Approximately one third of the human immunodeficiency virus (HIV)-infected population in Europe and the United States is coinfected with the hepatitis C virus (HCV), and this is increasing as a result of an epidemic of acute HCV infection among HIV-infected homosexual men.[1, 2]
HCV coinfection in HIV-positive individuals is associated with significantly faster rates of fibrosis and higher associated liver morbidity and mortality than in hepatitis C alone.[3-5]
However, a considerable proportion of HIV+ patients manage to overcome acute hepatitis C (ACH) spontaneously, indicating that even HIV-infected individuals can eliminate HCV through their residual immune system. Accumulating evidence suggests that innate immunity, in particular, natural killer (NK) cells, plays an important role in this context.[6-8]
NK cells constitute a major component of the intrahepatic lymphocyte pool. In contrast to the peripheral blood, which contains approximately 5%-10% NK cells, intrahepatic lymphocytes comprise approximately 30% NK cells, and the percentage of intrahepatic NK cells may increase to >50% in liver diseases.
Infections with viruses such as HCV result in activation of NK cells, which can kill virus-infected cells without previous immunization. In addition, NK cells produce proinflammatory cytokines, which induce an antiviral state of host cells and play a critical role in the recruitment of T cells to sites of inflammation. Moreover, NK cells enhance the interactions between antigen-presenting cells and T cells and directly modulate the function of both CD4+ and CD8+ T cells.[10, 11]
Function of NK cells is regulated by interactions of NK cell receptors (NKRs), which, in general, can be divided in activating and inhibitory NKRs, and their respective ligands. Activating NKRs include NKG2C and NKG2D, and the “natural cytotoxicity receptors,” NKp30/44/46. Inhibitory receptors comprise NKG2A and members of the “killer immunglobuline-like” receptor family.
Immunogenetic studies indicate that NK cells may influence the outcome of acute HCV infection as well as immunopathogenesis in chronic hepatitis C (CHC).[13-16] Accordingly, in vitro studies suggest that NK cells are able to recognize and kill HCV-infected hepatocytes.[17, 18] Recent studies in HCV monoinfection indicated that NK cells are activated in the acute phase of HCV infection.[6-8] More important, these studies provided the first data indicating that NK cells might affect the natural course of AHC. However, it remained unclear whether and how NK cells display activity against HCV or rather indirectly modulate HCV antibody (anti-HCV) immune responses. Moreover, regulation of NK cell activity may be even more complex in HIV-coinfected patients because HIV monoinfection has been shown to be associated with significant alterations of the NK cell pool.
In the present study, we show that during AHC, NK cells display a significant in vitro anti-HCV activity, even in HIV-coinfected patients. More important, we demonstrate, for the first time, that effective interferon-gamma (IFN-γ)-mediated inhibition of HCV by NK cells is associated with a self-limited course of acute HCV infection.
A substantial proportion of de novo HCV-infected patients are able to spontaneously eliminate the virus, even in the presence of HIV coinfection, but the factors determining the natural course of AHC are only incompletely defined.
Increasing data indicate an important role for NK cells,[6-8, 13-16, 20-22] and recent studies suggested that distinct populations of NK cells may be involved in the early control and clearance of HCV infection.[6-8]
Here, we show that during the acute phase of HCV infection, NK cells effectively block HCV replication in vitro. More important, we provide first evidence that anti-HCV NK cell activity during the acute phase of hepatitis C is associated with different outcomes of the infection, because we found NK cells from patients clearing the virus to be significantly more effective in blocking viral replication, as compared to NK cells obtained from patients subsequently progressing to CHC.
This is in contrast to previous studies in HCV+/HIV− patients[6-8] that observed significant alterations of NK cell phenotype and activity, but did not find any significant associations between outcome of acute HCV infection and NK cell functions. However, these studies used the hematopoietic K562 and/or 221 cell lines as targets,[6-8] which might not be ideal for studying NK cell activity against a hepatotropic virus such as HCV. To overcome this obstacle, we used the well-established HuH7A2HCVreplicon cell system.
Noncytolytic effector functions are considered critical for antiviral activity of HCV-specific CD8+ T cells. In line with these data, we recently demonstrated a role for IFN-γ secretion in NK cell-mediated inhibition of viral replication in CHC. In the current study, we present several lines of evidence indicating that IFN-γ secretion by NK cells may also play an important role in modulating the outcome of acute HCV infection.
First, we show that NK cells from resolvers display a significantly stronger IFN-γ secretion after coincubation with HuH7A2HCVreplicon cells than patients with a chronic course of infection. Second, we found the frequency of IFN- γ+ NK cells to be positively correlated with antiviral activity of NK cells, whereas no such correlation was observed for NK cell degranulation. Third, we demonstrated that supernatants of NK cells effectively block HCV replication, indicating that anti-HCV NK cell activity is not dependent on direct cell-cell contact. Fourth, we could show that blocking of IFN- γ with a specific Ab significantly reduced the antiviral function of NK cells. Of note, blocking of IFN- γ neutralized the differences in anti-HCV activity between NK cells from patients who were going to clear the virus and those developing CHC.
Taken together, these data suggest that a robust IFN-γ secretion enables NK cell to effectively fight HCV, thereby favoring the self-limited course of AHC.
This resembles findings in studies on HCV-specific CD8+ T cells showing that during AHC, the first decline of HCV RNA correlates with the initiation of IFN-γ production of virus-specific CD8+ T lymphocytes and the appearance of IFN-γ-producing CD8+ T cells in the liver.[25, 26] Of note, intrahepatic accumulation of IFN-γ-secreting HCV-specific CD8+ T cells has also been observed in the absence of significant liver disease, which further supports the noncytopathic nature of this pathway.
On the other hand, we found patients with AHC to display significantly higher levels of alanine aminotransferase (ALT) and AST, respectively, compared to patients with chronic HCV infection, indicating the presence of cytolytic effector functions. However, in our in vitro studies, a strong NK cell-mediated inhibition of HCV replication was observed in the presence of only minimal cytolysis of HuH7A2HCVreplicon cells and mild AST elevations, even at the highest E:T ratio, suggesting a limited role of NK cell cytotoxicity in AHC. Accordingly, patients with a self-limiting and chronic course of acute HCV infection did not differ significantly with respect to serum levels of AST and ALT, respectively.
However, NK cells have been suggested to be more activated in the intrahepatic compartment, and cytotoxic activity of liver-infiltrating has been proposed to be associated with liver cell damage in CHC. Of note, CD56Bright NK cells, which represent the dominant intrahepatic NK cell subset, displayed a higher degranulation in patients with self-limited infection, as compared to patients developing CHC. Thus, we cannot completely exclude a role for intrahepatic CD56Bright NK cells in liver inflammation during the acute phase of HCV infection.
We and others recently showed that the activating NK cell receptor, NKp46, might be involved in antiviral NK cell activity in patients chronically infected with HCV and demonstrated intrahepatic frequency of NKp46-expressing NK cells to be inversely correlated with HCV loads.[20, 28] In line with these data, we found that during AHC, the frequency of NKp46+ NK cells was positively correlated with in vitro inhibition of HCV replication. Furthermore, surface expression of NKp46 was higher in patients that were able to clear the virus, as compared to those that became chronically infected, although this effect was statistically significant only in the CD56Bright NK cell subset. Furthermore, blocking of NKp46 significantly reduced the capability of circulating NK cells to block HCV replication, suggesting a role of NKp46 in the regulation of anti-HCV immune responses also in the acute phase of HCV infection.
However, the strongest association between NKR expression and outcome of AHC was found for the activating NKG2D receptor with significantly higher surface expression and frequency of NKG2D+ NK cells in patients with a self-limited course of HCV infection. In addition, there was a strong positive correlation between NKG2D expression and IFN-γ secretion as well as inhibition of HCV replication. Accordingly, we found that blocking of NKG2D significantly reduced antiviral NK cell activity, supporting an important role of NKG2D in NK cell-mediated immune responses against HCV. Of note, major histocompatibility complex class I chain-related molecules, the natural ligands for NKG2D, have been shown to be induced on HCV-infected hepatocytes. Thus, it is conceivable that NK cells can identify and kill HCV-infected hepatocytes in a NKG2D-dependent fashion. Moreover, in chronic HCV monoinfection, Sène et al. recently identified HCV NS5A-induced down-regulation of NKG2D, resulting in impaired NK cell cytolytic activity and IFN-γ secretion, as a mechanism potentially involved in viral escape. However, reports on NKG2D in CHC are controversial, and thus further studies are needed to clearly delineate the role of this NK cell receptor in HCV infection.[29-32]
Both patient subgroups (resolvers and chronics) did not differ significantly with respect to age, gender distribution, route of HCV transmission, CD4+ T cell counts, viral loads, or comorbidities. Patients with chronic course of HCV infection comprised a higher number of individuals with a HCV genotype 4 infection, but a lower number of individuals carrying an IL28B C/C genotype. However, none of these parameters was associated with NK cell phenotype and functions, respectively (data not shown). Thus, it remained unclear why patients with a self-limited and chronic course of AHC displayed the observed differences in NK cell phenotype and function.
Another important issue relates to the question of whether our results are specific for HIV/HCV coinfection or can be extrapolated to patients infected with HCV alone. In line with previous reports, we observed HIV monoinfection to be associated with significant phenotypic and functional alterations of the NK cell compartment. Thus, some of our observations may, at least in part, be attributable to HIV coinfection, despite the fact that all our HIV- patients were HIV RNA negative under efficient HAART and displayed robust CD4+ T cell counts above 400 cells/μL. Indeed, increased expression of NKG2D and/or NKp46 as a potential predictor of self-limited course of AHC is somewhat in contrast to reports in HCV monoinfected patients[6-8] and therefore may represent an HIV-associated phenomenon. Accordingly, dys-regulated expression of NKG2D and NKp46 has been proposed as a mechanism potentially involved in impaired NK cell functions in HIV RNA+ patients,[33, 34] and effective antiretroviral therapy has been shown to be associated with normalization of NK cell receptor expression. Thus, higher frequencies of NKG2D and/or NKp46-expressing NK cells in resolvers may characterize HIV patients that are more likely to clear HCV as the result of a better reconstitution of NK cell functions under HAART. However, data obtained in HCV monoinfection are still conflicting. For instance, Alter et al. found low expression of NKG2D to be associated with spontaneous viral clearance, whereas Amadei et al. did not. Moreover, functional data obtained in monoinfected patients support a role for these two receptors in modulating anti-HCV immune responses irrespective of HIV coinfection.[20, 28, 29, 35]
Taken together, our data indicate that during the acute phase of hepatitis C, a robust IFN-γ-mediated inhibition of HCV replication by NK cells is associated with spontaneous clearance of HCV infection, underpinning the important role of NK cells.