & AIMS: Biomarkers reflecting disease activity and prognosis in primary sclerosing cholangitis (PSC) have not been firmly established. Enhanced liver fibrosis (ELF) test was previously reported to predict outcome in PSC. We aimed to validate the prognostic utility of ELF test in an independent, multi-centre, retrospective PSC study population. METHODS: We collected serum samples from PSC patients from seven countries. We estimated rates of transplant-free survival by the Kaplan-Meier method, used Cox proportional hazards regression to explore the association between ELF test and clinical outcome and determined prognostic performance of ELF test by computing the area under the receiver operating characteristic (AUC-ROC) curve. RESULTS: The final analysis included 534 PSC patients (61% males). Features of autoimmune hepatitis or concomitant inflammatory bowel disease affected 44 (8%) and 379 (71%) patients respectively. ELF test levels were higher in patients reaching the combined endpoint liver transplantation or death (median 10.9 [Interquartile range (IQR): 9.8-12.1]; n=24 deaths, 79 liver transplantations) compared to those censored (8.8 [IQR: 8.0-9.8]); P<.001. ELF test expressed as mild, moderate and severe fibrosis was significantly associated with the risk of reaching the endpoint (P<.001). ELF test independently predicted clinical outcome (Hazard ratio 1.31; 95% confidence interval [1.05-1.65]; P=.018), and enabled good discrimination between PSC patients with and without endpoint (AUC-ROC: 0.79). CONCLUSION: Our retrospective data validates the predictive utility of ELF test for clinical outcomes in PSC. The clinical utility of biomarkers for fibrosis in patients with PSC should be assessed in prospective patient cohorts.

miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation.

Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24-dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.

Forkhead box A2 regulates biliary heterogeneity and senescence during cholestatic liver injury in mice‡.

Biliary-committed progenitor cells (small mouse cholangiocytes; SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, forkhead box A2 (FoxA2), is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Messenger RNA expression in SMCCs and LGCCs was assessed by polymerase chain reaction (PCR) array analysis. Liver tissues and hepatic stellate cells (HSCs) from primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients were tested by real-time PCR for methylation, senescence, and fibrosis markers. Bile duct ligation (BDL) and multidrug resistance protein 2 (MDR2) knockout mice (MDR2-/- ) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes, DNA methyltransferase (DNMT) 1 and DNMT3B. Serum alanine aminotransferase and aspartate aminotransferase levels in nonobese diabetic/severe combined immunodeficiency mice engrafted with SMCCs post-BDL showed significant changes compared to vehicle-treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2-/- mouse liver as well as in isolated HSCs by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. CONCLUSION: The definitive endoderm marker and the positive regulator of biliary development, FoxA2, mediates the therapeutic effect of biliary-committed progenitor cells during cholestatic liver injury. (Hepatology 2017;65:544-559).