Hepatocyte Adenosine Kinase Promotes Excessive Fat Deposition and Liver Inflammation.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease is highly associated with obesity and progresses to nonalcoholic steatohepatitis when the liver develops overt inflammatory damage. While removing adenosine in the purine salvage pathway, adenosine kinase (ADK) regulates methylation reactions. We aimed to study whether hepatocyte ADK functions as an obesogenic gene/enzyme to promote excessive fat deposition and liver inflammation. METHODS: Liver sections of human subjects were examined for ADK expression using immunohistochemistry. Mice with hepatocyte-specific ADK disruption or overexpression were examined for hepatic fat deposition and inflammation. Liver lipidomics, hepatocyte RNA sequencing (RNA-seq), and single-cell RNA-seq for liver nonparenchymal cells were performed to analyze ADK regulation of hepatocyte metabolic responses and hepatocyte-nonparenchymal cells crosstalk. RESULTS: Whereas patients with nonalcoholic fatty liver disease had increased hepatic ADK levels, mice with hepatocyte-specific ADK disruption displayed decreased hepatic fat deposition on a chow diet and were protected from diet-induced excessive hepatic fat deposition and inflammation. In contrast, mice with hepatocyte-specific ADK overexpression displayed increased body weight and adiposity and elevated degrees of hepatic steatosis and inflammation compared with control mice. RNA-seq and epigenetic analyses indicated that ADK increased hepatic DNA methylation and decreased hepatic Ppara expression and fatty acid oxidation. Lipidomic and single-cell RNA-seq analyses indicated that ADK-driven hepatocyte factors, due to mitochondrial dysfunction, enhanced macrophage proinflammatory activation in manners involving increased expression of stimulator of interferon genes. CONCLUSIONS: Hepatocyte ADK functions to promote excessive fat deposition and liver inflammation through suppressing hepatocyte fatty acid oxidation and producing hepatocyte-derived proinflammatory mediators. Therefore, hepatocyte ADK is a therapeutic target for managing obesity and nonalcoholic fatty liver disease.

Sex-Dependent Differences in Cholestasis: Why Estrogen Signaling May Be a Key Pathophysiological Driver.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are cholestatic liver diseases that have significant clinical impact with debilitating symptoms and mortality. While PBC is predominantly seen in perimenopausal and postmenopausal women, men who are diagnosed with PBC have worse clinical outcomes and all-cause mortality. In contrast, 60% to 70% of patients with PSC are men; the data indicate that female sex may be an independent factor against PSC-related complications. These findings suggest a sex-dependent biological basis for these differences. Estrogen has been implicated in the pathogenesis of intrahepatic cholestasis of pregnancy and may induce cholestasis through a variety of interactions. However, it is unclear why some sexual dimorphic features may provide a protective effect despite known estrogen models that induce cholestasis. This article provides a brief introductory background and discusses the sexual dimorphism in clinical presentation in PSC and PBC. It also explores the role of estrogen signaling in pathogenesis and how it relates to intrahepatic cholestasis of pregnancy. Studies have already targeted certain molecules involved in estrogen signaling, and this review discusses these studies that identify estrogen-related receptor, estrogen receptor-α, estrogen receptor-ß, farnesoid X receptor, and mast cells as possible targets, in addition to long noncoding RNA H19-induced cholestasis and sexual dimorphism. It also explores these interactions and their role in the pathogenesis of PBC and PSC.

Development of Scaffold-Free Three-Dimensional Cholangiocyte Organoids to Study the Progression of Primary Sclerosing Cholangitis.

Organoids are novel in vitro models to study intercellular cross talk between the different types of cells in disease pathophysiology. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), scaffold-free multicellular three-dimensional cholangiocyte organoids (3D-CHOs) were developed using primary liver cells derived from normal subjects and patients with PSC. Human liver samples from healthy donors and patients with PSC were used to isolate primary cholangiocytes [epithelial cell adhesion molecule (EpCam)+/ cytokeratin-19+], liver endothelial cells (CD31+), and hepatic stellate cells (HSCs; CD31-/CD68-/desmin+/vitamin A+). 3D-CHOs were formed using cholangiocytes, HSCs, and liver endothelial cells, and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence, quantitative RT-PCR, and transmission electron microscopy. Transcription profiles for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, and SCTR), fibrosis (ACTA2, COL1A1, DESMIN, and TGFß1), angiogenesis (PECAM, VEGF, CDH5, and vWF), and inflammation (IL-6 and TNF-α) confirmed PSC phenotypes of 3D-CHOs. Because cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal immunofluorescence was used to demonstrate localization of the neurokinin-1 receptor within cytokeratin-19+ cholangiocytes and desmin+ HSCs. Moreover, 3D-CHOs from patients with PSC confirmed PSC phenotypes with up-regulated neurokinin-1 receptor, tachykinin precursor 1, and down-regulated membrane metalloendopeptidase. Scaffold-free multicellular 3D-CHOs showed superiority as an in vitro model in mimicking PSC in vivo phenotypes compared with two-dimensional cell culture, which can be used in PSC disease-related research.

Prolonged administration of a secretin receptor antagonist inhibits biliary senescence and liver fibrosis in Mdr2 -/- mice.

BACKGROUND AND AIMS: Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2 -/- [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5-27 treatment in Mdr2 -/- mice. APPROACH AND RESULTS: In vivo studies were performed in male wild-type and Mdr2 -/- mice treated with saline or SCT 5-27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2 -/- mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-ß1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2 -/- mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2 -/- compared with wild-type mice, which was reversed by long-term SCT 5-27 treatment. In vitro , SCT 5-27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-ß1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5-27-induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. CONCLUSIONS: Modulating the SCT/SR axis may be critical for managing PSC.

p16 INK4A drives nonalcoholic fatty liver disease phenotypes in high fat diet fed mice through biliary E2F1/FOXO1/IGF-1 signaling.

BACKGROUND AND AIMS: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. APPROACH AND RESULTS: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. CONCLUSION: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Loss of apical sodium bile acid transporter alters bile acid circulation and reduces biliary damage in cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout (Mdr2-/-) mice were treated with control or ASBT Vivo-Morpholino (VM). We measured 1) ASBT expression and MC presence in liver/ileum; 2) liver damage/DR; 3) hepatic fibrosis/inflammation; 4) biliary inflammation/histamine serum content; and 5) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in Mdr2-/- mice decreased 1) biliary ASBT expression, 2) PSC phenotypes, 3) hepatic TBA, and 4) gut barrier integrity compared with control. We found alterations between wild-type (WT) and Mdr2-/- mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA-stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC.NEW & NOTEWORTHY We evaluated knockdown of the apical sodium bile acid transporter (ASBT) using Vivo-Morpholino in Mdr2KO mice. ASBT inhibition decreases primary sclerosing cholangitis (PSC) pathogenesis by reducing hepatic mast cell infiltration, altering bile acid species/cholehepatic shunt, and regulating gut inflammation/dysbiosis. Since a large cohort of PSC patients present with IBD, this study is clinically important. We validated findings in human PSC and PSC-IBD along with studies in novel human 3-D organoids formed from human PSC livers.

The Functional Roles of Immune Cells in Primary Liver Cancer.

Primary liver cancer includes hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Incidence of liver cancer has been increasing in recent years, and the 5-year survival is <20%. HCC and CCA are often accompanied with a dense stroma coupled with infiltrated immune cells, which is referred to as the tumor microenvironment. Populations of specific immune cells, such as high density of CD163+ macrophages and low density of CD8+ T cells, are associated with prognosis and survival rates in both HCC and CCA. Immune cells in the tumor microenvironment can be a therapeutic target for liver cancer treatments. Previous studies have introduced immunotherapy using immune checkpoint inhibitors, pulsed dendritic cells, or transduced T cells, to enhance cytotoxicity of immune cells and inhibit tumor growth. This review summarizes current understanding of the roles of immune cells in primary liver cancer covering HCC and CCA.

Development and Characterization of Human Primary Cholangiocarcinoma Cell Lines.

Cholangiocarcinoma (CCA) is the second most common primary liver tumor and is associated with late diagnosis, limited treatment options, and a 5-year survival rate of around 30%. CCA cell lines were first established in 1971, and since then, only 70 to 80 CCA cell lines have been established. These cell lines have been essential in basic and translational research to understand and identify novel mechanistic pathways, biomarkers, and disease-specific genes. Each CCA cell line has unique characteristics, reflecting a specific genotype, sex-related properties, and patient-related signatures, making them scientifically and commercially valuable. CCA cell lines are crucial in the use of novel technologies, such as three-dimensional organoid models, which help to model the tumor microenvironment and cell-to-cell crosstalk between tumor-neighboring cells. This review highlights crucial information on CCA cell lines, including: i) type of CCA (eg, intra- or extrahepatic), ii) isolation source (eg, primary tumor or xenograft), iii) chemical digestion method (eg, trypsin or collagenase), iv) cell-sorting method (colony isolation or removal of fibroblasts), v) maintenance-medium choice (eg, RPMI or Dulbecco's modified Eagle's medium), vi) cell morphology (eg, spindle or polygonal shape), and vii) doubling time of cells.

Melatonin receptor 1A, but not 1B, knockout decreases biliary damage and liver fibrosis during cholestatic liver injury.

BACKGROUND AND AIMS: Melatonin reduces biliary damage and liver fibrosis in cholestatic models by interaction with melatonin receptors 1A (MT1) and 1B (MT2). MT1 and MT2 can form heterodimers and homodimers, but MT1 and MT2 can heterodimerize with the orphan receptor G protein-coupled receptor 50 (GPR50). MT1/GPR50 dimerization blocks melatonin binding, but MT2/GPR50 dimerization does not affect melatonin binding. GPR50 can dimerize with TGFß receptor type I (TGFßRI) to activate this receptor. We aimed to determine the differential roles of MT1 and MT2 during cholestasis. APPROACH AND RESULTS: Wild-type (WT), MT1 knockout (KO), MT2KO, and MT1/MT2 double KO (DKO) mice underwent sham or bile duct ligation (BDL); these mice were also treated with melatonin. BDL WT and multidrug resistance 2 KO (Mdr2-/- ) mice received mismatch, MT1, or MT2 Vivo-Morpholino. Biliary expression of MT1 and GPR50 increases in cholestatic rodents and human primary sclerosing cholangitis (PSC) samples. Loss of MT1 in BDL and Mdr2-/- mice ameliorated biliary and liver damage, whereas these parameters were enhanced following loss of MT2 and in DKO mice. Interestingly, melatonin treatment alleviated BDL-induced biliary and liver injury in BDL WT and BDL MT2KO mice but not in BDL MT1KO or BDL DKO mice, demonstrating melatonin's interaction with MT1. Loss of MT2 or DKO mice exhibited enhanced GPR50/TGFßR1 signaling, which was reduced by loss of MT1. CONCLUSIONS: Melatonin ameliorates liver phenotypes through MT1, whereas down-regulation of MT2 promotes liver damage through GPR50/TGFßR1 activation. Blocking GPR50/TGFßR1 binding through modulation of melatonin signaling may be a therapeutic approach for PSC.

Three-Dimensional Organoids as a Model to Study Nonalcoholic Fatty Liver Disease.

Despite the rising prevalence of nonalcoholic fatty liver disease (NAFLD), the underlying disease pathophysiology remains unclear. There is a great need for an efficient and reliable "human" in vitro model to study NAFLD and the progression to nonalcoholic steatohepatitis (NASH), which will soon become the leading indication for liver transplantation. Here, we review the recent developments in the use of three-dimensional (3D) liver organoids as a model to study NAFLD and NASH pathophysiology and possible treatments. Various techniques that are currently used to make liver organoids are discussed, such as the use of induced pluripotent stem cells versus primary cell lines and human versus murine cells. Moreover, methods for inducing lipid droplet accumulation and fibrosis to model NAFLD are explored. Finally, the limitations specific to the 3D organoid model for NAFLD/NASH are reviewed, highlighting the need for further development of multilineage models to include hepatic nonparenchymal cells and immune cells. The ultimate goal is to be able to accurately recapitulate the complex liver microenvironment in which NAFLD develops and progresses to NASH.

Mast Cells Regulate Ductular Reaction and Intestinal Inflammation in Cholestasis Through Farnesoid X Receptor Signaling.

BACKGROUND AND AIMS: Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/FGF15. Patients with primary sclerosing cholangitis (PSC) typically present with inflammatory bowel disease (IBD). Mast cells (MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In bile-duct-ligated (BDL) MC-deficient mice (B6.Cg-KitW-sh /HNihrJaeBsmJ [KitW-sh ]), ductular reaction (DR) and liver fibrosis decrease compared with BDL wild type, and MC injection exacerbates liver damage in normal mice. APPROACH AND RESULTS: In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, and hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared with control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in patients PSC and PSC-IBD compared with healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence-associated secretory phenotype (SASP), fibrosis, and histamine in KitW-sh mice. Inhibition of MC-FXR before injection reduced these parameters. BDL and KitW-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15, and intestinal inflammation, which decreased in BDL KitW-sh and KitW-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in KitW-sh mice that was reduced following FXR inhibition. BDL and multidrug resistance 2/ATP-binding cassette family 2 member 4 knockout (Mdr2-/- ) mice, models of PSC, displayed increased intestinal MC infiltration and FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in Mdr2-/- mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic stellate cell activation. CONCLUSIONS: Our studies demonstrate that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling.

Inhibition of Secretin/Secretin Receptor Axis Ameliorates NAFLD Phenotypes.

BACKGROUND AND AIMS: Human NAFLD is characterized at early stages by hepatic steatosis, which may progress to NASH when the liver displays microvesicular steatosis, lobular inflammation, and pericellular fibrosis. The secretin (SCT)/secretin receptor (SCTR) axis promotes biliary senescence and liver fibrosis in cholestatic models through down-regulation of miR-125b signaling. We aim to evaluate the effect of disrupting biliary SCT/SCTR/miR-125b signaling on hepatic steatosis, biliary senescence, and liver fibrosis in NAFLD/NASH. APPROACH AND RESULTS: In vivo, 4-week-old male wild-type, Sct-/- and Sctr-/- mice were fed a control diet or high-fat diet (HFD) for 16 weeks. The expression of SCT/SCTR/miR-125b axis was measured in human NAFLD/NASH liver samples and HFD mouse livers by immunohistochemistry and quantitative PCR. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were evaluated in mouse liver and human NAFLD/NASH liver samples. miR-125b target lipogenesis genes in hepatocytes were screened and validated by custom RT2 Profiler PCR array and luciferase assay. Biliary SCT/SCTR expression was increased in human NAFLD/NASH samples and in livers of HFD mice, whereas the expression of miR-125b was decreased. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were observed in human NAFLD/NASH samples as well as HFD mice, which were decreased in Sct-/- and Sctr-/- HFD mice. Elovl1 is a lipogenesis gene targeted by miR-125b, and its expression was also decreased in HFD mouse hepatocytes following Sct or Sctr knockout. Bile acid profile in fecal samples have the greatest changes between wild-type mice and Sct-/- /Sctr-/- mice. CONCLUSION: The biliary SCT/SCTR/miR-125b axis promotes liver steatosis by up-regulating lipid biosynthesis gene Elovl1. Targeting the biliary SCT/SCTR/miR-125b axis may be key for ameliorating phenotypes of human NAFLD/NASH.

Mast Cells Promote Nonalcoholic Fatty Liver Disease Phenotypes and Microvesicular Steatosis in Mice Fed a Western Diet.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. APPROACH AND RESULTS: Wild-type (WT) and KitW-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and KitW-sh WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulin-like growth factor 1 expression that promotes MC migration/activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in KitW-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WD-induced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in KitW-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in KitW-sh WD mice and was found to target ALDH1A3. CONCLUSIONS: MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment.

The Apelin-Apelin Receptor Axis Triggers Cholangiocyte Proliferation and Liver Fibrosis During Mouse Models of Cholestasis.

BACKGROUND AND AIMS: Apelin (APLN) is the endogenous ligand of its G protein-coupled receptor, apelin receptor (APJ). APLN serum levels are increased in human liver diseases. We evaluated whether the APLN-APJ axis regulates ductular reaction and liver fibrosis during cholestasis. APPROACH AND RESULTS: We measured the expression of APLN and APJ and serum APLN levels in human primary sclerosing cholangitis (PSC) samples. Following bile duct ligation (BDL) or sham surgery, male wild-type (WT) mice were treated with ML221 (APJ antagonist) or saline for 1 week. WT and APLN-/- mice underwent BDL or sham surgery for 1 week. Multidrug resistance gene 2 knockout (Mdr2-/- ) mice were treated with ML221 for 1 week. APLN levels were measured in serum and cholangiocyte supernatants, and cholangiocyte proliferation/senescence and liver inflammation, fibrosis, and angiogenesis were measured in liver tissues. The regulatory mechanisms of APLN-APJ in (1) biliary damage and liver fibrosis were examined in human intrahepatic biliary epithelial cells (HIBEpiCs) treated with APLN and (2) hepatic stellate cell (HSC) activation in APLN-treated human HSC lines (HHSteCs). APLN serum levels and biliary expression of APLN and APJ increased in PSC samples. APLN levels were higher in serum and cholangiocyte supernatants from BDL and Mdr2-/- mice. ML221 treatment or APLN-/- reduced BDL-induced and Mdr2-/- -induced cholangiocyte proliferation/senescence, liver inflammation, fibrosis, and angiogenesis. In vitro, APLN induced HIBEpiC proliferation, increased nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, reactive oxygen species (ROS) generation, and extracellular signal-regulated kinase (ERK) phosphorylation. Pretreatment of HIBEpiCs with ML221, diphenyleneiodonium chloride (Nox4 inhibitor), N-acetyl-cysteine (NAC, ROS inhibitor), or PD98059 (ERK inhibitor) reduced APLN-induced cholangiocyte proliferation. Activation of HHSteCs was induced by APLN but reduced by NAC. CONCLUSIONS: The APLN-APJ axis induces cholangiocyte proliferation through Nox4/ROS/ERK-dependent signaling and HSC activation through intracellular ROS. Modulation of the APLN-APJ axis may be important for managing cholangiopathies.

Mast Cells Induce Ductular Reaction Mimicking Liver Injury in Mice Through Mast Cell-Derived Transforming Growth Factor Beta 1 Signaling.

BACKGROUND AND AIMS: Following liver injury, mast cells (MCs) migrate into the liver and are activated in patients with cholestasis. Inhibition of MC mediators decreases ductular reaction (DR) and liver fibrosis. Transforming growth factor beta 1 (TGF-ß1) contributes to fibrosis and promotes liver disease. Our aim was to demonstrate that reintroduction of MCs induces cholestatic injury through TGF-ß1. APPROACH AND RESULTS: Wild-type, KitW-sh (MC-deficient), and multidrug resistance transporter 2/ABC transporter B family member 2 knockout mice lacking l-histidine decarboxylase were injected with vehicle or PKH26-tagged murine MCs pretreated with 0.01% dimethyl sulfoxide (DMSO) or the TGF-ß1 receptor inhibitor (TGF-ßRi), LY2109761 (10 µM) 3 days before sacrifice. Hepatic damage was assessed by hematoxylin and eosin (H&E) and serum chemistry. Injected MCs were detected in liver, spleen, and lung by immunofluorescence (IF). DR was measured by cytokeratin 19 (CK-19) immunohistochemistry and F4/80 staining coupled with real-time quantitative PCR (qPCR) for interleukin (IL)-1ß, IL-33, and F4/80; biliary senescence was evaluated by IF or qPCR for p16, p18, and p21. Fibrosis was evaluated by sirius red/fast green staining and IF for synaptophysin 9 (SYP-9), desmin, and alpha smooth muscle actin (α-SMA). TGF-ß1 secretion/expression was measured by enzyme immunoassay and qPCR. Angiogenesis was detected by IF for von Willebrand factor and vascular endothelial growth factor C qPCR. In vitro, MC-TGF-ß1 expression/secretion were measured after TGF-ßRi treatment; conditioned medium was collected. Cholangiocytes and hepatic stellate cells (HSCs) were treated with MC-conditioned medium, and biliary proliferation/senescence was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and qPCR; HSC activation evaluated for α-SMA, SYP-9, and collagen type-1a expression. MC injection recapitulates cholestatic liver injury characterized by increased DR, fibrosis/TGF-ß1 secretion, and angiogenesis. Injection of MC-TGF-ßRi reversed these parameters. In vitro, MCs induce biliary proliferation/senescence and HSC activation that was reversed with MCs lacking TGF-ß1. CONCLUSIONS: Our study demonstrates that reintroduction of MCs mimics cholestatic liver injury and that MC-derived TGF-ß1 may be a target in chronic cholestatic liver disease.

Organoids and Spheroids as Models for Studying Cholestatic Liver Injury and Cholangiocarcinoma.

Cholangiopathies, such as primary sclerosing cholangitis, biliary atresia, and cholangiocarcinoma, have limited experimental models. Not only cholangiocytes but also other hepatic cells including hepatic stellate cells and macrophages are involved in the pathophysiology of cholangiopathies, and these hepatic cells orchestrate the coordinated response against diseased conditions. Classic two-dimensional monolayer cell cultures do not resemble intercellular cell-to-cell interaction and communication; however, three-dimensional cell culture systems, such as organoids and spheroids, can mimic cellular interaction and architecture between hepatic cells. Previous studies have demonstrated the generation of hepatic or biliary organoids/spheroids using various cell sources including pluripotent stem cells, hepatic progenitor cells, primary cells from liver biopsies, and immortalized cell lines. Gene manipulation, such as transfection and transduction can be performed in organoids, and established organoids have functional characteristics which can be suitable for drug screening. This review summarizes current methodologies for organoid/spheroid formation and a potential for three-dimensional hepatic cell cultures as in vitro models of cholangiopathies.

Amelioration of Large Bile Duct Damage by Histamine-2 Receptor Vivo-Morpholino Treatment.

Histamine binds to one of the four G-protein-coupled receptors expressed by large cholangiocytes and increases large cholangiocyte proliferation via histamine-2 receptor (H2HR), which is increased in patients with primary sclerosing cholangitis (PSC). Ranitidine decreases liver damage in Mdr2-/- (ATP binding cassette subfamily B member 4 null) mice. We targeted hepatic H2HR in Mdr2-/- mice using vivo-morpholino. Wild-type and Mdr2-/- mice were treated with mismatch or H2HR vivo-morpholino by tail vein injection for 1 week. Liver damage, mast cell (MC) activation, biliary H2HR, and histamine serum levels were studied. MC markers were determined by quantitative real-time PCR for chymase and c-kit. Intrahepatic biliary mass was detected by cytokeratin-19 and F4/80 to evaluate inflammation. Biliary senescence was determined by immunofluorescence and senescence-associated ß-galactosidase staining. Hepatic fibrosis was evaluated by staining for desmin, Sirius Red/Fast Green, and vimentin. Immunofluorescence for transforming growth factor-ß1, vascular endothelial growth factor-A/C, and cAMP/ERK expression was performed. Transforming growth factor-ß1 and vascular endothelial growth factor-A secretion was measured in serum and/or cholangiocyte supernatant. Treatment with H2HR vivo-morpholino in Mdr2-/--mice decreased hepatic damage; H2HR protein expression and MC presence or activation; large intrahepatic bile duct mass, inflammation and senescence; and fibrosis, angiogenesis, and cAMP/phospho-ERK expression. Inhibition of H2HR signaling ameliorates large ductal PSC-induced damage. The H2HR axis may be targeted in treating PSC.

Knockout of the Tachykinin Receptor 1 in the Mdr2-/- (Abcb4-/-) Mouse Model of Primary Sclerosing Cholangitis Reduces Biliary Damage and Liver Fibrosis.

Activation of the substance P (SP)/neurokinin 1 receptor (NK1R) axis triggers biliary damage/senescence and liver fibrosis in bile duct ligated and Mdr2-/- (alias Abcb4-/-) mice through enhanced transforming growth factor-ß1 (TGF-ß1) biliary secretion. Recent evidence indicates a role for miR-31 (MIR31) in TGF-ß1-induced liver fibrosis. We aimed to define the role of the SP/NK1R/TGF-ß1/miR-31 axis in regulating biliary proliferation and liver fibrosis during cholestasis. Thus, we generated a novel model with double knockout of Mdr2-/- and NK1R-/ (alias Tacr1-/-) to further address the role of the SP/NK1R axis during chronic cholestasis. In vivo studies were performed in the following 12-week-old male mice: (i) NK1R-/-; (ii) Mdr2-/-; and (iii) NK1R-/-/Mdr2-/- (Tacr1-/-/Abcb4-/-) and their corresponding wild-type controls. Liver tissues and cholangiocytes were collected, and liver damage, changes in biliary mass/senescence, and inflammation as well as liver fibrosis were evaluated by both immunohistochemistry in liver sections and real-time PCR. miR-31 expression was measured by real-time PCR in isolated cholangiocytes. Decreased ductular reaction, liver fibrosis, biliary senescence, and biliary inflammation were observed in NK1R-/-/Mdr2-/- mice compared with Mdr2-/- mice. Elevated expression of miR-31 was observed in Mdr2-/- mice, which was reduced in NK1R-/-/Mdr2-/- mice. Targeting the SP/NK1R and/or miR-31 may be a potential approach in treating human cholangiopathies, including primary sclerosing cholangitis.

Kupffer Cells: Inflammation Pathways and Cell-Cell Interactions in Alcohol-Associated Liver Disease.

Chronic alcohol consumption is linked to the development of alcohol-associated liver disease (ALD). This disease is characterized by a clinical spectrum ranging from steatosis to hepatocellular carcinoma. Several cell types are involved in ALD progression, including hepatic macrophages. Kupffer cells (KCs) are the resident macrophages of the liver involved in the progression of ALD by activating pathways that lead to the production of cytokines and chemokines. In addition, KCs are involved in the production of reactive oxygen species. Reactive oxygen species are linked to the induction of oxidative stress and inflammation in the liver. These events are activated by the bacterial endotoxin, lipopolysaccharide, that is released from the gastrointestinal tract through the portal vein to the liver. Lipopolysaccharide is recognized by receptors on KCs that are responsible for triggering several pathways that activate proinflammatory cytokines involved in alcohol-induced liver injury. In addition, KCs activate hepatic stellate cells that are involved in liver fibrosis. Novel strategies to treat ALD aim at targeting Kupffer cells. These interventions modulate Kupffer cell activation or macrophage polarization. Evidence from mouse models and early clinical studies in patients with ALD injury supports the notion that pathogenic macrophage subsets can be successfully translated into novel treatment options for patients with this disease.