The Epigenetic Controller Lysine-Specific Demethylase 1 (LSD1) Regulates the Outcome of Hepatitis C Viral Infection.

Hepatitis C virus (HCV) alters gene expression epigenetically to rearrange the cellular microenvironment in a beneficial way for its life cycle. The host epigenetic changes induced by HCV lead to metabolic dysfunction and malignant transformation. Lysine-specific demethylase 1 (LSD1) is an epigenetic controller of critical cellular functions that are essential for HCV propagation. We investigated the putative role of LSD1 in the establishment of HCV infection using genetic engineering and pharmacological inhibition to alter endogenous LSD1 levels. We demonstrated for the first time that HCV replication was inhibited in LSD1-overexpressing cells, while specific HCV proteins differentially fine-tuned endogenous LSD1 expression levels. Electroporation of the full-length HCV genome and subgenomic replicons in LSD1 overexpression enhanced translation and partially restored HCV replication, suggesting that HCV might be inhibited by LSD1 during the early steps of infection. Conversely, the inhibition of LSD1, followed by HCV infection in vitro, increased viral replication. LSD1 was shown to participate in an intriguing antiviral mechanism, where it activates endolysosomal interferon-induced transmembrane protein 3 (IFITM3) via demethylation, leading endocytosed HCV virions to degradation. Our study proposes that HCV-mediated LSD1 oscillations over countless viral life cycles throughout chronic HCV infection may promote epigenetic changes related to HCV-induced hepatocarcinogenesis.

HCV-Induced Immunometabolic Crosstalk in a Triple-Cell Co-Culture Model Capable of Simulating Systemic Iron Homeostasis.

Iron is crucial to the regulation of the host innate immune system and the outcome of many infections. Hepatitis C virus (HCV), one of the major viral human pathogens that depends on iron to complete its life cycle, is highly skilled in evading the immune system. This study presents the construction and validation of a physiologically relevant triple-cell co-culture model that was used to investigate the input of iron in HCV infection and the interplay between HCV, iron, and determinants of host innate immunity. We recorded the expression patterns of key proteins of iron homeostasis involved in iron import, export and storage and examined their relation to the iron regulatory hormone hepcidin in hepatocytes, enterocytes and macrophages in the presence and absence of HCV. We then assessed the transcriptional profiles of pro-inflammatory cytokines Interleukin-6 (IL-6) and interleukin-15 (IL-15) and anti-inflammatory interleukin-10 (IL-10) under normal or iron-depleted conditions and determined how these were affected by infection. Our data suggest the presence of a link between iron homeostasis and innate immunity unfolding among liver, intestine, and macrophages, which could participate in the deregulation of innate immune responses observed in early HCV infection. Coupled with iron-assisted enhanced viral propagation, such a mechanism may be important for the establishment of viral persistence and the ensuing chronic liver disease.

The Hepatitis C virus NS5A and core proteins exert antagonistic effects on HAMP gene expression: the hidden interplay with the MTF-1/MRE pathway.

Hepcidin, a 25-amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and macrophages, is a mediator of innate immunity and the central iron-regulatory hormone. Circulating hepcidin controls iron efflux by inducing degradation of the cellular iron exporter ferroportin. HCV infection is associated with hepatic iron overload and elevated serum iron, which correlate with poor antiviral responses. The HCV nonstructural NS5A protein is known to function in multiple aspects of the HCV life cycle, probably exerting its activity in concert with cellular factor(s). In this study, we attempted to delineate the effect of HCV NS5A on HAMP gene expression. We observed that transient transfection of hepatoma cell lines with HCV NS5A resulted in down-regulation of HAMP promoter activity. A similar effect was evident after transduction of Huh7 cells with a recombinant baculovirus vector expressing NS5A protein. We proceeded to construct an NS5A-expressing stable cell line, which also exhibited down-regulation of HAMP gene promoter activity and significant reduction of HAMP mRNA and hepcidin protein levels. Concurrent expression of HCV core protein, a well-characterized hepcidin inducer, revealed antagonism between those two proteins for hepcidin regulation. In attempting to identify the pathways involved in NS5A-driven reduction of hepcidin levels, we ruled out any NS5A-induced alterations in the expression of the well-known hepcidin inducers SMAD4 and STAT3. Further analysis linked the abundance of intracellular zinc ions and the deregulation of the MTF-1/MRE/hepcidin axis with the observed phenomenon. This effect could be associated with distinct phases in HCV life cycle.

Hepatocyte autotaxin expression promotes liver fibrosis and cancer.

Autotaxin (ATX) is a secreted lysophospholipase D that catalyzes the production of lysophosphatidic acid (LPA), a pleiotropic growth-factor-like lysophospholipid. Increased ATX expression has been detected in various chronic inflammatory disorders and different types of cancer; however, little is known about its role and mode of action in liver fibrosis and cancer. Here, increased ATX expression was detected in chronic liver disease (CLD) patients of different etiologies, associated with shorter overall survival. In mice, different hepatotoxic stimuli linked with the development of different forms of CLDs were shown to stimulate hepatocyte ATX expression, leading to increased LPA levels, activation of hepatic stellate cells (HSCs), and amplification of profibrotic signals. Hepatocyte-specific, conditional genetic deletion and/or transgenic overexpression of ATX established a liver profibrotic role for ATX/LPA, whereas pharmacological ATX inhibition studies suggested ATX as a possible therapeutic target in CLDs. In addition, hepatocyte ATX ablation and the consequent deregulation of lipid homeostasis was also shown to attenuate hepatocellular carcinoma (HCC) development, thus implicating ATX/LPA in the causative link of cirrhosis and HCC. CONCLUSION: ATX is a novel player in the pathogenesis of liver fibrosis and cancer and a promising therapeutic target. (Hepatology 2017;65:1369-1383).

Alterations in the iron homeostasis network: A driving force for macrophage-mediated hepatitis C virus persistency.

Mechanisms that favor Hepatitis C virus (HCV) persistence over clearance are unclear, but involve defective innate immunity. Chronic infection is characterized by hepatic iron overload, hyperferraemia and hyperferittinaemia. Hepcidin modulates iron egress via ferroportin and its storage in ferritin. Chronic HCV patients have decreased hepcidin, while HCV replication is modified by HAMP silencing. We aimed to investigate interactions between HCV and hepcidin, during acute and chronic disease, and putative alterations in cellular iron homeostasis that enhance HCV propagation and promote viral persistence. Thus, we used HCV JFH-1-infected co-cultures of Huh7.5 hepatoma and THP-1 macrophage cells, HCV patients' sera and Huh7 hepcidin-expressing cells transfected with HCV replicons. Hepcidin levels were elevated in acutely infected patients, but correlated with viral load in chronic patients. HAMP expression was up-regulated early in HCV infection in vitro, with corresponding changes in ferritin and FPN. Hepcidin overexpression enhanced both viral translation and replication. In HCV-infected co-cultures, we observed increased hepcidin, reduced hepatoma ferritin and a concurrent rise in macrophaghic ferritin over time. Altered iron levels complemented amplified replication in hepatoma cells and one replication round in macrophages. Iron-loading of macrophages led to enhancement of hepatic HCV replication through reversed ferritin "flow." Viral transmissibility from infected macrophages to naïve hepatoma cells was induced by iron. We propose that HCV control over iron occurs both by intracellular iron sequestration, through hepcidin, and intercellular iron mobilisation via ferritin, as means toward enhanced replication. Persistence could be achieved through HCV-induced changes in macrophagic iron that enhances viral replication in these cells.

A complex signaling network involving protein kinase CK2 is required for hepatitis C virus core protein-mediated modulation of the iron-regulatory hepcidin gene expression.

Hepatitis C virus (HCV) infection is associated with hepatic iron overload and elevated serum iron that correlate to poor antiviral responses. Hepcidin (HAMP), a 25-aa cysteine-rich liver-specific peptide, controls iron homeostasis. Its expression is up-regulated in inflammation and iron excess. HCV-mediated hepcidin regulation remains controversial. Chronic HCV patients possess relatively low hepcidin levels; however, elevated HAMP mRNA has been reported in HCV core transgenic mice and HCV replicon-expressing cells. We investigated the effect of HCV core protein on HAMP gene expression and delineated the complex interplay of molecular mechanisms involved. HCV core protein up-regulated HAMP promoter activity, mRNA, and secreted protein levels. Enhanced promoter activity was abolished by co-transfections of core with HAMP promoter constructs containing mutated/deleted BMP and STAT binding sites. Dominant negative constructs, pharmacological inhibitors, and silencing experiments against STAT3 and SMAD4 confirmed the participation of both pathways in HAMP gene regulation by core protein. STAT3 and SMAD4 expression levels were found increased in the presence of HCV core, which orchestrated SMAD4 translocation into the nucleus and STAT3 phosphorylation. To further understand the mechanisms governing the core effect, the role of the JAK/STAT-activating kinase CK2 was investigated. A CK2-dominant negative construct, a CK2-specific inhibitor, and RNAi interference abrogated the core-induced increase on HAMP promoter activity, mRNA, and protein levels, while CK2 acted in synergy with core to significantly enhance HAMP gene expression. Therefore, HCV core up-regulates HAMP gene transcription via a complex signaling network that requires both SMAD/BMP and STAT3 pathways and CK2 involvement.

Hepcidin and the iron enigma in HCV infection.

An estimated 30-40% of patients with chronic hepatitis C have elevated serum iron, transferrin saturation, and ferritin levels. Clinical data suggest that iron is a co-morbidity factor for disease progression following HCV infection. Iron is essential for a number of fundamental metabolic processes in cells and organisms. Mammalian iron homeostasis is tightly regulated and this is maintained through the coordinated action of sensory and regulatory networks that modulate the expression of iron-related proteins at the transcriptional and/or posttranscriptional levels. Disturbances of iron homeostasis have been implicated in infectious disease pathogenesis. Viruses, similarly to other pathogens, can escape recognition by the immune system, but they need iron from their host to grow and spread. Hepcidin is a 25-aa peptide, present in human serum and urine and represents the key peptide hormone, which modulates iron homeostasis in the body. It is synthesized predominantly by hepatocytes and its mature form is released in circulation. In this review, we discuss recent advances in the exciting crosstalk of molecular mechanisms and cell signaling pathways by which iron and hepcidin production influences HCV-induced liver disease.

The mannosylated extracellular domain of Her2/neu produced in P. pastoris induces protective antitumor immunity.

BACKGROUND: Her2/neu is overexpressed in various human cancers of epithelial origin and is associated with increased metastatic potential and poor prognosis. Several attempts have been made using the extracellular domain of Her2/neu (ECD/Her2) as a prophylactic vaccine in mice with no success in tumor prevention. METHODS: The extracellular domain of Her2/neu (ECD/Her2) was expressed in yeast P. pastoris, in a soluble highly mannosylated form. The immune response of the immunization with this recombinant ECD/Her2 was analyzed using immunoprecipitation and western blot analysis, proliferation and cytotoxicity assays as well as specific tumor growth assays. RESULTS: Mannosylated ECD/Her2 elicited a humoral response with HER2/neu specific antibodies in vaccinated mice, which were able to reduce the proliferation rate of cancer cells in vitro. Moreover, it elicited a cellular response with Her2/neu-specific CTL capable of lysing tumor cells, in vitro. When immunized Balb/c and HHD mice were challenged with Her2/neu-overexpressing cells, tumor growth was inhibited. CONCLUSION: Here we report on the efficacy of the extracellular domain of human Her2/neu produced in yeast P. pastoris, which confers mannosylation of the protein, to act as a potent anti-tumor vaccine against Her2/neu overexpressing tumors. Specific cellular and humoral responses were observed as well as efficacy.