Interfering with interferon: Re-igniting the debate on calcineurin inhibitor choice and antiviral therapy for hepatitis C virus recurrence†
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With end-stage liver disease due to chronic hepatitis C virus (HCV) infection being the leading indication for liver transplantation worldwide, management and treatment of recurrent disease remains one of the major clinical challenges today. Although the combination therapy of pegylated interferon-α (IFN-α) and ribavirin has some efficacy, the sustained virological response rate is far from satisfactory. The study by Hirano and coworkers1 in this issue of Liver Transplantation sheds new light on how immunosuppression may affect the antiviral activity of IFN-α by demonstrating that the commonly used calcineurin inhibitor, FK506 (tacrolimus), inhibits IFN-α signaling. This study may re-ignite a smoldering debate on the differential effect of calcineurin inhibitors on HCV replication and antiviral therapy.
Abbreviations
CsA, cyclosporine A; CyP, cyclophilin; HCV, hepatitis C virus; IFN, interferon; ISG, interferon-stimulating gene; ISGF-3; interferon-stimulated gene factor 3; ISRE, interferon-stimulated regulatory element; JAK, Janus kinase; NS5B, nonstructural protein 5B; PKR, double-stranded RNA–dependent protein kinase; STAT, signal transducer and activator of transcription; SVR, sustained virologic response.
The success of transplantation for HCV is compromised by an accelerated recurrence of liver cirrhosis, which reduces patient and graft survival. After organ transplantation, the HCV viral load increases by approximately 1 log, and this is generally considered an effect of the immunosuppression. Several studies have shown that the problem of HCV recurrence has worsened within the past decades. It has been speculated that changes in the type and intensity of immunosuppressive therapy, among other factors, could be reasons for this deterioration.2 The discovery that cyclosporine A (CsA) can inhibit HCV replication in vitro sparked a clinical debate on the possible differential impact of the calcineurin inhibitors tacrolimus and CsA on HCV recurrence.
The primary cellular targets of the calcineurin inhibitors are immunophilins. CsA binds to cyclophilins (CyPs), and tacrolimus binds to FK binding proteins; both events result in a profound inhibition of the phosphatase activity of calcineurin. This inhibition of calcineurin by CsA and tacrolimus results in the blocking of interleukin-2 production that is required for T cell activation. Independent of its immunosuppressive activity, CsA has potent antiviral effects on several viruses in vitro, including human immunodeficiency virus-1, vaccinia virus, and vesicular stomatitis virus. These viruses incorporate cellular CyPA into their viral particles during their life cycles and take advantage of CyPA for replication. As shown by many independent research groups, CsA has a potent antiviral effect on HCV replication in vitro,3-9 whereas tacrolimus does not exhibit any anti-HCV activity. The maximum antiviral effect of CsA, observed on HCV genotype 1b at approximately 1 μg/mL, was similar to the reduction seen with IFN-α. These concentrations of CsA are feasible in the clinical setting because the intrahepatic concentrations are up to 10 times higher than those in peripheral blood.10 The antiviral activity of CsA is not mediated by an IFN response and is independent of calcineurin signaling. Recent publications suggest that the anti-HCV activity of CsA acts via the inhibition of CyP, particularly CyPB.11, 12 CyPB has been shown to be an important functional regulator of HCV RNA polymerase [nonstructural protein 5B (NS5B)], which plays a central role in HCV genome replication. CyPB interacts with NS5B and stimulates its RNA binding activity. By disrupting this association, CsA inhibits NS5B RNA binding and thereby suppresses the level of HCV replication.
Despite the clear antiviral effect of CsA in vitro, there is still controversy about the effect of CsA on HCV replication, in vivo, in the setting of clinical organ transplantation. Although many studies have compared CsA and tacrolimus as maintenance immunosuppression following liver transplantation, data on their effects on HCV recurrence are limited. What are available are conflicting reports on the effects of these calcineurin inhibitors on HCV viral load or fibrosis progression. A recent study by Levy and colleagues13 showed that time to biopsy-proven HCV recurrence was significantly longer with CsA in comparison with tacrolimus. The relevance of this clinical parameter is not clear because this study and another by Berenguer et al.14 did not observe a difference in the incidence of severe HCV recurrence or fibrosis between treatment groups. Therefore, on the basis of current data, it is not possible to conclude that CsA by itself has a profound effect on viral replication or on the course of HCV recurrence after transplantation. However, as with many antiviral compounds, in vivo efficacy can be observed only when it is used in combination with other antiviral drugs. An example of this can be seen with ribavirin. The nucleoside analog ribavirin has, by itself, at best only a limited and transient antiviral effect in vivo.15 In contrast to this limited potency with ribavirin monotherapy, when it is combined with pegylated IFN-α, a remarkable 3-fold increase in sustained virologic response (SVR) is observed in chronic HCV patients. Similarly, in vitro evidence suggests that cotreatment of CsA with low-dose IFN-α results in greater inhibition of HCV replication in comparison with the effects of CsA or IFN-α alone.6 Most studies even suggest that the combined effect of CsA and IFN-α is synergistic.5, 7, 9
Despite the relative success of IFN-α and ribavirin combination therapy in chronic HCV patients, results of antiviral therapy after transplantation are generally disappointing. Although the combination therapy is somewhat effective, the SVR rate is far from satisfactory. The fact that CsA and IFN exert a complementary inhibitory effect on HCV replication in vitro raises the question of whether CsA can influence the efficacy of antiviral therapy in transplant recipients. Although the number of studies that have specifically addressed this question are limited, one study in which chronic HCV patients were treated with IFN-α2b therapy either in the presence or absence of CsA treatment16 observed a significantly higher SVR rate in the CsA group versus the group with IFN alone (55% versus 32%). Moreover, in liver transplant recipients, a retrospective study suggests that the SVR to IFN-α and ribavirin combination therapy was significantly lower in patients receiving tacrolimus-based immunosuppression in comparison with patients receiving CsA (27% versus 46%).6 Perhaps the most compelling anecdotal evidence comes from a study by Sugawara and colleagues,17 in which HCV-positive transplant patients received tacrolimus plus steroids and were treated preemptively with IFN-α2b and ribavirin. Within 2 years, 34% of the patients showed an SVR. When 8 of the 21 nonresponders were converted to CsA with continuing antiviral therapy, 5 patients' serum HCV became undetectable within 3 months, with no acute rejection or change in liver or renal function. Are these studies proof that the antiviral properties of CsA truly make an impact in vivo when it is combined with IFN-based anti-HCV therapy? The clinical data to support this may still be too limited. However, it is conceivable that the gross antiviral effect, as observed in HCV replicon models in vitro, is countered in patients by the inhibition of the antiviral immune response by CsA.
In the current issue of Liver Transplantation, an article by Hirano and coworkers1 now reveals another aspect of calcineurin inhibitors and their impact on the treatment for HCV. They report that tacrolimus and CsA have different effects on the molecular pathways involved in IFN signaling and speculate that immunosuppression is part of the reason that IFN resistance is observed during posttransplantation antiviral therapy. Very little is known about the effect of immunosuppressants on IFN signaling. So far, it has been shown only that CsA and glucocorticoids can inhibit the cellular responses to IFN-γ;18, 19 nothing was known about the responses to IFN-α. The authors examined the IFN-α signal transduction, using cell lines in vitro. IFN-α signaling is triggered by the binding of IFN-α to its receptor on the cell surface, which causes 2 tyrosine phosphorylations of Janus kinase (JAK) in the cell. Phosphorylated JAK can, in turn, phosphorylate signal transducer and activator of transcription 1 (STAT-1) and STAT-2, and this induces the formation of interferon-stimulated gene factor 3 (ISGF-3) consisting of STAT-1, STAT-2, and p48. ISGF-3 is translocated from the cytoplasm to the nucleus, where it can interact with interferon-stimulated regulatory elements (ISREs) in the promoter region of a large number of interferon-stimulating genes (ISGs). Double-stranded RNA–dependent protein kinase (PKR) is one such ISG that is important for the IFN-induced antiviral effect against HCV. The authors examined IFN-induced activation of this signaling cascade during treatment with tacrolimus or CsA. They found that the activation of ISRE-mediated transcription and the resulting up-regulation of ISGs such as PKR and STAT-1 were inhibited by treatment with tacrolimus but not with CsA. Similarly, phosphorylation of STAT-1 and STAT-2 and translocation of STAT-1 into the nucleus were attenuated following the treatment with tacrolimus, but not CsA. As the tyrosine phosphorylation of JAK-1 was not altered, tacrolimus seems to block the IFN-α signaling pathway at the step between the phosphorylation of JAK and that of STAT. Thus, tacrolimus suppresses the IFN signaling, but does this affect the antiviral activity of IFN-α? They show in Fig. 7 that pretreatment of HCV replicon cells with tacrolimus did not affect HCV replication but indeed partly reduced the antiviral effect of IFN-α in this system. On the contrary, pretreatment with CsA reduced HCV replication and when combined with IFN-α showed some additional inhibition. Although the lack of statistical analysis in this experiment should lead to caution in interpretation, this evidence could suggest that tacrolimus interferes with the antiviral function of IFN-α in vitro.
The arguments regarding choice of calcineurin inhibitor for HCV-related transplantation patients has come from clear evidence in basic research, but currently there is still no reliable clinical evidence to settle this debate. It is hoped that upcoming prospective clinical trails may provide the missing clinical knowledge on optimal immunosuppression for posttransplantation antiviral therapy.