Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) deletion in myeloid cells augments cholestatic liver injury.

Ductular reactive (DR) cells exacerbate cholestatic liver injury and fibrosis. Herein, we posit that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) emanates from recruited macrophages and restrains DR cell expansion, thereby limiting cholestatic liver injury. Wild type (WT), Trailfl/fl and myeloid-specific Trail deleted (TrailΔmye) C57BL/6 mice were exposed to DDC diet-induced cholestatic liver injury, which induced hepatomegaly and liver injury as compared to control diet-fed mice. However, parameters of liver injury, fibrosis, and inflammation were all increased in the TrailΔmye mice as compared to the WT and Trailfl/fl mice. High dimensional mass cytometry indicated that cholestasis resulted in increased hepatic recruitment of subsets of macrophages and neutrophils in the TrailΔmye mice. Spatial transcriptomics analysis revealed that the PanCK+ cholangiocytes from TrailΔmye mice had increased expression of the known myeloid attractants S100a8, Cxcl5, Cx3cl1, and Cxcl1. Additionally, in situ hybridization of Cxcl1, a potent neutrophil chemoattractant, demonstrated an increased expression in CK19+ cholangiocytes of TrailΔmye mice. Collectively, these data suggest that TRAIL from myeloid cells, particularly macrophages, restrains a subset of DR cells (i.e., Cxcl1 positive cells), limiting liver inflammation and fibrosis. Reprogramming macrophages to express TRAIL may be salutary in cholestasis.

Macrophage RAGE activation is proinflammatory in NASH.

Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte-derived macrophages. The receptor for advanced glycation endproducts (RAGE) is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that RAGE-expressing macrophages are proinflammatory and mediate liver inflammation in NASH. Compared with healthy controls, RAGE expression was increased in liver biopsies from patients with NASH. In a high-fat, -fructose, and -cholesterol-induced (FFC)-induced murine model of NASH, RAGE expression was increased, specifically on recruited macrophages. FFC mice that received a pharmacological inhibitor of RAGE (TTP488), and myeloid-specific RAGE KO mice (RAGE-MKO) had attenuated liver injury associated with a reduced accumulation of RAGE+ recruited macrophages. Transcriptomics analysis suggested that pathways of macrophage and T cell activation were upregulated by FFC diet, inhibited by TTP488 treatment, and reduced in RAGE-MKO mice. Correspondingly, the secretome of ligand-stimulated BM-derived macrophages from RAGE-MKO mice had an attenuated capacity to activate CD8+ T cells. Our data implicate RAGE as what we propose to be a novel and potentially targetable mediator of the proinflammatory signaling of recruited macrophages in NASH.

Rho-associated protein kinase 1 inhibition in hepatocytes attenuates nonalcoholic steatohepatitis.

BACKGROUND: NASH is the progressive form of NAFLD characterized by lipotoxicity, hepatocyte injury, tissue inflammation, and fibrosis. Previously, Rho-associated protein kinase (ROCK) 1 has been implicated in lipotoxic signaling in hepatocytes in vitro and high-fat diet-induced lipogenesis in vivo. However, whether ROCK1 plays a role in liver inflammation and fibrosis during NASH is unclear. Here, we hypothesized that pathogenic activation of ROCK1 promotes murine NASH pathogenesis. METHODS AND RESULTS: Patients with NASH had increased hepatic ROCK1 expression compared with patients with fatty liver. Similarly, hepatic ROCK1 levels and activity were increased in mice with NASH induced by a western-like diet that is high in fat, fructose, and cholesterol (FFC). Hepatocyte-specific ROCK1 knockout mice on the FFC diet displayed a decrease in liver steatosis, hepatic cell death, liver inflammation, and fibrosis compared with littermate FFC-fed controls. Mechanistically, these effects were associated with a significant attenuation of myeloid cell recruitment. Interestingly, myeloid cell-specific ROCK1 deletion did not affect NASH development in FFC-fed mice. To explore the therapeutic opportunities, mice with established NASH received ROCKi, a novel small molecule kinase inhibitor of ROCK1/2, which preferentially accumulates in liver tissue. ROCK inhibitor treatment ameliorated insulin resistance and decreased liver injury, inflammation, and fibrosis. CONCLUSIONS: Genetic or pharmacologic inhibition of ROCK1 activity attenuates murine NASH, suggesting that ROCK1 may be a therapeutic target for treating human NASH.

Calcium-Dependent Translocation of S100B Is Facilitated by Neurocalcin Delta.

S100B is a calcium-binding protein that governs calcium-mediated responses in a variety of cells-especially neuronal and glial cells. It is also extensively investigated as a potential biomarker for several disease conditions, especially neurodegenerative ones. In order to establish S100B as a viable pharmaceutical target, it is critical to understand its mechanistic role in signaling pathways and its interacting partners. In this report, we provide evidence to support a calcium-regulated interaction between S100B and the neuronal calcium sensor protein, neurocalcin delta both in vitro and in living cells. Membrane overlay assays were used to test the interaction between purified proteins in vitro and bimolecular fluorescence complementation assays, for interactions in living cells. Added calcium is essential for interaction in vitro; however, in living cells, calcium elevation causes translocation of the NCALD-S100B complex to the membrane-rich, perinuclear trans-Golgi network in COS7 cells, suggesting that the response is independent of specialized structures/molecules found in neuronal/glial cells. Similar results are also observed with hippocalcin, a closely related paralog; however, the interaction appears less robust in vitro. The N-terminal region of NCALD and HPCA appear to be critical for interaction with S100B based on in vitro experiments. The possible physiological significance of this interaction is discussed.

Knockout of sulfatase 2 is associated with decreased steatohepatitis and fibrosis in a mouse model of nonalcoholic fatty liver disease.

Sulfatase 2 (SULF2) is a heparan sulfate editing enzyme that regulates the milieu of growth factors and cytokines involved in a variety of cellular processes. We used a murine model of diet-induced steatohepatitis to assess the effect of SULF2 downregulation on the development of nonalcoholic steatohepatitis (NASH) and liver fibrosis. Wild-type B6;129 mice (WT) and Sulf2-knockout B6;129P2-SULF2Gt(PST111)Byg mice (Sulf2-KO) were fed a fast-food diet (FFD) rich in saturated fats, cholesterol, and fructose or a standard chow diet (SC) ad libitum for 9 mo. WT mice on FFD showed a threefold increase in hepatic Sulf2 mRNA expression, and a 2.2-fold increase in hepatic SULF2 protein expression compared with WT mice on SC. Knockout of Sulf2 led to a significant decrease in diet-mediated weight gain and dyslipidemia compared with WT mice on FFD. Knockout of Sulf2 also abrogated diet-induced steatohepatitis and hepatic fibrosis compared with WT mice on FFD. Furthermore, expression levels of the profibrogenic receptors TGFßR2 and PDGFRß were significantly decreased in Sulf2-KO mice compared with WT mice on FFD. Together, our data suggest that knockout of Sulf2 significantly downregulates dyslipidemia, steatohepatitis, and hepatic fibrosis in a diet-induced mouse model of NAFLD, suggesting that targeting of SULF2 signaling may be a potential therapeutic mechanism in NASH.NEW & NOTEWORTHY We report for the first time that in wild-type (WT) mice, fast-food diet (FFD) induced a threefold increase in hepatic Sulf2 mRNA and a 2.2-fold increase in sulfatase 2 (SULF2) protein expression compared with WT mice on standard chow diet (SC). We showed that knockout of SULF2 ameliorates FFD-induced obesity, hyperlipidemia, steatohepatitis, and fibrosis. These data, along with work from other laboratories, suggest that SULF2 may be critical to the ability of the liver to progress to nonalcoholic steatohepatitis and fibrosis in conditions of overnutrition.

IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis.

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1αΔhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4µ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Deficiency Promotes the Ductular Reaction, Macrophage Accumulation, and Hepatic Fibrosis in the Abcb4-/- Mouse.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr-/-) mice were crossbred with ATP binding cassette subfamily B member 4-deficient (Abcb4-/-, alias Mdr2-/-) mice. Male and female wild-type, Tr-/-, Mdr2-/-, and Tr-/-Mdr2-/- mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2-/- and Tr-/-Mdr2-/- mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2-/- mice, compared with extensive bridging fibrosis in Tr-/-Mdr2-/- mice. DR cell population was greatly expanded in the Tr-/-Mdr2-/- versus Mdr2-/- mice. The expanded DR cell population in Tr-/-Mdr2-/- mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr-/-Mdr2-/- versus Mdr2-/- mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2-/- mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2-/- model of cholestasis.

Targeted Apoptosis of Ductular Reactive Cells Reduces Hepatic Fibrosis in a Mouse Model of Cholestasis.

BACKGROUND AND AIMS: In cholestatic liver diseases, ductular reactive (DR) cells extend into the hepatic parenchyma and promote inflammation and fibrosis. We have previously observed that multidrug-resistant 2 (Mdr2-/- ) double knockout (DKO) mice lacking tumor necrosis factor-related apoptosis-inducing ligand receptor (Tr-/- ) display a more extensive ductular reaction and hepatic fibrosis compared to Mdr2-/- mice. This observation suggests that the magnitude of the DR-cell population may be regulated by apoptosis. APPROACH AND RESULTS: To examine this concept, we cultured epithelial cell adhesion molecule-positive reactive cholangioids (ERCs) obtained from wild-type (WT), Tr-/- , Mdr2-/- and DKO mice. Single-cell transcriptomics and immunostaining of both WT and DKO ERCs confirmed their DR-cell phenotype. Moreover, DKO ERCs displayed a unique translational cluster with expression of chemokines, indicating a reactive state. Incubation with the myeloid cell leukemia 1 (MCL1) inhibitor S63845, a proapoptotic BH3-mimetic therapy, significantly decreased DKO and Mdr2-/- ERC viability compared to WT. Intravenous administration of S63845 significantly reduced the DR-cell population and markers of inflammation and liver fibrosis in Mdr2-/- and DKO mice. Furthermore, DKO mice treated with S63845 displayed a significant decrease in hepatic B lymphocytes compared to untreated mice as assessed by high-definition mass cytometry by time-of-flight. Coculture of bone marrow-derived macrophages with ERCs from DKO mouse livers up-regulated expression of the B cell-directed chemokine (C-C motif) ligand 5. Finally, DR cells were noted to be primed for apoptosis with Bcl-2 homologous antagonist/killer activation in vitro and in vivo in primary sclerosing cholangitis liver specimens. CONCLUSIONS: DR cells appear to play a key role in recruiting immune cells to the liver to actively create an inflammatory and profibrogenic microenvironment. Pharmacologic targeting of MCL1 in a mouse model of chronic cholestasis reduces DR-cell and B-cell populations and hepatic fibrosis.

Diet Mimicking "Fast Food" Causes Structural Changes to the Retina Relevant to Age-Related Macular Degeneration.

Introduction: Metabolic syndrome is a disorder characterized by a constellation of findings including truncal obesity, elevated blood pressure, abnormal cholesterol levels, and high blood glucose. Recent evidence suggests that metabolic syndrome may be associated with increased risk of age-related macular degeneration (AMD) and other eye diseases. Recently, C57BL/6J wild-type mice fed with a "fast food" diet consisting of high fat, cholesterol, and fructose-supplemented water showed unique systemic pathology consistent with metabolic syndrome and nonalcoholic steatohepatitis. Additionally, these mice showed higher levels of fibrosis, inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction compared to mice fed with only a high-fat diet alone. Since similar pathways are activated in AMD, we sought to determine whether mice fed a "fast food" diet exhibited retinal changes.Methods: 3-month-old wild-type mice were randomized to a standard chow (n = 11) or a "fast food" (n = 18) diet and fed for 9 months. At 1 year of age, tissues were collected and retinas were analyzed using transmission electron microscopy. Quantitative measures of Bruch's membrane thickness and retinal pigment epithelium (RPE) cell counts were performed.Results: "Fast food" fed mice showed ocular pathology relevant to various stages of AMD including basal laminar deposits, focal thickening of Bruch's membrane, and a significant loss of RPE cells.Discussion/conclusion: A wild-type mouse model of metabolic syndrome fed a "fast food" diet developed changes to the retina similar to some of the pathologic features seen in AMD. Further investigations into this and similar animal models as well as further epidemiological studies are needed to more clearly define the association between metabolic syndrome and AMD.

Activated cholangiocytes release macrophage-polarizing extracellular vesicles bearing the DAMP S100A11.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/ß inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Integrin ß1-enriched extracellular vesicles mediate monocyte adhesion and promote liver inflammation in murine NASH.

BACKGROUND & AIMS: Hepatic recruitment of monocyte-derived macrophages (MoMFs) contributes to the inflammatory response in non-alcoholic steatohepatitis (NASH). However, how hepatocyte lipotoxicity promotes MoMF inflammation is unclear. Here we demonstrate that lipotoxic hepatocyte-derived extracellular vesicles (LPC-EVs) are enriched with active integrin ß1 (ITGß1), which promotes monocyte adhesion and liver inflammation in murine NASH. METHODS: Hepatocytes were treated with either vehicle or the toxic lipid mediator lysophosphatidylcholine (LPC); EVs were isolated from the conditioned media and subjected to proteomic analysis. C57BL/6J mice were fed a diet rich in fat, fructose, and cholesterol (FFC) to induce NASH. Mice were treated with anti-ITGß1 neutralizing antibody (ITGß1Ab) or control IgG isotype. RESULTS: Ingenuity® Pathway Analysis of the LPC-EV proteome indicated that ITG signaling is an overrepresented canonical pathway. Immunogold electron microscopy and nanoscale flow cytometry confirmed that LPC-EVs were enriched with activated ITGß1. Furthermore, we showed that LPC treatment in hepatocytes activates ITGß1 and mediates its endocytic trafficking and sorting into EVs. LPC-EVs enhanced monocyte adhesion to liver sinusoidal cells, as observed by shear stress adhesion assay. This adhesion was attenuated in the presence of ITGß1Ab. FFC-fed, ITGß1Ab-treated mice displayed reduced inflammation, defined by decreased hepatic infiltration and activation of proinflammatory MoMFs, as assessed by immunohistochemistry, mRNA expression, and flow cytometry. Likewise, mass cytometry by time-of-flight on intrahepatic leukocytes showed that ITGß1Ab reduced levels of infiltrating proinflammatory monocytes. Furthermore, ITGß1Ab treatment significantly ameliorated liver injury and fibrosis. CONCLUSIONS: Lipotoxic EVs mediate monocyte adhesion to LSECs mainly through an ITGß1-dependent mechanism. ITGß1Ab ameliorates diet-induced NASH in mice by reducing MoMF-driven inflammation, suggesting that blocking ITGß1 is a potential anti-inflammatory therapeutic strategy in human NASH. LAY SUMMARY: Herein, we report that a cell adhesion molecule termed integrin ß1 (ITGß1) plays a key role in the progression of non-alcoholic steatohepatitis (NASH). ITGß1 is released from hepatocytes under lipotoxic stress as a cargo of extracellular vesicles, and mediates monocyte adhesion to liver sinusoidal endothelial cells, which is an essential step in hepatic inflammation. In a mouse model of NASH, blocking ITGß1 reduces liver inflammation, injury and fibrosis. Hence, ITGß1 inhibition may serve as a new therapeutic strategy for NASH.

Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.

BACKGROUND & AIMS: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.

YAP-associated chromosomal instability and cholangiocarcinoma in mice.

Deregulated Hippo pathway signaling is associated with aberrant activation of the downstream effector yes-associated protein (YAP), an emerging key oncogenic mediator in cholangiocarcinoma (CCA). In our prior work, we have demonstrated that biliary transduction of YAP along with Akt as a permissive factor induces CCA in mice. To further delineate the mechanisms associated with YAP-associated biliary oncogenesis, we have established seven malignant murine cell lines from our YAP-driven murine CCA model. These cells express the CCA markers SRY (Sex Determining Region Y)-Box 9 (SOX9), cytokeratin (CK)-7 and 19 but lack hepatocyte nuclear factor 4 alpha and alpha-smooth muscle actin, markers of hepatocellular carcinoma and cancer-associated fibroblasts, respectively. Notably, the murine CCA cells can be readily implanted into mouse livers with resultant orthotopic tumor formation. In this unique syngeneic orthotopic murine model, tumors exhibit histopathologic features resembling human CCA. We analyzed transcriptome data from YAP-associated parent CCA tumor nodules and identified a gene expression pattern associated with chromosomal instability, known as CIN25. Similarly, mate-pair sequencing of the murine CCA cells revealed chromosomal missegregation with gains and losses of several whole chromosomes demonstrating aneuploidy. Of the CIN25 genes, forkhead box M1 (Foxm1), a key cell cycle regulator, was the most significantly upregulated CIN25 gene product. Accordingly, small interfering RNA (siRNA)-mediated silencing of YAP as well as FOXM1 inhibition with thiostrepton induced CCA cell death. These preclinical data imply a role for YAP-mediated chromosomal instability in cholangiocarcinoma, and suggest FOXM1 inhibition as a therapeutic target for CCA.

Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ~5000 cells with a central lumen of ~150 µm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~80%. Cholangioids exposed to hydrogen peroxide exhibited cellular senescence and the senescence-associated secretory phenotype (SASP; increased IL-6, p21, SA-ß-Gal, yH2A.x and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater number of extracellular vesicles (EVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~2-fold increase in macrophage migration. In summary, we developed a method to generate normal and diseased cholangioids, characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

A longitudinal study of whole body, tissue, and cellular physiology in a mouse model of fibrosing NASH with high fidelity to the human condition.

The sequence of events that lead to inflammation and fibrosing nonalcoholic steatohepatitis (NASH) is incompletely understood. Hence, we investigated the chronology of whole body, tissue, and cellular events that occur during the evolution of diet-induced NASH. Male C57Bl/6 mice were assigned to a fast-food (FF; high calorie, high cholesterol, high fructose) or standard-chow (SC) diet over a period of 36 wk. Liver histology, body composition, mitochondrial respiration, metabolic rate, gene expression, and hepatic lipid content were analyzed. Insulin resistance [homeostasis model assessment-insulin resistance (HOMA-IR)] increased 10-fold after 4 wk. Fibrosing NASH was fully established by 16 wk. Total hepatic lipids increased by 4 wk and remained two- to threefold increased throughout. Hepatic triglycerides declined from sixfold increase at 8 wk to threefold increase by 36 wk. In contrast, hepatic cholesterol levels steadily increased from baseline at 8 wk to twofold by 36 wk. The hepatic immune cell population altered over time with macrophages persisting beyond 16 wk. Mitochondrial oxygen flux rates of FF mice diet were uniformly lower with all the tested substrates (13-276 pmol·s-1·ml-1 per unit citrate synthase) than SC mice (17-394 pmol·s-1·ml-1 per unit citrate synthase) and was accompanied by decreased mitochondrial:nuclear gene copy number ratios after 4 wk. Metabolic rate was lower in FF mice. Mitochondrial glutathione was significantly decreased at 24 wk in FF mice. Expression of dismutases and catalase was also decreased in FF mice. The evolution of NASH in the FF diet-induced model is multiphasic, particularly in terms of hepatic lipid composition. Insulin resistance precedes hepatic inflammation and fibrosis. Mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent. Collectively, these observations provide a unique overview of the sequence of changes that coevolve with the histological evolution of NASH.NEW & NOTEWORTHY This study demonstrates in a first of kind longitudinal analysis, the evolution of nonalcoholic steatohepatitis (NASH) on a fast-food diet-induced model. Key findings include 1) hepatic lipid composition changes in a multiphasic fashion as NASH evolves; 2) insulin resistance precedes hepatic inflammation and fibrosis, answering a longstanding chicken-and-egg question regarding the relationship of insulin resistance to liver histology in NASH; and 3) mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent.

Biliary tract instillation of a SMAC mimetic induces TRAIL-dependent acute sclerosing cholangitis-like injury in mice.

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease of unknown etiopathogenesis characterized by fibrous cholangiopathy of large and small bile ducts. Systemic administration of a murine TNF-related apoptosis-inducing ligand (TRAIL) receptor agonist induces a sclerosing cholangitis injury in C57BL/6 mice, suggesting endogenous TRAIL may contribute to sclerosing cholangitis syndromes. Cellular inhibitor of apoptosis proteins (cIAP-1 and cIAP-2) are negative regulators of inflammation and TRAIL receptor signaling. We hypothesized that if endogenous TRAIL promotes sclerosing cholangitis, then cIAP depletion should also induce this biliary tract injury. Herein, we show that cIAP protein levels are reduced in the interlobular bile ducts of human PSC livers. Downregulation of cIAPs in normal human cholangiocytes in vitro by use of a SMAC mimetic (SM) induces moderate, ripoptosome-mediated apoptosis and RIP1-independent upregulation of proinflammatory cytokines and chemokines. Cytokine and chemokine expression was mediated by the non-canonical activation of NF-κB. To investigate whether downregulation of cIAPs is linked to generation of a PSC-like phenotype, an SM was directly instilled into the mouse biliary tree. Twelve hours after biliary instillation, TUNEL-positive cholangiocytes were identified; 5 days later, PSC-like changes were observed in the SM-treated mice, including a fibrous cholangiopathy of the interlobular bile ducts, portal inflammation, significant elevation of serum markers of cholestasis and cholangiographic evidence of intrahepatic biliary tract injury. In contrast, TRAIL and TRAIL-receptor deficient mice showed no sign of cholangiopathy following SM intrabiliary injection. We conclude that in vivo antagonism of cIAPs in mouse biliary epithelial cells is sufficient to trigger cholangiocytes apoptosis and a proinflammatory response resulting in a fibrous cholangiopathy resembling human sclerosing cholangitis. Therefore, downregulation of cIAPs in PSC cholangiocytes may contribute to the development of the disease. Our results also indicate that inhibition of TRAIL signaling pathways may be beneficial in the treatment of PSC.

Prohibitin 1 suppresses liver cancer tumorigenesis in mice and human hepatocellular and cholangiocarcinoma cells.

Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c-MYC interacts directly with both proteins. Furthermore, c-MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c-MYC and PHB1's role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down-regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each other's expression, whereas PHB1 negatively regulates the expression of c-MYC, MAFG, and c-MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E-box element, and repress E-box promoter activity. PHB1 promoter contains a repressive E-box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c-MYC, c-MAF, and MAFG in cancer cells and human HCC/CCA. All 8-month-old liver-specific Phb1 knockout mice developed HCC, and one developed CCA. Five-month-old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. CONCLUSION: We have identified that PHB1, down-regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E-box and positively regulates MAT1A while suppressing c-MYC, MAFG, and c-MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis-induced CCA. (Hepatology 2017;65:1249-1266).

Data on the identity and myristoylation state of recombinant, purified hippocalcin.

In this data article we report on the purity and post translation modification of bacterially expressed and purified recombinant hippocalcin (HPCA): a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. MALDI-TOF in source decay (ISD) analysis was used to identify both the myristoylated or non-myristoylated forms of the protein. MALDI-TOF ISD data on the identity of the protein, amino acid sequence and myristoylation efficiency are provided. This data relates to the article "Single-Column Purification of the Tag-free, Recombinant Form of the Neuronal Calcium Sensor Protein, Hippocalcin Expressed in Eschericia coli" [1].

Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells.

BACKGROUND & AIMS: Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34(+) hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. METHODS: The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah(-/-) mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. RESULTS: Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah(-/-) mice by 5-100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. CONCLUSIONS: Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. LAY SUMMARY: Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.

Datasets depicting mobility retardation of NCS proteins observed upon incubation with calcium, but not with magnesium, barium or strontium.

In this data article we show the specificity of the Ca(2+)-induced mobility shift in three proteins that belong to the neuronal calcium sensor (NCS) protein family: Hippocalcin, GCAP1 and GCAP2. These proteins did not display a shift in mobility in native gels when incubated with divalent cations other than Ca(2+) - such as Mg(2+), Ba(2+), and Sr(2+), even at 10× concentrations. The data is similar to that obtained with another NCS protein, neurocalcin delta (Viviano et al., 2016, "Electrophoretic Mobility Shift in Native Gels Indicates Calcium-dependent Structural Changes of Neuronal Calcium Sensor Proteins", [1]).