Natural killer cells are polarized toward cytotoxicity in chronic hepatitis C in an interferon-alfa-dependent manner

Abstract

Background & aims: Patients with chronic hepatitis C virus (HCV) infection display great variability in disease activity and progression. Although virus-specific adaptive immune responses have been characterized extensively and found to be impaired in chronic hepatitis C, the role of innate immune responses in disease activity and progression of chronic hepatitis C is not well understood.

Methods: We studied 42 HCV-infected patients and 12 healthy uninfected controls.

Results: We found an increased frequency of natural killer (NK) cells expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), NKp44, NKG2C, and CD122 in chronic hepatitis C as compared with healthy controls (P < .05 for all markers) and stronger activation of NK cells in the liver than in the blood (P < .05). This NK cell phenotype was associated with polarization of NK cell function toward CD107a expression as a marker of degranulation, but with not increased interferon (IFN)-gamma production of CD56(dim) NK cells. The polarized NK cell phenotype correlated with alanine aminotransferase levels (r(2) = 0.312, P = .03). To investigate whether in vivo exposure of NK cells to HCV-induced type I IFN was causing this NK cell phenotype, peripheral blood mononuclear cells from 10 healthy controls and 8 HCV-infected patients were stimulated in the presence of IFN-alfa, which resulted in increased NK cell expression of TRAIL and CD107a (P < .001), but not IFN-gamma.

Conclusions: Collectively, these results describe a polarized NK cell phenotype induced by chronic exposure to HCV-induced IFN-alfa. This phenotype may contribute to liver injury through TRAIL expression and cytotoxicity, whereas the lacking increase in IFN-gamma production may facilitate the inability to clear HCV.

Fig. 1. Multicolor flow cytometry to analyze NK cells in PBMC

(A) Gating strategy. (B-C) Representative dot plots showing the expression of cell surface markers on NK cells from healthy controls (B) and chronic HCV patients (C).

Fig. 2. Phenotype of peripheral blood NK cells

Frequency (A) and MFI (B) of NK cells displaying the indicated surface markers in the blood of patients with chronic HCV infection and healthy, uninfected controls. Horizontal lines indicate the mean. n.s., not significant.

Fig. 3. Comparison of NK cell phenotypes in blood and liver in chronic hepatitis C

Frequency and MFI of CD3-CD56+ NK cells (A-B), CD3-CD56^dim NK cells (C-D) and CD3-CD56^bright NK cells (E-F) with the indicated surface markers in blood and liver of patients with chronic HCV infection.

Fig. 4. NK cell degranulation, a marker for cytotoxicity, correlates to liver injury in chronic hepatitis C

(A, B) The frequency of degranulating (CD107a+) NK cells within all CD3-CD56+ NK cells (left panel), the CD3-CD56^dim NK cell subset (middle panel) and the CD3-CD56^brightNK cell subset (right panel) is higher in HCV-infected patients with elevated ALT levels than in those with normal ALT levels. NK cells were exposed to MHC-negative K562 cells (A) or to MHC-negative K562 cells in the presence of IL-12 and IL-15 (B) to induce degranulation. (C) The frequency of degranulating (CD107a+) NK cells within all CD3-CD56+ NK cells (left panel) and within the CD3-CD56^dim NK cell subset (right panel) correlates to ALT levels. Shaded areas indicate the range of normal (< 41 U/l) ALT levels. Horizontal lines indicate the mean.

Fig. 5. NK cell IFN-γ secretion does not increase in patients with chronic hepatitis C

The frequency of IFN-γ-producing NK cells within all CD3-CD56+ NK cells (left panel), the CD3-CD56^dim NK cell subset (middle panel) and the CD3-CD56^bright NK cell subset (right panel) is not higher in HCV-infected patients with elevated ALT levels than in those with normal ALT levels. IFN-γ secretion was induced by stimulation with IL-12 and IL-15 (A) or with IL-12 and IL-18 (B). Only significant p-values (<0.05) are indicated. Horizontal lines indicate the mean.

Fig. 6. NK cells of healthy, uninfected control subjects upregulate markers of cytotoxicity (CD107a, TRAIL) but not IFN-γ-production after in vitro exposure to IFN-α

CD3-CD56^dim (A, B) and CD3-CD56^bright (C, D) NK cells increase TRAIL and CD107a expression (A, C) but not IFN-γ-production (B, D) after in vitro exposure to IFN-α. n.s., not significant.

Fig. 7. NK cells of chronic HCV patients upregulate markers of cytotoxicity (CD107a, TRAIL) but not IFN-γ-production after in vitro exposure to IFN-α

CD3-CD56^dim (A, B) and CD3-CD56^bright (C, D) NK cells increase TRAIL and CD107a expression (A, C) but not IFN-γ-production (B, D) after in vitro exposure to IFN-α. n.s., not significant.

Fig. 8. Model

Chronic exposure to HCV-induced IFN-α induces a polarized NK cell phenotype that contributes to liver inflammation due to increased cytotoxicity but not to viral clearance due to lack of IFN-γ production (see discussion for details).