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Natural killer (NK) cells are large granular lymphocytes that play an important role in the control of viral infections. NK cells do have direct antiviral effects, mediated by cytolytic (eg, TNF-related apoptosis inducing ligand (TRAIL) or perforin mediated) or non-cytolytic (eg, IFN-γ mediated) effector functions as well as regulatory effects.1––3 The latter may be mediated by lysing of virus-infected antigen-presenting cells, activated CD4 T cells or virus-specific CD8 T cells.4
Several lines of evidence support an important role of NK cells in hepatitis C virus (HCV) infection. First, genetic studies have demonstrated that genes encoding the inhibitory NK cell receptor KIR2DL3 and its human leukocyte antigen C group 1 (HLA-C1) ligand directly influence resolution of HCV infection.5 Second, an activated multifunctional NK cell response (cytotoxicity and IFN-γ production) is detectable early after HCV exposure in healthcare workers and injecting drug users who do not develop acute infection suggesting an important contribution to the prevention of high-level viremia.6 ,7 Third, highly activated NK cells with increased degranulation and significant cytokine production are present during acute HCV infection although irrespective of the outcome of infection.8 ,9 Fourth, NK cells can inhibit HCV replication in vitro by IFN-γ mediated non-cytoytic and by perforin/granzyme and TRAIL-mediated cytotoxic mechanisms.1 In this context, it is interesting to note that HCV-infected hepatocytes upregulate expression of TRAIL receptors.10 Fifth, in chronic HCV infection, NK cells are activated and may display alterations in phenotype and function.1 ,2 Finally, upon initiation of antiviral IFN-based therapy, NK cells are further activated and may contribute to HCV clearance by primarily cytotoxic TRAIL-mediated effector functions.11 Interestingly, NK cells from patients with a greater than 2 log(10) first-phase HCV RNA decline to IFN-α-based therapy also display strong pSTAT1 induction in vivo, whereas NK cells from patients with a less than 2 log(10) first-phase HCV RNA decline exhibit lower pSTAT1 induction in vivo, supporting an important role of IFN-α-induced modulation of STAT1/4 phosphorylation in NK cell activation.12
There is growing interest in the mechanism by which NK cells are regulated during chronic HCV infection. NK cells are controlled by the integration of signals from activating and inhibitory receptors which include the inhibitory C-type lectin receptor NKG2A, the activating C-type lectin receptors NKG2C, NKG2D and the activating natural cytotoxicity receptors NKp30, NKp44 and NKp46. NKp46 is a particularly interesting molecule since it is considered to be the main activating NK cell receptor that also has an important role in the regulation of adaptive immunity. It has also been shown to play a prominent role in HCV infection.13 For example, a high expression of NKp46 defines a specific human NK cell subset that is characterised by a high functional capacity (eg, high cytotoxicity and IFN-γ production) and a high antiviral activity in vitro.14 ,15
By analysing the phenotype and function of blood and intrahepatic NK cells prior and serially after initiation of antiviral therapy, Pembroke et al16 add important new insights into the biological role of NKp46 in HCV infection. Indeed, in agreement with previous studies14 ,17 and in comparison with patients with other chronic liver diseases, they found significant higher levels of NKp46 expression in the livers of patients chronically infected with HCV, while other activating receptors, such as CD16, NKG2D or NKp30 are down-regulated. By contrast with previous studies, however, NKp46 expression was also found to be highly expressed in the peripheral blood.
Interestingly, and in agreement with the higher cytolytic activity of NKp46+ cells observed in vitro,16 a high (>80%) frequency of intrahepatic NKp46+ cells was positively associated with liver inflammation in histological examinations. Noteworthy, the positive association between NKp46 expression and liver inflammation was only found in patients chronically infected with HCV, but not in persons with other chronic liver diseases indicating an HCV-specific effect of NKp46+ NK cells. This may be explained by an HCV-specific upregulation of the still unknown ligand for NKp46, as has been shown in vitro by hepatoma cell lines transfected with the JFH-1 HCV strain. It is important to note that a recent study by Krämer et al14 also observed an accumulation of NKp46+ cells in the liver that, however, correlated negatively with viral load but not with liver inflammation. Also, in that study, a similar enrichment of NKp46+ cells was found in the livers of patients with other chronic liver diseases, such as autoimmunehepatitis or non-alcoholic steatohepatitis. The reasons for these differences are not entirely clear, but may reflect the further separation of NKp46 cells into NKp46high and NKp46dim expressing cells in that study.
Another important finding of the study by Pembroke et al18 that extends their own previous observations and findings by Bozzano et al19 is that the expression of NKp46 may predict a sustained virological response to IFN therapy. Indeed, they observed that patients chronically infected with HCV, who achieved a sustained virological response after IFN-based therapy, displayed a lower mean frequency of activated NKp46+ cells (72%) compared with patients who did not respond to therapy (80.5%).16 These results indicate that in vivo activated NK cells, characterised by a high NKp46 expression, are more resistant to IFN-induced activation and induction of their effector functions. This can be recapitulated in vitro where a better functional responsiveness of NK cells after IFN stimulation, for example, a higher degranulation, is inversely linked to the frequency of NKp46+ NK cells. On the one hand, these results indicate that early IFN-induced NK cell cytotoxic effector functions may contribute to early viral control and are thus in agreement with a previous study by Ahlenstiel et al.11 On the other hand, these results suggest that expression of NKp46 may be used as a marker to predict responsiveness to IFN-based therapies. This is of clinical significance, since IFN will still constitute the backbone of antiviral therapy regiments in times of direct antiviral therapies.20 Interestingly, a similar prediction of IFN-responsiveness has been reported for other components of the innate immune system, such as interferon-stimulated genes (ISG) where high pretreatment ISG levels combined with a low inducibility predict a poor response to IFN therapy.21 These results clearly indicate that a high activation of innate immunity as indicated by ISGs and NKp46 is a signature for non-response to IFN therapy. The findings that NKp46 is a marker for IFN-unresponsiveness and liver inflammation represents indeed a clinical relevant ‘pradoxon’ since patients with higher activated NK cells and, consequently, more severe liver inflammation are less likely to respond to IFN-based antiviral therapy (figure 1).
The results of this study support an important contribution of the innate immune response to rapid early control of HCV during IFN-based therapy that may also have an impact on cellular immune responses. Indeed, the group by Godkin could previously show that IFN-mediated rapid innate control of viremia did compromise adaptive virus-specific T cell responses.18 This is in agreement with other studies showing lack of T cell restoration after IFN-induced viral clearance.22
However, important questions remain. For example, the mechanisms responsible for the lack of NKp46 down-regulation in the chronically HCV infected liver are unclear, the ligand of NKp46 still needs to be identified, the regulatory effect on adaptive immunity requires further investigation, and the predictive value of NKp46 for response to antiviral therapy needs to be clarified in prospective studies. This is also of importance since in contrast with the results reported by Pembroke et al, Golden-Mason et al15 reported a higher NKp46 expression in female Caucasian Americans compared with male African–Americans, and suggested that this may explain their higher response rate to antiviral therapy.
In sum, the results of this elegant study may have several clinical applications. First, NKp46 may be used as a biomarker for IFN responsiveness. Additionally, therapeutic blockade of NKp46 may, on one hand, reduce liver inflammation, and on the other hand, reduce NK cell activity prior IFN therapy to allow significant IFN-induced NK cell activation and rapid viral control.
Competing interests None.
Patient consent Obtained.
Provenance and peer review Commissioned; internally peer reviewed.
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