Tamoxifen alleviates hepatitis C virus‐induced inhibition of both toll‐like receptor 7 and JAK‐STAT signalling pathways in PBMCs of infected Egyptian females

Summary. Hepatitis C virus (HCV) is a major health concern in Egypt being highly prevalent among Egyptians. The two genders experience different responses to HCV infection and show variations in response to interferon (IFN)‐based therapy that may be attributed to sex hormones. We previously demonstrated the suppressive effect of 17β‐estradiol (E2) on the expression of the IFN‐stimulated gene MxA in HCV‐infected peripheral blood mononuclear cells (PBMCs). The selective oestrogen receptor (ER) modulator Tamoxifen has been shown to have an antiviral effect against HCV, but its effect on the host immune response is unknown. We investigated the effect of Tamoxifen on the IFN signalling pathways in PBMCs of HCV‐infected Egyptian females. We pooled PBMCs and treated then with exogenous interferon alpha (IFNα) or the TLR7 ligand, Imiquimod, and quantified the relative expressions of MxA using RTqPCR. Studies were performed with and without Tamoxifen pretreatment. Pretreatment with Tamoxifen reversed the suppressive effect of E2 on the JAK‐STAT pathway in IFNα‐treated PBMCs as indicated by a significant increase in MxA expression (P = 0.05*). Tamoxifen pretreatment also significantly upregulated MxA expression in Imiquimod‐treated PBMCs (P = 0.0011**), an effect not ascribed to ER blocking nor to an upregulation in TLR7 expression because Tamoxifen showed no potentiating effect on the expression of the receptor. In conclusion, our findings reveal that Tamoxifen has immunomodulatory effects whereby it enhances the host IFN signalling pathways during HCV infection.     

Current issues in the management of paediatric viral hepatitis

Viral hepatitis poses important problems for children. In preschoolers, hepatitis A virus (HAV) infection frequently causes acute liver failure. Vaccinating toddlers against HAV in countries with high endemicity is expected to decrease mortality. HAV vaccine demonstrates efficacy (comparable to immunoglobulin) as post‐exposure prophylaxis. A recently developed vaccine against hepatitis E virus (HEV) may benefit fetal health, because pregnant women are most prone to acute liver failure as a result of HEV. Hepatitis B vaccine continues to demonstrate value and versatility for preventing serious liver disease. With chronic infection, undetectable levels of serum HBV DNA complement e‐seroconversion as the preferred outcome measure; suppressed viral load correlates with long‐term complications better than HBeAg status. Among Taiwanese children, low pretreatment HBV DNA (<2 × 10⁸ copies/ml) strongly predicted response to interferon‐α. Future paediatric studies must incorporate HBV DNA levels. The rationale for routine treatment of immunotolerant hepatitis B during childhood remains uncertain. Any treatment of chronic hepatitis B in childhood requires consideration of the risks and benefits. Childhood hepatitis C virus (HCV) infection results mainly from mother‐to‐infant transmission. Babies of HCV‐infected women should be tested for serum HCV RNA at 1 month of age. If negative, confirmatory anti‐HCV antibody testing may be performed between 12 and 15 months of age. Children with chronic hepatitis C may develop progressive fibrosis/cirrhosis, particularly in the setting of obesity and insulin resistance. Treatment of children chronically infected with genotype 2 or 3 is highly successful: combination therapy of pegylated interferon‐α and ribavirin is well tolerated and superior to pegylated interferon‐α alone.     

IL‐28B (IFN‐λ3) and IFN‐α synergistically inhibit HCV replication

Genetic variation in the IL‐28B (interleukin‐28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon‐α and ribavirin. However, the mechanisms by which polymorphisms in the IL‐28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN‐λs and IFN‐α on HCV RNA replication. The anti‐HCV effect of IFN‐λ3 and IFN‐α in combination was also assessed. Changes in gene expression induced by IFN‐λ3 and IFN‐α were compared using cDNA microarray analysis. IFN‐λs at concentrations of 1 ng/mL or more exhibited concentration‐ and time‐dependent HCV inhibition. In combination, IFN‐λ3 and IFN‐α had a synergistic anti‐HCV effect; however , no synergistic enhancement was observed for interferon‐stimulated response element (ISRE) activity or upregulation of interferon ‐ stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN‐λ3‐induced gene expression occurred later and lasted longer than that induced by IFN‐α. In addition, although the genes upregulated by IFN‐α and IFN‐λ3 were similar to microarray analysis, interferon‐stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN‐α and IFN‐λ3 in combination showed synergistic anti‐HCV activity in vitro. Differences in time‐dependent upregulation of these genes might contribute to the synergistic antiviral activity.     

Long‐term stimulation of Toll‐like receptor 3 in primary human hepatocytes leads to sensitization for antiviral responses induced by poly I:C treatment

Chronic hepatitis C infection is associated with increased expression of interferon‐sensitive genes (ISGs) in the liver, which is, paradoxically, correlated with the nonresponse to interferon (IFN)‐based therapies. In the present study PHHs were isolated from HCV‐infected or uninfected patients and stimulated with the TLR1‐9 ligands for 6–24 h. Expression of cytokines and ISGs was determined by ELISA and qRT‐PCR. A comparative analysis was performed for TLR3 signalling, which was also correlated with single nucleotide polymorphisms (SNPs) related to HCV pathogenesis. TLR‐activated PHHs produced pro‐inflammatory and anti‐inflammatory cytokines, whereas IFNs were exclusively induced by TLR3 stimulation. Here, IL‐29 and IL‐28A were significantly highly expressed than IFN‐α and IFN‐β. TLR3‐induced IFN response was enhanced in hepatocytes isolated from patients with HCV infection. This hyper‐responsiveness could be mimicked in naïve PHHs consistently stimulated with low dose of poly I:C, but not Guardiquimod. The higher responsiveness in PHH isolated from HCV‐infected patients could be partially explained by higher frequencies of unfavourable SNP alleles of different SNPs associated with HCV progression and treatment outcome. These data suggest that durable activation of TLR3 but not TLR7, by low doses of viral replicative intermediates, increases the sensitivity to viral invasion. These findings shed new light on the relevance of TLR3 in the pathogenesis of HCV and may provide a possible explanation for the increased ISG expression during chronic HCV infection, the so‐called IFN paradox.     

The development of systemic lupus erythematosus following interferon‐α therapy for hepatitis C infection

Interferon‐alpha (IFN‐α) therapy has been associated with de novo development of systemic lupus erythematosus (SLE), and discontinuation of IFN‐α resulted in disease resolution in most patients. Recurrence of SLE in the absence of concomitant IFN‐α therapy has not been reported. We present here a woman who developed overt clinical manifestations of SLE one year after withdrawal of IFN‐α therapy for hepatitis C virus (HCV) infection. This report highlights the importance of long‐term follow‐up for the belated development of SLE in patients who have experienced IFN‐α induced autoimmune phenomena.     

Adaptive immune responses during acute uncomplicated and fulminant hepatitis E

Background and Aim: Hepatitis E virus (HEV) infection is endemic in several developing countries. Clinical manifestations of this infection vary widely from asymptomatic infection to uncomplicated acute viral hepatitis and fulminant hepatic failure. The pathogenesis of this disease and the reason of varying disease severity remain unknown. In viral infections, tissue injury can be caused either by virus itself or by host immune responses directed against infected cells. We therefore studied adaptive immune responses to HEV antigens in patients with hepatitis E of varying disease severity and healthy controls. Methods: Cytokine secreting CD4+ T cells and antibody‐producing B cells specific for HEV were enumerated through intracellular cytokine staining and enzyme‐linked immunosorbent spot assay, respectively. Results: Patients with fulminant hepatitis E had a less marked expansion of HEV‐specific interferon‐γ or tumor necrosis factor‐α secreting CD4+ T cells than patients with uncomplicated hepatitis E and healthy controls. These patients also had fewer CD4+ T cells that produce γ‐interferon or tumor necrosis factor‐α upon in vitro polyclonal stimulation. In addition, patients with fulminant disease had a more marked expansion of B cells that can secrete immunoglobulin G anti‐HEV than patients with uncomplicated infection and control patients. Conclusion: These findings suggest that less‐marked antiviral cellular immune responses and heightened antiviral humoral responses are associated with a more severe disease during HEV infection.     

High incidence of type 1 diabetes mellitus during or shortly after treatment with pegylated interferon α for chronic hepatitis C virus infection

Background: Development of diabetes mellitus (DM) during or shortly after treatment with interferon α (IFN‐α) in patients with chronic hepatitis C virus (HCV) infection has been reported sporadically. We prospectively screened for DM during and after IFN‐α therapy for chronic HCV infection. Methods: Blood glucose levels of patients with chronic HCV infection were routinely assessed at all outpatient visits during and after treatment with pegylated‐IFN‐α (Peg‐IFN‐α) and ribavirin (Riba). Results: Between December 2002 and October 2005, 189 non‐diabetic patients were treated with Peg‐IFN‐α/Riba, of whom five developed type 1 DM (2.6%), three type 2 DM (1.6%) and one an indeterminate type of DM. Classical symptoms of DM were present in three patients who developed DM shortly after cessation of Peg‐IFN‐α/Riba. In the other patients, symptoms of DM were either indistinguishable from side effects caused by Peg‐IFN‐α/Riba or absent. Conclusion: Our study showed a high incidence of type 1 DM during Peg‐IFN‐α/Riba therapy for chronic HCV infection. Symptoms of DM may be absent or mistaken for Peg‐IFN‐α/Riba‐associated side effects. To diagnose DM without delay, we propose routine assessment of blood glucose at all outpatient visits during and after Peg‐IFN‐α/Riba treatment in chronic HCV patients.     

Induction of interferon‐λ contributes to Toll‐like receptor‐3‐activated hepatic stellate cell–mediated hepatitis C virus inhibition in hepatocytes

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.     

Protein phosphatase 2A impairs IFNα‐induced antiviral activity against the hepatitis C virus through the inhibition of STAT1 tyrosine phosphorylation

Mammalian cells have developed several mechanisms to sense viruses and initiate adequate responses such as production of interferons. Interferons activate the antiviral response through the Jak‐STAT signalling pathway. To establish a chronic infection, viruses need to counteract this barrier of defence. The hepatitis C and hepatitis B viruses are known to up‐regulate the expression of protein phosphatase 2A (PP2A). In this study, we show that PP2Ac associates with Jak1/Tyk2/STAT1 and reduces Jak1/Tyk2/STAT1 phosphorylation resulting in an impairment of the IFNα‐induced HCV antiviral response. Using the fully infectious HCV cell culture system (HCVcc), we demonstrate that the PP2A catalytic activity is not required to block the antiviral effect of IFNα, although it is needed to support HCVcc replication. Our data suggest an important contribution of virus‐induced PP2Ac up‐regulation in the establishment of a chronic infection.     

IFN‐free therapy is associated with restoration of type I IFN response in HIV‐1 patients with acute HCV infection who achieve SVR

Interferon (IFN)‐free direct‐acting antiviral agents (DAAs) have revolutionized chronic hepatitis C virus (HCV) treatment; early studies suggest excellent efficacy in acute HCV. However, changes in innate immune responses during DAA therapy for acute HCV are unknown. We studied interferon‐stimulated gene (ISG) expression and related cytokines/chemokines in HIV‐infected patients with acute HCV receiving sofosbuvir plus ribavirin (SOF+RBV) as part of the A5327 clinical trial. ISG expression was determined from PBMCs, and circulating cytokines/chemokines were quantified from serum from study participants. The overall sustained virologic response (SVR) was 57%; all treatment failures were due to virologic relapse. Apart from NOS2a, baseline ISG/chemokine/cytokine levels were similar irrespective of treatment outcome. Downregulation of ISGs was observed at treatment week four and end of treatment (EOT), implicating HCV in establishing elevated ISGs early during HCV infection. Levels of many of these ISGs increased at post‐treatment week 12 (PTW12) in relapsers only, coinciding with recurrent HCV RNA. Eleven ISGs were differentially expressed in responders vs relapsers. On‐treatment viral suppression was also associated with a reduction in IP‐10, CXCL11 and MIP‐1β levels. In contrast, circulating IFN‐α levels were significantly higher at EOT and PTW12 in responders vs relapsers. Upregulation of peripheral ISG expression is established early in the course of HCV infection during acute HCV infection, but did not predict subsequent treatment outcome with SOF+RBV. ISGs were downregulated during therapy and increased post‐therapy in relapsers. IFN‐α levels were higher in responders at EOT/PTW12, suggesting that impaired type I IFN production/secretion may contribute to relapse.     

Hepatitis C: viral and host factors associated with non‐response to pegylated interferon plus ribavirin

Treatment for chronic hepatitis C virus (HCV) infection has evolved considerably in the last years. The standard of care (SOC) for HCV infection consists in the combination of pegylated interferon (PEG‐IFN) plus ribavirin. However, it only induces a sustained virological response (SVR) in half of genotype 1‐infected patients. Several viral and host factors have been associated with non‐response: steatosis, obesity, insulin resistance, age, male sex, ethnicity and genotypes. Many studies have demonstrated that in non‐responders, some interferon‐stimulated genes were upregulated before treatment. Those findings associated to clinical, biochemical and histological data may help detect responders before starting any treatment. This is a very important issue because the standard treatment is physically and economically demanding. The future of HCV treatment would probably consist in the addition of specifically targeted antiviral therapy for HCV such as protease and/or polymerase inhibitors to the SOC. In genotype 1 patients, very promising results have been reported when the protease inhibitor telaprevir or boceprevir is added to the SOC. It increases the SVR rates from approximately 50% (PEG‐IFN plus ribavirin) to 70% (for patients treated with a combination of PEG‐IFN plus ribavirin plus telaprevir). Different elements are associated with non‐response: (i) viral factors, (ii) host factors and (iii) molecular mechanisms induced by HCV proteins to inhibit the IFN signalling pathway. The goal of this review is to present the mechanisms of non‐response, to overcome it and to identify factors that can help to predict the response to anti‐HCV therapy.     

Hepatitis C virus expression suppresses interferon signaling by degrading STAT1

BACKGROUND & AIMS: The molecular mechanisms by which hepatitis C virus (HCV) antagonizes the antiviral actions of interferon (IFN) have not been fully characterized. Specifically, how HCV proteins impact on IFN signaling components has yet to be elucidated. We used an HCV cell-based expression model to examine the interaction between HCV protein expression and host type I IFN signaling components in the Jak-STAT kinase pathway. METHODS: Full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into Huh-T7 cells. HCV expression was monitored by an HCV core antigen enzyme-linked immunosorbent assay. STAT1, P-STAT1, and HCV protein expression was investigated with immunoprecipitation and Western blots. RESULTS: Overexpression and small interfering RNA studies showed that STAT1 was indispensable for control of HCV expression. STAT1 and P-STAT1 expression were markedly reduced in HCV-transfected cells. Full-length HCV, HCV core/E1/E2, and NS3-4A were each associated with decreased STAT1 expression, which was attributable to proteasome-dependent degradation of STAT1. HCV core, but not HCV E1, E2, NS3, NS4, or NS5, bound to STAT1. STAT2 expression was not affected by HCV. CONCLUSIONS: HCV expression selectively degrades STAT1 and reduces P-STAT1 accumulation in the nucleus in a proteasome-dependent manner. HCV core protein binds STAT1, suggesting that this viral protein is associated with STAT1 degradation. STAT1 plays an indispensable role in innate antiviral immunity against HCV expression. In turn, HCV subverts the Jak-STAT kinase by selectively inducing STAT1 degradation.     

Interferon-α suppressed granulocyte colony stimulating factor production is reversed by CL097, a TLR7/8 agonist

Background and Aim: Neutropenia, a major side‐effect of interferon‐α (IFN‐α) therapy can be effectively treated by the recombinant form of granulocyte colony stimulating factor (G‐CSF), an important growth factor for neutrophils. We hypothesized that IFN‐α might suppress G‐CSF production by peripheral blood mononuclear cells (PBMCs), contributing to the development of neutropenia, and that a toll‐like receptor (TLR) agonist might overcome this suppression. Methods: Fifty‐five patients who were receiving IFN‐α/ribavirin combination therapy for chronic hepatitis C virus (HCV) infection were recruited. Absolute neutrophil counts (ANC), monocyte counts and treatment outcome data were recorded. G‐CSF levels in the supernatants of PBMCs isolated from the patients and healthy controls were assessed by enzyme‐linked immunosorbent assay following 18 h of culture in the absence or presence of IFN‐ α or the TLR7/8 agonist, CL097. Results: Therapeutic IFN‐α caused a significant reduction in neutrophil counts in all patients, with 15 patients requiring therapeutic G‐CSF. The reduction in ANC over the course of IFN‐α treatment was paralleled by a decrease in the ability of PBMCs to produce G‐CSF. In vitro G‐CSF production by PBMCs was suppressed in the presence of IFN‐α; however, co‐incubation with a TLR7/8 agonist significantly enhanced G‐CSF secretion by cells obtained both from HCV patients and healthy controls. Conclusions: Suppressed G‐CSF production in the presence of IFN‐α may contribute to IFN‐α‐induced neutropenia. However, a TLR7/8 agonist elicits G‐CSF secretion even in the presence of IFN‐α, suggesting a possible therapeutic role for TLR agonists in treatment of IFN‐α‐induced neutropenia.     

Baseline sensitivity of T cells to alpha‐IFN correlates with sustained virological response to IFN‐based triple therapy in HCV infection

Chronic infection with HCV is a public health problem with approximately 170 million people infected worldwide. Interferon alpha (IFNα) sensitivity in liver and IL28B genotype has been identified as important determinants of HCV clearance in the setting of pegylated interferon/ribavirin treatment. Herein, we explored IFNα sensitivity in PBMC from 21 healthy donors and 21 HCV‐infected patients treated with pegylated interferon/ribavirin and HCV nonstructural protein‐3 inhibitors (i.e. telaprevir/boceprevir). We explored phospho‐STAT1 level as read‐out for IFN signalling pathway activation in PBMC, T cells and monocytes and correlated results with virological response. We found that PBMC from healthy donors are desensitized to IFNα after priming and challenged with IFNα, with a subsequent decrease of phospho‐STAT1 and interferon‐stimulated genes. Furthermore, we show that CD3+ T cells, but not monocytes, become desensitized after 4 weeks of treatment, with a significant decrease of phospho‐STAT1 after ex vivo IFNα stimulation. Finally, we identified baseline phospho‐STAT1 level in CD3+ T cells as a potential biomarker of sustained virological response, regardless of the IL28B genotype. In the upcoming costly era of IFN‐sparing regimen, baseline IFNα sensitivity could act as biomarker to define cost‐effectiveness strategies of treatment by identifying patients who will or will not respond to IFN‐based treatments.     

The combination of type I interferon and ribavirin has an inhibitory effect on mouse hepatitis virus infection

Aim: To determine the differences in efficacy between therapy using IFN‐β and ribavirin, and using IFN‐α and ribavirin. Methods: We studied the effect of combination therapy consisting of IFN‐β and ribavirin on mouse hepatitis virus (MHV) infection in mice. Results: Combination treatment of ribavirin and IFN‐α was more effective than IFN‐α mono‐treatment in the MHV‐mouse system, and combination treatment with ribavirin and IFN‐β was more effective than IFN‐β mono‐treatment in the MHV‐mouse system. Furthermore, administering IFN‐β once or twice one day before combination treatment using ribavirin and IFN‐α was more effective than administering IFN‐α once or twice one day before the combination treatment using ribavirin and IFN‐α. Conclusion: These data indicate that this MHV‐infection system is a good animal model to assess anti‐HCV activity for therapy using IFN and ribavirin, and suggest that administering IFN‐β before the start of combination therapy with ribavirin and IFN‐α promotes the therapeutic effects of combination treatment with ribavirin and IFN‐α ?in chronic hepatitis C patients.     

Anti‐ulcer agent teprenone inhibits hepatitis C virus replication: potential treatment for hepatitis C

Background: Previously we reported that 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors, statins, inhibited hepatitis C virus (HCV) RNA replication. Furthermore, recent reports revealed that the statins are associated with a reduced risk of hepatocellular carcinoma and lower portal pressure in patients with cirrhosis. The statins exhibited anti‐HCV activity by inhibiting geranylgeranylation of host proteins essential for HCV RNA replication. Geranylgeranyl pyrophosphate (GGPP) is a substrate for geranylgeranyltransferase. Therefore, we examined the potential of geranyl compounds with chemical structures similar to those of GGPP to inhibit HCV RNA replication. Methods: We tested geranyl compounds [geranylgeraniol, geranylgeranoic acid, vitamin K₂ and teprenone (Selbex)] for their effects on HCV RNA replication using genome‐length HCV RNA‐replicating cells (the OR6 assay system) and a JFH‐1 infection cell culture system. Teprenone is the major component of the anti‐ulcer agent, Selbex. We also examined the anti‐HCV activities of the geranyl compounds in combination with interferon (IFN)‐α or statins. Results: Among the geranyl compounds tested, only teprenone exhibited anti‐HCV activity at a clinically achievable concentration. However, other anti‐ulcer agents tested had no inhibitory effect on HCV RNA replication. The combination of teprenone and IFN‐α exhibited a strong inhibitory effect on HCV RNA replication. Although teprenone alone did not inhibit geranylgeranylation, surprisingly, statins' inhibitory action against geranylgeranylation was enhanced by cotreatment with teprenone. Conclusions: The anti‐ulcer agent teprenone inhibited HCV RNA replication and enhanced statins' inhibitory action against geranylgeranylation. This newly discovered function of teprenone may improve the treatment of HCV‐associated liver diseases as an adjuvant to statins.