Integrated Multiomics Reveals Glucose Use Reprogramming and Identifies a Novel Hexokinase in Alcoholic Hepatitis.

BACKGROUND & AIMS: We recently showed that alcoholic hepatitis (AH) is characterized by dedifferentiation of hepatocytes and loss of mature functions. Glucose metabolism is tightly regulated in healthy hepatocytes. We hypothesize that AH may lead to metabolic reprogramming of the liver, including dysregulation of glucose metabolism. METHODS: We performed integrated metabolomic and transcriptomic analyses of liver tissue from patients with AH or alcoholic cirrhosis or normal liver tissue from hepatic resection. Focused analyses of chromatin immunoprecipitation coupled to DNA sequencing was performed. Functional in vitro studies were performed in primary rat and human hepatocytes and HepG2 cells. RESULTS: Patients with AH exhibited specific changes in the levels of intermediates of glycolysis/gluconeogenesis, the tricarboxylic acid cycle, and monosaccharide and disaccharide metabolism. Integrated analysis of the transcriptome and metabolome showed the used of alternate energetic pathways, metabolite sinks and bottlenecks, and dysregulated glucose storage in patients with AH. Among genes involved in glucose metabolism, hexokinase domain containing 1 (HKDC1) was identified as the most up-regulated kinase in patients with AH. Histone active promoter and enhancer markers were increased in the HKDC1 genomic region. High HKDC1 levels were associated with the development of acute kidney injury and decreased survival. Increased HKDC1 activity contributed to the accumulation of glucose-6-P and glycogen in primary rat hepatocytes. CONCLUSIONS: Altered metabolite levels and messenger RNA expression of metabolic enzymes suggest the existence of extensive reprogramming of glucose metabolism in AH. Increased HKDC1 expression may contribute to dysregulated glucose metabolism and represents a novel biomarker and therapeutic target for AH.

Kinase analysis in alcoholic hepatitis identifies p90RSK as a potential mediator of liver fibrogenesis.

OBJECTIVE: Alcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order to discover novel molecular targets. DESIGN: Extensive phosphoprotein analysis by reverse phase protein microarrays was performed in AH (n=12) and normal human livers (n=7). Ribosomal S6 kinase (p90RSK) hepatic expression was assessed by qPCR, Western blot and immunohistochemistry. Kaempferol was used as a selective pharmacological inhibitor of the p90RSK pathway to assess the regulation of experimentally-induced liver fibrosis and injury, using in vivo and in vitro approaches. RESULTS: Proteomic analysis identified p90RSK as one of the most deregulated kinases in AH. Hepatic p90RSK gene and protein expression was also upregulated in livers with chronic liver disease. Immunohistochemistry studies showed increased p90RSK staining in areas of active fibrogenesis in cirrhotic livers. Therapeutic administration of kaempferol to carbon tetrachloride-treated mice resulted in decreased hepatic collagen deposition, and expression of profibrogenic and proinflammatory genes, compared to vehicle administration. In addition, kaempferol reduced the extent of hepatocellular injury and degree of apoptosis. In primary hepatic stellate cells, kaempferol and small interfering RNA decreased activation of p90RSK, which in turn regulated key profibrogenic actions. In primary hepatocytes, kaempferol attenuated proapoptotic signalling. CONCLUSIONS: p90RSK is upregulated in patients with chronic liver disease and mediates liver fibrogenesis in vivo and in vitro. These results suggest that the p90RSK pathway could be a new therapeutic approach for liver diseases characterised by advanced fibrosis.

Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcoholic hepatitis.

UNLABELLED: Alcoholic hepatitis (AH) frequently progresses to multiple organ failure (MOF) and death. However, the driving factors are largely unknown. At admission, patients with AH often show criteria of systemic inflammatory response syndrome (SIRS) even in the absence of an infection. We hypothesize that the presence of SIRS may predispose to MOF and death. To test this hypothesis, we studied a cohort including 162 patients with biopsy-proven AH. The presence of SIRS and infections was assessed in all patients, and multivariate analyses identified variables independently associated with MOF and 90-day mortality. At admission, 32 (19.8%) patients were diagnosed with a bacterial infection, while 75 (46.3%) fulfilled SIRS criteria; 58 patients (35.8%) developed MOF during hospitalization. Short-term mortality was significantly higher among patients who developed MOF (62.1% versus 3.8%, P < 0.001). The presence of SIRS was a major predictor of MOF (odds ratio = 2.69, P = 0.025) and strongly correlated with mortality. Importantly, the course of patients with SIRS with and without infection was similar in terms of MOF development and short-term mortality. Finally, we sought to identify serum markers that differentiate SIRS with and without infection. We studied serum levels of high-sensitivity C-reactive protein, procalcitonin, and lipopolysaccharide at admission. All of them predicted mortality. Procalcitonin, but not high-sensitivity C-reactive protein, serum levels identified those patients with SIRS and infection. Lipopolysaccharide serum levels predicted MOF and the response to prednisolone. CONCLUSION: In the presence or absence of infections, SIRS is a major determinant of MOF and mortality in AH, and the mechanisms involved in the development of SIRS should be investigated; procalcitonin serum levels can help to identify patients with infection, and lipopolysaccharide levels may help to predict mortality and the response to steroids.

The biliary epithelium gives rise to liver progenitor cells.

UNLABELLED: Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1ß). However, HNF1ß expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)-positive hepatocytes. In order to investigate the role of HNF1ß-expressing cells we used a tamoxifen-inducible Hnf1ßCreER/R26R(Yfp/LacZ) mouse to lineage-trace Hnf1ß(+) biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1ß(+) cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1ß(+) cells to hepatocyte was detected. We next assessed the contribution of Hnf1ß(+) -derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl-1,4-dihydro-collidin (DDC)-diet and a choline-deficient ethionine-supplemented (CDE)-diet. The contribution of Hnf1ß(+) cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC-diet treatment, mice fed with a CDE-diet showed a small population of hepatocytes derived from Hnf1ß(+) cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome-wide expression profile of Hnf1ß(+) -derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways. CONCLUSION: HNF1ß(+) biliary duct cells are the origin of LPC. HNF1ß(+) cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration.

CCL20 mediates lipopolysaccharide induced liver injury and is a potential driver of inflammation and fibrosis in alcoholic hepatitis.

OBJECTIVE: Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury. DESIGN: CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo. RESULTS: CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs. CONCLUSIONS: Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH.

Human and experimental evidence supporting a role for osteopontin in alcoholic hepatitis.

UNLABELLED: We identified, in the transcriptome analysis of patients with alcoholic hepatitis (AH), osteopontin (OPN) as one of the most up-regulated genes. Here, we used a translational approach to investigate its pathogenic role. OPN hepatic gene expression was quantified in patients with AH and other liver diseases. OPN protein expression and processing were assessed by immmunohistochemistry, western blotting and enzyme-linked immunosorbent assay. OPN gene polymorphisms were evaluated in patients with alcoholic liver disease. The role of OPN was evaluated in OPN(-/-) mice with alcohol-induced liver injury. OPN biological actions were studied in human hepatic stellate cells (HSCs) and in precision-cut liver slices. Hepatic expression and serum levels of OPN were markedly increased in AH, compared to normal livers and other types of chronic liver diseases, and correlated with short-term survival. Serum levels of OPN also correlated with hepatic expression and disease severity. OPN was mainly expressed in areas with inflammation and fibrosis. Two proteases that process OPN (thrombin and matrix metalloproteinase 7) and cleaved OPN were increased in livers with AH. Patients with AH had a tendency of a lower frequency of the CC genotype of the +1239C single-nucleotide polymorphism of the OPN gene, compared to patients with alcohol abuse without liver disease. Importantly, OPN(-/-) mice were protected against alcohol-induced liver injury and showed decreased expression of inflammatory cytokines. Finally, OPN was induced by lipopolysaccharide and stimulated inflammatory actions in HSCs. CONCLUSION: Human and experimental data suggest a role for OPN in the pathogenesis of AH. Further studies should evaluate OPN as a potential therapeutic target.

Transcriptome analysis identifies TNF superfamily receptors as potential therapeutic targets in alcoholic hepatitis.

OBJECTIVE: Alcoholic hepatitis (AH) is a severe clinical condition that needs novel therapies. The identification of targets for therapy is hampered by the lack of animal models of advanced AH. The authors performed a translational study through a transcriptome analysis in patients with AH to identify new molecular targets. DESIGN: Hepatic gene expression profiling was assessed by DNA microarray in patients with AH (n=15) and normal livers (n=7). Functional analysis was assessed by gene set enrichment analysis. Quantitative PCR was performed in patients with AH (n=40), hepatitis C (n=18), non-alcoholic steatohepatitis (n=20) and in mouse models of acute and chronic liver injury. Protein expression was assessed by immunohistochemistry and western blotting. RESULTS: Gene expression analysis showed 207 genes >5-fold differentially expressed in patients with AH and revealed seven pathways differentially regulated including 'cytokine-cytokine receptor interaction'. Several tumour necrosis factor (TNF) superfamily receptors, but not ligands, were overexpressed in AH. Importantly, Fn14 was the only TNF superfamily receptor exclusively upregulated in AH compared with other liver diseases and correlated with both 90-day mortality and severity of portal hypertension. Fn14 protein expression was detected in areas of fibrogenesis and in a population of hepatocytes. Fn14 expression was increased in experimental models of liver injury and was detected in progenitor cells. CONCLUSION: Translational research revealed that TNF superfamily receptors are overexpressed in AH. Fn14, the receptor for TNF-like weak inducer of apoptosis, is selectively upregulated in patients with AH. TNF superfamily receptors could represent a potential target for therapy.

Interleukin-22 treatment ameliorates alcoholic liver injury in a murine model of chronic-binge ethanol feeding: role of signal transducer and activator of transcription 3.

UNLABELLED: Interleukin-22 (IL-22), a recently identified member of the IL-10 family of cytokines that is produced by Th17 and natural killer cells, plays an important role in controlling bacterial infection, homeostasis, and tissue repair. Here, we tested the effect of IL-22 on alcohol-induced liver injury in a murine model of chronic-binge ethanol feeding. Feeding male C57BL/6 mice with a Lieber-DeCarli diet containing 5% ethanol for 10 days, followed by a single dose of ethanol (5 g/kg body weight) by gavage, induces significant fatty liver and liver injury with peak serum levels of approximately 250 IU/L alanine aminotransferase and 420 IU/L aspartate aminotransferase 9 hours after gavage. Moreover, chronic-binge ethanol administration increases expression of hepatic and serum inflammatory cytokines and hepatic oxidative stress. Using this model, we demonstrate that treatment with IL-22 recombinant protein activates hepatic signal transducer and activator of transcription 3 (STAT3) and ameliorates alcoholic fatty liver, liver injury, and hepatic oxidative stress. Administration with IL-22 adenovirus also prevents alcohol-induced steatosis and liver injury. Deletion of STAT3 in hepatocytes abolishes the hepatoprotection provided by IL-22 in alcoholic liver injury. In addition, IL-22 treatment down-regulates the hepatic expression of fatty acid transport protein, but up-regulates several antioxidant, antiapoptotic, and antimicrobial genes. Finally, expression of IL-22 receptor 1 is up-regulated whereas IL-22 is undetectable in the livers from mice with chronic-binge ethanol feeding or patients with alcoholic hepatitis. CONCLUSION: Chronic-binge ethanol feeding may be a useful model to study the early stages of alcoholic liver injury. IL-22 treatment could be a potential therapeutic option to ameliorate alcoholic liver disease, due to its antioxidant, antiapoptotic, antisteatotic, proliferative, and antimicrobial effects with the added benefit of potentially few side effects.

Endothelial dysfunction markers predict short-term mortality in patients with severe alcoholic hepatitis.

OBJECTIVES: Alcoholic hepatitis (AH) is a severe condition characterized by a marked inflammatory response and high short-term mortality. Endothelial dysfunction (ED) is an early event in vascular and inflammatory disorders. The aim of this study is to evaluate ED in AH patients. METHODS: Prognostic value of ED biomarkers was evaluated in patients with severe AH (n = 67), compensated alcoholic cirrhosis (n = 15), heavy drinkers without liver disease (n = 15) and controls (n = 9), and in a validation cohort of 50 patients with AH. Gene expression of ED markers was analyzed in liver tissue. RESULTS: Plasma levels of ED markers such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin and von Willebrand factor (vWF) increased along alcohol-related liver disease (ALD) progression. Intergroup analysis showed a significant increase of these markers in AH patients. In addition, VCAM-1 showed a positive correlation with Maddrey, MELD and ABIC scores and inflammation parameters (i.e. C-reactive protein and LPS levels). Importantly, levels of VCAM-1 were higher in patients with increased mortality and were independently associated with short-term survival (90-day) when adjusted by ABIC score. These results were confirmed in an independent cohort of AH patients. In addition, severe AH patients showed altered hepatic expression of ED markers. CONCLUSIONS: In this study we show that advanced ALD and particularly severe AH is associated with an increase of ED biomarkers, which correlate with patient outcomes. These results suggest that ED may be a pathogenic event in AH and highlight endothelial factors as potential biomarkers in AH.

LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis.

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development.

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis.

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.

Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells.

Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.

GSSG/GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug induced oxidative stress.

The formation of reactive oxygen species (ROS) could cause cellular damage and eventually lead to apoptosis and necrosis. The ratio between oxidized glutathione and reduced glutathione (GSSG-to-GSH ratio) has been used as an important in vitro and in vivo biomarker of the redox balance in the cell and consequently of cellular oxidative stress. This paper optimizes a LC-MS/MS method for the simultaneous determination of GSH and GSSG. The proposed method is based on the derivatization of reduced GSH using iodoacetic acid (IAA) in order to prevent its rapid oxidation to GSSG during sample preparation. The optimized analytical method was applied to evaluate the effect of different pharmaceutical agents on GSSG-to-GSH ratio in cryopreserved rat and human hepatocytes in culture. Hepatocyte viabilities were also determined at the same time by using the WST-1 assay as a direct measurement of cell mitochondrial respiration. The results obtained demonstrate that cryopreserved rat and human hepatocytes in culture are reliable in vitro models for the evaluation of cellular oxidative stress. In addition, the GSSG-to-GSH ratio measurements could be a biomarker of hepatotoxicity providing similar results to those of cytotoxicity assay.