HCV-infected Human Hepatocytes Research Articles

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

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).

Induction of C/EBPb-Thr266 Phosphorylation by HCV Infection is Indispensable for the Zone-3 Phenotype and Proliferation in Cultured Human Hepatocytes

Liver acinar zonation allows for diverse functions to be performed on demand by specialized hepatocytes. Therefore, understanding how hepatitis C virus (HCV) infection may modulate the acinar zone phenotypes should provide insights into the pathophysiology of HCV infection. We assessed the effects HCV infection in the highlydifferentiated primary human hepatocyte culture that we have developed for the efficient replication of intact HCV virions. We found that HCV genotype 1 infection stimulated the zone-3 phenotype after 72 hr since the cultured hepatocytes expressed GS, β-Catenin and HIF-1α , markers of zone-3 hepatocytes. HCV infection also induced phosphorylated-C/EBPβ-Thr266 and proliferation in these cultured hepatocytes. A peptide designed to selectively inhibit phosphorylation of C/EBPβ-Thr266 blocked this phosphorylation, the zone-3 phenotype and hepatocyte proliferation induced by HCV infection in these cultured human hepatocytes. In addition, we found that in normal, uninfected human hepatocytes, unphosphorylated-C/EBPβ-Thr266 was associated with protein von Hippel Lindau (pVHL) and Axin (inhibitors of HIF-1α and β-catenin activation, respectively). These associations were blocked by the HCV infection since phosphorylated C/EBPβ-Thr266 was not associated with either pVHL or Axin. The inhibition of C/EBPβ-Thr266 phosphorylation normalized the association of unphosphorylatable C/EBPβ-Thr266 with pVHL and Axin, which is expected to inhibit the activation of HIF-1α and β−catenin. Collectively, these results strongly suggest that C/EBPβ-Thr266 phosphorylation is indispensable for the activation of GS, HIF-1α and β-catenin, and the induction of zone-3 phenotype in HCV-infected human hepatocytes.

Viral non-coding RNA inhibits HNF4α expression in HCV associated hepatocellular carcinoma.

BackgroundHepatitis C virus (HCV) infection is an established cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC); however, it is unclear if the virus plays a direct role in the development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is critical determinant of epithelial architecture and hepatic development; depletion of HNF4α is correlated with oncogenic transformation. We explored the viral role in the inhibition of HNF4α expression, and consequent induction of tumor-promoting genes in HCV infection-associated HCC.MethodsWestern blot analysis was used to monitor the changes in expression levels of oncogenic proteins in liver tissues from HCV-infected humanized mice. The mechanism of HNF4α depletion was studied in HCV-infected human hepatocyte cultures in vitro. Targeting of HNF4α expression by viral non-coding RNA was examined by inhibition of Luciferase HNF4α 3’-UTR reporter. Modulation of invasive properties of HCV-infected cells was examined by Matrigel cell migration assay.ResultsResults show inhibition of HNF4α expression by targeting of HNF4α 3’-UTR by HCV-derived small non-coding RNA, vmr11. Vmr11 enhances the invasive properties of HCV-infected cells. Loss of HNF4α in HCV-infected liver tumors of humanized mice correlates with the induction of epithelial to mesenchymal transition (EMT) genes.ConclusionsWe show depletion of HNF4α in liver tumors of HCV-infected humanized mice by HCV derived small non-coding RNA (vmr11) and resultant induction of EMT genes, which are critical determinants of tumor progression. These results suggest a direct viral role in the development of hepatocellular carcinoma.

Loss of nuclear PTEN in HCV-infected human hepatocytes.

BackgroundHepatitis C virus (HCV) infection is a major risk factor for chronic hepatitis and hepatocellular carcinoma (HCC); however, the mechanism of HCV-mediated hepatocarcinogenesis is not well understood. Insufficiency of PTEN tumor suppressor is associated with more aggressive cancers, including HCC. We asked whether viral non-coding RNA could initiate oncogenesis in HCV infected human hepatocytes. The results presented herein suggest that loss of nuclear PTEN in HCV-infected human hepatocytes results from depletion of Transportin-2, which is a direct target of viral non-coding RNA, vmr11.MethodsThe intracellular distribution of PTEN in HCV-infected cells was monitored by immunostaining and Western blots of nuclear and cytoplasmic proteins. Effects of PTEN depletion were examined by comparing expression arrays of uninfected cells with either HCV-infected or vmr11-transfected cells. Target genes suggested by array analyses were validated by Western blot. The influence of nuclear PTEN deficiency on virus production was determined by quantitative analysis of HCV genomic RNA in culture media of infected hepatocytes.ResultsImport of PTEN to the nucleus relies on the interaction of Transportin-2 and PTEN proteins; we show that depletion of Transportin-2 by HCV infection or by the introduction of vmr11 in uninfected cells results in reduced nuclear PTEN. In turn, nuclear PTEN insufficiency correlates with increased virus production and the induction of γ-H2AX, a marker of DNA double-strand breaks and genomic instability.ConclusionAn HCV-derived small non-coding RNA inhibits Transportin-2 and PTEN translocation to the nucleus, suggesting a direct viral role in hepatic oncogenesis.

HCV-infected hepatocytes drive CD4+ CD25+ Foxp3+ regulatory T-cell development through the Tim-3/Gal-9 pathway.

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T-cell Ig and mucin domain protein-3 (Tim-3) and galectin-9 (Gal-9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg-cell development, here we provide pilot data showing that HCV-infected human hepatocytes express higher levels of Gal-9 and TGF-β, and upregulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured human CD4(+) T cells, driving conventional CD4(+) T cells into CD25(+) Foxp3(+) Treg cells. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4(+) T cells into Foxp3(+) Treg cells in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg-cell induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate human Foxp3(+) Treg-cell development and function during HCV infection.

ME3738 enhances the effect of interferon and inhibits hepatitis C virus replication both in vitro and in vivo

ME3738 (22β-methoxyolean-12-ene-3β, 24-diol), a derivative of soyasapogenol B, attenuates liver disease in several animal models of acute and chronic liver injury. ME3738 is thought to inhibit replication of hepatitis C virus (HCV) by enhancing interferon (IFN)-β production, as determined using the HCV full-length binary expression system. We examined the effect of ME3738 combined with IFN-α on HCV replication using the genotype 1b subgenomic replicon system and an in vivo mouse HCV model. HCV replicon cells (ORN/3-5B/KE cells and Con1 cells) were incubated with ME3738 and/or IFN-α, and then intracellular IFN-stimulated genes (ISGs) and HCV RNA replication were analyzed by reverse-transcription-real time polymerase chain reaction and luciferase reporter assay. HCV-infected human hepatocyte chimeric mice were also treated with ME3738 and/or IFN-α for 4 weeks. Mouse serum HCV RNA titer, HCV core antigen, and ISGs expression in the liver were measured. ME3738 induced gene expression of oligoadenylate synthetase 1 and inhibited HCV replication in both HCV replicon cells. The drug enhanced the effect of IFN to significantly increase ISG expression levels, inhibit HCV replication in replicon cells, and reduce mouse serum HCV RNA and core antigen levels in mouse livers. The combination treatment was not hepatotoxic as evident histologically and did not reduce human serum albumin in mice. ME3738 inhibited HCV replication, enhancing the effect of IFN-α to increase ISG expression both in vitro and in vivo, suggesting that the combination of ME3738 and IFN might be useful therapeutically for patients with chronic hepatitis C.

Primary cultures of human hepatocytes isolated from hepatitis C virus‐infected cirrhotic livers as a model to study hepatitis C infection

Since the discovery of hepatitis C virus (HCV), researchers have encountered difficulties with in vitro models. The aim of this study was to determine whether HCV-infected human primary hepatocytes, isolated from cirrhotic livers at liver transplantation, can be used as a model to study HCV infection. Hepatocytes were isolated with collagenase and cultured over a 20-day period on different matrices. Viral kinetics was monitored with/without treatment by real-time polymerase chain reaction. Cell yield and viability were higher with uninfected/non-cirrhotic livers (77.2+/-1.8%) in comparison with HCV-infected cirrhotic livers (68.8+/-12%). HCV-infected hepatocytes behaved similar to non-infected cells and expressed albumin and cytochrome P4502E1. HCV-positive strand was identified in supernatants and cell lysates. HCV-negative strand was only found inside cells and correlated with viral RNA recovery in the medium. Improvement in the degree of hepatocyte differentiation was associated with better HCV recovery. Antiviral treatment with interferon-alpha, EX4 and cyclosporine A induced significant reductions in HCV RNA. Primary cultures of HCV-infected human hepatocytes from end-stage cirrhotic livers is feasible, represents an excellent model to study specific virus-host interactions and can be used to assess viral replication.

HCV Induces Oxidative and ER Stress, and Sensitizes Infected Cells to Apoptosis in SCID/Alb-uPA Mice

Hepatitis C virus (HCV) is a blood-borne pathogen and a major cause of liver disease worldwide. Gene expression profiling was used to characterize the transcriptional response to HCV H77c infection. Evidence is presented for activation of innate antiviral signaling pathways as well as induction of lipid metabolism genes, which may contribute to oxidative stress. We also found that infection of chimeric SCID/Alb-uPA mice by HCV led to signs of hepatocyte damage and apoptosis, which in patients plays a role in activation of stellate cells, recruitment of macrophages, and the subsequent development of fibrosis. Infection of chimeric mice with HCV H77c also led an inflammatory response characterized by infiltration of monocytes and macrophages. There was increased apoptosis in HCV-infected human hepatocytes in H77c-infected mice but not in mice inoculated with a replication incompetent H77c mutant. Moreover, TUNEL reactivity was restricted to HCV-infected hepatocytes, but an increase in FAS expression was not. To gain insight into the factors contributing specific apoptosis of HCV infected cells, immunohistological and confocal microscopy using antibodies for key apoptotic mediators was done. We found that the ER chaperone BiP/GRP78 was increased in HCV-infected cells as was activated BAX, but the activator of ER stress–mediated apoptosis CHOP was not. We found that overall levels of NF-κB and BCL-xL were increased by infection; however, within an infected liver, comparison of infected cells to uninfected cells indicated both NF-κB and BCL-xL were decreased in HCV-infected cells. We conclude that HCV contributes to hepatocyte damage and apoptosis by inducing stress and pro-apoptotic BAX while preventing the induction of anti-apoptotic NF-κB and BCL-xL, thus sensitizing hepatocytes to apoptosis.

Culture and characterization of human hepatocytes isolated from hepatitis C virus infected liver: Effect of collagen-coated surface and FBS-supplemented medium

The aim of this study is to maintain and prolong the viability and metabolic functions of hepatocytes isolated from hepatitis C virus (HCV)-infected human livers. Collagen-coated surface and 10% fetal bovine serum (FBS) supplemented culture media were studied. Metabolic functions and attachment ability of human hepatocytes in these conditions were compared with normalized conditions (Petri dish, medium without FBS). Our results showed that collagen-coated dishes with 10% FBS-supplemented culture medium increased urea production and albumin synthesis. Urea concentration remained stable (30 pg/cell/h) during the first 3-days when human hepatocytes were cultured in collagen-coated dishes. These hepatocytes showed higher urea production when compared with hepatocytes cultured in normal Petri dishes producing 36.4 and 31.7 pg/cell/h concentrations of urea, respectively. Albumin secretion rate was maximal on day 3 after plating (0.86 pg/cell/h). When using culture medium supplemented with 10% FBS, the metabolic functions of hepatocytes were higher compared to when using normal culture medium. This study demonstrates that human hepatocytes can be isolated from HCV-infected livers via surgical biopsies with high cell yield rate, and their metabolic functions remain stable when cultured in collagen-coated dishes with FBS-rich medium for several days. Base on these results, HCV-infected human hepatocytes can be used in the study of clinical diseases and in the performance of physiological and pharmacological liver experiments in vitro.

A reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus RNA.

We have developed a reliable internally controlled RT-nested PCR method for the detection of hepatitis C virus (HCV) RNA using in vitro synthesized Renilla luciferase (Rluc) RNA as an internal control. Using this method, the 5'-noncoding region of HCV RNA (144 nucleotides) and Rluc RNA (276 nucleotides) were efficiently amplified in a single tube, and the sensitivity and specificity of this method were comparable to standard RT-nested PCR. This method was successfully performed on RNA specimens obtained from in vitro HCV-infected human hepatocyte PH5CH8 cells, which support HCV replication. In addition, we demonstrated that this method was useful for the evaluation of antiviral reagents by confirming the anti-HCV activity of bovine lactoferrin, which we previously found to be a new inhibitor of HCV infection. Therefore, this method may be useful for the studies of not only HCV but also of other viruses.

Iron enhances hepatitis C virus replication in cultured human hepatocytes.

Iron overload in the presence of increasing concentrations of iron is one of the indicators of poor response to interferon therapy in chronic hepatitis C. In order to analyze the effect of iron on hepatitis C virus (HCV) replication, we measured replication in an HCV-infected cell line. Cells from a non-neoplastic HCV-infected human hepatocyte line (PH5CH8) susceptible to HCV infection and supportive of HCV replication were used in this study. The replication of HCV RNA was measured by reverse transcription-nested polymerase chain reaction (RT-nested PCR). PH5CH8 cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. PH5CH8 cells were incubated with 0, 1, 10, 50, and 100 microM of FeSO4 at 37 degrees C with 5% CO2. Forty-eight hours after iron supplementation, the quantity of HCV RNA in the cells incubated in 50 and 100 microM of FeSO4 was approximately ten times that of the cells with no iron supplementation. Similar changes were observed beginning at 12 h from supplementation with FeSO4 and continued for at least 72 h after supplementation. MTT assay indicated that iron did not have cytotoxic effects on the PH5CH8 cells. Iron enhances HCV replication in a hepatocyte cell line. The results suggest that iron deposition in hepatocytes could facilitate HCV infection in the liver.