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BACKGROUND Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors known to regulate glucose and fatty acid metabolism, inflammation, endothelial function and fibrosis. PPAR isoforms have been extensively studied in metabolic diseases, including type 2 diabetes and cardiovascular diseases. Recent data extend the key role of PPARs to liver diseases coursing with vascular dysfunction, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). AIM This review summarises and discusses the pathobiological role of PPARs in cardiovascular diseases with a special focus on their impact and therapeutic potential in NAFLD and NASH. RESULTS AND CONCLUSIONS PPARs may be attractive for the treatment of NASH due to their liver-specific effects but also because of their efficacy in improving cardiovascular outcomes, which may later impact liver disease. Assessment of cardiovascular disease in the context of NASH trials is, therefore, of the utmost importance, both from a safety and efficacy perspective.

A joint meeting of the European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Diseases (AASLD) was held in London on September 30 and October 1, 2017. The goals of the meeting were to identify areas of broad agreement and disagreement, develop consensus, and determine future directions to ultimately reduce the burden, morbidity, and mortality of alcohol-related liver disease (previously termed alcoholic liver disease). The specific aims of the meeting were to identify unmet needs and areas for future investigation, in order to reduce alcohol consumption, develop markers for diagnosis and prognosis of disease, and create a framework to test novel pharmacological agents with pre-specified treatment endpoints. A table summary of these goals and aims is provided in the context of epidemiology, current management strategies, next steps for future trials and translational science.

Exosomes, membranous nanovesicles, naturally carry bio-macromolecules and play pivotal roles in both physiological intercellular crosstalk and disease pathogenesis. Here, we showed that B cell-derived exosomes can function as vehicles to deliver exogenous miRNA-155 mimic or inhibitor into hepatocytes or macrophages, respectively. Stimulation of B cells significantly increased exosome production. Unlike in parental cells, baseline level of miRNA-155 was very low in exosomes derived from stimulated B cells. Exosomes loaded with a miRNA-155 mimic significantly increased miRNA-155 levels in primary mouse hepatocytes and the liver of miRNA-155 knockout mice. Treatment of RAW macrophages with miRNA-155 inhibitor loaded exosomes resulted in statistically significant reduction in LPS-induced TNFα production and partially prevented LPS-induced decrease in SOCS1 mRNA levels. Furthermore, exosome-mediated miRNA-155 inhibitor delivery resulted in functionally more efficient inhibition and less cellular toxicity compared to conventional transfection methods. Similar approaches could be useful in modification of target biomolecules in vitro and in vivo.

Recognition of hepatitis C virus (HCV)-infected hepatocyes and interferon (IFN) induction are critical in antiviral immune response. We hypothesized that cell-cell contact between pDCs and HCV-infected cells was required for IFNα induction via involvement of cell surface molecules. Co-culture of human peripheral blood mononuclear cells (PBMCs) with genotype 1a full length HCV genomic replicon cells (FL) or genotype 2a JFH-1 virus infected hepatoma cells (JFH-1), not with uninfected hepatoma cells (Huh7.5), induced IFNα production. Depletion of pDCs from PBMCs attenuated IFNα release and purified pDCs produced high levels of IFNα after co-culture with FL replicons or JFH-1 infected cells. IFNα induction by HCV-containing hepatoma cells required viral replication, direct cell-cell contact with pDCs, and receptor-mediated endocytosis. We determined that the tetraspanin proteins, CD81 and CD9 and not other HCV entry receptors were required for IFNα induction in pDCs by HCV infected hepatoma cells. Disruption of cholesterol-rich membrane microdomains, the localization site of CD81 or inhibition of CD81 downstream molecule, Rac GTPase, inhibited IFNα production from co-cultures. IFNα production by HCV infected hepatoma cells was decreased in pDCs from HCV infected patients compared to normal controls. We found that pre-exposure of normal PBMCs to HCV viral particles attenuated IFNα induction by HCV infected hepatoma cells or TLR ligands and this inhibitory effect could be prevented by an anti-HCV E2 blocking antibody. In conclusion, our novel data show that recognition of HCV-infected hepatoma cells by pDCs involves CD81/CD9-associated membrane microdomains and induces potent IFNα production.

This presentation describes educational programs for trainees and faculty that are offered through the UMCCTS.

Background: MicroRNAs (miRs) are small non-coding molecules that regulate gene expression. MiRs expression levels change not only in diseased tissues but also in circulation. Further, miRs are stable in frozen samples that make them attractive for biomarker discovery. Recent reports suggest altered expression of circulating miRNAs in various diseases. MiR-122 is highly abundant in hepatocytes where it regulates different metabolic pathways while miR-155 is a central regulator of inflammation. The aim of this study was to evaluate circulating miRNAs as potential markers of hepatocyte damage and inflammation in liver diseases. Methods: Serum/plasma and liver samples were collected from C57/BL6 mice after: 1. Chronic alcohol feeding with Lieber-deCarli diet containing alcohol or pair-fed diet for 5 weeks 2. Acetaminophen (APAP) administration. 3. TLR9/4 administration. 4. CCL4 administration. Serum/plasma ALT was evaluated and total RNA was analyzed for miRNAs expression with TaqMan MicroRNA assay. Non-parametric Mann-Whitney test was used for statistics. Results: The alcohol, APAP, TLR9/TLR4 and CCL4, -induced liver injury models all resulted in ALT increase and more important, in increased serum/plasma miR-122 levels compared to control mice. There was a linear correlation between miR-122 and ALT levels. After CCL4 treatment, serum miR-122 was upregulated as early as one week over controls and it remained elevated. No increase in serum miR-122 in Toll like receptor 4 or NADPH oxidase–deficient mice was found after alcohol feeding as these KO mice were protected from alcohol-induced liver injury and inflammation. Alcohol-, APAP, TLR9/TLR4 and CCL4-induced liver damage all involve in activation of the inflammatory cascade. Consistent with this, we found increased serum miR-155 levels. Conclusion: Our novel results show that serum/plasma miR-122 up-regulation correlates with ALT, thus, miR-122 could be a useful biomarker in acute and chronic liver injury. We also report that serum miR-155 is increased in liver disease with inflammation.

Background: Inflammation and oxidative stress are key factors in the pathogenesis of non-alcoholic steatohepatitis (NASH). Polymorphonuclear neutrophils are capable to produce significant amounts of reactive oxygen species (ROS) via the NADPH oxidase complex. Increased hepatic neutrophil infiltration has been described in steatohepatitis. We aimed to investigate the in vitro ROS generation by neutrophils of NASH patients and the hepatic NADPH oxidase activity in murine steatohepatitis. Material and methods: PMNs were isolated from peripheral blood of NASH patients (n=16) and healthy controls (n=16). In vitro ROS production was measured by luminol chemiluminescence after phorbol myristate acetate (PMA) or opsonized zymosan stimulation. Hepatic lipid peroxidation and NADPH oxidase activation were measured in mice fed with methionine-choline-deficient (MCD) or -supplemented (MCS) diets. Results: PMA activated oxidative burst both in patients and controls. However, ROS production was significantly increased in non-diabetic NASH patients (n=9) compared to controls 30 min after the PMA stimulation. PMNs from NASH patients with diabetes mellitus (n=7) did not have higher ROS production after PMA-stimulation compared to controls. The PMA-induced peak chemiluminescence was significantly higher in the non-diabetic NASH patients compared to controls and diabetic NASH patients. No significant difference was observed without any stimulation and in opsonized zymosan induced chemiluminescence. Consistent with the increased oxidative capacity of PMNs in NASH patients, we found increased hepatic lipid peroxidation, higher expression and activation of the NADPH oxidase complex in MCD-steatohepatitis. Conclusion: Our finding supports the role of neutrophil oxidative stress in NASH. Our novel data suggests that the increased oxidative capacity of the PMNs it is not only localized to the liver but can have systemic effects and serve as a potential biomarker of NASH.

Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of pro-inflammatory cytokines, including interleukin (IL)-1β. IL-1β, Type-I IL-1 receptor (IL1R1) and IL-1 receptor antagonist (IL-1Ra) are all part of the IL-1 superfamily that play a role in inflammation. IL-1β maturation is dependent on Caspase-1. Using wild-type (WT), Caspase-1-, IL-1R1- and IL-1Ra deficient mice fed with Lieber-DeCarli alcohol or control diet, we have identified that signaling mediated by the active IL-1β was required for development of alcohol-induced steatosis, inflammation and injury. Increased IL-1β was due to upregulation of Caspase-1 activity and inflammatory activation. The pathogenic role of IL-1 signaling in ALD was attributable to the presence of IL-1R1 on liver parenchymal cells. Importantly, in vivo intervention with recombinant IL-1Ra, Anakinra, which blocks IL-1 signaling, significantly attenuated both liver steatosis and inflammation. In primary hepatocytes, physiological doses of IL-1β induced steatosis and upregulated the inflammatory and pro-steatotic chemokine MCP-1. MCP-1, but not IL-1β induced hepatocyte cytotoxicity at concentrations found in ALD. In conclusion, we demonstrate that Caspase-1-dependent upregulation of IL-1β and signaling mediated by IL-1 is crucial in the pathogenesis of ALD in a cell specific manner. Our findings suggest a potential role of IL-1Ra in the treatment of ALD.