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Although antiviral drugs can effectively inhibit hepatitis B virus (HBV) replication, the maintenance of chronic inflammation in the liver is still considered to be an important cause for the progression of HBV-related liver disease to liver fibrosis and advanced liver disease. As an endogenous inhibitory receptor of IL-1R and TLR signaling pathways, single immunoglobulin interleukin-1-related receptor (SIGIRR) has been proven to reduce inflammation in tissues to maintain system homeostasis. However, the relationship between SIGIRR expression and HBV replication and inflammatory pathway activation in hepatocytes remains unclear. In this study, hepatitis B virus X protein (HBx) upregulated MyD88 in liver cells, promoting NF-κB signaling and inflammatory factor production with LPS treatment, and the cell supernatant accelerated the activation and collagen secretion of hepatic stellate cells. However, SIGIRR overexpression suppressed HBx-mediated MyD88/NF-κB inflammatory signaling activation and inflammatory cytokine production induced by LPS in hepatocytes and HBV replication hepatocytes. Although we did not find any effect of SIGIRR on HBV replication in vitro, this study investigated the role of SIGIRR in blocking the proinflammatory function of HBx, which may provide a new idea for the treatment of chronic hepatitis B., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background & Aims: Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-β1 signaling., Methods: The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice., Results: We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-β1 (TGF-β1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-β1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-β1-induced fibrogenesis., Conclusions: Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-β1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection., Impact and Implications: HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-β1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-β1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-β1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-β1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-β1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-β1-induced OCT4/Nanog-dependent pathway., (Copyright © 2022 by The American Association of Immunologists, Inc.)

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

Primary human hepatocytes (PHHs) are considered the gold standard for the in vitro study of HBV replication as they directly reflect the metabolism and functionality of the human liver. However, several limitations of this system include PHH donor-to-donor variability, limited life span and low permissiveness to HBV infection, which precludes long-term infection studies and viral passaging. Here, an easy-to-set-up co-culture platform that combines PHH with hepatic stellate cells (HSCs) was developed. This platform does not rely on chemical supplementation to sustain robust HBV replication and viral antigen secretion making it a more physiologically relevant system for in vitro HBV infection studies compared to the traditional short-lived PHH monocultures., (Copyright © 2021. Published by Elsevier Inc.)

While the interactions between HIV and various liver cell populations have been explored, the relevance of these interactions when patients are well-controlled on ART is less clear. Therefore, we focus this perspective on HIV-related alterations that may drive hepatic inflammation and fibrosis in aviremic patients, with a focus on Kupffer cells and Hepatic Stellate Cells. Persistent CD4+ T cell depletion in the gut resulting in increased gut permeability has been postulated to play a role in systemic immune activation in HIV patients. The liver, with its unique location, remains the gatekeeper between the gut and the systemic circulation. The resident liver macrophage, Kupffer cell, is responsible for clearing and responding to these products. We propose that changes in Kupffer cell biology, in the context of HIV infection, creates a mileu that drives hepatic inflammation and fibrosis in response to microbial translocation. Targeting these pathways may be helpful in improving liver-related outcomes in HIV patients., (Copyright © 2020 Zhang and Bansal.)

Liver fibrosis is a manifestation of chronic liver injury. It leads to hepatic dysfunction and is a critical element in the pathogenesis of cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSC) plays a central role in liver fibrogenesis of different etiologies. To elucidate the molecular mechanism of this phenomenon, it is important to analyze the changes in gene expression that accompany the HSC activation process. In this study, we isolated quiescent and activated HSCs from control mice and mice with CCl4-induced liver fibrosis, respectively, and performed RNA sequencing to compare the differences in gene expression patterns between the two types of HSCs. We also reanalyzed public gene expression data for fibrotic liver tissues isolated from patients with HBV infection, HCV infection, and nonalcoholic fatty liver disease to investigate the gene expression changes during liver fibrosis of these three etiologies. We detected 146 upregulated and 18 downregulated genes in activated HSCs, which were implicated in liver fibrosis as well. Among the overlapping genes, seven transcription factor-encoding genes, ARID5B, GATA6, MITF, PBX1, PLAGL1, SOX4, and SOX9, were upregulated, while one, RXRA, was downregulated. These genes were suggested to play a critical role in HSC activation, and subsequently, in the promotion of liver fibrosis. We undertook the RNA sequencing of quiescent and activated HSCs and analyzed the expression profiles of genes associated with HSC activation in liver fibrotic tissues from different liver diseases, and also aimed to elucidate the changes in gene expression patterns associated with HSC activation and liver fibrosis., Competing Interests: The authors have declared that no competing interests exist.

In an era of improved survival due to modern antiretroviral therapy, liver disease has become a major cause of morbidity and mortality, resulting in death in 15-17% of human immunodeficiency virus (HIV)-infected patients. Alcohol enhances HIV-mediated liver damage and promotes the progression to advanced fibrosis and cirrhosis. However, the mechanisms behind these events are uncertain. Here, we hypothesize that ethanol metabolism potentiates accumulation of HIV in hepatocytes, causing oxidative stress and intensive apoptotic cell death. Engulfment of HIV-containing apoptotic hepatocytes by non-parenchymal cells (NPCs) triggers their activation and liver injury progression. This study was performed on primary human hepatocytes and Huh7.5-CYP cells infected with HIV-1ADA, and major findings were confirmed by pilot data obtained on ethanol-fed HIV-injected chimeric mice with humanized livers. We demonstrated that ethanol exposure potentiates HIV accumulation in hepatocytes by suppressing HIV degradation by lysosomes and proteasomes. This leads to increased oxidative stress and hepatocyte apoptosis. Exposure of HIV-infected apoptotic hepatocytes to NPCs activates the inflammasome in macrophages and pro-fibrotic genes in hepatic stellate cells. We conclude that while HIV and ethanol metabolism-triggered apoptosis clears up HIV-infected hepatocytes, continued generation of HIV-expressing apoptotic bodies may be detrimental for progression of liver inflammation and fibrosis due to constant activation of NPCs.

Retrospective studies indicate that co-infection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) accelerates hepatic fibrosis progression. We have developed a co-culture system (MLH) comprising primary macrophages, hepatic stellate cells (HSC, LX-2), and hepatocytes (Huh-7), permissive for active replication of HCV and HIV, and assessed the effect of these viral infections on the phenotypic changes and fibrogenic gene expression in LX-2 cells. We detected distinct morphological changes in LX-2 cells within 24 hr post-infection with HCV, HIV or HCV/HIV in MLH co-cultures, with migration enhancement phenotypes. Human fibrosis microarrays conducted using LX-2 cell RNA derived from MLH co-culture conditions, with or without HCV and HIV infection, revealed novel insights regarding the roles of these viral infections on fibrogenic gene expression in LX-2 cells. We found that HIV mono-infection in MLH co-culture had no impact on fibrogenic gene expression in LX-2 cells. HCV infection of MLH co-culture resulted in upregulation (>1.9x) of five fibrogenic genes including CCL2, IL1A, IL1B, IL13RA2 and MMP1. These genes were upregulated by HCV/HIV co-infection but in a greater magnitude. Conclusion: Our results indicate that HIV-infected macrophages accelerate hepatic fibrosis during HCV/HIV co-infection by amplifying the expression of HCV-dependent fibrogenic genes in HSC.

Hepatitis C virus (HCV) treatment in HIV/HCV co-infected individuals has renewed relevance given the ongoing opioid crisis and rise of new HIV and HCV infections associated with injection drug use. Patients co-infected with HIV and HCV demonstrate increased rates of hepatic fibrosis, progression to liver failure, and liver-related mortality. HIV co-infection does not impact outcomes of current HCV treatments, and patients should be treated the same as HCV mono-infected persons, though attention to drug:drug interactions is required. In this review, we discuss the mechanisms mediating injury to the liver in HIV mono-infection and HIV/HCV co-infection, and present the landmark trials of HCV treatment in HIV-infected individuals.

Hepatocyte apoptosis is a crucially important mechanism for liver disease pathogenesis, and the engulfment of apoptotic bodies (AB) by non-parenchymal cells serves as a leading mechanism of inflammation and fibrosis progression. Previously, we have shown that hepatitis C virus (HCV) and alcohol metabolites induce massive apoptosis in hepatocytes and the spread of HCV-infection to the neighboring uninfected cells. Here, we hypothesize that the capturing of AB by non-parenchymal cells, macrophages and hepatic stellate cells (HSC) changes their phenotype to promote inflammation and fibrosis. In this regard, we generated AB from Huh7.5 CYP2E1 (RLW) cells also treated with an acetaldehyde-generating system (AGS) and incubated them with human monocyte-derived macrophages (MDMs) and HSC (LX2 cells). Activation of inflammasomes and pro-fibrotic markers has been tested by RT-PCR and linked to HCV expression and AGS-induced lipid peroxidation in RLW cells. After exposure to AB we observed activation of inflammasomes in MDMs, with a higher effect of AB HCV+, further enhanced by incubation of MDMs with ethanol. In HSC, activation of inflammasomes was modest; however, HCV and AGS exposure induced pro-fibrotic changes. We conclude that HCV as well as lipid peroxidation-adducted proteins packaged in AB may serve as a vehicle for delivery of parenchymal cell cargo to non-parenchymal cells to activate inflammasomes and pro-fibrotic genes and promote liver inflammation and fibrosis.

Hepatitis B virus (HBV) infection is a liver disorder that can result in cirrhosis, liver failure and hepatocellular carcinoma. HBV infection remains a major global health problem, as it affects more 350 million people chronically and kills roughly 600,000 people annually. Drugs currently used against HBV include IFN-α that decreases viremia, inflammation and the growth of liver fibrosis, and adefovir that decreases the viral load. Each of these drugs can have severe side-effects. In the present paper, we consider the treatment of chronic HBV by a combination of IFN-α and adefovir, and raise the following question: What should be the optimal ratio between IFN-α and adefovir in order to achieve the best 'efficacy' under constraints on the total amount of the drugs; here the efficacy is measured by the reduction of the levels of inflammation and of fibrosis? We develop a mathematical model of HBV pathogenesis by a system of partial differential equations (PDEs) and use the model to simulate a 'synergy map' which addresses the above question.

Cirrhosis develops from liver fibrosis and is the severe pathological stage of all chronic liver injury. Cirrhosis caused by hepatitis B virus and hepatitis C virus infection is especially common. Liver fibrosis and cirrhosis involve excess production of extracellular matrix, which is closely related to liver sinusoidal endothelial cells (LSECs). Damaged LSECs can synthesize transforming growth factor-beta and platelet-derived growth factor, which activate hepatic stellate cells and facilitate the synthesis of extracellular matrix. Herein, we highlight the angiogenic cytokines of LSECs related to liver fibrosis and cirrhosis at different stages and focus on the formation and development of liver fibrosis and cirrhosis. Inhibition of LSEC angiogenesis and antiangiogenic therapy are described in detail. Targeting LSECs has high therapeutic potential for liver diseases. Further understanding of the mechanism of action will provide stronger evidence for the development of anti-LSEC drugs and new directions for diagnosis and treatment of liver diseases., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interests related to this article.

Background: Liver fibrosis which mainly occurs upon chronic hepatitis virus infection potentially leads to portal hypertension, hepatic failure and hepatocellular carcinoma. However, the immune status of Th17 and Treg cells in liver fibrosis is controversial and the exact mechanisms remain largely elusive., Methods: Liver tissues and peripheral blood were obtained simultaneously from 32 hepatitis B virus infected patients undergoing surgery for hepatocellular carcinoma at the medical center of Sun Yat-sen University. Liver tissues at least 3 cm away from the tumor site were used for the analyses. Levels of Th17 cells and regulatory T cells were detected by flow cytometry analysis and immunohistochemistry. In vitro experiment, we adopted magnetic cell sorting to investigate how hepatic stellate cells regulate the levels of Th17 cells and regulatory T cells., Results: We found that hepatic Th17 cells and regulatory T cells were increased in patients with advanced stage HBV-related liver fibrosis. Hepatic stellate cells upregulated the levels of Th17 cells and regulatory T cells via PGE2/EP2 and EP4 pathway., Conclusions: We found that the increased levels of Th17 cells and regulatory T cells were upregulated by hepatic stellate cells. These results may provide insight into the role of hepatic stellate cells and Th17 cells and regulatory T cells in the persistence of fibrosis and into the occurrence of hepatocellular carcinoma following cirrhosis.

Reelin is a secreted extracellular glycoprotein that plays a critical role during brain development. Several studies have described Reelin expression in hepatic stellate cells of the human liver. In order to investigate the possible role of Reelin in the process of hepatic fibrogenesis, in this study we investigated Reelin expression in the liver tissue of patients infected with the Hepatitis C Virus (HCV). On this basis, Reelin expression was analysed by immunohistochemistry during liver biopsies of 81 patients with HCV-related chronic hepatitis. A Knodell score was used to stage liver fibrosis. Hepatic stellate cells/myofibroblast immunohistochemical markers (CRBP-1, alpha-SMA) were also evaluated. As further confirmed by co-localization experiments (Reelin +CRBP-1), Reelin protein was expressed by hepatic stellate cells/myofibroblasts, and a significant positive correlation was found between Reelin expression and the stage of liver fibrosis (P=0.002). Moreover, Reelin correlated with CRBP-1 positive cells (P=0.002), but not with alpha-SMA, suggesting that Reelin should not be regarded as a marker of hepatic stellate cells/myofibroblasts differentiation but rather as a functional protein expressed during some phases of liver fibrosis. Furthermore, Disabled-1 (Dab1), a Reelin adaptor protein, was expressed in cells of ductular reaction suggesting a paracrine role for Reelin with regards these elements. In conclusion, Reelin was expressed by human hepatic stellate cells/myofibroblasts and the number of these cells increased significantly in the lobule as the liver fibrosis progressed, suggesting a role for Reelin in the activation of hepatic stellate cells/myofibroblasts during liver injury. Reelin may potentially be incorporated into liver injury evaluations in combination with other histological data.

Most hepatitis C virus (HCV) infection results in persistent infection. Significant portion of chronic HCV-infected patients develop hepatocellular carcinoma (HCC). Chronic hepatitis C is also associated with extrahepatic manifestations, including cryoglobulinemia, lymphoma, insulin resistance, type 2 diabetes, and neurological disorders. The molecular mechanisms of how HCV infection causes liver cancer are largely unknown. HCV replication or viral proteins may perturb cellular hemostasis and induce the generation of reactive oxygen species (ROS); viral components or viral replication products act as agonist to trigger innate immune response and cause chronic inflammation. Within the liver, non-hepatocytes such as hepatic stellate cell (HSC) are activated upon HCV infection to provide the major source of extracellular proteins and play important roles in fibrogenesis. With the great achievements of HCV treatment, especially the direct-acting antivirals (DAAs) against HCV, HCV eradication is possible. However, until now there are only very limited data on the effect of DAA-based anti-HCV treatment on HCC patients.

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta-1 (TGFβ1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFβ1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells., Conclusion: HIV accentuates an HCV-driven profibrogenic program in hepatocyte and hepatic stellate cell lines through ROS, NFκB, and TGFβ1 up-regulation; coculture of hepatocyte and hepatic stellate cell lines significantly increased expression of CoL1A1 and TIMP1; and our novel coculture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses and may provide important insights into hepatic fibrosis. (Hepatology 2016;64:1951-1968)., (© 2016 by the American Association for the Study of Liver Diseases.)

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver diseases, but its involvement in hepatic fibrogenesis remains unclear. Special AT-rich binding protein 1 (SATB1) has been implicated in reprogramming chromatin organization and transcription profiles in many cancers and non-cancer-related conditions. We found that hepatic SATB1 expression was significantly up-regulated in fibrotic tissues from chronic hepatitis B virus (HBV)-infected patients and HBV transgenic (HBV-Tg) mouse model. Knockdown of SATB1 in the liver significantly alleviated CCl4-induced fibrosis in HBV-Tg mouse model. Moreover, we suggested HBV encoded x protein (HBx) induced SATB1 expression through activation of JNK and ERK pathways. Enforced expression of SATB1 in hepatocytes promoted the activation and proliferation of hepatic stellate cells (HSCs) by secretion of connective tissue growth factor (CTGF), Interleukin-6 (IL-6) and platelet derived growth factor-A (PDGF-AA). Our findings demonstrated that HBx upregulated hepatic SATB1 which exerted pro-fibrotic effects by paracrine activation of stellate cells in HBV-related fibrosis.

Highly active antiretroviral therapy has significantly improved the prognosis of HIV-infected subjects. However, patients treated long term still manifest increased mortality and, even with undetectable plasma viremia, often experience persistent immune activation. Furthermore, liver-related mortality is now the most common cause of non-AIDS-related death in HIV-infected individuals on highly active antiretroviral therapy through accelerated fibrosis progression. TLRs are the first line of the host response to pathogens and play an important role in human host defense against viruses through sensing of viral structural proteins. Growing evidence points to TLR4 as a key player in chronic immune activation, HIV recognition/replication, and liver fibrosis progression, suggesting that HIV triggering of TLR4 may dictate some aspects of the multifaceted AIDS pathogenesis. In this study, we provide evidence for an interplay between host TLR4 and HIV-1 gp120 in human monocyte-derived macrophages and hepatic stellate cells, leading to intracellular pathways and biologic activities that mediate proinflammatory and profibrogenic signals. Finally, we hypothesize that CCR5 and TLR4 are likely part of a common receptor cluster, as the blocking of CCR5 by specific antagonists impairs the macrophage capacity to produce chemokines in response to LPS. Chronic immune activation and liver fibrosis remain important obstacles for highly active antiretroviral therapy success. Thus, the identification of gp120-TLR4 axis as a novel determinant of immune system and hepatic stellate cell biology opens new perspectives to the management of HIV infection and disease., (© Society for Leukocyte Biology.)

Accelerated fibrosis in patients co-infected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has been a major cause of mortality in the highly active anti-retroviral therapy (HAART) era. However, the role of co-infection in accelerating the progression of liver fibrosis, particularly with regard to the effects of co-infection on hepatic stellate cells (HSCs), remains unclear. We hypothesized that HIV and HCV induce liver fibrosis synergistically by altering the regulation of epimorphin production, and thereby indirectly alter HSC function. Here, we examined the effects of epimorphin on HSC proliferation and invasion, and the changes in fibrogenesis-related gene activity in HSCs (LX2) in the presence of inactivated CXCR4-tropic HIV and HCV (JFH1). The combination of HIV and HCV significantly increased epimorphin expression, which increased the proliferation and invasion capabilities of HSCs. Epimorphin also induced the expression of profibrogenic tissue inhibitor of metalloproteinase 1 (TIMP1) in an extracellular signal-regulated kinase (ERK)-dependent manner. These data indicated that the effects of HIV/HCV co-infection on hepatic fibrosis might be mediated in part by EPM. Strategies to limit the expression of EPM might represent a novel therapeutic approach to prevent the progression of hepatic fibrosis during HIV/HCV co-infection.

Hepatitis C virus (HCV) infection is associated with increased thrombotic risk. Several mechanisms are involved including direct endothelial damage by the HCV virus, with activation of tissue factor, altered fibrinolysis and increased platelet aggregation and activation. In advanced stages, chronic HCV infection may evolve to liver cirrhosis, a condition in which alterations in the portal microcirculation may also ultimately lead to thrombin activation, platelet aggregation, and clot formation. Therefore in advanced HCV liver disease there is an increased prevalence of thrombotic phenomena in portal vein radicles. Increased thrombin formation may activate hepatic stellate cells and promote liver fibrosis. In addition, ischemic changes derived from vascular occlusion by microthrombi favor the so called parenchymal extinction, a process that promotes collapse of hepatocytes and the formation of gross fibrous tracts. These reasons may explain why advanced HCV infection may evolve more rapidly to end-stage liver disease than other forms of cirrhosis.

Objective: The aim of this study was to longitudinally evaluate and analyze the role of interleukin-22-producing CD4 positive cells (IL-22) in the pathogenesis of Hepatitis C Virus recurrence after Orthotopic Liver Transplantation (HCV-OLT)., Methods: 15 HCV-OLT, 15 age- and gender- matched non-HCV post-OLT (OLT) and 15 hepatitis C virus infected (HCV) patients were enrolled into our study from the liver transplantation and research center at Beijing 302 Hospital. We determined the frequencies of IL-22 using flow cytometry and expression of IL-22 mRNA using PCR in peripheral blood and liver tissue. We also divided HCV-OLT patients into rapid fibrosis progression (RFP) and slow fibrosis progression (SFP), examined IL-22 cells and analyzed the correlations between IL-22 frequencies and liver injury, fibrosis and clinical parameters. Moreover, we investigated the role of IL-22 in Human Hepatic Stellate Cells (HSCs)., Results: The levels of serum IL-22, frequencies of IL-22 producing cells in peripheral blood mononuclear cells, and expression of IL-22 mRNA and protein in the liver in the HCV-OLT group were significantly higher than that in the HCV and OLT groups. Furthermore, eight (53.3%) patients developed RFP after two years; another three patients were diagnosed liver cirrhosis. The frequencies of IL-22 were much higher in RFP compared with SFP, while no significant difference existed between OLT and SFP. Intrahepatic IL-22 positive cells were located in fibrotic areas and significantly correlated with α-smooth muscle actin (α-SMA) and fibrosis staging scores, not with grading scores and HCRVNA. In vitro, IL-22 administration prevented HSCs apoptosis, promoted HSCs proliferation and activation, up-regulated the expression of HSC-sourced growth factors including α-SMA, TGF-β and TIMP-1, and increased the production of liver fibrosis markers including laminin, hyaluronic acid and collagen type IV., Conclusion: Peripheral and intrahepatic IL-22 is up-regulated and plays a pathological role in exacerbating liver fibrosis by activating HSCs in HCV-OLT patients, which may predict RFP and serve as an attractive target for anti-fibrotic therapy.

Background: The induction of apoptosis in hepatic stellate cells (HSCs) is a promising therapeutic strategy against hepatitis B virus (HBV)-related hepatic fibrosis. The underlying mechanisms of apoptosis in HSCs, however, are unknown under consideration of HBV infection. In this study, the effects of HBV on apoptosis and endoplasmic reticulum (ER) stress signaling in HSCs were examined., Methods: The effects of conditioned media (CM) from HepG2.2.15 on apoptosis induced by the proteasome inhibitor MG132 in LX-2 and HHSteC were studied in regard to c-Jun. In combination with c-Fos, c-Jun forms the AP-1 early response transcription factor, leading to AP-1 activation, signal transduction, endoplasmic reticulum (ER) stress and apoptosis., Results: In LX-2 cells, MG132 treatment was associated with the phosphorylation of c-Jun, activation of AP-1 and apoptosis. However, in the presence of CM from HepG2.2.15, these phenomena were attenuated. In HHSteC cells, similar results were observed. HBV genomic DNA is not involved in the process of HSC apoptosis. It is possible that HBeAg has an inhibitory effect on MG132-induced apoptosis in LX-2. We also observed the upregulation of several ER stress-associated genes, such as cAMP responsive element binding protein 3-like 3, inhibin-beta A and solute carrier family 17-member 2, in the presence of CM from HepG2.2.15, or CM from PXB cells infected with HBV., Conclusions: HBV inhibits the activation of c-Jun/AP-1 in HSCs, contributing to the attenuation of apoptosis and resulting in hepatic fibrosis. HBV also up-regulated several ER stress genes associated with cell growth and fibrosis. These mechanistic insights might shed new light on a treatment strategy for HBV-associated hepatic fibrosis.

The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-β) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-β cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

Background & Aims: Acute liver failure (ALF) is a condition with high mortality and morbidity. Fibrosis in chronic liver disease was extensively researched, whereas fibrosis and underlying mechanism in acute liver failure remains unclear., Methods: Hepatitis B virus related ALF patients were recruited to investigate if there was ongoing fibrosis by liver histology and liver stiffness measurement(LSM) analysis as well as fibrosis markers assay. Sera HMGB1 were kinetically detected in progression and remission stage of ALF. Hepatic stellate cell(HSC) activation by HMGB1 was explored by testing mRNA and protein level of α-SMA and collagen 1a1 by using qPCR and western blot. Autophagy induction by HMGB1 was explored by LC3-II conversion, autophagy flux and fluorescence., Results: Firstly, ongoing fibrosis in progression stage of ALF was confirmed by histological analysis, LS measurement as well as fibrosis markers detection. HSC activation and autophagy induction in explanted liver tissue also revealed. Next, kinetic monitoring sera HMGB1 revealed elevated HMGB1 in progression stage of ALF vs HBsAg carrier, and drop back to base level in remission stage. Thirdly, rHMGB1 dose dependently activated HSCs, as indicated by increased mRNA and proteins level in α-SMA and collagen 1a1. Moreover, autophagy was induced in HSC treated with rHMGB1, as illustrated by increased LC3 lipidation, elevated autophagy flux and GFP-LC3 puncta., Conclusions: Acute liver failure is accompanied by ongoing fibrosis, HSC activation and autophagy induction. Increased HMGB1 activates HSC via autophagy induction. Those findings integrate HMGB1, HSCs activation, autophagy into a common framework that underlies the fibrosis in ALF., (© 2014 The Authors. Liver International Published by John Wiley & Sons Ltd.)

Background: Chronic HCV infection is associated with the development of hepatic fibrosis. The direct role of HCV in the fibrogenic process is unknown. Specifically, whether HCV is able to infect hepatic stellate cells (HSCs) is debated., Objective: To assess whether human HSCs are susceptible to HCV infection., Design: We combined a set of original HCV models, including the infectious genotype 2a JFH1 model (HCVcc), retroviral pseudoparticles expressing the folded HCV genotype 1b envelope glycoproteins (HCVpp) and a subgenomic genotype 1b HCV replicon, and two relevant cellular models, primary human HSCs from different patients and the LX-2 cell line, to assess whether HCV can infect/replicate in HSCs., Results: In contrast with the hepatocyte cell line Huh-7, neither infectious HCVcc nor HCVpp infected primary human HSCs or LX-2 cells. The cellular expression of host cellular factors required for HCV entry was high in Huh-7 cells but low in HSCs and LX-2 cells, with the exception of CD81. Finally, replication of a genotype 2a full-length RNA genome and a genotype 1b subgenomic replicon was impaired in primary human HSCs and LX-2 cells, which expressed low levels of cellular factors known to play a key role in the HCV life-cycle, suggesting that human HSCs are not permissive for HCV replication., Conclusions: Human HSCs are refractory to HCV infection. Both HCV entry and replication are deficient in these cells, regardless of the HCV genotype and origin of the cells. Thus, HCV infection of HSCs does not play a role in liver fibrosis. These results do not rule out a direct role of HCV infection of hepatocytes in the fibrogenic process., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease (CLD) and is frequently linked to intrahepatic microvascular disorders. Activation of hepatic stellate cells (HSC) is a central event in liver damage, due to their contribution to hepatic renewal and to the development of fibrosis and hepatocarcinoma. During the progression of CLDs, HSC attempt to restore injured tissue by stimulating repair processes, such as fibrosis and angiogenesis. Because HSC express the key vascular receptor Tie2, among other angiogenic receptors and mediators, we analyzed its involvement in the development of CLD., Methods: Tie2 expression was monitored in HSC cultures that were exposed to media from HCV-expressing cells (replicons). The effects of Tie2 blockade on HSC activation by either neutralizing antibody or specific signaling inhibitors were also examined., Results: Media from HCV-replicons enhanced HSC activation and invasion and upregulated Tie2 expression. Notably, the blockade of Tie2 receptor (by a specific neutralizing antibody) or signaling (by selective AKT and MAPK inhibitors) significantly reduced alpha-smooth muscle actin (α-SMA) expression and the invasive potential of HCV-conditioned HSC., Conclusions: These findings ascribe a novel profibrogenic function to Tie2 receptor in the progression of chronic hepatitis C, highlighting the significance of its dysregulation in the evolution of CLDs and its potential as a novel therapeutic target.

Background: Insulin resistance is highly prevalent in patients with chronic hepatitis C (CHC) and to some extent accounts for fibrosis and reducing viral eradication. Activated cannabinoid 1 receptor (CB1R) signaling has been implicated in the development of phenotypes associated with insulin resistance and steatosis. We investigated the role of the endocannabinoid system in glucose metabolism disorders induced by hepatitis C virus (HCV) replication., Methods: Human hepatic stellate cells (HSC; LX-2 cells) were co-cultured with Huh-7.5 cells or Huh-7.5 cells harboring HCV replicon (replicon cells). Endocannabinoid levels were then measured by liquid chromatography/mass spectrometry. The expression of CB1R and its downstream glucose metabolism genes in hepatocytes were determined by real-time PCR and Western blot. Glucose uptake by hepatocytes and glucose production were measured. Glucose metabolism tests and measurements of HCV RNA levels and nonstructural protein 5A (NS5A) levels were taken after treatment with CB1R agonist arachidonyl-2-chloroethanolamide (ACEA) or antagonist AM251., Results: Compared to the co-culture with Huh-7.5 cells, the level of 2-arachidonoylglycerol (2-AG) and the CB1R mRNA and protein levels increased in the co-culture of LX-2 cells with replicon cells. The activation of CB1R decreased AMP-activated protein kinase (AMPK) phosphorylation, inhibited cell surface expression of glucose transporter 2 (GLUT2), and suppressed cellular glucose uptake; furthermore, it increased cyclic AMP response element-binding protein H (CREBH), then up-regulated phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes and down-regulated the glucokinase (GK) gene, thus promoting glucose production. Interferon treatment restored the aforementioned changes. CB1R antagonist improved glucose metabolism disorders by an increase in glucose uptake and a decrease in glucose production, and inhibited HCV replication., Conclusions: HCV replication may not only increase the 2-AG content, but may also up-regulate the expression of CB1R of hepatocytes, then change the expression profile of glucose metabolism-related genes, thereby causing glucose metabolism disorders of hepatocytes and promoting HCV replication. Treatment with CB1R antagonist improved glucose metabolism disorders and inhibited viral genome replication., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hepatic fibrosis is caused by an imbalance between production and dissolution of extracellular matrix after chronic and inflmmatory injury, when hepatic stellate cells are stimulated to proliferate and secret extracellular matrix. The most common causes of liver fibrosis are chronic viral hepatitis B and C. Cirrhosis is the most advanced stage of fibrosis, which usually develop into hepatocellular carcinoma (HCC). microRNAs participate the pathogenesis of hepatic fibrosis and cirrhosis or even the onset of HCC. In this review, we will summarize the role of miRNA in the pathogenesis of viral hepatitis fibrosis, non-alcoholic steatohepatitis fibrosis, primary biliary cirrhosis and HCC onset, especially in the regulation of stellate cells.

Background: Although accumulating data has implicated a role for the Apelinergic system in cirrhosis, the role of Apelin during different stages of fibrogenesis has not been clarified, whereas no studies have been conducted on its expression in human hepatocellular carcinoma (HCC). This study aimed to elucidate its role in hepatic remodelling and carcinogenesis., Methods: Immunolocalization of Apelin was compared during different stages of HCV-induced liver disease (n=123)., Results: Apelin level in hepatic stellate cells (HSC), portal fibroblast and endothelial cells was significantly elevated in F3 stage .In cirrhosis, the expression was markedly striking and extended as linear staining in the cirrhotic septa and proliferated capillaries. In liver cirrhosis with high grade dysplastic nodule (HGDN) group and liver cirrhosis with HCC group, Apelin was constantly expressed in the hepatocytes with the exemption of non-parenchymatous cells. Apelin gradually increased in HGDN, HCC grade-I and HCC grade II (P<0.0001). In contrast, Apelin gradually decreased in the cirrhosis adjacent to HGDN, HCC grade-I and HCC grade II (P<0.0001). The gradual incline in Apelin expression in dysplastic and malignant cells was paralleled by a decline in their adjacent cirrhotic liver (P=0.013)., Conclusion: In HCV chronic hepatitis, Apelin seems to manipulate the differentiation of HSC ending in hepatic remodelling. The uptake of Apelin from the stromal components by the epithelial cells may initiate the transformation of adjacent epithelial cells and supports the evolution and progression of dysplastic nodules and hepatocellular carcinoma. These findings could have both prognostic and therapeutic applications.

Objectives: The risk of porcine endogenous retrovirus infection is a major barrier for pig-to-human xenotransplant. Porcine endogenous retrovirus, present in porcine cells, can infect many human and nonhuman primate cells in vitro, but there is no evidence available about in vitro infection of human liver cells. We investigated the susceptibility of different human liver cells to porcine endogenous retrovirus., Materials and Methods: The supernatant from a porcine kidney cell line was added to human liver cells, including a normal hepatocyte cell line (HL-7702 cells), primary hepatocytes (Phh cells), and a liver stellate cell line (Lx-2 cells), and to human embryonic kidney cells as a reference control. Expression of the porcine endogenous retrovirus antigen p15E in the human cells was evaluated with polymerase chain reaction, reverse transcription-polymerase chain reaction, and Western blot., Results: The porcine endogenous retrovirus antigen p15E was not expressed in any human liver cells (HL-7702, Phh, or Lx-2 cells) that had been exposed to supernatants from porcine kidney cell lines. Porcine endogenous retrovirus-specific fragments were amplified in human kidney cells., Conclusions: Human liver cells tested were not susceptible to infection by porcine endogenous retrovirus. Therefore, not all human cells are susceptible to porcine endogenous retrovirus.

Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

There is limited information about the role of hepatic stellate cells (HSC) in liver innate immunity against hepatitis C virus (HCV). We thus examined whether HSC can produce antiviral factors that inhibit HCV replication in human hepatocytes. HSC expressed functional Toll-like receptor 3 (TLR-3), which could be activated by its ligand, polyinosine-polycytidylic acid (poly I:C), leading to the induction of interferon-λ (IFN-λ) at both mRNA and protein levels. TLR-3 signalling of HSC also induced the expression of IFN regulatory factor 7 (IRF-7), a key regulator of IFN signalling pathway. When HCV JFH-1-infected Huh7 cells were co-cultured with HSC activated with poly I:C or incubated in media conditioned with supernatant (SN) from poly I:C-activated HSC, HCV replication was significantly suppressed. This HSC SN action on HCV inhibition was mediated through IFN-λ, which was evidenced by the observation that antibody to IFN-λ receptors could neutralize the HSC-mediated anti-HCV effect. The role of IFN-λ in HSC-mediated anti-HCV activity is further supported by the observation that HSC SN treatment induced the expression of IRF-7 and IFN-stimulated genes (ISGs), OAS-1 and MxA in HCV-infected Huh7 cells. These observations indicate that HSC may be a key regulatory bystander, participating in liver innate immunity against HCV infection using an IFN-λ-dependent mechanism., (© 2013 John Wiley & Sons Ltd.)

Hepatitis C virus core protein (HCVcp), which is secreted by infected cells, is reported as an immunomodulator in immune cells. However, the effects of HCVcp on hepatic stellate cells (HSCs), the key cells in liver fibrosis, still remain unclear. In this study, we investigated the effects of HCVcp on obese receptor (ObR) related downstream signaling pathways and fibrogenic gene expression in HSCs. LX-2, a human HSC line, was incubated with HCVcp. Inhibitors and short interfering RNAs were used to interrogate the mechanisms of HCVcp action on HSCs. HCVcp (20-100 ng/ml) concentration-dependently stimulated α-smooth muscle actin (α-SMA) protein expression and mRNA expression of α-SMA, procollagen α2(I) and TGF-β1 genes, with a plateau of 220% of controls at 100 ng/ml. HCVcp induced mRNA and protein expression of ObR. Blocking of Ob-Rb with a neutralizing antibody inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and AMPKα stimulated by HCVcp. Furthermore, knockdown of Ob-Rb down-regulated HCVcp-induced STAT3, AKT, and AMPKα phosphorylation, and reversed HCVcp-suppressed mRNA expression of matrix metalloproteinase (MMP)-1, peroxisome proliferator-activated receptor (PPAR)γ and sterol regulatory element binding protein-1c (SREBP-1c) genes. AMPKα signaling blockade reversed HCVcp-suppressed SREBP-1c mRNA expression. HCVcp stimulated reactive oxygen species formation and gp91(phox) (a component of NADPH oxidase) protein expression, together with AKT phosphorylation, leading to suppression of PPARγ and SREBP-1c genes. Our results provide a new finding that HCVcp induced ObR-dependent Janus Kinase (JAK) 2-STAT3, AMPKα, and AKT signaling pathways and modulated downstream fibrogenetic gene expression in HSCs., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: Our aim was to establish a correlation between hepatic stellate cells (HSCs) activity within different areas of hepatic tissue and the degree of liver fibrosis, necroinflammation, and steatosis in patients with viral C chronic liver disease., Patients and Methods: We prospectively included 41 liver biopsies from patients with chronic hepatitis C or liver cirrhosis, prior to antiviral treatment. Our control group consisted of seven tissue samples, obtained from 10 to 38 weeks old embryos. We assessed the alpha-smooth muscle actin (α-SMA) expression using immunohistochemistry and a semi-quantitative scoring method., Results: We found a strong correlation (p<0.0001) between the number of activated HSCs and the stage of fibrosis in all examined areas. Necroinflammatory activity was significantly correlated (p<0.05) with the number of activated HSCs in perivenular area (p=0.014) and intermediate area (p=0.018), and strongly correlated (p<0.0001) in periportal and portal tracts and fibrous septa areas. We found no correlation between the degree of steatosis and the number of activated HSCs in the perivenular area (p=0.25), intermediate area (p=0.166) and in the periportal area (p=0.154); however, in the portal tracts and fibrous septa area we observed a significant correlation (p=0.022)., Conclusions: The analysis of HSCs activity within specified areas of liver tissue may lead to new perspectives in early diagnosis of liver fibrosis and in the development of future antifibrinogenic therapies.

Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-β1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-β1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-β1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-β1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-β1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-β1 is critical for the induction of alpha smooth muscle actin (α-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-β1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-β1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-β1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection.

The activation of hepatic stellate cells (HSCs) is closely associated with liver fibrosis in chronic hepatitis B virus (HBV) infection. However, the molecular mechanisms leading to HSC activation remain unclear. It has been reported that the platelet-derived growth factor-B (PDGF-B)/PDGF receptor-β (PDGFR-β) signaling pathway is involved in this process. Thus, we investigated whether HBV and its protein contribute to HSC proliferation by the PDGF-B/PDGFR-β signaling pathway. HBV particles were purified from the supernatant of HepG2.2.15 cells by ultracentrifugation and the cell lines carrying HBV preS, e, c or x genes were obtained. After incubation with HBV particles or co-cultured with the cell lines expressed in the viral protein, the proliferation of LX-2 cells, an HSC cell line, were detected by flow cyto-metry and real-time PCR and the expression of molecules related to the PDGF-B/PDGFR-β signaling pathway were further measured. Our results indicated that HBV particles, c and x proteins promoted LX-2 proliferation and increased the mRNA levels of PDGF-B, PDGFR-β, collagen-I and α-smooth muscle actin (α-SMA), as well as the phosphorylation of PDGFR-β; however, the expression protein levels of PDGF-B and PDGFR-β remained unchanged. In conclusion, HBV particles and HBV c and x proteins promote HSC proliferation and fibrogenesis in vitro and the PDGF-B/PDGFR-β signaling pathway is important in this process.

Unlabelled: Although annular fibrosis is the hallmark of cirrhosis, other microscopic changes that affect liver function such as sinusoid capillarization or loss of metabolic zonation are common. A sustained virological response (SVR) may halt fibrosis deposition in hepatitis C virus (HCV)-infected patients, but its impact on the other cirrhosis-associated lesions is unknown. The aim of this study was to assess the impact of an SVR on cirrhosis-related histopathological features. Paired pre- and posttreatment liver biopsies from 38 HCV patients with cirrhosis with an SVR were analyzed. Fibrosis was staged using the METAVIR scoring system, and the area of fibrosis was measured using morphometry. Ductular proliferation, metabolic zonation, sinusoid capillarization, and hepatic stellate cell activation were assessed by anti-cytokeratin-7, anti-glutamine synthetase (GS), anti-cytochrome P4502E1 (CYP2E1), anti-CD34, and anti α-smooth muscle actin (αSMA). After 61 months from an SVR, cirrhosis regression was observed in 61%, and the collagen content decreased in 89%. Although periportal and lobular necroinflammation vanished, portal inflammation persisted in 66%. Ductular proliferation decreased in 92%. Before treatment, metabolic zonation was lost, as shown by GS and CYP2E1, in 71% and 88%, respectively, with normalization in 79% and 73%, after an SVR. Conversely, no changes in sinusoidal capillarization were observed after treatment, as assessed by CD34 (P = 0.41) and αSMA (P = 0.95). Finally, no differences in all the immunohistochemical scores emerged whether or not cirrhosis persisted., Conclusion: Cirrhosis regression and decreased fibrosis are frequently observed among HCV patients with cirrhosis with an SVR. Despite ductular proliferation vanishing and lobular zonation restoration, portal inflammation and sinusoidal capillarization may not regress after viral eradication., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Notwithstanding numerous evidences implicating toll-like receptor-4 (TLR4) in the pathogenesis of chronic hepatitis C virus (HCV) infection, the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. We aimed to evaluate, by means of immunohistochemistry and real-time PCR (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Fifty patients who had undergone liver biopsy and 11 patients transplanted because of chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by immunohistochemistry for TLR4, α-smooth muscle actin and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. Immunohistochemistry for α-smooth muscle actin was used to derive a score of activation of hepatic stellate cells and portal/septal myofibroblasts, while immunohistochemistry for CK-7 was used to evaluate and count hepatic progenitor cells, interlobular bile ducts and intermediate hepatocytes. In patients, the parenchymal elements responsible for the highest TLR4 level of expression were hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts. Double-labeling experiments between anti-TLR4 and anti-CK7, anti-CD133, anti-CD44, anti-neural cell adhesion molecule, anti-epithelial cell adhesion molecule and anti-sex determining region Y-box 9, confirmed these findings. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of liver disease (P<0.001), the grade of inflammation (P<0.001), and the activity of portal/septal myofibroblasts (P<0.001). rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (P<0.001). TLR4 expression positively correlated with fibrosis (P<0.05) and inflammation (P<0.05). The present results suggest that TLR4 expression by hepatic progenitor cells and biliary epithelial cells contributes to the progression of liver damage in the course of chronic HCV-related infection.

Background & Aims: Collagen I deposition contributes to liver fibrosis, yet little is known about other factors that mediate this process. Fibromodulin is a liver proteoglycan that regulates extracellular matrix organization and is induced by fibrogenic stimuli. We propose that fibromodulin contributes to the pathogenesis of fibrosis by regulating the fibrogenic phenotype of hepatic stellate cells (HSCs)., Methods: We analyzed liver samples from patients with hepatitis C-associated cirrhosis and healthy individuals (controls). We used a coculture model to study interactions among rat HSCs, hepatocytes, and sinusoidal endothelial cells. We induced fibrosis in livers of wild-type and Fmod(-/-) mice by bile duct ligation, injection of CCl(4), or administration of thioacetamide., Results: Liver samples from patients with cirrhosis had higher levels of fibromodulin messenger RNA and protein than controls. Bile duct ligation, CCl(4), and thioacetamide each increased levels of fibromodulin protein in wild-type mice. HSCs, hepatocytes, and sinusoidal endothelial cells produced and secreted fibromodulin. Infection of HSCs with an adenovirus that expressed fibromodulin increased expression of collagen I and α-smooth muscle actin, indicating increased activation of HSCs and fibrogenic potential. Recombinant fibromodulin promoted proliferation, migration, and invasion of HSCs, contributing to their fibrogenic activity. Fibromodulin was sensitive to reactive oxygen species. HepG2 cells that express cytochrome P450 2E1 produced fibromodulin, and HSCs increased fibromodulin production in response to pro-oxidants. In mice, administration of an antioxidant prevented the increase in fibromodulin in response to CCl(4). Coculture of hepatocytes or sinusoidal endothelial cells with HSCs increased the levels of reactive oxygen species in the culture medium, along with collagen I and fibromodulin proteins; this increase was prevented by catalase. Fibromodulin bound to collagen I, but the binding did not prevent collagen I degradation by matrix metalloproteinase 13. Bile duct ligation caused liver fibrosis in wild-type but not Fmod(-/-) mice., Conclusions: Fibromodulin levels are increased in livers of patients with cirrhosis. Hepatic fibromodulin activates HSCs and promotes collagen I deposition, which leads to liver fibrosis in mice., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2(-/-)/p19(ARF-/-), Stat3(Δhc)/Mdr2(-/-)) and a model of experimental induced fibrosis (CCl(4)) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.

Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.

Background and Aims: Hepatic stellate cells, the major producers of extracellular matrix in the liver, and hepatocytes bear CXCR4 and CCR5, the two main co-receptors for entry of the human immunodeficiency virus (HIV). In vitro studies suggest that HIV-envelope proteins can modulate the replication of hepatitis C virus (HCV) and fibrogenesis. We investigated the influence of HIV tropism on liver fibrosis and the concentration of HCV RNA in HIV-HCV co-infected patients., Methods: We used a phenotypic assay to assess HIV tropism in 172 HCV-HIV co-infected patients: one group (75 patients) had mild fibrosis (score ≤F2) and the other (97 patients) had severe fibrosis (score >F2). We also assessed the relationship between HIV tropism and HCV RNA concentration in all these patients. We also followed 34 of these patients for 3 years to determine the evolution of HIV tropism and liver fibrosis, estimated by liver stiffness., Results: Initially, most patients (91.8%) received a potent antiretroviral therapy. CXCR4-using viruses were found in 29% of patients. The only factor associated with a CXCR4-using virus infection in multivariate analysis was the nadir of CD4 cells: <200/mm(3) (OR: 3.94, 95%CI: 1.39-11.14). The median HCV RNA concentrations in patients infected with R5 viruses, those with dual-mixed viruses and those with X4 viruses, were all similar. The prevalence of CXCR4-using viruses in patients with mild fibrosis (≤F2) (31%) and those with severe fibrosis (F3-F4) (28%, p = 0.6) was similar. Longitudinal analyses showed that the presence of CXCR4-using viruses did not increase the likelihood of fibrosis progression, evaluated by measuring liver stiffness., Conclusions: The presence of CXCR4-using viruses in patients receiving a potent antiretroviral therapy does not influence HCV RNA concentration or liver fibrosis.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, including chronic hepatitis, fibrosis, and cirrhosis. Fibrosis often develops in HCV-infected livers and ultimately leads to cirrhosis and carcinoma. During fibrosis, hepatic stellate cells (HSC) play important roles in the control of extracellular matrix synthesis and degradation in fibrotic livers. In this study, we established a subgenomic replicon (SGR) cell line with human hepatic stellate cells to investigate the effect of HCV RNA replication on HSC. Isolated SGR clones contained HCV RNA copy numbers ranging from 10(4) to 10(7) per μg total RNA, and long-term culture of low-copy number SGR clones resulted in markedly increased HCV RNA copy numbers. Furthermore, HCV RNA replication affected gene expression of extracellular matrix-related molecules in both hepatic stellate cells and hepatic cells, suggesting that HCV RNA replication and/or HCV proteins directly contribute to liver fibrosis. The HCV RNA-replicating hepatic stellate cell line isolated in this study will be useful for investigating hepatic stellate cell functions and HCV replication machinery., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Introduction: Hepatic stellate cells (HSC) are key-players in the pathogenesis of liver fibrosis, inducing collagen deposition and abnormal extracellular matrix remodeling., Aim: The purpose of this study was to identify the stellate cells using immunohistochemical techniques and to establish if there is a correlation between the expression of stellate cells and the clinical and histological parameters in patients with chronic viral hepatitis C., Materials and Methods: The studied group included 30 patients with chronic viral hepatitis C genotype 1, in whom a liver biopsy was performed previous to the antiviral treatment. After the histological analyze, the biopsy was stained with an anti-SMA antibody (Dako, Carpinteria, CA). The amount of positive stained area was determined using an arbitrary semiquantitative score from 1 to 4., Results: Our observations suggest that there is a strong correlation between the stellate cells activity, evaluated using a semiquantitative score, and the stage of liver fibrosis (rs=0.76, p<0.001). Also, our study revealed a direct correlation, but less intense, with the necro-inflammatory activity (rs=0.39, p=0.03), the steatosis degree (rs=0.428, p=0.01) and the value of alanine aminotransferase (rs=0.4, p=0.03). The age and the viremia level were not correlated with the activity of the stellate cells., Conclusions: This study suggests that the transition of stellate cells from inactivated state to the state of highly fibrogenic cell is influenced mainly by the histological liver modifications (necroinflammatory activity and steatosis) and less by clinical parameters (age, sex) or the viremia level.

Chronic infection of hepatitis C virus (HCV) leads to hepatic fibrosis and subsequently cirrhosis, although the underlying mechanisms have not been established. Previous studies have indicated that the binding of HCV E2 protein and CD81 on the surface of hepatic stellate cells (HSCs) lead to the increased protein level and activity of matrix metallopeptidase (MMP) 2, indicating that E2 may involve in the HCV-induced fibrosis. This study was designed to investigate the involvement of HCV E2 protein in the hepatic fibrogenesis. Results showed that E2 protein may promote the expression levels of α-smooth muscle actin (α-SMA) and collagen α(I). Furthermore, several pro-fibrosis or pro-inflammatory cytokines, including transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF), interleukin (IL)-6 and IL-1β, were significantly increased in E2 transfected-HSC cell lines, while the expression of MMP-2 are also considerably increased. Moreover, the significant increases of CTGF and TGF-β1 in a stable E2-expressing Huh7 cell line were also observed the same results. Further molecular studies indicated that the impact of E2 protein on collagen production related to higher production of ROS and activated Janus kinase (JAK)1, JAK2 and also enhance the activation of ERK1/2 and p38, while catalase and inhibitors specific for JAK, ERK1/2, and p38 abolish E2-enhanced expression of collagen α(I). Taken together, this study indicated that E2 protein involve in the pathogenesis of HCV-mediated fibrosis via an up-regulation of collagen α(I) and oxidative stress, which is JAK pathway related.

Unlabelled: Patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) develop more rapid fibrosis than those infected with HCV only. In HIV/HCV-coinfected patients, fibrosis progression correlates with HIV RNA levels, suggesting a direct role of HIV in liver fibrogenesis. Chemokine (C-C motif) receptor 5 (CCR5) and cysteine-X-cysteine receptor 4 (CXCR4), the two major coreceptors required for HIV entry into cells, are expressed on activated hepatic stellate cells (HSCs), the principle fibrogenic cell type in the liver. We therefore examined whether HIV can infect HSCs, explored the potential mechanisms of viral entry, and assessed the impact of infection as reflected by the ability of HSCs to transfer virus to T lymphocytes and elicit a proinflammatory and profibrogenic response. We report that the laboratory-adapted viruses HIV-IIIB (CXCR4-tropic or X4) and HIV-BaL (CCR5-tropic or R5) and primary HIV isolates can infect both a human stellate cell line, LX-2, and primary human HSCs. HIV entry and gene expression in HSCs was confirmed using HIV-green fluorescent protein (GFP) expression viral constructs in the presence or absence of the reverse-transcriptase inhibitor azidothymidine. CD4 expression on a subset of primary HSCs was demonstrated using fluorescence-activated cell sorting and immunofluorescence staining. Blocking experiments in the presence of anti-CD4, anti-CXCR4, and anti-CCR5 revealed that HIV entry into HSCs is predominantly CD4/chemokine coreceptor-independent. HIV infection promoted HSC collagen I expression and secretion of the proinflammatory cytokine monocyte chemoattractant protein-1. Furthermore, infected LX-2 cells were capable of transferring GFP-expressing virus to T lymphocytes in a coculture system., Conclusion: Taken together, our results suggest a potential role of HIV in liver fibrosis/inflammation mediated through effects on HSCs. The role of early highly active antiretroviral therapy initiation in patients with HIV/HCV coinfection warrants further investigation.

Objective: The study objective was an in-depth ultrastructural analysis of intermediate hepatocyte-like cells (IHCs), constituting a subpopulation of liver progenitor/oval cells, in children with chronic hepatitis B viral (HBV) infection., Methods: Ultrastructural investigations were conducted on liver biopsy material, fixed in a solution of 2.5% glutaraldehyde, 2% paraformaldehyde, and 0.1 mol/l cacodylate buffer, obtained from 40 children, aged 3-16 years, with chronic hepatitis B., Results: Transmission-electron microscopic analysis of liver progenitor/oval cells showed, apart from a morphologically unchanged population of oval cells, the presence of IHCs displaying variously pronounced ultrastructural changes, including degeneration. Interesting was that damaged IHCs were mainly observed in patients with a coexisting advanced liver fibrosis, where they frequently adhered to bundles of collagen fibers. Submicroscopic abnormalities in these cells referred mainly to mitochondria and granular endoplasmic reticulum. The most pronounced mitochondrial alterations observed in degenerating IHCs in the course of chronic HBV infection were characterized by distinct swelling, loss of mitochondrial crests, and the presence of myelin structures within the matrix. In granular endoplasmic reticulum, shortening and segmental degranulation of the reticulum were observed. The above changes were accompanied by the appearance of primitive phagosome-like structures with absorbed biliary pigment. In the vicinity of altered IHCs, transitional hepatic stellate cells could be found., Conclusion: Our study seems to suggest that chronic HBV infection, lasting from childhood and coexisting with intensive fibrosis may, with the involvement of other carcinogenic factors, promote degenerating IHCs towards neoplastic transformation in adulthood.