Although new therapies based on small molecule inhibitors of HCV protease and polymerase look promising for HCV infection, it is likely that they will be used in combination with, rather than instead of, IFN-α
-based regimens. Therefore, an improved understanding of the mechanisms of action and resistance to IFN in HCV infection will be critical to improve outcomes as well as for the development of new therapeutic approaches. In this study, we have performed an extensive examination of the responses to 3 different forms of IFN in the PBMCs and liver of the chimpanzee, which is the only reliable animal model of HCV infection. We showed that, although less responsive than humans to hIFN, chimpanzees are able to respond to hIFN-α
, and consensus IFN in both PBMCs and liver. The patterns of ISG induction in response to the various IFNs were similar to those previously reported in cell culture,22
Although consensus IFN is derived from type I IFNs, it has been shown to induce both type I- and type II-specific ISGs and was shown to be more potent than either IFN-α
in ISG induction. This effect is a result of the higher binding affinity of consensus IFN to IFNAR than IFN-α5,28
and the cross interaction of the signal transduction pathways between the 2 types of IFNs.
Our data are consistent with the recently reported microarray data showing strong ISG induction after IFN-α
treatment of naive chimpanzees.29
More importantly, for the first time, we showed that HCV-infected chimpanzees had a blunted response to IFN therapy. Although a lower ISG induction by all 3 forms of IFN was observed in PBMCs, the most notable difference was seen in the liver. Despite high doses of all 3 forms of IFN, little or no hepatic ISG induction was seen in the infected animals. Consistent with the lack of detectable IFN response, there was little or no change in HCV viral load. The basal levels of ISG and phosphorylated STAT1 expression were higher in infected than naive chimpanzees, indicating that HCV infection led to a type I IFN response. This observation is consistent with previous studies in chimpanzees26,27
and raises the possibility that the ISGs were already maximally induced in infected chimpanzees such that exogenous IFNs did not lead to any further ISG induction. However, the increased basal level in the infected chimpanzees does not account entirely for the reduced ISG induction because the absolute levels of ISGs after IFN treatment were substantially lower in infected animals than those in naive animals (10- to 100-fold lower). This suggests that, in infected chimpanzees, there is interference with ability to respond to IFN. Two possibilities could explain this blunted IFN response. During chronic infection, the continuous but ineffective IFN action leads to a relatively resistant state of the liver to further exposure of IFN. Continuous IFN action could lead to IFN receptor down-regulation, a phenomenon indeed observed in ligand-receptor interaction. We found no decrease in IFNAR in either chimpanzees or humans (data not shown) before or during IFN treatment, consistent with previous in vitro data.32
Alternatively, chronic HCV infection, either through a direct viral mechanism or indirect actions, may activate negative regulator(s) of IFN leading to an IFN resistant state in the liver. This concept is supported by the observation that chronic hepatitis C patients may be more susceptible to other hepatic viral infections.33,34
The observation that hepatic phosphorylated STAT1 showed a paradoxical decline after IFN treatment in the infected animals strongly supports the activation of an IFN-inhibitory pathway, as postulated above. By assessing the status of the various IFN-signaling inhibitors in the liver biopsy samples, we found that, following IFN administration, the expression of SOCS3 was significantly up-regulated. It appears that the induction of SOCS3 expression was much more dramatic at the mRNA () than the protein level () for both infected chimpanzees. This difference might be due to the unstable nature of the SOCS proteins and the lack of samples at earlier time points, which made it difficult to compare the 2 levels. SOCS3 acts by interacting with the JAKs, resulting in impaired STAT1 and STAT3 phosphorylation. This leads to reduced STAT1 nuclear translocation, lack of binding to the ISRE, and ultimately decreased ISG expression.24
Being IFN inducible, SOCS3 expression is also induced by IL-6, IL-10, TNF-α
, and toll-like receptors 4 and 9.35
A previous study using an HCV transgenic mouse model showed a similar inhibition of JAK-STAT signaling by HCV expression; however, no induction of SOCS1 or SOCS3 was detected.36
On the other hand, HCV core protein has been shown to induce SOCS3 expression in several cell lines, resulting in impaired IFN and specifically STAT1 signaling.15,37,38
In addition, Zhu et al32
also showed that cells harboring HCV replicons resistant to IFN therapy produced higher levels of SOCS3. With silencing of SOCS3, IFN sensitivity was partially restored.32
Our in vivo finding of increased SOCS3 expression and reduced phosphorylated STAT1 after IFN treatment is consistent with the known functional mechanism of this IFN-inhibitory pathway. This pathway could provide a plausible explanation for a direct viral mechanism of IFN resistance in vivo.
If SOCS3 expression were induced by viral proteins, only infected cells would be resistant to IFN. However, our in vivo data suggest a general IFN-resistant state in the infected liver (). Alternatively, activation of IFN inhibitory pathway(s) may occur through the production of soluble factor(s) that act in an autocrine/paracrine fashion in the liver and to a lesser extent PBMCs as they traverse the liver in blood. To examine the possible soluble factor(s), we studied a panel of cytokine/chemokine levels in the serum of HCV-infected and naive chimpanzees in response to IFN treatment. Several cytokines have been reported to be induced by HCV proteins, such as IL-6, IL-8, TNF-α
, MCP-1, and RANTES.39–43
Among them, IL-8 has been shown to have anti-IFN activity through inhibition of ISGF3 assembly and interaction with the ISRE. We found no induction of IL-8 by IFN in infected chimpanzees. However, IL-6 was induced in response to IFN in both serum and liver tissue of the infected chimpanzees with a time course similar to SOCS3 induction. Other cytokines known to modulate SOCS3 expression, such as IL-10 and TNF-α
, were not induced by IFN in the infected animals. IL-6 mRNA was not detectable in the livers of treated patients in our cohort; however, biopsies were performed 24 hours after IFN treatment compared with 8 hours in the chimpanzees, and, consequently, IL-6 expression may have been missed, particularly given the low levels seen in chimpanzees suggesting that the peak may already have been missed at 8 hours.
Chen et al reported that human nonresponsders to IFN-α
-based therapy had a higher expression of numerous ISGs in pretreatment liver biopsy samples as compared with those who achieved a sustained virologic response.44
The opposite pattern was seen with the hepatic SOCS3 expression in our cohort, with ~11-fold higher expression in control patients who responded to treatment (RR group) compared with the nonresponders (SR group). As a negative regulator of IFN, high SOCS3 levels prior to treatment would block ISG expression. With IFN-α
treatment, SOCS3 declines, allowing for ISG induction. The degree of ISG induction may be more important than the absolute level of ISG expression for viral clearance. Consequently, the baseline level of SOCS3 expression and the degree to which SOCS3 changes after IFN treatment determine the pretreatment level of ISG expression and the degree to which ISGs can be induced by IFN-α
therapy. In our human study, the SOCS3 expression declined much less in the SR than the RR in response to IFN-α
. This difference could contribute to a blunted ISG induction in the human nonresponders. Infected chimpanzees, perhaps as an extreme example of the human nonresponders, actually have a rise in hepatic SOCS3 with IFN therapy and therefore exhibit little or no ISG induction and thus no decline in viremia.
Based on these observations, we propose a model () in which HCV infection leads to endogenous IFN production and also to an increased expression of SOCS3, either directly by a viral mechanism or indirectly by the induction of a soluble factor, possibly IL-6. As a consequence of SOCS3 induction and other mechanisms (eg, interaction of HCV NS5A or E2 protein with double-strand RNA-dependent protein kinase [PKR]), the antiviral activity of IFN is impaired, allowing HCV to establish persistent infection. In chimpanzees, IFN treatment leads to further SOCS3 induction, which prevents ISG activation and markedly blunts the IFN response. As described above in humans, SOCS3 declines but much greater in RR than in SR after IFN treatment. This proposed inhibitory pathway is variably operational in HCV-infected humans because of the genetic heterogeneity in the human population, thus accounting for the diverse infection outcomes and treatment responses.
Figure 8 Model of IFN resistance in chronic HCV infection. HCV infection leads to endogenous IFN production and also to increased expression of SOCS3, either directly by a viral protein (eg, core) or indirectly by an IFN inhibitory factor (eg, IL-6 or other soluble (more ...)
In summary, we have shown that chimpanzees respond to hIFNs with ISG production in both PBMCs and liver. HCV infection leads to the production of type I IFN resulting in sustained activation of the JAK-STAT pathway and ISG induction in the liver. Typical ISG responses were seen in treated naive chimpanzees; however, ISG production was reduced in PBMCs and almost completely abrogated in the liver of HCV-infected animals. SOCS3 expression was markedly increased in the livers of infected chimpanzees after IFN treatment and was associated with a decrease in phosphorylated STAT1. A similar pattern of SOCS3 expression was seen in patients associated with treatment response. This negative regulator of IFN, possibly mediated by the production of IL-6, may be an important mechanism of IFN nonresponse in chronic HCV infection. Further studies to elucidate the biologic importance of this interaction may have crucial implications for the improvement of current HCV therapy.