| Hepatitis C virus (HCV) is estimated to infect more than 170 million people worldwide. HCV infection usually causes chronic hepatitis in approximately 80% of infected individuals with 20% progressing to cirrhosis and 1-2% developing hepatocellular carcinoma. Currently, Interferon-α (IFN-α) is one of the most efficient drugs for the treatment of chronic hepatitis C and the standard treatment for HCV infection is IFN-α in combination with ribavirin. Unfortunately, more than 60% of patients with chronic HCV infection will experience either no control of viral replication by therapy (nonresponders) or a relapse when therapy is stopped (relapsers). The molecular mechanisms underlying failure of IFN-α treatment are not well understood. As our previous research found no correlation between viral genomic sequence and outcome of IFN-α treatment, host factors associated with IFN therapy, were explored in this study.It has been proposed that HCV has the ability of interfering with type I interferon (IFN-α/β) production, blocking JAK-STAT signaling transduction, inhibiting expression or activity of ISGs (interferon -stimulated genes), which may support HCV replication and persistence as well as resistance to IFN-α therapy. Host gene expression is the outcome of host reaction to infection and viral interference with host, and may correlate with efficiency of IFN-α treatment. To identify signature or pattern of host gene expression which can distinguish different outcome of therapy, expression of type I interferon, interferon regulated factors (IRFs), positive and negative regulatory factors of IFN signaling, classical antiviral effectors and whole cellular genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 34 chronic hepatitis C (CHC) patients before, during and after IFN/ribavirin treatment byreal-time quantitative RT-PCR and microarray. Among them, 6 of 34 were nonresponders (NR) and 28 of 34 were sustained responders (SR) according to serum HCV RNA test at the end of treatment and 6 months post-treatment. Gene expression was compared between these two groups.To identify the correlation between gene expression of type I interferon, IRFs and outcome of IFN therapy, gene expression level of IFN-α/β, IRF-1, IRF-3, IRF-5 and IRF-7 were quantified in CHC patients. Result showed that, IFN-α and IFN-β expression level in the patients and donors were not significantly different (P: 0.927 and 0.937). However, IRF family members were differentially expressed in the two patient groups. IRF-3 expression was not influenced by chronic infection (P=0.980) and IRF-7 expression was not significantly different between patients and donors(P=0.071); IRF-1 expression levels were obviously enhanced in patients(P<0.001) and IRF-5 expression level was remarkably lower than that of the donors (P=0.002). The relevance analysis of gene expression showed that, while IRF-1 expression and the IFN-α/β expression did not have relevance, IRF-7 expression and IFN-α expression were correlated (R= 0.453; P= 0.007) and IRF-5 expression correlated with expression of both IFN-α and IFNβ (R=0.686, 0.621; P <0.001, 0.001). This result may reflect the mechanism of low expression of host type I interferon, which might be associated with low expression of key factors that regulated interferon's synthesis. However, further analysis did not reveal any correlation between gene expression of IFN-α/β, IRF-1, IRF-3, IRF-5 and IRF-7 before treatment and responses of host to interferon treatment. In order to confirm the influence of HCV persistent infection on IRF and type I interferon, HCV replicon cell (Huh7 cell line harboring subgenomic HCV replicons) was used. Result showed that, under NDV attack, IRF-7 promoter activity or the endogenous IRF-7 and IFN-α expression level of HCV replicon cells was lower than that of Huh7 cell (fold activity of IRF-7 promoter: 2.54 vs. 4.20, P<0.001; endogenous IRF-7mRNA expression level: 0.137 vs. 1.671, P<0.001; endogenous IFN-α mRNA expression level: 0.246 vs 2.207, P<0.001). On inhibition of HCV replication by ribavirin or IFN-α treatment, both IRF-7 promoter activity and expression of endogenous IRF-7and IFN-α were restored. This result may suggest a new mechanism of inhibition of type I interferon by HCV.As the interferon-induced intracellular antiviral effect depends largely on efficiency of signal transduction (JAK-STAT pathway), the effect of HCV chronicinfection on expression of positive and negative regulatory genes of JAK-STAT pathway in patients' PBMCs was further analyzed. Results showed that, compared to healthy donors, the expressions of positive regulatory genes (STAT1 and P48) and negative regulatory genes (SOCS1 and SOCS3) were markedly enhanced in CHC patients (STAT1: CHC vs Donors 96.5 vs. 69, P=0.039; P48: CHC vs. Donors 121.5 vs. 66.9, P=0.013; SOCS1: CHC vs. Donors 5.96 vs. 2.13, P=0.005; SOCS3: CHC vs. Donors 78.3 vs. 18.9, P<0.001). Expression level of SOCS1 and SOCS5 before treatment was negatively correlated with serum HCV RNA level (R= -0.427 and -0.385; P= 0.013 and 0.027). As SOCS1 and the SOCS5 are mainly expressed in the Thl cell, the above result indicated that HCV replication might be able to suppress the Thl immunity. Further analysis of relevance between expression of these key factors of signaling pathway and outcome of IFN therapy showed high SOCS3 expression was predictive of no response to IFN therapy (NR vs. SR 203.0 vs. 64.1, P=0.007). It indicates that SOCS3 expression level may be used as marker for prediction of IFN-α therapy. Using flow cytometer to analyze the cytokine secreting cells, we found a switch-over of cytokine synthesis from Th2 to Th1 (IFN-γ for Th1, IL-4 for Th2) (Th1: 0 week vs. 12 weeks 4.28±2.4% vs. 7.63±3.5%, P<0.01; Th2: 0 week vs. 12 weeks 0.66±0.52% vs. 0.37±0.53%, P= 0.013) during IFN-α therapy, which was paralleled to up-regulation of SOCS1 and SOCS5 level and down-regulation of SOCS3 level. This result indicated that SOCS expression level in PBMC might be used to evaluate immune regulatory effect of IFN-α treatment. In vitro assay in cell line further showed that the expression of SOCS1 and SOCS3 in Huh7 cell line were induced by HCV Core protein, but not by other HCV non-structural proteins.As antiviral effect of interferon mainly depends on induction of antiviral effectors molecule, gene expression of classical antiviral effectors MxA, PKR and 2 ' 5 ' OAS before and during treatment and its correlation with outcome of therapy were further analyzed. Results showed that, compared with healthy donors, MxA, PKR and 2 ' 5 ' OAS expression in patients were obviously enhanced (MxA: CHC vs. Donors 141.4 vs. 5.5, P<0.001; PKR: CHC vs. Donors 75.7 vs. 12.3, P<0.001; 2'5'OAS: CHC vs. Donors 72.6 vs. 4.18, P<0.001) and were further elevated by IFN-α treatment. However, there was no correlation of both basal expression levels and fold induction during treatment of the above genes with outcome of IFN treatment, which suggest that MxA, PKR and 2'5' OAS gene expression cannot be used as predictive marker for IFN therapy. DNA microarrays(Affymetrix Com, HG U-133A, containing 13000cellular genes and EST) were further used to analyze the global gene expression of PBMCs in NR and SR groups. Comparing the fold of induction of genes from these two groups, we found that 16 genes were differentially expressed between NR and SR. These genes included ISGs (STAT1, GBP1, IFIT1, GIP3 and IFIT2), chemokines (CXCL10 and CCL8), genes related with signaling pathway (STK3, RASGRP3, RIN2 and LEPR), metabolism-correlated gene (APOBEC3A, MANEA and ZC3HAV1) and several genes with uncertain function (REC8L1 and C6orf62).In summary, our research results indicated that HCV chronic infection has the influence on expression of type I interferon and its related genes in PBMCs. Under HCV chronic infection situation, while expression of type I interferon and its key regulatory factor was suppressed, the expression of the positive and negative regulatory factors of interferon signaling pathway were strengthened. Furthermore, while the expression of type I interferon, its key regulatory factor and MxA, PKR, 2'5'OAS before treatment have no relationship with outcome of IFN-α treatment, there was correlation of expression of negative factor SOCS3 with outcome of IFN-α treatment. In addition, 16 cellular genes in PBMCs were differentially expressed between NR and SR. Further study with HCV replicon cells is needed to understand the molecular mechanism underlying intervention of host cell gene expression by HCV chronic infection. The possible application of host gene expression pattern in guiding clinical interferon treatment such as predicting responses to IFN treatment need to be further investigated in a larger cohort of CHC patients. These findings, on the one hand, can enrich our knowledge about interaction between HCV and host defense system; on the other hand, may help to optimize clinical treatment and improve efficacy of IFN, and finally realize the individualized interferon treatment for HCV patients. |
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