Natural killer cells and hepatitis C: Natural killer p46 expression linked to antiviral and antifibrotic activity
Authors: Maximilian Heeg, Robert Thimme
Journal: Hepatology (2012)

See Articles on Pages 1201 and 1214. Natural killer (NK) cells play an important role in innate immune response and are essential in the host's first-line defense against viral infections. A major hallmark of NK cells is their ability to kill infected cells without requiring previous immunization and to produce large amounts of antiviral effector cytokines, including interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). NK cells also play an important role in the priming and regulation of adaptive immune responses. For example, NK cells can regulate T-cell responses by lysing virus-infected antigen-presenting cells or by cytolytically eliminating activated CD4 T cells that affect CD8 T-cell function and exhaustion, as has been recently demonstrated in the lymphocytic choriomeningitis virus mouse model.1, 2 Accordingly, in that model, NK-cell depletion causes enhanced T-cell immunity that may lead to rapid virus control and prevention of chronic infection.1 Abbreviations: AA, African Americans; CA, Caucasian Americans; HCV, hepatitis C virus; HLA, human leukocyte antigen; HSCs, hepatic stellate cells; IFN-γ, interferon gamma; KIRs, killer cell immunoglobulin-like receptors; IL, interleukin; NK, natural killer; SVR, sustained virological response; TNF-α, tumor necrosis factor alpha; Based on to the intensity of CD56 expression, NK cells can be divided into two functional different subsets. CD56dim NK cells represent approximately 90% of the circulating NK-cell population and predominantely mediate cytotoxic effector functions. CD56bright NK cells contribute up to 10% of the peripheral blood NK-cell population and their primary function is cytokine production. However, recent studies have challenged this simple dichotomy by showing that CD56bright, as well as CD56dim, cells are capable of exerting both functions.3 NK-cell activation and function is tightly regulated by multiple activating and inhibitory receptors. NK receptors include (1) the killer cell immunoglobulin-like receptors (KIRs) that recognize human leukocyte antigen (HLA) class I molecules, (2) the C-type lectin receptors, including the activating receptors, NKG2C, NKG2D, and NKG2E, and the inhibitory receptor, NKG2A, and (3) the activating natural cytotoxicity receptors, such as NKp30, NKp44, and NKp46. In hepatitis C virus (HCV) infection, the essential role of NK cells has been shown in several studies. For example, Khakoo et al. found that KIR2DL3, an inhibitory NK-cell receptor, and its HLA-C1 ligand directly influence the outcome of HCV infection.4 During acute infection, NK cells are activated, irrespective of the later outcome of infection,5 and they produce higher amounts of IFN-γ and are more cytotoxic, compared to NK cells obtained from healthy controls. Peak NK-cell activity either precedes or coincides with peak T-cell responses, supporting an indirect role of NK cells in priming and regulating adaptive immune responses.6 During chronic HCV infection, pertubations in NK-cell frequency, phenotype, and function have been reported, as reviewed elsewhere.7, 8 Indeed, peripheral blood NK-cell frequencies are reduced in chronic HCV infection, compared to healthy individuals. In addition, an impaired production of the TH1 polarizing cytokine, IFN-γ, and an increased production of immunoregulatory cytokines, such as interleukin (IL)-10 and transforming growth factor beta, has been reported.9, 10 In contrast, cytotoxicity of NK cells is increased and correlates with the degree of liver inflammation.10, 11 This polarization toward cytotoxicity may be induced by IFN-α.11, 12 Given their important role in the regulation of NK cells, several studies have analyzed the expression of inhibitory and activating receptors on NK cells during acute and chronic HCV infection. Most, but not all, of these studies have revealed an increase in the expression of the inhibitory receptor, NKG2A, and the activating receptors, such as NKp30, NKp44, and NKp46.13, 14 NKp46 is a particularly interesting molecule. It is considered to be the main activating NK-cell receptor and has been shown to be involved in tumor eradication and the killing of virus-infected cells by interacting with viral proteins, and up-regulation of NKp46 in response to IFN-γ can predict a sustained virological response (SVR) in chronic HCV infection.15 In this issue of HEPATOLOGY, two elegant studies from the laboratories of Jacob Nattermann and Hugo Rosen give important new insights into the biological role of NKp46 in HCV infection.16, 17 Indeed, by using different experimental models and different study cohorts, both studies come to similar conclusions. This itself is a remarkable finding in a field where studies examining the phenotype and function of NK cells have often yielded diverging data. The first important finding of these studies is that high expression of NKp46 (NKp46high) defines a specific human NK-cell subset. Indeed, in comparison to NKp46dim cells, NKp46high NK cells are characterized by a higher expression of immature differentiation markers, such as CD127, CD62L, and CD27,17 a higher functional ability (e.g., a higher target cell cytotoxicity) and a higher IFN-γ production after stimulation with IL-12 and IL-1516, 17 as well as a stronger up-regulation of genes involved in cytotoxicicty after stimulation with Toll-like receptor ligands.16 Although the majority of the NKp46high NK-cell subset is also CD56bright, differences in functional and phenotypical properties indicate that NKp46 expression defines a unique NK-cell subset. This is further supported by microarray analysis that showed a differential regulation of more than 800 genes in NKp46high versus CD56bright NK cells.17 Importantly, by using NK cells from chronically HCV-infected patients17 or from healthy donors16 and by using the replicon system17 or the Huh7.5 Japanese fulmanant hepatitis type 1 in vitro infection system16 as a readout, both studies show that NKp46high cells have an increased anti-HCV activity. Most likely, combined noncytolytic and cytolytic effector functions contribute to the antiviral activity of NKp46high NK cells. Indeed, Krämer et al. provide evidence that soluble factors, specifically IFN-γ, contribute to the antiviral effect, because incubation of HCV-replicating Huh7 cells with supernatants from NKp46high cells led to a significant inhibition of HCV replication and because this inhibition could be effectively blocked by the addition of anti-IFN-γ.17 This is in agreement with previous studies that have shown a control of HCV replication by NK-cell IFN-γ secretion in vitro18, 19 and after adoptive transfer of NK/NK T cells after liver transplantation in vivo.20 The contribution of cytolytic effector mechanisms in NKp46-mediated antiviral activity is supported by studies showing that cytotoxicity is the major mechanism involved in NK-cell-mediated elimination of HCV-infected hepatocytes21 and that NK cells can kill HCV-infected hepatocytes by perforin/granzyme and TNF-related apoptosis-inducing ligand–mediated mechanisms.12, 22 Importantly, both studies also present evidence that NKp46 is indeed mechanistically involved in the observed antiviral effects: Blocking of NKp46 led to significantly reduced antiviral effects as well as lower IFN-γ secretion and degranulation,17 and treatment with an agonist NKp46 antibody resulted in a significantly reduced HCV copy number.16 The immunobiological relevance of these elegant in vitro studies is further supported by several important findings in humans. For example, Krämer et al. found a higher frequency of NKp46high cells in the blood of chronically HCV-infected patients, compared to healthy controls. In addition, NKp46high NK cells accumulate in the liver of HCV-infected patients and this correlates negatively with viral load, supporting their direct involvement in viral control.17 However, in this context it is important to note that an accumulation of NKp46high cells was also found in the livers of patients with nonalcoholic steatohepatitis and autoimmune hepatitis, suggesting that NKp46high cells might play a crucial role in other liver diseases as well. In contrast to NKp46high cells, NKp46dim NK cells are not enriched in the intrahepatic compartment and indeed display a lower frequency in the liver, compared to the peripheral blood.17 The involvement of NKp46 in anti-HCV immunity is further supported by the finding by Golden-Mason et al. that female Caucasian Americans (CA) have a higher expression of NKp46, in comparison to male African Americans (AA).16 Accordingly, NK cells from female CAs display a higher cytotoxicity. These findings are of relevance because previous epidemiological studies have clearly shown that HCV-infected patients with female gender and CA race are more likely to spontaneously clear the virus and to achieve an SVR during antiviral therapy with pegylated IFN-α and ribavirin, compared to male AA.23-25 Thus, these results clearly link race- and gender-related variations in NKp46 expression to differential anti-HCV immunity and, more important, to differential HCV natural history and treatment response, strongly supporting the biological relevance of NKp46 in HCV infection. Interestingly, Krämer et al. also observed an inverse association between the frequency of NKp46high NK cells and the stage of liver fibrosis. Mechanistically, this may be explained by the higher cytotoxic activity of NKp46high cells against human hepatic stellate cells (HSCs), compared to the NKp46dim subset.17 This finding is of relevance because activated HSCs are critically involved in the development of liver fibrosis and because the NKp46-mediated killing of HSCs has been recently shown to attenuate liver fibrosis.26 Thus, NKp46 expression is linked to both, antiviral as well as antifibrotic activity. Clearly, these two studies have provided significant new insights into HCV immunobiology. However, several important questions remain that need to be addressed in future studies. For example, the ligand by which HCV interacts with NKp46 remains elusive. In other viral infections, it has been shown that NKp46 interacts with viral hemagglutinins.27 Noteworthy, Golden-Mason et al. demonstrate an up-regulation of the NKp46 ligand on the surface of HCV-infected hepatocytes.16 However, whether this is the result of a specific HCV component or an unspecific stress response is not yet clear. This is a clinically relevant question because NKp46 may represent a promising target in HCV therapy. It will also be important to better characterize the factors that regulate the expansion, maintenance, and liver homing of this specific NK-cell subset. Furthermore, the mechanisms that contribute to the failure of these cells to control HCV in all patients require further investigation to better define their role in HCV immunopathogenesis. Finally, because NK cells can modulate T-cell responses by cytolytically eliminating CD4+ and CD8+ T cells and because NKp46high cells are characterized by a high cytotoxic potential, their effect on the maturation and development of adaptive immune responses will be of interest. In summary, both studies clearly underline the importance of NK cells as key players in HCV immunity and point toward a pivotal role of NKp46. The NKp46high subset is defined by both the production of IFN-γ and high cytolytic activity and has the potential to effectively control HCV replication in vitro by cytolytic and noncytolytic effector mechanisms and to kill HSCs (Fig. 1). Thus, NKp46high NK cells may have an important role in the control of viral replication and the modulation of liver fibrosis. These findings may be an important step toward the development of novel approaches to HCV therapy. NKp46high NK cells are characterized by a high IFN-γ production and a high cytotoxicity. By using these effector mechanisms, NKp46high cells can mediate strong antiviral, as well as antifibrotic, activities. A proposed mechanism for the antifibrotic effect is the killing of HSCs by NKp46high NK cells. The antiviral effect is mediated by noncytolytic as well as cytolytic effector mechanisms.