Interferon-alpha therapy does not modulate hepatic expression of classical type I interferon inducible genes

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Treatment with interferon-alpha(2) (IFN-alpha(2)) can induce viral clearance and marked biochemical and histological improvement. IFN-alpha(2) treatment has been shown to stimulate the expression of type I IFN regulated genes in peripheral blood mononuclear cells (PBMCs) of hepatitis C patients; however, whether it affects hepatic expression remains unknown. This study thus aimed at comparing hepatic gene expression with particular emphasis on type I IFN inducible genes in patients with chronic hepatitis C before and during an IFN-alpha(2) monotherapy. Responsiveness to IFN-alpha(2) therapy was monitored by determining serum and hepatic viral load. Differential gene expression analysis was performed by two different techniques, namely suppression subtractive hybridization (SSH) and differential display (DD). Expression of two prototype type I IFN regulated genes was quantified in further PBMC and liver samples. Among different genes found to be up-regulated during an effective, that is, virus clearing, IFN-alpha treatment, only a single one was identified which can be accounted to type I IFN responsive genes. Parallel quantitative real time PCR analyses demonstrated significant induction of the type I IFN regulated genes MxA and PKR in PBMC, but not in the liver. Taken together, while IFN-alpha treatment leads to the induction of type I IFN regulated genes in PBMC, such an induction appears not to occur in the liver of hepatitis C patients. The mechanism by which IFN-alpha treatment causes viral clearance might be independent of hepatic activation of type I IFN regulated genes.     

Upregulation of endogenous intrahepatic interferon stimulated genes during chronic hepatitis C virus infection

The success of interferon-alpha and ribavirin combination therapy for the treatment of chronic hepatitis C viral infection differs between patients. In an attempt to identify predictors of host response to therapy, the levels of mRNA for interferon (IFN) stimulated genes: MxA, PKR, 2'5' OAS, ISG15, and interleukin 8 (IL-8), were examined in liver by real-time RT-PCR prior to commencement of therapy. The levels of intrahepatic classical IFN stimulated genes, but not IL-8, in chronic HCV disease (n = 44) were found to be significantly upregulated (P < 0.001) compared to the control cohort (n = 12). The genotype of the infecting HCV strain did not influence IFN stimulated gene expression. These results suggest that the endogenous type 1 IFN antiviral effector pathway is broadly activated during chronic HCV disease, although the levels of mRNA for any of the IFN-stimulated genes tested did not predict the outcome of combination therapy.     

Co-infection by serologically-silent hepatitis B virus may contribute to poor interferon response in patients with chronic hepatitis C by down-regulation of type-I interferon receptor gene expression in the liver

Intrahepatic mRNA levels of type-I interferon (IFN) receptor genes have been shown to correlate with the clinical efficacy of IFN therapy in patients with chronic hepatitis C. Recently, co-infection by serologically-silent hepatitis B virus (HBV) has been assumed to be associated with the poor IFN response in patients with chronic hepatitis C. The aim of this study was to investigate the relationship between the co-infection of serologically-silent HBV and type-I IFN receptor gene expression in the liver of patients with chronic hepatitis C. The intrahepatic mRNA levels of IFNAR2, one of the two subunits of the type-I IFN receptor, were quantified and compared with both the prevalence of HBV DNA and the hepatitis C virus (HCV) genotype in 45 patients with chronic hepatitis C, who were negative for hepatitis B surface antigen. Co-infection, as evaluated by a nested polymerase chain reaction, was present in 22 patients (48.9%), with dominance of the HCV genotype 1b (65.2%) over genotype 2a (31.8%). Co-infection was associated with lower IFNAR2 mRNA levels, higher levels of serum HCV RNA, and a poor IFN response, regardless of the HCV genotype. The findings suggest the possibility that co-infection by serologically-silent HBV is one of the factors that can lead to an unfavorable IFN response in chronic hepatitis C by down-regulation of IFN receptor gene expression in the liver.     

Proteasome activator and antigen-processing aminopeptidases are regulated by virus-induced type I interferon in the hepatitis C virus-infected liver.

Many components of the class I antigen-processing pathway are thought to be regulated solely by interferon-gamma (IFN-gamma). Herein, we report type I IFN-mediated induction of proteasome activator (PA28) subunits alpha and beta, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and leucine aminopeptidase (LAP). This mechanism was initiated by either synthetic RNA (poly(I-C)) or by hepatitis C virus (HCV) RNA-mediated induction of type I IFN and abrogated by blocking of type I IFN. In serial liver biopsies of chimpanzees with acute HCV infection, increases in PA28 subunit and aminopeptidase mRNA levels correlated with intrahepatic type I IFN responses and preceded intrahepatic IFN-gamma responses by several weeks. Thus, viral RNA-induced type I IFN regulates the antigen-processing machinery early during viral infection and prior to IFN-gamma response. This mechanism may contribute to the high effectiveness of type I IFN-based therapies if administered early during acute HCV infection.     

How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A

Interferons (IFNs) induce an antiviral state in the cell through complex and indirect mechanisms, which culminate in a direct inhibition of viral replication and stimulation of the host adaptive responses. Viruses often counteract with elaborate strategies to interfere with the induction as well as action of IFN effector molecules. This evolutionary battle between viruses and IFN components is a subject of intense research aimed at understanding the immunopathogenesis of viruses and the molecular basis of IFN signaling and action. In the case with hepatitis C virus (HCV), this may have profound implications for the therapeutic use of recombinant IFN in treating chronic hepatitis C. Depending on the subtype of HCV, current IFN-based treatment regimens are effective for only a small subset of chronic hepatitis C patients. Thus, one of the Holy Grails in HCV research is to understand the mechanisms by which the virus may evade IFN antiviral surveillance and establish persistent infection, which may eventually provide insights into new avenues for better antiviral therapy. Despite the lack of an efficient tissue culture system and an appropriate animal model for HCV infection, several mechanisms have been proposed based on clinical studies and in vitro experiments. This minireview focuses on the HCV NS5A nonstructural protein, which is implicated in playing a role in HCV tolerance to IFN treatment, possibly in part through its ability to inhibit the cellular IFN-induced PKR protein kinase.     

Genotyping of Canadian hepatitis C virus isolates by PCR.

We used PCR for hepatitis C virus (HCV) genotyping with type-specific primers from the core and NS5 genes. Type I was predominant in the general population (58% in blood donors) as well as in different risk groups, such as intravenous drug abusers (58%), blood transfusion recipients (64%), hemophiliacs (62%), and patients with HCV chronic liver disease (76%). Types II, III, and IV were less prevalent in Canada, being found in 10.92, 6.72, and 5.88% of the population, respectively. The type II core primer was not type specific and reacted with the majority of our type I HCV samples, suggesting a false-positive dual infection with two different genotypes (I and II). Digestion of these amplified type I and type II products with restriction endonuclease AccI proved to be very useful in the exclusion of false-positive dual type I and type II infections.     

Global transcriptional profiling demonstrates the combination of type I and type II interferon enhances antiviral and immune responses at clinically relevant doses.

A role for type II interferon (IFN-gamma) in resolving viral infection is suggested by the correlation of hepatitis C virus (HCV) clearance with enhancement of IFN-gamma-producing activated T cells in the resolution of acute HCV infection. Using vesicular stomatitis virus (VSV), a synergistic direct antiviral effect was documented using IFN-gamma1b and a potent, consensus type I IFN (IFN alfacon-1). Global expression profiling following EC50 exposure to IFN alfacon-1, IFN-gamma1b, or a cocktail of the two allowed the antiviral state to be correlated with induction of a subset of IFN-stimulated genes (ISGs). Genes identified through this analysis corresponded to classic antiviral components, ISGs more recently associated with direct antiviral functions, as well as expressed sequence tags (ESTs) and hypothetical proteins. The magnitude of these antiviral EC50-correlated expression events in human hepatoma (Huh7) cells exposed to clinically relevant doses of IFN alfacon-1, IFN-gamma1b, or a cocktail of the two was also probed because the standard of care for patients with chronic hepatitis C is type I IFN-containing regimens. Relative to type I IFNs used alone, the addition of type II IFN caused enhanced expression not only of many of the genes correlated with the direct antiviral state but also of genes involved in (1) antigen presentation to cytotoxic T lymphocytes (CTLs), (2) macrophage, natural killer (NK), and T helper 1 (Th1) cell recruitment and activation, (3) complement system function, (4) apoptosis, and (5) ISGs with unknown functions. As many of these processes are correlated clinically with resolution of chronic HCV infection, the combined use of these IFNs could display a beneficial effect on viral clearance in patients infected with HCV and other viruses through enhancement of one of these processes or of the direct antiviral state.     

Expression of interferon alpha/beta receptor in the liver of chronic hepatitis C patients

Interferon (IFN) demonstrates antiviral activity by binding to receptors on the cell surface. Expression of the IFN receptor in hepatocytes may be directly associated with a hepatitis C virus (HCV) infection and the response to IFN therapy. A competitive PCR method was developed to measure IFN alpha/beta (α/β) receptor mRNA in liver samples obtained by needle biopsy. Thirty-one patients with chronic hepatitis C (21 without cirrhosis, 10 with cirrhosis) and six normal subjects were used. Eighteen of the 21 patients without cirrhosis received the IFN therapy. Competitive PCR was carried out using IFN α/β receptor gene-specific primers and a specific competitor. Expression of the receptor was detected in all liver samples. There was no association between the expression level and serum alanine aminotransferase level, serum (2′–5′) oligo (A) synthetase level, amount of serum HCV RNA, or HCV genotype. The expression level in patients with chronic hepatitis was significantly higher than that in normal livers (P < 0.05) and in cirrhotic livers (P < 0.01). Seven of the 18 patients treated with IFN demonstrated a sustained response to IFN (sustained responders), and the remaining 11 did not (nonsustained responders). The expression level of IFN α/β receptor mRNA in the sustained responders was significantly higher than that in the nonsustained responders (P < 0.01). Thus, the expression of IFN α/β receptor mRNA may be one of the host factors influencing the response to IFN therapy. J. Med. Virol. 56:217–223, 1998. © 1998 Wiley-Liss, Inc.     

Hepatitis C virus genotypes,reactivity to recombinant immunoblot assay 2 antigens and liver disease

To clarify the relationship between hepatitis C virus (HCV) genotypes and liver disease, we typed HCV genomes in the sera of 151 blood donors, 180 patients with type C chronic liver disease (CLD), and 30 haemophiliacs residing in Hiroshima, Japan. All of the subjects were positive for anti-HCV and HCV-RNA, and were examined for seroreactivity to HCV-specific antigens. The HCV genotypes were determined by polymerase chain reaction (PCR) with type-specific primers deduced from the putative core region of the HCV genome. Significantly more (P< 0.001) type II HCV was found in the samples from the CLD patients (80%) than in those from the blood donors (55%). Significantly more (P< 0.001) type III HCV was found in the samples from the blood donors (29.1%) than in those from the CLD patients (11.7%). There was no significant difference in the distribution of the HCV types among the patients with chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. A four-antigen recombinant immunoblot assay (RIBA-2) assay was used to compare the serum samples for their reactivity to a range of structural and nonstructural peptides specific for HCV (5–1–1, C100-3, C33c, and C22-3). The frequency of serop-ositivity to 5–1–1 and C100-3 was significantly higher (P < 0.001) in type II HCV-infected blood donors than in type III HCV-infected donors (68.2% and 65.9% vs. 4.5% and 22.7%, respectively). Among the type III HCV-infected individuals, the CLD patients had a significantly higher (P<0.01) frequency of seropositivity to 5–1–1 than the blood donors (33.3% vs. 4.5%). These results suggest that type II HCV is more likely than type III HCV to induce clinical disease, and further, that the difference in the extent of synthesis of the viral protein (5–1–1) between these types may play a role in the pathogenicity of HCV. © 1994 Wiley-Liss, Inc.     

干扰素治疗相关的自身免疫性副作用研究进展

干扰素(IFNs)是一种广泛存在的具有强烈抗病毒作用的细胞因子,IFN-α被认为是目前慢性乙型肝炎和丙型肝炎的标准治疗方法.病毒感染时,尤其是丙型肝炎病毒(HCV)感染,包含核酸的免疫复合物诱导的IFN-α产生增加,持续性的免疫系统激活诱导自身免疫性损伤.IFN为基础的治疗可以加重这种自身免疫损伤.IFNs可以影响多种类型的细胞,使多个系统受到影响.因此,用IFN-α治疗的患者可以出现广谱的自身免疫性疾病,如自身免疫性甲状腺、风湿性关节炎、冷球蛋白血症、结节病、系统性红斑狼疮、1型糖尿病和重症肌无力等.     

Interferon-inducible gene expression in chimpanzee liver infected with hepatitis C virus.

The molecular host response to hepatitis C virus (HCV) infection was examined by isolation of HCV-induced genes from a cDNA library constructed from chimpanzee liver during the acute phase of hepatitis C. Two cDNA clones, 130-7 and 130-51, were obtained by differential hybridization with cDNA probes prepared from poly(A)+ RNAs of infected and uninfected livers. Northern blot analysis revealed that the 130-7 and 130-51 cDNAs were expressed as 1.5- and 1.0-kb products, respectively, in chimpanzee liver and that their induction rates of the two were 20 and 4, respectively. Nucleotide sequence analyses of these cDNA inserts showed that the sequence of cDNA 130-7 was that of a class I major histocompatibility antigen and that the sequence of cDNA 130-51 was 98% homologous with a human interferon-inducible mRNA. These results suggest that HCV infection may actively induce interferon, which in turn induces the expressions of these interferon-inducible genes. Furthermore, the high expression of HLA class I antigen in the acute phase of hepatitis C suggests that liver cell injury in HCV infection may be mediated by cytotoxic T cells that recognize viral antigen in association with HLA class I antigen.     

Emerging roles of interferon-stimulated genes in the innate immune response to hepatitis C virus infection

Infection with hepatitis C virus (HCV), a major viral cause of chronic liver disease, frequently progresses to steatosis and cirrhosis, which can lead to hepatocellular carcinoma. HCV infection strongly induces host responses, such as the activation of the unfolded protein response, autophagy and the innate immune response. Upon HCV infection, the host induces the interferon (IFN)-mediated frontline defense to limit virus replication. Conversely, HCV employs diverse strategies to escape host innate immune surveillance. Type I IFN elicits its antiviral actions by inducing a wide array of IFN-stimulated genes (ISGs). Nevertheless, the mechanisms by which these ISGs participate in IFN-mediated anti-HCV actions remain largely unknown. In this review, we first outline the signaling pathways known to be involved in the production of type I IFN and ISGs and the tactics that HCV uses to subvert innate immunity. Then, we summarize the effector mechanisms of scaffold ISGs known to modulate IFN function in HCV replication. We also highlight the potential functions of emerging ISGs, which were identified from genome-wide siRNA screens, in HCV replication. Finally, we discuss the functions of several cellular determinants critical for regulating host immunity in HCV replication. This review will provide a basis for understanding the complexity and functionality of the pleiotropic IFN system in HCV infection. Elucidation of the specificity and the mode of action of these emerging ISGs will also help to identify novel cellular targets against which effective HCV therapeutics can be developed.     

Anti-extractable nuclear antigens (ENA) antibodies in patients with chronic hepatitis C before and after treatment with interferon

A high prevalence of serological markers classically associated with autoimmune hepatitis or other autoimmune diseases has been reported in patients with chronic hepatitis C. However, the prevalence of antibodies to extractable nuclear antigens (anti-ENA) are rarely reported in such patients and the effect of treatment with interferon (IFN) on their prevalence is not known. In the present study, serum samples collected from 44 patients with chronic hepatitis C and 44 patients with non-hepatitis C virus (HCV) infected liver diseases were tested for anti-ENAs (U1 RNP, Sm, Ro/SS-A, La/SS-B and Scl-70) antibodies by enzyme-linked immunosorbent assay (ELISA). In 26 patients with chronic hepatitis C who received IFN treatment, serum samples were also collected just after completion of IFN treatment, and/or at 6-40 months after completion of the treatment, and tested for these antibodies. Sixteen (36%) of 44 sera from patients with chronic hepatitis C were positive for at least one of the above anti-ENA antibodies, whereas only 7 (16%) of 44 sera from patients with non-HCV infected liver diseases were positive for such antibodies (p = 0.0290). There was no significant difference in the prevalence of each of anti-ENA antibody between men and women. Results of anti-ENA antibodies in most IFN-treated patients with chronic hepatitis C did not change after treatment. However, in some cases serum anti-U1 RNP, anti-La/SS-B and anti-Scl-70 became negative or converted to the gray zone after completion of IFN treatment regardless of HCV elimination. Our results showed that the overall prevalence of anti-ENA antibodies was significantly higher in patients with chronic hepatitis C than in those with non-HCV-infected liver diseases. However, the disappearance of anti-ENA antibodies after IFN treatment in patients with chronic hepatitis C may be due to the immunomodulating effects of IFN rather than HCV elimination.     

Anti-extractable Nuclear Antigens (ENA) Antibodies in Patients with Chronic Hepatitis C before and after Treatment with Interferon

A high prevalence of serological markers classically associated with autoimmune hepatitis or other autoimmune diseases has been reported in patients with chronic hepatitis C. However, the prevalence of antibodies to extractable nuclear antigens (anti-ENA) are rarely reported in such patients and the effect of treatment with interferon (IFN) on their prevalence is not known. In the present study, serum samples collected from 44 patients with chronic hepatitis C and 44 patients with non-hepatitis C virus (HCV) infected liver diseases were tested for anti-ENAs (U1 RNP, Sm, Ro/SS-A, La/SS-B and Scl-70) antibodies by enzyme-linked immunosorbent assay (ELISA). In 26 patients with chronic hepatitis C who received IFN treatment, serum samples were also collected just after completion of IFN treatment, and/or at 6–40 months after completion of the treatment, and tested for these antibodies. Sixteen (36%) of 44 sera from patients with chronic hepatitis C were positive for at least one of the above anti-ENA antibodies, whereas only 7 (16%) of 44 sera from patients with non-HCV infected liver diseases were positive for such antibodies (p=0.0290). There was no significant difference in the prevalence of each of anti-ENA antibody between men and women. Results of anti-ENA antibodies in most IFN-treated patients with chronic hepatitis C did not change after treatment. However, in some cases serum anti-U1 RNP, anti-La/SS-B and anti-Scl-70 became negative or converted to the gray zone after completion of IFN treatment regardless of HCV elimination. Our results showed that the overall prevalence of anti-ENA antibodies was significantly higher in patients with chronic hepatitis C than in those with non-HCV-infected liver diseases. However, the disappearance of anti-ENA antibodies after IFN treatment in patients with chronic hepatitis C may be due to the immunomodulating effects of IFN rather than HCV elimination.     

检测丙型肝炎患者NS5区抗体的临床意义探讨

目的检测慢性丙型肝炎患者和１４例ＨＣＶ原发感染后ＮＳ５抗体长达一年半的动态变化，探讨ＮＳ５抗体的临床意义。方法应用重组ＮＳ５抗原，建立ＥＩＡ方法进行检测。结果慢性丙型肝炎患者抗－ＮＳ５抗体阳性率为６０．４８％，ＨＣＶ感染后一月抗－ＮＳ５抗体阳性率为１６．３５％，三月为７５％。结论抗－ＮＳ５抗体无早期诊断价值。抗－ＮＳ５抗体持续阳性者，血清ＡＬＴ多明显升高，抗－ＮＳ５抗体与肝脏疾病活动性相关。丙型肝炎患者中存在抗－Ｃ、抗－ＮＳ３、抗－ＮＳ４抗体阴性，而抗－ＮＳ５抗体单独阳性，表明检测抗－ＮＳ５抗体具有独特的诊断价值     

Expression of interferon receptor genes in the liver as a predictor of interferon response in patients with chronic hepatitis C.

Interferon (IFN) receptor mRNA expression patterns in the liver have been shown to correlate with the effectiveness of IFN therapy in patients with hepatitis C virus (HCV) infection. However, it is not clear to what extent this factor contributes to the short (primary)- and long (sustained)-term results of IFN treatment with respect to biochemical and virological remission. Eighty-two patients who subsequently received lymphoblastoid IFN-alpha therapy underwent liver biopsies before IFN therapy. Possible factors that might correlate with IFN response were chosen and analyzed. The primary biochemical and virological responses at the end to treatment (24 weeks) were 63% and 43% vs. 46% and 32% for sustained biochemical and virological remission at the end of follow-up (48 weeks), respectively. In univariate analysis, the absence of HCV genotype 1b, a low titer of HCV RNA, and the expression of IFN receptor mRNA were significantly correlated with sustained biochemical and virological responses to IFN therapy. Multiple logistic regression analysis showed that IFN receptor mRNA expression and the absence of genotype 1b were significant predictors of the sustained biochemical and virological effectiveness of IFN therapy. IFN receptor mRNA expression predicted a sustained virological response to IFN therapy with a positive predictive value of 100% with genotype non-1b and had a negative predictive value of 97% with genotype 1b. It is concluded that expression of IFN receptor genes in the liver is a useful index for predicting the short- and long-term efficacy of IFN therapy in patients with chronic HCV infection.     

Expression of thioredoxin and thioredoxin-binding protein-2 in the liver of patients with chronic hepatitis C as a predictor of response to interferon therapy

Oxidative stress contributes to the pathogenesis of various hepatic injuries. Thioredoxin (TRX) is an indicator of oxidative stress, reported to be increased in the serum of patients with chronic hepatitis C with the progression of fibrosis. The aim of this study was to evaluate the clinical significance of the expression of TRX and thioredoxin-binding protein-2 (TBP-2), which is a negative regulator of TRX function, in the liver of patients with chronic hepatitis C and the relationship of this to the efficacy of interferon (IFN) treatment. A retrospective study was performed using the liver biopsy specimens obtained before IFN treatment from 69 patients with chronic serotype 1 hepatitis C virus (HCV) infection. TRX and TBP-2 mRNA levels in the liver biopsy specimens were amplified by real-time RT-PCR. The serum TRX protein level was estimated with a sandwich enzyme-linked immunosorbent assay kit, and the expression of TRX protein in the liver was examined immunohistochemically in 19 patients. There was no association between the serum TRX level and the TRX level in the liver. There was a significant correlation between the expression level of TRX protein in the liver and the TRX mRNA level in the liver. TRX and TBP-2 levels in the liver tended to decrease slightly with increased fibrosis stage, although not significantly. The TRX level in the liver tended to increase with hepatitis activity index, although not significantly. TBP-2 mRNA levels in the liver were significantly higher in responders than non-responders to the IFN therapy (p<0.05). Among patients who had a high viral load of >850 KIU/ml, the TRX level in the livers of non-responders was significantly lower than that in the livers of responders (p<0.05). TRX and TBP-2 mRNA levels in the liver before IFN therapy may predict the outcome of IFN therapy in patients with chronic serotype 1 HCV infection.     

Tissue localization of Toll-like receptors in biopsy specimens of liver from children infected with hepatitis C virus

Toll-like receptors (TLR) are important tools of innate immunity, localized mainly on cells of the immune system, but also have been shown on cells of other origin. In the current study, they have been searched in biopsy specimens of liver from children bearing chronic viral hepatitis of C type (HCV). TLR2, TLR3 and TLR4 were traced by means of polyclonal antibodies and avidin-biotin complex (ABC) immunohistochemistry. Besides, mRNA for TLR was looked for using specific primers and polymerase chain reaction. Several controls, including neutralization of primary antibody with respective blocking peptide, confirmed the specificity of the immunohistochemical reaction. All TLR tested could be visualized in a focal distribution in single hepatocytes and some cells of inflammatory infiltrates. There was no reaction whatsoever in liver samples not infected with hepatotropic virus. In molecular studies, mRNA for TLR2 and TLR4 was detected in both noninfected and hepatitis B virus-infected established cell lines of human hepatoma as well as in HCV(+) biopsy samples. These data indicate that TLR can be traced in liver cells, both at the protein and at the mRNA level. Their irregular and focal distribution in HCV(+), but not in HCV(-), liver suggests some role of TLR in the pathogenesis of chronic viral hepatitis, at least in children.