Increased expression of long non-coding RNA FIRRE promotes hepatocellular carcinoma by HuR-CyclinD1 axis signaling.

There is a critical need to understand the disease processes and identify improved therapeutic strategies for hepatocellular carcinoma (HCC). The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. The aim of this study was to identify a lncRNA as a potential biomarker of HCC and investigate the mechanisms by which the lncRNA promotes HCC progression using human cell lines and in vivo. Using RNA-Seq analysis, we found that lncRNA FIRRE was significantly upregulated in hepatitis C virus (HCV) associated liver tissue and identified that lncRNA FIRRE is significantly upregulated in HCV-associated HCC compared to adjacent non-tumor liver tissue. Further, we observed that FIRRE is significantly upregulated in HCC specimens with other etiologies, suggesting this lncRNA has the potential to serve as an additional biomarker for HCC. Overexpression of FIRRE in hepatocytes induced cell proliferation, colony formation, and xenograft tumor formation as compared to vector-transfected control cells. Using RNA pull-down proteomics, we identified HuR as an interacting partner of FIRRE. We further showed that the FIRRE-HuR axis regulates cyclin D1 expression. Our mechanistic investigation uncovered that FIRRE is associated with an RNA-binding protein HuR for enhancing hepatocyte growth. Together, these findings provide molecular insights into the role of FIRRE in HCC progression.

Hepatitis C virus modified sE2F442NYT as an antigen in candidate vaccine facilitates human immune cell activation.

The rational selection of hepatitis C virus (HCV) vaccine antigen will aid in the prevention of future chronic liver disease burden and associated healthcare costs. We have previously shown that HCV E2 glycoprotein is not highly immunogenic, and the modification of E2 reduced CD81 binding and displayed altered cytokine and protective immune responses in vitro and in a surrogate mouse model. Here, we compared the influence of a parental and a modified sE2F442NYT glycoprotein region from HCV genotype 1a for the activation of peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), CD4+T cells, and B cells. Modified sE2F442NYT, when incubated with DCs, induced a higher number of CD86-positive cells. The sE2F442NYT or parental sE2 encapsulated as mRNA-lipid nanoparticle (sE2F442NYT mRNA-LNP) primed DCs co-cultured with autologous CD4+T cells did not induce CD25 or forkhead box P3 expression. PBMC-derived CD4+T cells treated with sE2F442NYT exhibited enhanced signal transducer and activator of transcription (Stat)1/Stat4 phosphorylation in response to anti-CD3/CD28 stimulation in comparison to parental sE2 treatment and facilitated isotype switching in B cells, leading to the generation of a broader subclass of antibodies. Cells treated with modified sE2F442NYT displayed an increase in activated Stat3 and extracellular signal-regulated kinase (ERK). Likewise, PBMC-derived naïve B cells upon in vitro stimulation with sE2F442NYT induced an increased proliferation, Stat3 and ERK activation, and protein kinase B (Akt) suppression. Thus, the modified sE2F442NYT antigen from HCV facilitates improved DC, CD4+T, and B cell activation compared to parental sE2 to better induce a robust protective immune response, supporting its selection as an HCV candidate vaccine antigen for preclinical and clinical HCV vaccine trials.IMPORTANCEThe nature of an enhanced immune response induced by sE2F442NYT will help in the selection of a broad cross-protective antigen from hepatitis C virus genotypes, and the inclusion of relatively conserved sE1 with sE2F442NYT may further strengthen the efficacy of the candidate vaccine in evaluating it for human use.

Targeting Lin28 axis enhances glypican-3-CAR T cell efficacy against hepatic tumor initiating cell population.

Overexpression of Lin28 is detected in various cancers with involvement in the self-renewal process and cancer stem cell generation. In the present study, we evaluated how the Lin28 axis plays an immune-protective role for tumor-initiating cancer cells in hepatocellular carcinoma (HCC). Our result using HCC patient samples showed a positive correlation between indoleamine 2,3-dioxygenase-1 (IDO1), a kynurenine-producing enzyme with effects on tumor immune escape, and Lin28B. Using in silico prediction, we identified a Sox2/Oct4 transcriptional motif acting as an enhancer for IDO1. Knockdown of Lin28B reduced Sox2/Oct4 and downregulated IDO1 in tumor-initiating hepatic cancer cells. We further observed that inhibition of Lin28 by a small-molecule inhibitor (C1632) suppressed IDO1 expression. Suppression of IDO1 resulted in a decline in kynurenine production from tumor-initiating cells. Inhibition of the Lin28 axis also impaired PD-L1 expression in HCC cells. Consequently, modulating Lin28B enhanced in vitro cytotoxicity of glypican-3 (GPC3)-chimeric antigen receptor (CAR) T and NK cells. Next, we observed that GPC3-CAR T cell treatment together with C1632 in a HCC xenograft mouse model led to enhanced anti-tumor activity. In conclusion, our results suggest that inhibition of Lin28B reduces IDO1 and PD-L1 expression and enhances immunotherapeutic potential of GPC3-CART cells against HCC.

Modified E2 Glycoprotein of Hepatitis C Virus Enhances Proinflammatory Cytokines and Protective Immune Response.

Hepatitis C virus (HCV) is characterized by a high number of chronic cases owing to an impairment of innate and adaptive immune responses. CD81 on the cell surface facilitates HCV entry by interacting with the E2 envelope glycoprotein. In addition, CD81/E2 binding on immunity-related cells may also influence host response outcome to HCV infection. Here, we performed site-specific amino acid substitution in the front layer of E2 sequence to reduce CD81 binding and evaluate the potential of the resulting immunogen as an HCV vaccine candidate. The modified sE2 protein (F442NYT), unlike unmodified sE2, exhibited a significant reduction in CD81 binding, induced higher levels of proinflammatory cytokines, repressed anti-inflammatory response in primary monocyte-derived macrophages as antigen-presenting cells, and stimulated CD4+ T cell proliferation. Immunization of BALB/c mice with an E1/sE2F442NYT nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccine resulted in improved IgG1-to-IgG2a isotype switching, an increase in neutralizing antibodies against HCV pseudotype virus, a B and T cell proliferative response to antigens, and improved protection against infection with a surrogate recombinant vaccinia virus-expressing HCV E1-E2-NS2aa134-966 challenge model compared to E1/unmodified sE2 mRNA-LNP vaccine. Further investigation of the modified E2 antigen may provide helpful information for HCV vaccine development. IMPORTANCE Hepatitis C virus (HCV) E2-CD81 binding dampens protective immune response. We have identified that an alteration of amino acids in the front layer of soluble E2 (sE2) disrupts CD81 interaction and alters the cytokine response. Immunization with modified sE2F442NYT (includes an added potential N-linked glycosylation site and reduces CD81 binding activity)-mRNA-LNP candidate vaccine generates improved proinflammatory response and protective efficacy against a surrogate HCV vaccinia challenge model in mice. The results clearly suggested that HCV E2 exhibits immunoregulatory activity that inhibits induction of robust protective immune responses. Selection of engineered E2 antigen in an mRNA-LNP platform amenable to nucleic acid sequence alterations may open a novel approach for multigenotype HCV vaccine development.

SARS-CoV-2 Spike Protein Induces Paracrine Senescence and Leukocyte Adhesion in Endothelial Cells.

Increased mortality in COVID-19 cases is often associated with microvascular complications. We have recently shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein promotes an inflammatory cytokine interleukin 6 (IL-6)/IL-6R-induced trans signaling response and alarmin secretion. Virus-infected or spike-transfected human epithelial cells exhibited an increase in senescence, with a release of senescence-associated secretory phenotype (SASP)-related inflammatory molecules. Introduction of the bromodomain-containing protein 4 (BRD4) inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP-related inflammatory molecule release in TMNK-1 or EAhy926 (representative human endothelial cell lines), when cells were exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein. Cells also exhibited a senescence phenotype with enhanced p16, p21, and senescence-associated ß-galactosidase (SA-ß-Gal) expression and triggered SASP pathways. Inhibition of IL-6 trans signaling by tocilizumab and inhibition of inflammatory receptor signaling by the Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike-transfected and endothelial cells exposed to spike-transfected CM. ROS generation in endothelial cell lines was reduced after treatment with tocilizumab and zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which have in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that human endothelial cells exposed to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. IMPORTANCE The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein-associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture medium (CM). Endothelial cell lines (TMNK-1 or EAhy926) treated with CM exhibited an increase in cellular senescence markers by a paracrine mode and led to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence and a potential contribution to microvascular complication in productively SARS-CoV-2-infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6, and BRD4) showed a reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2-associated pathogenesis.

Hepatitis C Virus Evades Interferon Signaling by Suppressing Long Noncoding RNA Linc-Pint Involving C/EBP-ß.

Hepatitis C virus (HCV) regulates many cellular genes in modulating the host immune system for benefit of viral replication and long-term persistence in a host for chronic infection. Long noncoding RNAs (lncRNAs) play an important role in the regulation of many important cellular processes, including immune responses. We recently reported that HCV infection downregulates lncRNA Linc-Pint (long intergenic non-protein-coding RNA p53-induced transcript) expression, although the mechanism of repression and functional consequences are not well understood. In this study, we demonstrate that HCV infection of hepatocytes transcriptionally reduces Linc-Pint expression through CCAAT/enhancer binding protein ß (C/EBP-ß). Subsequently, we observed that the overexpression of Linc-Pint significantly upregulates interferon alpha (IFN-α) and IFN-ß expression in HCV-replicating hepatocytes. Using unbiased proteomics, we identified that Linc-Pint associates with DDX24, which enables RIP1 to interact with IFN-regulatory factor 7 (IRF7) of the IFN signaling pathway. We furthermore observed that IFN-α14 promoter activity was enhanced in the presence of Linc-Pint. Together, these results demonstrated that Linc-Pint acts as a positive regulator of host innate immune responses, especially IFN signaling. HCV-mediated downregulation of Linc-Pint expression appears to be one of the mechanisms by which HCV may evade innate immunity for long-term persistence and chronicity. IMPORTANCE The mechanism by which lncRNA regulates the host immune response during HCV infection is poorly understood. We observed that Linc-Pint was transcriptionally downregulated by HCV. Using a chromatin immunoprecipitation (ChIP) assay, we showed inhibition of transcription factor C/EBP-ß binding to the Linc-Pint promoter in the presence of HCV infection. We further identified that Linc-Pint associates with DDX24 for immunomodulatory function. The overexpression of Linc-Pint reduces DDX24 expression, which in turn results in the disruption of DDX24-RIP1 complex formation and the activation of IRF7. The induction of IFN-α14 promoter activity in the presence of Linc-Pint further confirms our observation. Together, our results suggest that Linc-Pint acts as a positive regulator of host innate immune responses. Downregulation of Linc-Pint expression by HCV helps in escaping the innate immune system for the development of chronicity.

SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells.

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.

Inhibition of Long Noncoding RNA Linc-Pint by Hepatitis C Virus in Infected Hepatocytes Enhances Lipogenesis.

BACKGROUND AND AIMS: HCV often causes chronic infection in liver, cirrhosis, and, in some instances, HCC. HCV encodes several factors' those impair host genes for establishment of chronic infection. The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. However, their role in virus replication and underlying diseases is poorly understood. In this study, we have shown that HCV exploits lncRNA long intergenic nonprotein-coding RNA, p53 induced transcript (Linc-Pint) in hepatocytes for enhancement of lipogenesis. APPROACH AND RESULTS: We identified a lncRNA, Linc-Pint, which is significantly down-regulated in HCV-replicating hepatocytes and liver specimens from HCV infected patients. Using RNA pull-down proteomics, we identified serine/arginine protein specific kinase 2 (SRPK2) as an interacting partner of Linc-Pint. A subsequent study demonstrated that overexpression of Linc-Pint inhibits the expression of lipogenesis-related genes, such as fatty acid synthase and ATP-citrate lyase. We also observed that Linc-Pint significantly inhibits HCV replication. Furthermore, HCV-mediated enhanced lipogenesis can be controlled by exogenous Linc-Pint expression. Together, our results suggested that HCV-mediated down-regulation of Linc-Pint enhances lipogenesis favoring virus replication and liver disease progression. CONCLUSIONS: We have shown that SRPK2 is a direct target of Linc-Pint and that depletion of SRPK2 inhibits lipogenesis. Our study contributes to the mechanistic understanding of the role of Linc-Pint in HCV-associated liver pathogenesis.

Molecular Changes in Relation to Alcohol Consumption and Hepatocellular Carcinoma.

Alcohol is the one of the major causes of liver diseases and promotes liver cirrhosis and hepatocellular carcinoma (HCC). In hepatocytes, alcohol is converted to acetaldehyde, which causes hepatic steatosis, cellular apoptosis, endoplasmic reticulum stress, peroxidation, production of cytokines and reduces immune surveillance. Endotoxin and lipopolysaccharide produced from intestinal bacteria also enhance the production of cytokines. The development of hepatic fibrosis and the occurrence of HCC are induced by these alcohol metabolites. Several host genetic factors have recently been identified in this process. Here, we reviewed the molecular mechanism associated with HCC in alcoholic liver disease.

Hepatitis C Virus Mediated Inhibition of miR-181c Activates ATM Signaling and Promotes Hepatocyte Growth.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection promotes hepatocyte growth and progress to hepatocellular carcinoma. We previously observed that HCV infection of hepatocytes transcriptionally down-regulates miR-181c expression through CCAAT/enhancer binding protein ß (C/EBP-ß). Here, we examined the role of miR-181c in the regulation of cell cycle progression in relation to HCV infection. In silico analysis suggested that ataxia-telangiectasia mutated (ATM) protein, a protein kinase, is a direct target of miR-181c. ATM is a central mediator of response for cellular DNA double-strand break. APPROACH AND RESULTS: Our results demonstrated that ATM expression is higher in HCV-infected hepatocytes and chronic HCV-infected liver biopsy specimens. We have shown a direct interaction of miR-181c with the 3' untranslated region of ATM, and the presence of ATM in miR-181c-associated RNA-induced silencing complex. Exogenous expression of miR-181c inhibited ATM expression and activation of its downstream molecules, Chk2 and Akt. On the other hand, introduction of anti-miR-181c restored ATM and phosphorylated Akt. Furthermore, introduction of miR-181c significantly inhibited phospho-cyclin-dependent kinase 2 (CDK2) and cyclin-A expression, arresting cell cycle progression, whereas overexpression of miR-181c promoted apoptosis of HCV-infected hepatocytes and can be inhibited by overexpression of ATM from a clone lacking miR-181c binding sites. In addition, miR-181c significantly regressed tumor growth in the xenograft human hepatocellular carcinoma mouse model. CONCLUSIONS: Together, our results suggest that HCV infection suppresses miR-181c in hepatocytes, resulting in ATM activation and apoptosis inhibition for promotion of cell cycle progression. The results provide mechanistic insight into understanding the role of miR-181c in HCV-associated hepatocyte growth promotion, and may have the potential for therapeutic intervention.

Establishment of a Patient-Derived Xenograft Tumor From Hepatitis C-Associated Liver Cancer and Evaluation of Imatinib Treatment Efficacy.

BACKGROUND AND AIMS: Chronic hepatitis C virus (HCV) infection is one of the major causal factors for hepatocellular carcinoma (HCC). The treatment options for HCC are limited for lack of a convenient animal model for study in HCV infection and liver pathogenesis. This study aimed to develop a patient-derived xenograft (PDX) tumor in mice by using a tumor from a patient with HCV-associated HCC and evaluating this model's therapeutic potential. APPROACH AND RESULTS: After resection of the primary tumor from the patient liver, excess viable tumor was implanted into highly immunodeficient mice. A mouse xenograft tumor line was developed, and the tumor was successfully passaged for at least three rounds in immunodeficient mice. The patient's primary tumor and the mouse xenografts were histologically similar. Genetic profiling by short-tandem-repeat analysis verified that the HCC-PDX model was derived from the HCC clinical specimen. HCV RNA present in the patient liver specimen was undetectable after passage as xenograft tumors in mice. Human albumin, α1 -antitrypsin, glypican-3, α-smooth muscle actin, and collagen type 1A2 markers were detected in human original tumor tissues and xenograft tumors. Both the patient primary tumor and the xenograft tumors had a significantly higher level of receptor tyrosine kinase (c-Kit) mRNA. Treatment of HCC-PDX xenograft tumor-bearing mice with the c-Kit inhibitor imatinib significantly reduced tumor growth and phospho-Akt and cyclin D1 expression, as compared with untreated control tumors. CONCLUSIONS: Our results demonstrated establishment of an HCV-associated HCC-PDX model as a powerful tool for evaluating candidate drugs. Information on molecular changes in cancer-specific gene expression facilitates efficient targeted therapies and treatment strategies.

Exosomes from COVID-19 Patients Carry Tenascin-C and Fibrinogen-ß in Triggering Inflammatory Signals in Cells of Distant Organ.

SARS-CoV-2 infection can cause cytokine storm and may overshoot immunity in humans; however, it remains to be determined whether virus-induced soluble mediators from infected cells are carried by exosomes as vehicles to distant organs and cause tissue damage in COVID-19 patients. We took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and COVID-19 patients. Our results revealed that tenascin-C (TNC) and fibrinogen-ß (FGB) are highly abundant in exosomes from COVID-19 patients' plasma compared with that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via the Nuclear factor-κB (NF-κB) pathway, we examined the status of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-C motif chemokine ligand 5 (CCL5) expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase compared with that from healthy subjects. Together, our results demonstrate that TNC and FGB are transported through plasma exosomes and potentially trigger pro-inflammatory cytokine signaling in cells of distant organ.

Transforming Growth Factor ß Acts as a Regulatory Molecule for Lipogenic Pathways among Hepatitis C Virus Genotype-Specific Infections.

Hepatitis C virus (HCV) infection promotes metabolic disorders, and the severity of lipogenic disease depends upon the infecting virus genotype. Here, we have examined HCV genotype 1-, 2-, or 3-specific regulation of lipid metabolism, involving transforming growth factor ß (TGF-ß)-regulated phospho-Akt (p-Akt) and peroxisome proliferator-activated receptor alpha (PPARα) axes. Since HCV core protein is one of the key players in metabolic regulation, we also examined its contribution in lipid metabolic pathways. The expression of regulatory molecules, TGF-ß1/2, phospho-Akt (Ser473), PPARα, sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FASN), hormone-sensitive lipase (HSL), and acyl dehydrogenases was analyzed in virus-infected hepatocytes. Interestingly, HCV genotype 3a exhibited much higher activation of TGF-ß and p-Akt, with a concurrent decrease in PPARα expression and fatty acid oxidation. A significant and similar decrease in HSL, unlike in HCV genotype 1a, was observed with both genotypes 2a and 3a. Similar observations were made from ectopic expression of the core genomic region from each genotype. The key role of TGF-ß was further verified using specific small interfering RNA (siRNA). Together, our results highlight a significant difference in TGF-ß-induced activity for the HCV genotype 2a- or 3a-induced lipogenic pathway, exhibiting higher triglyceride synthesis and a decreased lipolytic mechanism. These results may help in therapeutic modalities for early treatment of HCV genotype-associated lipid metabolic disorders.IMPORTANCE Hepatic steatosis is a frequent complication associated with chronic hepatitis C virus (HCV) infection and is a key prognostic indicator for progression to fibrosis and cirrhosis. Several mechanisms are proposed for the development of steatosis, especially with HCV genotype 3a. Our observations suggest that transforming growth factor ß (TGF-ß) and peroxisome proliferator-activated receptor alpha (PPARα)-associated mechanistic pathways in hepatocytes infected with HCV genotype 2a and 3a differ from those in cells infected with genotype 1a. The results suggest that a targeted therapeutic approach for enhanced PPARα and lipolysis may reduce HCV genotype-associated lipid metabolic disorder in liver disease.

Hepatitis C Virus Manipulates Humans as its Favorite Host for a Long-Term Relationship.

Chronic hepatitis C virus (HCV) infection-associated liver disease is a global health problem. HCV often causes silent disease, and eventually progresses to end-stage liver disease. HCV infects hepatocytes; however, initial manifestation of liver disease is mostly displayed in hepatic stellate cells (HSCs), causing fibrosis/cirrhosis, and is believed to occur from inflammation in the liver. It remains unclear why HCV is not spontaneously cleared from infected liver in the majority of individuals and develops chronic infection with progressive liver disease. Direct-acting antivirals (DAAs) show excellent results in controlling viremia, although beneficial consequence in advanced liver disease remains to be understood. In this review, we highlight the current knowledge that has contributed to our understanding of the role of HCV in inflammation, immune evasion, metabolic disorders, liver pathogeneses, and efforts in vaccine development.

Hepatitis C Virus E2 Envelope Glycoprotein Induces an Immunoregulatory Phenotype in Macrophages.

A comprehensive strategy to control hepatitis C virus (HCV) infection needs a vaccine. Our phase I study with recombinant HCV E1/E2 envelope glycoprotein (EnvGPs) as a candidate vaccine did not induce a strong immune response in volunteers. We analyzed the interactions of HCV EnvGPs with human monocyte-derived macrophages as antigen-presenting cells. HCV E2 induced immune regulatory cytokine interleukin (IL)-10 and soluble CD163 (sCD163) protein expression in macrophages from 7 of 9 blood donors tested. Furthermore, HCV E2 enhanced Stat3 and suppressed Stat1 activation, reflecting macrophage polarization toward M2 phenotype. E2-associated macrophage polarization appeared to be dependent of its interaction with CD81 leading endothelial growth factor receptor (EGFR) activation. Additionally, E2 suppressed the expression of C3 complement, similar to HCV-exposed dendritic cells (DCs), implying potential impairment of immune cell priming. Conclusion: Our results suggest that E2 EnvGP may not be an ideal candidate for HCV vaccine development, and discrete domains within E2 may prove to be more capable of elliciting a protective immune response. (Hepatology 2018).

Association between MicroRNA-373 and Long Noncoding RNA NORAD in Hepatitis C Virus-Infected Hepatocytes Impairs Wee1 Expression for Growth Promotion.

Chronic hepatitis C virus (HCV) infection may lead to end-stage liver disease, including hepatocellular carcinoma (HCC). We have shown previously that microRNA-373 (miR-373) is upregulated in HCV-infected human liver biopsy specimens. To gain insight into the role of miR-373 in HCV-mediated pathogenesis, we investigated its interacting partner for hepatocyte growth regulation. Transcriptome sequencing (RNA-seq) data revealed that Wee1 is associated with miR-373 and is a direct target. Interestingly, higher expression of Wee1 was noted in HCV-infected hepatocytes than in uninfected hepatocytes, suggesting that other factors may block miR-373-mediated Wee1 inhibition. We subsequently found an association between the long noncoding RNA NORAD (LINC00657) and miR-373, and we demonstrated that NORAD binds to miR-373 and Wee1 independently. However, the high level of Wee1 expression in HCV-infected hepatocytes suggested that miR-373 forms a complex with NORAD. Depletion of miR-373 or the inhibitor Wee1 reduces the growth of Huh7.5 cells harboring the HCV genome as well as reducing Wee1 expression. Taken together, our data demonstrate a novel mechanism of hepatocyte growth promotion during HCV infection involving a miR-373-NORAD-Wee1 axis, which may be a target for future therapy against HCV-associated HCC.IMPORTANCE The mechanism of HCV-mediated liver pathogenesis is poorly understood. In this study, we observed that HCV infection upregulates miR-373 and Wee1, a pivotal player in the G2 checkpoint in the cell cycle, although Wee1 is a direct target for miR-373. Subsequent investigation demonstrated that miR-373 forms a complex with the long noncoding RNA NORAD, resulting in the release of their common target, Wee1, in HCV-infected cells, which, in turn, favors uncontrolled cell growth. Our study suggested a previously unknown mechanism for hepatocyte growth promotion following HCV infection, and this pathway can be targeted for future therapy against HCV-mediated liver pathogenesis.

Rapid hepatitis C virus clearance by antivirals correlates with immune status of infected patients.

Altered immune parameters associated with hepatitis C virus (HCV) genotype 1b infection and their correlation with virus eradication in direct-acting antivirals (DAA)-treated patients were examined. Thirty-one HCV-infected patients were treated with DAAs for 12 weeks. Pre-DAA-treatment and post-DAA-treatment sera were analyzed for cytokines/chemokines using MILLIPLEX MAP. Serum complement level and antibody neutralization activity were measured separately. Sera from 11 spontaneously cleared HCV subjects were included for comparison. Rapid virological responders (RVR) or end-of-treatment responders (EOTR) were defined as patients with HCV RNA negative at week 4 or positive at week 4 and negative at week 12, respectively. HCV RNA eradication and a decrease in liver fibrosis-related cytokines after treatment were observed when compared with pretreatment sera from RVR and EOTR. In pretreatment sera, interferons and T-helper 1 or 2 cell-associated cytokines/chemokines were significantly higher among RVR as compared with EOTR. Furthermore, serum complement and virus neutralizing antibody levels were higher in pretreatment RVR sera. Eradication of HCV RNA by DAA decreased liver fibrosis-related cytokines. Pretreatment sera from RVR displayed an enhanced cytokine/chemokine, complement and virus neutralizing antibody response as compared with EOTR sera. Our results suggested that enhanced host immune status may play an additive role on HCV RNA clearance by DAA.

Hepatitis C Virus Core Protein Modulates Endoglin (CD105) Signaling Pathway for Liver Pathogenesis.

Endoglin is part of the TGF-ß receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In a previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition (EMT) state and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV-associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of an HCV core-expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV-infected cells or viral core gene-transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase 1 (ALK1) Fc blocking peptide and endoglin small interfering RNA (siRNA). Further, follow-up from in vitro analysis suggested that the antiapoptosis Bcl2 protein, proliferation-related cyclin D1 protein, and CSC-associated Hes1, Notch1, Nanog, and Sox2 proteins are enhanced during infection or ectopic expression of HCV core protein.IMPORTANCE Endoglin plays a crucial role in fibrogenesis and angiogenesis and is an important protein for tumor growth, survival, and cancer cell metastasis. Endoglin enhances ALK1-SMAD1/5 signaling in different cell types, leading to increased proliferation and migration responses. We have observed endoglin expression on the HCV core-expressing cell surface of human hepatocyte origin and activation of phospho-SMAD1/5 and ID1 downstream signaling molecules. ID1 protein plays a role in CSC properties, and we found that this pathway is important for antiapoptotic and cell proliferation signaling. Blocking of endoglin-ALK1-SMAD1/5 might be a good candidate for therapy for liver cancer stem cells together with liver cirrhosis.

Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells.

Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor ß (TGF-ß) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3-TGF-ß pathway in HSC. This study contributes to the understanding of intercellular communication in the pathogenesis of liver disease during HCV infection.

Hepatitis C virus-induced tumor-initiating cancer stem-like cells activate stromal fibroblasts in a xenograft tumor model.

Hepatitis C virus (HCV) often causes persistent infection and is an increasingly important factor in the etiology of fibrosis/cirrhosis and hepatocellular carcinoma, although the mechanisms for the disease processes remain unclear. We have shown previously that HCV infection generates an epithelial-mesenchymal transition state and tumor-initiating cancer stem-like cells in human hepatocytes. In this study, we investigated whether HCV-induced tumor-initiating cancer stem-like cells when implanted into mice activate stromal fibroblasts. A number of fibroblast activation markers, including matrix metalloproteinase 2, were significantly increased at the mRNA or protein level in the xenograft tumors, suggesting the presence of tumor-associated fibroblasts. Fibroblast activation markers of murine origin were specifically increased in tumor, suggesting that fibroblasts migrate to form stroma. Next, we demonstrated that conditioned medium from HCV-infected human hepatocytes activates fibrosis-related markers in hepatic stellate cells. We further observed that these HCV-infected hepatocytes express transforming growth factor beta, which activates stromal fibroblast markers. Subsequent analysis suggested that anti-transforming growth factor beta neutralizing antibody, when incubated with conditioned medium from HCV-infected hepatocytes, inhibits fibrosis marker activation in primary human hepatic stellate cells. CONCLUSION: HCV-infected hepatocytes induce local fibroblast activation by secretion of transforming growth factor beta, and a preneoplastic or tumor state of the hepatocytes influences the network for the tumor-associated fibroblast environment. (Hepatology 2017;66:1766-1778).