Interferon signaling in chronic hepatitis C : mechanisms and implications for therapy

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide and can lead to liver cirrhosis and hepatocellular carcinoma. The current standard therapy of chronic hepatitis C (CHC) consists of a combination of pegylated interferon alpha (pegIFNα) and ribavirin. However, sustained viral clearance is achieved in only 50-60% of patients. The underlying mechanism of failure of pegIFNα based therapy remains unknown and no molecular or genetic markers have been identified that could predict the treatment outcome. The overall aim of the study described in this thesis is to understand the molecular basis for failure of IFNα based therapies in patients with CHC. The study has focused on the IFNinduced Jak-STAT (janus kinase-signal transducer and activator of transcription) signaling pathway. To address the molecular basis of treatment response to IFN therapy, three experimental approaches have been employed. The first approach involved the analysis of IFNα signaling and expression of interferon stimulated genes (ISGs) in liver biopsies and peripheral blood mononuclear cells (PBMCs) of HCV patients undergoing pegIFNα treatment. Paired liver biopsies and PBMCs from 16 patients were collected before andhours after the first injection of pegIFNα, and were subjected to analysis of global gene expression using Affymetrix arrays. Further, activation of the IFN-induced Jak-STAT signaling pathway was analyzed by immunoblotting, immunohistochemistry and gel shift assays. The correlation of these biochemical and molecular data with the clinical response to treatment demonstrated that in the liver of patients with a rapid response pegIFNα induced a strong upregulation of ISGs, whereas in patients that did not respond to therapy, induction of IFN-dependent gene expression was impaired. Surprisingly, the non-responders had maximally induced ISG expression already before treatment with pegIFNα. Furthermore, the analyses of STAT1 phosphorylation, nuclear localization and DNA binding confirmed that the endogenous IFN signaling pathway in non-responders is pre-activated and refractory to further stimulation. In contrast to liver samples, ISG expression in PBMCs was stimulated by pegIFNα in both responders and nonresponders, indicating that PBMCs are not a good surrogate marker for IFNα responses in the liver and that chronic HCV infection has strong local effects on the IFN system in liver. Our findings support an interesting concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection, but may also impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway in the liver. In the second approach we addressed the mechanisms underlying the pre-activation of the endogenous IFN system in a defined group of HCV patients (future non-responders). For this purpose, we analyzed ISG expression by quantitative RT-PCR and nuclear localization of STAT1 by immunohistochemistry in a cohort of 112 patients with CHC. By subdividing this cohort according to the HCV genotype (GT), we discovered that patients infected with HCV GT 1 and 4 more often show hepatic ISG preactivation than GT 2 and 3 patients, thus providing an explanation for the poor response to IFN therapy seen in GT 1/4 patients. We analyzed the possible involvement of viral sensory pathways in type I IFN production and ISG upregulation. Previously, the viral HCV NS3-4A protease was shown to interfere with viral sensory pathways by cleaving and thereby inactivating an important adaptor molecule, Cardif. We therefore assessed Cardif cleavage in liver biopsies of HCV patients and found that cleavage more often occurred in patients infected with HCV GTs 2 and 3. Our findings support a concept that the success of the virus in preventing the induction of the endogenous IFN system in the livers of these patients would, however, come at the cost of being more susceptible to IFNα therapies as is the case with GT 2/3 patients. In the third approach we designed an experimental model to study the molecular basis of refractoriness of IFN signaling in vivo. Previously, cell culture experiments demonstrated a long lasting desensitization period, which followed the initial activation of the IFNα signaling pathway. In the approach used here, we established a mouse model in which continuous presence of IFNα in vivo was achieved by multiple subcutaneous injections, mimicking the constitutively high serum levels achieved by pegIFNα in patients. Interestingly, this resulted in refractoriness of IFNα signaling. Activation of STAT1 and STAT2, but not STAT3, in the mouse liver was desensitized by continuous IFNα stimulation. To elucidate the mechanism of this refractoriness, the role of negative regulators of the Jak- STAT signaling pathway was investigated. IFN signaling remained refractory in mice deficient in suppressor of cytokine signaling (SOCS) 3 and persisting refractoriness was also observed in mice deficient in IL-10, a strong inducer of SOCS3. Ubiquitin specific peptidase 18 (USP18/UBP43) was recently identified as novel negative regulator of IFNα signal transduction. Interestingly, refractoriness could be overcome in USP18/UBP43 knockout mice. These data strongly indicate that UBP43 is the decisive factor in inducing a refractory state in the IFNα signaling pathway in vivo

Dysregulation of IFN System Can Lead to Poor Response to Pegylated Interferon and Ribavirin Therapy in Chronic Hepatitis C

Despite being expensive, the standard combination of pegylated interferon (Peg-IFN)- α and ribavirin used to treat chronic hepatitis C (CH) results in a moderate clearance rate and a plethora of side effects. This makes it necessary to predict patient outcome so as to improve the accuracy of treatment. Although the antiviral mechanism of genetically altered IL28B is unknown, IL28B polymorphism is considered a good predictor of IFN combination treatment outcome

MicroRNA profile before and after antiviral therapy in liver transplant recipients for Hepatitis C virus cirrhosis

BACKGROUND AND AIM: Management of Hepatitis C virus (HCV) recurrence is a major challenge after liver transplantation. Significant dysregulated expression of HCV receptors (i.e. claudin-1, occludin, CD81, Scavenger-receptor Type B1) has been shown recently during HCV infection. This might facilitate hepatocytic entry and reinfection of HCV. MicroRNAs (miRs) play role in the regulation of gene expression. We aimed to characterize miR expression profiles related to HCV infection and antiviral therapy in adult liver transplant recipients, with special emphasis on microRNAs predicted to target HCV receptors. METHODS: Twenty-eight adult liver transplant recipients were enrolled in the study. Paired biopsies were obtained at the time of HCV recurrence and at the end of antiviral treatment. MicroRNAs for HCV receptors were selected using target prediction software. Expression levels of miR-21, miR-23a miR-34a, miR-96, miR-99a*, miR-122, miR-125b, miR-181a-2*, miR-194, miR-195, miR-217, miR-221 and miR-224 were determined by RT-qPCR. RESULTS: miR-99a* and miR-224 expressions were increased in HCV recurrence samples, while miR-21 and miR-194 were decreased in comparison to normal liver tissue. Increased expressions of miR-221, miR-224 and miR-217 were observed in samples taken after antiviral therapy when compared with HCV recurrence samples. High HCV titer at recurrence was associated with higher level of miR-122. CONCLUSIONS: Samples at recurrence of HCV and after antiviral therapy revealed distinct HCV-related microRNA expression profiles, with significant dysregulation of those miRNAs potentially targeting mRNAs of HCV receptors. In particular, miR-194 and miR-21 might be involved in the regulation of HCV receptor proteins' expression during HCV infection and antiviral therapy

Dysregulation of IFN System Can Lead to Poor Response to Pegylated Interferon and Ribavirin Therapy in Chronic Hepatitis C

Despite being expensive, the standard combination of pegylated interferon (Peg-IFN)- α and ribavirin used to treat chronic hepatitis C (CH) results in a moderate clearance rate and a plethora of side effects. This makes it necessary to predict patient outcome so as to improve the accuracy of treatment. Although the antiviral mechanism of genetically altered IL28B is unknown, IL28B polymorphism is considered a good predictor of IFN combination treatment outcome

S-Adenosyl-Methionine and Betaine Improve Early Virological Response in Chronic Hepatitis C Patients with Previous Nonresponse

Treatment of chronic hepatitis C (CHC) with pegylated interferon (pegIFN ) and ribavirin results in a sustained response in approximately half of patients. Viral interference with IFN signal transduction through the Jak-STAT pathway might be an important factor underlying treatment failure. S-adenosyl-L-methionine (SAMe) and betaine potentiate IFN signaling in cultured cells that express hepatitis C virus (HCV) proteins, and enhance the inhibitory effect of IFN on HCV replicons. We have performed a clinical study with the aim to evaluate efficacy and safety of the addition of SAMe and betaine to treatment of CHC with pegIFN /ribavirin

Analysis of microRNA turnover in mammalian cells following Dicer1 ablation

Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function

USP18-Based Negative Feedback Control Is Induced by Type I and Type III Interferons and Specifically Inactivates Interferon α Response

Type I interferons (IFN) are cytokines that are rapidly secreted upon microbial infections and regulate all aspects of the immune response. In humans 15 type I IFN subtypes exist, of which IFN α2 and IFN β are used in the clinic for treatment of different pathologies. IFN α2 and IFN β are non redundant in their expression and in their potency to exert specific bioactivities. The more recently identified type III IFNs (3 IFN λ or IL-28/IL-29) bind an unrelated cell-type restricted receptor. Downstream of these two receptor complexes is a shared Jak/Stat pathway. Several mechanisms that contribute to the shut down of the IFN-induced signaling have been described at the molecular level. In particular, it has long been known that type I IFN induces the establishment of a desensitized state. In this work we asked how the IFN-induced desensitization integrates into the network built by the multiple type I IFN subtypes and type III IFNs. We show that priming of cells with either type I IFN or type III IFN interferes with the cell's ability to further respond to all IFN α subtypes. Importantly, primed cells are differentially desensitized in that they retain sensitivity to IFN β. We show that USP18 is necessary and sufficient to induce differential desensitization, by impairing the formation of functional binding sites for IFN α2. Our data highlight a new type of differential between IFNs α and IFN β and underline a cross-talk between type I and type III IFN. This cross-talk could shed light on the reported genetic variation in the IFN λ loci, which has been associated with persistence of hepatitis C virus and patient's response to IFN α2 therapy

The treatment response of chronically hepatitis C virus-infected patients depends on interferon concentration but not on interferon gene expression in peripheral blood mononuclear cells.

International audienceThe current treatment of chronic hepatitis C is based on pegylated alpha interferon (PEG-IFN-α) and ribavirin. The aim of this study was to identify biological and clinical variables related to IFN therapy that could predict patient outcome. The study enrolled 47 patients treated with PEG-IFN and ribavirin combined therapy. The interferon concentration was measured in serum by a bioassay. The expression of 93 interferon-regulated genes in peripheral blood mononuclear cells was quantified by real-time quantitative reverse transcription-PCR (RT-PCR) before and after 1 month of treatment. The interferon concentration in the serum was significantly lower in nonresponders than in sustained virological responders. Moreover, a significant correlation was identified between interferon concentration and interferon exposition as well as body weight. The analysis of interferon-inducible genes in peripheral blood mononuclear cells among the genes tested did not permit the prediction of treatment outcome. In conclusion, the better option seems to be to treat patients with weight-adjusted PEG-IFN doses, particularly for patients with high weight who are treated with PEG-IFN-α2a. Although the peripheral blood mononuclear cell samples are the easiest to obtain, the measurement of interferon-inducible genes seems not be the best strategy to predict treatment outcome

Circulating sCD14 Is Associated with Virological Response to Pegylated-Interferon-Alpha/Ribavirin Treatment in HIV/HCV Co-Infected Patients

Microbial translocation (MT) through the gut accounts for immune activation and CD4+ loss in HIV and may influence HCV disease progression in HIV/HCV co-infection. We asked whether increased MT and immune activation may hamper anti-HCV response in HIV/HCV patients.98 HIV/HCV patients who received pegylated-alpha-interferon (peg-INF-alpha)/ribavirin were retrospectively analyzed. Baseline MT (lipopolysaccharide, LPS), host response to MT (sCD14), CD38+HLA-DR+CD4+/CD8+, HCV genotype, severity of liver disease were assessed according to Early Virological Response (EVR: HCV-RNA <50 IU/mL at week 12 of therapy or ≥2 log(10) reduction from baseline after 12 weeks of therapy) and Sustained Virological Response (SVR: HCV-RNA <50 IU/mL 24 weeks after end of therapy). Mann-Whitney/Chi-square test and Pearson's correlation were used. Multivariable regression was performed to determine factors associated with EVR/SVR.71 patients displayed EVR; 41 SVR. Patients with HCV genotypes 1-4 and cirrhosis presented a trend to higher sCD14, compared to patients with genotypes 2-3 (p = 0.053) and no cirrhosis (p = 0.052). EVR and SVR patients showed lower levels of circulating sCD14 (p = 0.0001, p = 0.026, respectively), but similar T-cell activation compared to Non-EVR (Null Responders, NR) and Non-SVR (N-SVR) subjects. sCD14 resulted the main predictive factor of EVR (0.145 for each sCD14 unit more, 95%CI 0.031-0.688, p = 0.015). SVR was associated only with HCV genotypes 2-3 (AOR 0.022 for genotypes 1-4 vs 2-3, 95%CI 0.001-0.469, p = 0.014).In HIV/HCV patients sCD14 correlates with the severity of liver disease and predicts early response to peg-INF-alpha/ribavirin, suggesting MT-driven immune activation as pathway of HIV/HCV co-infection and response to therapy