Terence N. Bukong

Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90.

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.

Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages.

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. From the clinical editor: In this study, exosome-based delivery of miRNA-155 mimicker or inhibitor was found to have significant biological response in hepatocytes and macrophages. Exosome-based approaches may be useful in the modification of other target biomolecules.

Ethanol facilitates hepatitis C virus replication via up‐regulation of GW182 and heat shock protein 90 in human hepatoma cells

Alcohol use and hepatitis C virus (HCV) infection synergize to cause liver damage, and microRNA‐122 (miR‐122) appears to play a key role in this process. Argonaute 2 (Ago2), a key component of the RNA‐induced silencing complex (RISC), has been shown to be important in modulating miR‐122 function during HCV infection. However, GW182, a critical component of processing bodies (GW bodies) that is recruited by Ago2 to target messenger RNA (mRNA), has not been assessed in HCV infection. To characterize the role of GW182 in the pathogenesis of HCV infection, we determined its transcription and protein expression in an HCV J6/JFH1 culture system. Transcript and protein levels of GW182 as well as HCV RNA and protein expression increased with alcohol exposure. Specific silencing of mRNA expression by small interfering RNA against GW182 significantly decreased HCV RNA and protein expression. Overexpression of GW182 significantly increased HCV RNA and protein expression in HCV J6/JFH1 infected Huh7.5 cells. Furthermore, GW182 colocalized and coimmunoprecipitated with heat shock protein 90 (HSP90), which increased upon alcohol exposure with and without HCV infection and enhanced HCV gene expression. The use of an HSP90 inhibitor or knockdown of HSP90 decreased GW182 and miR‐122 expression and significantly reduced HCV replication. Conclusion: Overall, our results suggest that GW182 protein that is linked to miR‐122 biogenesis and HSP90, which has been shown to stabilize the RISC, are novel host proteins that regulate HCV infection during alcohol abuse. (HEPATOLOGY 2013)

Human ezrin‐moesin‐radixin proteins modulate hepatitis C virus infection

Host cytoskeletal proteins of the ezrin‐moesin‐radixin (EMR) family have been shown to modulate single‐stranded RNA virus infection through regulating stable microtubule formation. Antibody engagement of CD81, a key receptor for hepatitis C virus (HCV) entry, induces ezrin phosphorylation. Here we tested the role of EMR proteins in regulating HCV infection and explored potential therapeutic targets. We show that HCV E2 protein induces rapid ezrin phosphorylation and its cellular redistribution with F‐actin by way of spleen tyrosine kinase (SYK). Therapeutically blocking the functional roles of SYK or F‐actin reorganization significantly reduced Huh7.5 cell susceptibility to HCV J6/JFH‐1 infection. Using gene regulation, real‐time quantitative polymerase chain reaction, western blot, and fluorescent microscopy analysis, we found that proteins of the EMR family differentially regulate HCV infection in the J6/JFH‐1/Huh7.5 cell system. Moesin and radixin, but not ezrin, expression were significantly decreased in chronic HCV J6/JFH‐1‐infected Huh7.5 cells and HCV‐infected patient liver biopsies compared to controls. The decreases in moesin and radixin in HCV J6/JFH‐1‐infected Huh7.5 cells were associated with a significant increase in stable microtubules. Ezrin knockdown inhibited immediate postentry events in HCV infection. Overexpression of moesin or radixin significantly reduced HCV protein expression. In contrast, transient knockdown of moesin or radixin augmented HCV infection. Making use of the Con1 HCV replicon system, we tested the effect of EMR proteins on HCV replication. We found that transient knockdown of moesin increased HCV RNA expression while overexpression of EMR showed no significant effect on HCV replication. Conclusion: Our findings demonstrate the important role of EMR proteins during HCV infection at the postentry level and highlight possible novel targets for HCV treatment. (Hepatology 2013;58:1569–1579)

Alcoholic hepatitis and HCV interactions in the modulation of liver disease.

Most HCV‐infected patients regularly consume alcohol. Alcoholic liver disease (ALD) and chronic hepatitis C virus (HCV) infection together are the most common causes of liver disease worldwide. Although both factors independently cause liver disease, they synergistically promote rapid liver disease progression with devastating outcomes for patients. This review focuses on the prevalence, clinical characteristics and molecular pathophysiologic mechanisms of HCV infection associated with alcohol abuse. Recent findings have centred on the synergistic effect of alcohol and HCV on viral replication, hepatocyte apoptosis, oxidative stress, alcohol‐induced ‘leaky gut’, miR‐122 and immune dysregulation. Clinical and basic research findings presented here summarize key scientific findings with the aim of highlighting potential areas for new therapies and identifying ways of optimizing current treatments for alcoholics with HCV infection.

Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes

Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites.

Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease

The spectrum of alcoholic liver disease (ALD) is a major cause of mortality with limited therapies available. Because alcohol targets numerous signaling pathways in hepatocytes and in immune cells, the identification of a master regulatory target that modulates multiple signaling processes is attractive. In this report, we assessed the role of spleen tyrosine kinase (SYK), a nonreceptor tyrosine kinase, which has a central modulatory role in multiple proinflammatory signaling pathways involved in the pathomechanism of ALD. Using mouse disease models that represent various phases in the progression of human ALD, we found that alcohol, in all of these models, induced SYK activation in the liver, both in hepatocytes and liver mononuclear cells. Furthermore, significant SYK activation also occurred in liver samples and peripheral blood mononuclear cells of patients with ALD/alcoholic hepatitis compared to controls. Functional inhibition of SYK activation in vivo abrogated alcohol‐induced hepatic neutrophil infiltration, resident immune cell activation, as well as inflammasome and extracellular signal‐regulated kinase 1 and 2‐mediated nuclear factor kappa B activation in mice. Strikingly, inhibition of SYK activation diminished alcohol‐induced hepatic steatosis and interferon regulatory factor 3‐mediated apoptosis. Conclusion: Our data demonstrate a novel, functional, and multicellular role for SYK phosphorylation in modulating immune cell‐driven liver inflammation, hepatocyte cell death, and steatosis at different stages of ALD. These novel findings highlight SYK as a potential multifunctional target in the treatment of alcoholic steatohepatitis. (Hepatology 2016;64:1057‐1071)

Cannabis use is associated with reduced prevalence of non-alcoholic fatty liver disease: A cross-sectional study

Cannabis use is associated with reduced prevalence of obesity and diabetes mellitus (DM) in humans and mouse disease models. Obesity and DM are a well-established independent risk factor for non-alcoholic fatty liver disease (NAFLD), the most prevalent liver disease globally. The effects of cannabis use on NAFLD prevalence in humans remains ill-defined. Our objective is to determine the relationship between cannabis use and the prevalence of NAFLD in humans. We conducted a population-based case-control study of 5,950,391 patients using the 2014 Healthcare Cost and Utilization Project (HCUP), Nationwide Inpatient Survey (NIS) discharge records of patients 18 years and older. After identifying patients with NAFLD (1% of all patients), we next identified three exposure groups: non-cannabis users (98.04%), non-dependent cannabis users (1.74%), and dependent cannabis users (0.22%). We adjusted for potential demographics and patient related confounders and used multivariate logistic regression (SAS 9.4) to determine the odds of developing NAFLD with respects to cannabis use. Our findings revealed that cannabis users (dependent and non-dependent) showed significantly lower NAFLD prevalence compared to non-users (AOR: 0.82[0.76–0.88]; p<0.0001). The prevalence of NAFLD was 15% lower in non-dependent users (AOR: 0.85[0.79–0.92]; p<0.0001) and 52% lower in dependent users (AOR: 0.49[0.36–0.65]; p<0.0001). Among cannabis users, dependent patients had 43% significantly lower prevalence of NAFLD compared to non-dependent patients (AOR: 0.57[0.42–0.77]; p<0.0001). Our observations suggest that cannabis use is associated with lower prevalence of NAFLD in patients. These novel findings suggest additional molecular mechanistic studies to explore the potential role of cannabis use in NAFLD development.

Alcohol and HCV: Implications for Liver Cancer

Liver cancers are one of the deadliest known malignancies which are increasingly becoming a major public health problem in both developed and developing countries. Overwhelming evidence suggests a strong role of infection with hepatitis B and C virus (HBV and HCV), alcohol abuse, as well as metabolic diseases such as obesity and diabetes either individually or synergistically to cause or exacerbate the development of liver cancers. Although numerous etiologic mechanisms for liver cancer development have been advanced and well characterized, the lack of definite curative treatments means that gaps in knowledge still exist in identifying key molecular mechanisms and pathways in the pathophysiology of liver cancers. Given the limited success with current therapies and preventive strategies against liver cancer, there is an urgent need to identify new therapeutic options for patients. Targeting HCV and or alcohol-induced signal transduction, or virus-host protein interactions may offer novel therapies for liver cancer. This review summarizes current knowledge on the mechanistic development of liver cancer associated with HCV infection and alcohol abuse as well as highlights potential novel therapeutic strategies.

A Novel Human Radixin Peptide Inhibits Hepatitis C Virus Infection at the Level of Cell Entry

Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin–moesin–radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and their functional mechanism we analyzed the anti-HCV properties of different human derived radixin peptides. Sixteen different radixin peptides were derived, synthesized and tested. Real-time quantitative PCR, cell toxicity assay, immuno-precipitation/western blot analysis and computational resource for drug discovery software were used for experimental analysis. We found that a human radixin hinge region peptide (Peptide1) can specifically block HCV J6/JFH-1 infection of Huh7.5 cells. Peptide 1 had no cell toxicity or intracellular uptake into Huh7.5 cells. Mechanistically, the anti-HCV activity of Peptide 1 extended to disruption of HCV engagement of CD81 thereby blocking downstream SYK activation, which we have recently demonstrated to be important for effective HCV infection of target hepatocytes. Our findings highlight a novel functional class of anti-HCV agents that can inhibit HCV infection, most likely by disrupting vital viral-host signaling interactions at the level of virus entry.