Project description:NK cells are believed to contribute to the control of hepatitis C virus infection and pathogenesis of liver disease. Standard treatment of both acute and chronic hepatitis C is based on the administration of interferon alpha, however, the effects of type I interferons on human NK cells have not been studied in the context of hepatitis C. We therefore first performed a microarray screen for genes differentially regulated in human NK cells after stimulation of PBMC with recombinant interferon alpha-2b. One of the genes upregulated was TRAIL which was confirmed in vitro on the protein level. Keywords: Interferon alpha; human NK cells; stimulation for 6 hours; cells sorted after stimulation of whole PBMC
Project description:NK cells are believed to contribute to the control of hepatitis C virus infection and pathogenesis of liver disease. Standard treatment of both acute and chronic hepatitis C is based on the administration of interferon alpha, however, the effects of type I interferons on human NK cells have not been studied in the context of hepatitis C. We therefore first performed a microarray screen for genes differentially regulated in human NK cells after stimulation of PBMC with recombinant interferon alpha-2b. One of the genes upregulated was TRAIL which was confirmed in vitro on the protein level. Keywords: Interferon alpha; human NK cells; stimulation for 6 hours; cells sorted after stimulation of whole PBMC NK cells of PBMC of five healthy controls have been either isolated directly or after culturing for 6h in media containing 10% human AB serum supplemented with 1 and 100 ng/ml recombinant IFNa-2b. RNA of NK cells of the healthy controls was isolated and RNA was pooled for the hybridization.
Project description:The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional responses of neonatal woodchucks with self-limiting and progressing persistent infection with woodchuck hepatitis virus (WHV). This revealed that WHV does not induce intrahepatic gene expression during the early acute stage of infection (8 weeks), suggesting it is a “stealth” virus. At the mid-acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T cell signature, with perforin and other markers of immune-mediated cytotolytic response being strongly expressed. Strikingly, this was accompanied by high level expression of PD-1 and various other inhibitory T cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. Conversely, self-limiting infection was not associated with a strong interferon-α/β (IFN-α/β) transcriptional response, while the IFN-γ signaling response (as measured by expression of CXCL9) in the mid-acute phase was comparable to that in chronically infected adult animals. Nevertheless, viperin and other antiviral genes were differentially expressed during resolving infection, suggesting that a subset of interferon-stimulated genes (ISGs) may play a role in the control of WHV replication. Conclusion: This study identifies new immune pathways associated with the clearance of hepadnavirus infection and reveals novel targets with potential for the treatment of chronic hepatitis B.
Project description:To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-alpha in woodchuck, the transcriptional profiles of woodchuck liver were studied by using RNASeq Woodchucks liver samples with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-alpha or with placebo for 26 weeks
Project description:Approximately 50% of patients with chronic hepatitis C (CHC) have a sustained virologic response (SVR) to treatment with pegylated interferon (pegINF)-α and ribavirin. Non-response to treatment is associated with constitutively increased expression of IFN-stimulated genes (ISGs) in the liver. Treatment of patients with acute hepatitis C (AHC) is more effective, with SVR rates >90%. We investigated mechanisms of the different responses of patients with CHC and AHC to pegIFN-α therapy. We analyzed IFN signaling and ISG expression in liver samples from patients with acute hepatitis C (AHC), patients with chronic hepatitis (CHC), and individuals without hepatitis C (controls) using microarray, immunohistochemical, and protein analyses. Findings were compared with those from primary human hepatocytes stimulated with IFN-α or IFN-γ, as reference sets. Expression levels of 100s of genes, primarily those regulated by IFN-γ, were altered in liver samples from patients with AHC compared with controls. Expression of IFN-γ–stimulated genes was induced in liver samples from patients with AHC, whereas expression of IFN-α–stimulated genes was induced in samples from patients with CHC. In an expression analysis of negative regulators of IFN-α signaling, we did not observe differences in expression of SOCS1 or SOCS3 between liver samples from patients with AHC and those with CHC. However, USP18 (another negative regulator of IFN-α signaling), was upregulated in liver samples of patients with CHC that did not respond to therapy, but not in AHC. In conclusion, differences in expression of ISGs might account for the greater response of patients with AHC, compared to those with CHC, to treatment with pegINF-α and ribavirin. Specifically, USP18 is upregulated in liver samples of patients with CHC that do not respond to therapy, but not in patients with AHC. (Interferon-γ Stimulated Genes, but not USP18, are Expressed in Livers of Patients with Acute Hepatitis C; Dill MT, Makowska Z et al, Gastroenterology 2012 (in press)) Primary human hepatocytes from 2 donors were analyzed. From each donor there are 5 samples: untreated cells, cells treated with interferon alpha (1000 IU/ml) for 6 and 24 hours and cells treated with interferon gamma (1000 IU/ml) for 6 and 24 hours.
Project description:Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFN-alpha) and ribavirin. It achieves a sustained viral clearance in only 50â??60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFN-alpha. In patients with a rapid virological response to treatment, pegIFN-alpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFN-alpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway. Experiment Overall Design: Total of 78 Samples (before and after interferon treatement) are analyzed using Affymetrix Human U133 Plus 2.0 Array.
Project description:The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional responses of neonatal woodchucks with self-limiting and progressing persistent infection with woodchuck hepatitis virus (WHV). This revealed that WHV does not induce intrahepatic gene expression during the early acute stage of infection (8 weeks), suggesting it is a M-bM-^@M-^\stealthM-bM-^@M-^] virus. At the mid-acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T cell signature, with perforin and other markers of immune-mediated cytotolytic response being strongly expressed. Strikingly, this was accompanied by high level expression of PD-1 and various other inhibitory T cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. Conversely, self-limiting infection was not associated with a strong interferon-M-NM-1/M-NM-2 (IFN-M-NM-1/M-NM-2) transcriptional response, while the IFN-M-NM-3 signaling response (as measured by expression of CXCL9) in the mid-acute phase was comparable to that in chronically infected adult animals. Nevertheless, viperin and other antiviral genes were differentially expressed during resolving infection, suggesting that a subset of interferon-stimulated genes (ISGs) may play a role in the control of WHV replication. Conclusion: This study identifies new immune pathways associated with the clearance of hepadnavirus infection and reveals novel targets with potential for the treatment of chronic hepatitis B. Neonatal woodchucks of both genders were infected at 3 days of age with the same WHV7P1 inoculum containing 5 x 106 WID50 of WHV strain WHV7-11. Custom microarrays were generated from sequences obtained in transcriptome sequencing of woodchuck liver and PBMCs, and were used to examine liver gene expression in animals which eventually become chronically infected with WHV (8 weeks, n=5; 14 weeks, n=9), animals that eventually resolve WHV infection (8 weeks, n=10;14 weeks, n=7) and uninfected animals (8 weeks, n=5;14 weeks,n=5).
Project description:Hepatitis C virus (HCV) chronically infects 170 million people worldwide and is a leading cause of liver-related mortality due to hepatocellular carcinoma and cirrhosis1. Standard-of-care treatment is shifting from interferon-alpha (IFNM-NM-1)-based to IFNM-NM-1-free directly acting antiviral (DAA) regimens, which demonstrate improved efficacy and tolerability in clinical trials2,3. Virologic relapse after completion of DAA therapy is a common cause of treatment failure, although mechanisms are unclear2,3. We conducted a clinical trial using the DAA sofosbuvir with ribavirin (SOF/RBV)4, and report here detailed mRNA expression analysis of pre- and end-of-treatment (EOT) liver biopsies and blood samples. On-treatment viral clearance was accompanied by rapid down-regulation of interferon-stimulated genes (ISGs) in liver and blood. Analysis of paired liver biopsies from patients who achieved a sustained virologic response (SVR) revealed that viral clearance was accompanied by decreased expression of ISGs, IFNG, and IFNLs, but increased expression of IFNA2. Patients who achieved SVR had higher expression of a hepatic type-I interferon gene signature in unpaired EOT liver biopsies than patients who later relapsed. Together, these results support a model whereby restoration of type-I intrahepatic interferon signaling at the EOT is associated with sustained hepatic HCV suppression and prevention of relapse upon withdrawal of SOF/RBV. Sustained Virologic Response for Chronic Hepatitis C Patients Treated with Sofosbuvir and Ribavirin
Project description:All major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo. Remarkably, HCV replication is not sensitive to IFN? in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 to identify effector genes of the IFN? response and thereby identified the DExD/H box helicase DDX60L as a restriction factor of HCV replication. DDX60L and its homolog DDX60 were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells, and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. DDX60L had no impact on replication of hepatitis A virus (HAV), but severely impaired production of lentiviral vectors, arguing for a potential antiretroviral activity. Detection of endogenous DDX60L protein turned out to be difficult due to instability. DDX60L knockdown did not alter interferon stimulated gene (ISG) induction after IFN treatment, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found no impact of DDX60L on translation or stability of HCV subgenomic replicons, nor additional impact on entry and assembly of infectious virus. Similar to its homolog DDX60, DDX60L had a moderate impact on retinoic acid-inducible gene I (RIG-I)-dependent activation of innate immunity arguing for additional functions in the sensing of viral RNA. Gene Expression was compared between two cell lines, Huh6 and Huh7, under interferon-gamma or interferon-alpha treatment. We intended to identify genes that are more strongly upregulated in Huh-7 than in Huh6 in response to interferon treatment.
Project description:Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFN-alpha) and ribavirin. It achieves a sustained viral clearance in only 50–60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFN-alpha. In patients with a rapid virological response to treatment, pegIFN-alpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFN-alpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway. Keywords: Comparison of human patient biopses before and after interferon treatment