Early changes in interferon signaling define natural killer cell response and refractoriness to interferon-based therapy of hepatitis C patients

Abstract

Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased interferon gamma (IFN-γ) production in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is caused by type I interferon (IFN)-induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN-α-based therapy of HCV. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN-α-based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (tumor necrosis factor-apoptosis-inducing ligand [TRAIL] expression and degranulation) and decreased IFN-γ production. NK cells from patients with a greater than 2 log(10) first-phase HCV RNA decline to IFN-α-based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log(10) first-phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN-α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN-α during the second-phase virological response.

Conclusion: These data show that IFN-α-induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN-γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN-α-based therapy.

Fig. 1. Increased expression of STAT1 in NK cells during HCV infection is further enhanced by PegIFN/RBV therapy

(A) In vivo STAT1 expression in CD3-CD56+ NK cells and their CD56^bright and CD56^dim subpopulations in HCV-infected patients and healthy, uninfected blood donors. (B–D) In vivo STAT1 expression levels of all CD3-CD56+ NK cells (B) and their CD56^bright (C) and CD56^dim (D) subpopulations during therapy with PegIFN/RBV. Mean ± SEM are shown for 14 patients undergoing PegIFN/RBV therapy.h, hour; wk, week. *** P<0.001 by repeated measures ANOVA.

Figure 2. PegIFN/RBV therapy results in preferential phosphorylation of STAT1 over STAT4 in NK cells

(A–B) Maximal changes in pSTAT1 (A) and pSTAT4 (B) expression levels in chronic HCV patients of total CD3-CD56+ NK cells (left panel) and their CD56^bright (middle panel) and CD56^dim (right panel) subsets prior to and following PegIFN/RBV therapy initiation. (C) pSTAT1/pSTAT4 ratio (MFI) throughout the first 48h of therapy. *P<0.05, **P<0.01, **P<0.001 (comparing the indicated individual time points to the 0h time point).

Figure 3. PegIFN/RBV-induced changes in pSTAT1 expression in NK cells correlate to changes in NK cell function

Changes in TRAIL production (A, left graph), degranulation (B, left graph) and IFN-γ production (C, left graph) in response to PegIFN/RBV therapy initiation in correlation to changes in pSTAT1 expression level from 0h to 6h (A–C right graphs). r: Spearman correlation coefficient.

Figure 4. Refractoriness of pSTAT 1 inducibility in NK cells during PegIFN/RBV therapy

(A) pSTAT1 levels in CD3-CD56+ NK cells after in vitro stimulation with IFN-α (pSTAT1 inducibility). (B) In vitro pSTAT1 inducibility in CD3-CD56+ NK cells at the indicated PegIFN/RBV time points relative to in vivo pSTAT1 expression at the same time points. Inducibility was calculated as the fold change in pSTAT1 MFI after in vitro treatment with IFN at each time point. Mean ± SEM are shown for 21 patients. *P 0.05, ** P≤0.01 by repeated measures ANOVA. (C) In vivo pSTAT1 levels in NK cells prior to and 6h following PegIFN injection at start (left panel) and 12 weeks (right panel) of PegIFN/RBV therapy. h, hour; wk, week.

Figure 5. Correlation of pSTAT1 expression and first phase virological response

(A) Fold change in in vivo pSTAT1 expression by CD3-CD56+ NK cells during the first 6h of PegIFN/RBV therapy in individual patients with (solid lines) and without (broken lines) a greater than 2 log₁₀ first phase decline in HCV RNA titer. (B–C) Fold change in in vivo pSTAT1 expression by CD3-CD56+ NK cells during the first 6h (B) and 24h (C) of PegIFN/RBV therapy in individual patients with and without a greater than 2 log₁₀ first phase decline in HCV RNA titer. (D) Fold change in pSTAT1 inducibility (pSTAT1 MFI after in vitro treatment with IFN normalized to in vivo levels prior to (0h) or 6h after initiation of PegIFN/RBV therapy) in individual patients with (solid lines) and without (broken lines) a greater than 2 log₁₀ first phase decline in HCV RNA titer. (E–F) Fold change in pSTAT1 inducibility in NK cells (pSTAT1 MFI after in vitro treatment with IFN normalized to in vivo levels prior to (0h) or 6h (panel E) or 24h (panel F) after initiation of PegIFN/RBV therapy) in individual patients with and without a greater than 2 log₁₀ first phase decline in HCV RNA titer.