Ribavirin improves the IFN-γ response of natural killer cells to IFN-based therapy of hepatitis C virus infection

Abstract

Ribavirin (RBV) is an important component of interferon (IFN)-based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host IFN response, has been proposed as RBV's mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN-α/β receptor signaling, as NK cell cytotoxicity and IFN-γ production are regulated by signal transducer and activator of transcription (STAT)1- and STAT4-phosphorylation, respectively. Specifically, pSTAT1-dependent NK cell cytotoxicity increases and pSTAT4-dependent IFN-γ production decreases in response to endogenous, virus-induced IFN-α and during IFN-α-based therapy. To assess whether RBV has a direct effect on NK cells and/or improves the IFN-γ response of NK cells in the presence of IFN-α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of RBV pretreatment, who all received subsequent pegylated (Peg)IFN/ribavirin combination therapy. During RBV pretreatment, both the frequency of CD56(dim) NK cells with cytotoxic effector functions and the frequency of CD56(bright) NK cells with the capacity to produce IFN-γ decreased (P = 0.049 and P = 0.001, respectively). In vitro or in vivo exposure of NK cells to RBV improved the pSTAT4 (P < 0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN-α. This was associated with an increase in IFN-γ production but not cytotoxicity of NK cells during subsequent IFN-α-based therapy. The frequency of IFN-γ-producing NK cells was greater in fast second-phase virological responders than in slow responders.

Conclusion: RBV enhances the pSTAT4 and IFN-γ response of NK cells to IFN-α-stimulation.

Fig. 1. A 4-week course of ribavirin pretreatment results in a modest reduction of serum HCV RNA and ALT levels

(A–B) Serum HCV RNA levels and (C–D) ALT activity of patients who did (A, C) or did not (B, D) receive a 4-week course of ribavirin pretreatment prior to PegIFN/ribavirin combination therapy. Week −4 marks the start and week 0 the end of ribavirin pretreatment. Statistical analysis: paired t-test. ALT, alanine aminotransferase; ns, not significant.

Fig. 2. The decrease in the frequency of CD56^dim cytotoxic NK cells during ribavirin pretreatment correlates with the decrease in ALT levels

(A–B) Changes in the frequency of CD56^dim NK cells, that upregulate the degranulation marker CD107a in vitro in response to K562 target cells, in patients who did (A) or did not (B) receive a 4-week course of ribavirin pretreatment. Week −4 marks the start and week 0 the end of ribavirin pretreatment. Statistical analysis: Wilcoxon signed rank test (left panel) and paired t-test (right panel) based on results of D’Agostino & Pearson omnibus normality test. (C–D) Correlation between changes in the frequency of CD107a+ CD56^dim NK cells and ALT levels in patients who did (C) or did not (D) receive a 4-week course of ribavirin pretreatment. Statistical analysis: Spearman non-parametric test. Rho: Spearman correlation coefficient. MFI, mean fluorescence intensity; ns, not significant.

Fig. 3. The decrease in the frequency of IFN-γ-producing CD56^bright NK cells during ribavirin pretreatment correlates with the decrease in viremia and in NK cell pSTAT4 levels

(A–B) Changes in the frequency of IFN-γ-producing CD56^bright NK cells in patients who did (A) or did not (B) receive a 4-week course of ribavirin pretreatment. Statistical analysis: Wilcoxon signed rank test (left panel) and paired t-test (right panel) based on results of D’Agostino & Pearson omnibus normality test. (C–D) Correlation between the change in the frequency of CD56^bright NK cells and the change in viremia in patients who did (C) or did not (D) receive a 4-week course of ribavirin pretreatment. Statistical analysis: Spearman non-parametric test. Rho: Spearman correlation coefficient. MFI, mean fluorescence intensity; ns, not significant. (E–F) Correlation between the change in the frequency of IFN-γ-producing CD56^bright NK cells and the change in NK cell ex vivo pSTAT4 levels in patients who did (E) or did not (F) receive a 4-week course of ribavirin pretreatment. Statistical analysis: Spearman non-parametric test.

Fig. 4. Ribavirin improves the induction of pSTAT4 in NK cells in the presence of IFN-α

(A–B) IFN-α-induced pSTAT1 (A) and pSTAT4 (B) expression in CD3-CD56+ NK cells and their CD56^bright and CD56^dim subpopulations from chronic HCV patients (chronic HCV, n=22) and healthy, uninfected blood donors (healthy controls, n=23). The pSTAT1 and pSTAT4 inducibility reflect the pSTAT1 and pSTAT4 MFI of NK cells after in vitro stimulation of PBMC with IFN-α normalized to the respective MFI of unstimulated cells. Statistical analysis: Mann-Whitney nonparametric two-sample rank test. (C) IFN-α-induced pSTAT1 and pSTAT4 expression in NK cells from chronic HCV patients (n=8) after in vitro treatment of PBMCs with ribavirin. The pSTAT1 and pSTAT4 inducibility reflects the pSTAT1 and pSTAT4 MFI of NK cells after in vitro stimulation of PBMC with IFN-α normalized to the respective MFI in unstimulated cells. Statistical analysis: paired t-test after analysis with the D’Agostino & Pearson omnibus normality test. (D) Inducibility of pSTAT4 in CD3-CD56+ NK cells from chronic HCV patients who did or did not receive a 4-week course of ribavirin pretreatment. PBMCs were isolated prior to (week −4) and after (week 0) of a 4-week course ribavirin pretreatment and subjected to in vitro stimulation with IFN-α. The pSTAT1 and pSTAT4 inducibility reflects pSTAT1 and pSTAT4 MFI in NK cells after in vitro stimulation of PBMCs with IFN-α normalized to the respective MFI in unstimulated cells. The comparison remains statistically significant if the outlier in the ribavirin-pretreated group at week 0 is excluded. Statistical analysis: Mann-Whitney nonparametric two-sample rank test after analysis with the D’Agostino & Pearson omnibus normality test. MFI, mean fluorescence intensity; ns, not significant.

Fig. 5. A 4-week course of ribavirin pretreatment increases the pSTAT4-dependent IFN-γ production of NK cells during subsequent PegIFN/ribavirin therapy

(A–B) Changes in the frequency of IFN-γ-producing CD3-CD56+ NK cells (A) and CD56^bright NK cells (B) during therapy with PegIFN/ribavirin of patients who either had (solid lines, n=22) or had not (broken lines, n=31) received a 4-week course of ribavirin pretreatment. The frequency of IFN-γ-producing NK cells was determined after in vitro stimulation. Mean values ± SEM are shown. Asterisks indicate a p≤0.05 by unpaired t-test for the indicated time point. The comparison of the results from both patient groups by serial measures ANOVA is significant (p=0.0206 for panel A, p<0.0001 for panel B). (C) Frequency CD56^bright NK cells that produced IFN-γ in response to in vitro stimulation in ribavirin-pretreated patients who mounted either a slow (open triangles) or fast (filled triangles) 2^nd phase virological response to subsequent PegIFN/ribavirin therapy. Statistical analysis: Mann-Whitney nonparametric two-sample rank test after analysis with the D’Agostino & Pearson omnibus normality test. Mean values ± SEM are shown.