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
