Control of HCV Infection by Natural Killer Cells and Macrophages

Abstract

Host defense against invading pathogens within the liver is dominated by innate immunity. Natural killer (NK) cells have been implicated at all stages of hepatitis C virus (HCV) infection, from providing innate protection to contributing to treatment-induced clearance. Decreased NK cell levels, altered NK cell subset distribution, activation marker expression, and functional polarization toward a cytolytic phenotype are hallmarks of chronic HCV infection. Interferon α (IFN-α) is a potent activator of NK cells; therefore, it is not surprising that NK cell activation has been identified as a key factor associated with sustained virological response (SVR) to IFN-α-based therapies. Understanding the role of NK cells, macrophages, and other innate immune cells post-SVR remains paramount for prevention of disease pathogenesis and progression. Novel strategies to treat liver disease may be aimed at targeting these cells.

Successful treatment with interferon (IFN)-based therapies is associated with decreased inhibitory receptor expression, normalization of natural killer (NK) cell levels in the periphery and in the liver as well as restoration of IFN-γ production. The development of direct-acting antivirals (DAAs) has revolutionized the treatment of chronic hepatitis C virus (HCV). On DAA treatment, disappearance of virus correlates with phenotypic changes in NK cell populations as early as 2 weeks into treatment. However, other phenotypic changes (activation markers) are not evident until several weeks after clearance; although total NK numbers do not change, the frequency of immature NK cells decrease by week 2. A direct role for NK cells in a DAA-mediated HCV cure has yet to be shown. Monocytes and macrophages play vital roles in the innate immune defense against pathogens, but also stimulate interleukin (IL)-1b to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These pathways can shape both innate and adaptive immunity by regulating both NK cell function and T-cell differentiation, which influences the outcome of HCV infection. Additionally, impaired macrophage and monocyte phagocytosis and differentiation may contribute to persistent HCV infection and subsequent uncontrolled inflammation thus promoting progressive liver damage. Cooperation between NKs and macrophages shapes the host response to HCV. Galectin-9 production by hepatic macrophages may help establish and maintain chronic HCV infection through inhibition of NK and T-cell responses. The many strategies HCV uses to perturb both NK cell and macrophage function and the consequent dysregulation of these populations highlights the important role that these innate cell populations play in controlling HCV infection.

THE LIVER IS AN INNATE IMMUNE ORGAN

Host defense against invading pathogens within the liver is dominated by innate immunity. The liver is highly enriched with innate immune cell populations including macrophages (Kupffer cells [KCs]) and NK cells, which represent two key cell populations critical in host defense against potentially harmful agents (Gao et al. 2008). KCs are the largest group of fixed macrophages in the body, accounting for ∼20% of nonparenchymal cells (NPCs) in the liver, whereas lymphocytes represent ∼25% of the NPC pool. The average human liver contains a population of ∼10¹⁰ lymphocytes (Racanelli and Rehermann 2006) and hepatic NKs comprise 30%–50% of hepatic lymphocytes (Doherty and O'Farrelly 2000; Gao et al. 2009; Zheng et al. 2018). The relative enrichment for NK and KCs reflects their critical role in immune surveillance and elimination of pathogens encountered in the liver (Gao et al. 2009). In normal liver, KCs are important for maintaining local and systemic tolerance (Roland et al. 1993; Knolle et al. 1995); however, they also play a significant role in defense against infections. These highly phagocytic cells express a high density of surface scavenger receptors and promote tolerance in the face of the constant immunogenic challenge provided by commensal bacterial products and food antigens (Tacke and Zimmermann 2014). KCs also play an important role in the elimination of pathogens producing proinflammatory cytokines such as IL-12 and IL-18, which activate and expand NK cells as well as other factors that promote the infiltration and activation of monocytes and neutrophils (Racanelli and Rehermann 2006). In addition, KCs can activate hepatic stellate cells (HSCs) promoting fibrosis (Pradere et al. 2013). NK cells are considered the principal innate effectors representing the first line of defense in the control of viral infections (Biron et al. 1996; Lanier 2008a; Vivier et al. 2008). NK cells can kill virus-infected cells without prior sensitization via the release of granzyme- and perforin-containing cytotoxic granules and/or engagement of death receptors such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Farag and Caligiuri 2006). In addition to their cytotoxic properties, NKs produce cytokines, predominantly IFN-γ, which limits viral replication (Moretta et al. 2002) and promotes a proinflammatory phenotype in macrophages (Tosello-Trampont et al. 2016). NKs also influence the activation and/or trafficking of other key immune cell populations (Trinchieri 1995b; Eberlein et al. 2010), including dendritic cells (DCs) (Fernandez et al. 2002) and T cells (Zingoni et al. 2005; Welsh and Waggoner 2013).

NATURAL KILLER CELL BIOLOGY

An appreciation of the role played by NK cells in the control of HCV infection requires a basic understanding of the biology of NK cells. Because of their potent cytotoxic and inflammatory properties, NK cells have the potential to eliminate virally infected cells but also to promote immune-mediated pathology in response to chronic viral infection (Tian et al. 2013; Zheng et al. 2018). The activities of NK cells are tightly controlled by integration of signals from an array of inhibitory and activating NK receptors (NKRs) expressed on their surface. Inhibition is the default in steady-state conditions. Negative signaling induced by inhibitory receptors opposes NK-cell activation signals, thus providing an important safeguard from NK-cell reactivity toward normal, healthy cells (Watzl and Long 2010). The balance favoring inhibition seen under normal conditions is shifted toward activation in the setting of viral infection (Vidal et al. 2011). NK-cell activation includes loss of constitutive inhibition through down-regulation of major histocompatibility complex (MHC) class I (Llano et al. 2003), up-regulation of activating receptors and/or their ligands (Vidal et al. 2011), cell adhesion, and response to inflammatory cytokines IL-2, IL-15, IL-12, IL-18, and type I IFNs induced by viral infection (Herberman et al. 1981; Herberman 1987; Caligiuri et al. 1990; Carson et al. 1994; Trinchieri 1995a; Bryceson et al. 2009).

NATURAL KILLER CELL RECEPTORS

Human NK cells are identified as large granular lymphocytes that are negative for the T-cell antigen CD3 and positive for the expression of CD56. They can be characterized as effector or immature subsets based on the density of CD56 (neural cell adhesion molecule [NCAM]) expression. CD56^dim NKs, which make up ∼90% of circulating NKs, represent the effector population with high cytotoxic activity and relatively low IFN-γ secretion. CD56^bright NKs are a more immature population that secrete relatively high levels of IFN-γ but display little cytotoxicity (Michel et al. 2016). The majority of activating NKRs function as coreceptors requiring a second signal provided by loss of inhibition, cytokine stimulation, or an additional activating receptor (Horng et al. 2007; Bryceson et al. 2009). Natural cytotoxicity receptors (NCRs) NKp46 (NCR1), NKp44 (NCR2), and NKp30 (NCR3) are involved in cytotoxic activation and promote the clearance of virus-infected cells (Moretta et al. 2000, 2001; Raulet 2004; Lanier 2005, 2008b; Koch et al. 2013). Among the NCRs, NKp46 is the only receptor that has an ortholog in other species suggesting that NKp46 is the primary NCR involved in pathogen recognition (Koch et al. 2013). The other dominant NK-activating signal is provided by the evolutionarily conserved natural killer group 2 (NKG2) C-type lectin-like receptor family member D (NKG2D). Through recognition of ligands induced by cellular stress or infection, NKG2D plays an important role in the control of viral infections (Bauer et al. 1999). Activated NK cells can express TRAIL, which is responsible for extrinsic induction of cell death. TRAIL and its receptors have been shown to play important roles in the immune response to viral infections (Falschlehner et al. 2009). The killer immunoglobulin-like receptors (KIRs) family represents the main class of NK inhibitory receptors. They are highly diverse and interact with polymorphic MHC class I ligands. Their primary function lies in constitutive inhibition and in the generation of diversity in immune responses to pathogens and as such have received considerable attention as potential disease association markers (Parham 2005). The other dominant NK inhibitory signal is provided by NKG2A, which forms dimers with CD94 and binds human leukocyte antigen (HLA)-E (Romero et al. 2008), which presents leader peptides derived from classical MHC class I molecules (Braud et al. 1998; Bryceson et al. 2009). CD94/NGK2A serves to monitor appropriate expression of MHC class I and senses changes in overall MHC class I expression that arise from viral infection (Biassoni 2009). NKs are highly responsive to their environment and express several cytokine receptors for innate cytokines including type I IFNs. Additional receptors not exclusive to NK cells are involved in activation or inhibition, for the most part promoting adhesion to target cells. These include DNAX accessory molecule-1 (DNAM-1) and NKp80, also known as killer cell lectin-like receptor subfamily F, member 1 (KLRF1), involved in epithelial and myeloid cell interactions (Fig. 1; Biassoni 2009).

Figure 1.

Natural killer receptors (NKRs). The activity of NK cells is determined by integration of signals arising from engagement of activating and inhibitory cell surface receptors. NKs respond to numerous cytokines and use several coreceptors not exclusively expressed on NKs. (KIR) killer immunoglobulin-like receptor, (HLA) human leukocyte antigen, (NKG2) natural killer group 2 D/A, (IFN-α) interferon α, (IL) interleukin, (DNAM) DNAX accessory molecule-1, (TIGIT) T-cell immunoreceptor with Ig and ITIM domains, (KLRG1) killer cell lectin-like receptor G1.

NATURAL KILLER CELLS AND HCV INFECTION

NKs have been implicated at all stages of HCV infection, from providing innate protection to contributing to treatment-induced clearance. Genetic studies have linked polymorphisms in MHC class I and KIR to HCV resistance after low-dose exposure (Khakoo et al. 2004; Romero et al. 2008). In other studies, HCV-exposed individuals (either through intravenous drug use or health care workers with accidental percutaneous exposure) who remained uninfected, increased NCR expression, and enhanced NK functionality were associated with protection from HCV infection (Golden-Mason et al. 2010; Werner et al. 2013). In the acute phase of HCV infection, NKs display an activated phenotype with enhanced cytotoxicity and IFN-γ production when compared with uninfected controls (Amadei et al. 2010; Pelletier et al. 2010; Alter et al. 2011). An increased frequency of immature/regulatory CD56^bright NKs early in acute HCV infection has been reported, which correlated with serum HCV core protein levels and normalized with spontaneous viral clearance (Golden-Mason et al. 2014). Natural cytotoxicity was reduced and did not recover after viral clearance. In vitro culture of purified CD56^bright NK cells with HCV-core protein in the presence of IL-15 maintained a significant proportion of NKs in the immature state. The in vitro effect of core closely correlates with NK characteristics measured directly ex vivo in acute HCV infection. Pathway analysis suggests that HCV-core protein attenuates NK IFN type I responses. This study suggests that HCV-core protein alters NK cell maturation and influences the outcome of acute infection (Golden-Mason et al. 2014). Spontaneous clearance of HCV is associated with lower levels of NKG2A (de Groen et al. 2017). The HCV serine protease NS3 may play a role in the abrogation of NK-cell functions in the early phase of infection through down-regulation of NKp46 and NKp30 receptors on NK cells (Yang et al. 2017). HCV reinfection in the early posttransplant period is delayed (>1 wk) in recipients of liver allografts containing a higher proportion of NKp46 NK cells, suggesting their involvement in control of HCV acutely (Tanimine et al. 2016). The above studies, although showing involvement of NK cells in the acute phase of HCV infection do not provide clear evidence for a direct role for NK cells in determining the outcome of infection. It is thought that an indirect effect on downstream T cells may be responsible for early control of HCV infection (Marras et al. 2011). A recent study suggests that NK cell activation may even favor progression to chronicity rather than viral control through elimination of helper T cells (Hengst et al. 2019). Emerging data in other viral models (e.g., LCMV) indicate that NK cells can negatively regulate viral-specific T cells via NCR1, protecting the host from an “overshooting” T-cell response (Pallmer et al. 2019).

Decreased NK cell levels, altered NK cell subset distribution, activation marker expression and function are hallmarks of chronic HCV infection. NK cell frequency is reduced in chronic HCV compared with healthy controls (Meier et al. 2005; Morishima et al. 2006; Bonorino et al. 2009; Oliviero et al. 2009). The reason for this decrease is currently unknown but is probably not caused by NK cell recruitment to and compartmentalization in the liver as hepatic NK cell levels are also decreased (Kawarabayashi et al. 2000; Deignan et al. 2002; Bonorino et al. 2009). Decreased levels of effector NKs and/or increased levels of immature NKs is a consistent finding across different chronic HCV patient cohorts (Golden-Mason and Rosen 2013). Changes in NK phenotype have been widely reported although inconsistent data exist with respect to specific NKR expression. These discrepancies could be explained by several factors including the use of fresh or frozen cells, different methodologies, the characteristics of the cohort, and relatively small sample size (Cheent and Khakoo 2011). The phenotype of NK cells provides us with several clues as to the involvement of this population in control of chronic HCV. In the chronic phase of infection, NKs display an activated phenotype; however, their functionality may be skewed away from IFN-γ production toward cytotoxicity, which favors viral persistence and liver damage (Morishima et al. 2006; Golden-Mason et al. 2008; Oliviero et al. 2009; Ahlenstiel et al. 2010; Dessouki et al. 2010; Mondelli et al. 2012). This phenomenon may be linked to the requirement for caspase activation for IFN-γ and TNF-α production, which is dispensable for cytotoxicity (Ussat et al. 2010). Insufficient IFN-γ responses may result in increased viral replication as IFN-γ has direct antiviral properties and controls viral replication in a dose-dependent manner in vitro (Wang et al. 2008; Crotta et al. 2010). In addition to antiviral activity, IFN-γ is important for activation of macrophages as well as the differentiation and trafficking of appropriate helper T-cell responses (Boehm et al. 1997; Rotondi and Chiovato 2011). The contribution of individual NKRs to viral clearance or persistence remains to be clarified. A consistent finding across several patient cohorts is increased expression of the inhibitory receptor NKG2A, which suggests functional inhibition of NK cells and increased risk of viral persistence (Jinushi et al. 2004; Takehara and Hayashi 2005; Nattermann et al. 2006; De Maria et al. 2007; Golden-Mason et al. 2008; Yoon et al. 2016). Alternatively, the increased expression of NKG2A may reflect altered subset distribution as immature NKs express high levels of this receptor.

The evidence with respect to NCR expression in chronic HCV is conflicting as both decreased (Nattermann et al. 2006) and increased expression (De Maria et al. 2007; Ahlenstiel et al. 2010; Harrison et al. 2010) have been reported. A significant role for the NKG2D pathway in the defense against HCV infection is suggested by several studies, although the overall contribution of the NKG2D pathway in the control of HCV infection is not fully elucidated (De Maria et al. 2007; Oliviero et al. 2009; Sene et al. 2010). The HCV-NS5A protein down-regulates expression of NKG2D on NK cells via the TLR4 pathway thus impairing their function. The suggested mechanism is that NS5A triggers IL-10 secretion from monocytes, which in turn promotes TGF-β production that leads to down-modulation of NKG2D expression and impaired effector functions both IFN-γ production and degranulation (Sene et al. 2010). The HCV protease NS3/4A reduces the expression of NKG2D ligands MICA and MICB (Wen et al. 2008). Direct contact with HCV-infected cells impaired NK cell degranulation, lysis activity, and IFN-γ production, and this inhibition was associated with down-regulation of NKG2D and NKp30 on NK cells. These observations suggest that direct cell-to-cell interaction between NK cells and HCV-infected hepatocytes may impair NK cell function in vivo and thereby contribute to the establishment of chronic infection (Yoon et al. 2011). Augmentation of NKG2D activity may enhance immunity to some cancers or infections. For this to be possible, more research is needed to further understand mechanisms that regulate NKG2D function, expression, and signaling (Burgess et al. 2008). NKG2D expression has been variously reported to be up- or down-regulated or unchanged in HCV infection (Jinushi et al. 2004; De Maria et al. 2007; Ahlenstiel et al. 2010; Sene et al. 2010; Golden-Mason and Rosen 2013) and its precise role in HCV remains undefined.

LIVER NK CELLS

Intrahepatic NK cells may behave differently to peripheral NK cells owing to the “tolerogenic” environment in the liver (Doherty and O'Farrelly 2000). Much of what we know about hepatic NK cells in HCV infection is inferred from our knowledge of the altered expression of important NK cell ligands in infected liver and cell culture systems. Direct interaction between NKs and infected hepatocytes down-regulates NKG2D and NKP30 and inhibits NK cell function (Yoon et al. 2011). HCV core protein can up-regulate MHC class I expression on hepatocytes (Herzer et al. 2003), which acts as a ligand for inhibitory KIR, another potential NK inhibitory strategy at play in the liver. There is impairment of MIC-A/B expression in HCV infection, which may result in lower levels of NK cell activation via the NKG2D receptor (Jinushi et al. 2003; Wen et al. 2008; Sene et al. 2010). The increased expression of NKG2A on hepatic NK cells (Bonorino et al. 2009) may be important as HCV can up-regulate HLA-E, the ligand for NKG2A, in vivo and in vitro thus representing a mechanism by which HCV may modulate the hepatic NK cell response (Jinushi et al. 2003; Nattermann et al. 2005). Liver NK cell levels are decreased in chronic HCV infection and in cirrhosis further decreased (Kawarabayashi et al. 2000; Deignan et al. 2002; Bonorino et al. 2009) but appear to have similar activation status, as evidenced by the expression of CD69. Increased proportions of immature NK cells and increased expression of NKG2A in the liver of chronic HCV-infected patients (compared with chronic hepatitis B virus (HBV) infection) has been shown, which inversely correlated with viral load (Bonorino et al. 2009). Histological parameters do not correlate with NK cell populations (Pernollet et al. 2002), which decrease with histological progression (Tran et al. 1997; Kawarabayashi et al. 2000), suggesting they may not be directly involved in liver damage. Compared with controls, hepatic NKs in HCV-infected patients show reduced expression of TRAIL and impaired degranulation (Varchetta et al. 2012). Increased NKp46 expression has been associated with HCV control in vitro (Golden-Mason et al. 2012). Accumulation of NKp46^High NK cells in the liver of HCV-infected subjects has been shown and the frequency of intrahepatic NKp46^High NK cells was inversely correlated with HCV-RNA levels and fibrosis stage (Kramer et al. 2012). This suggests that hepatic NK cell populations are actively involved in control of HCV and may even play an antifibrotic role.

NKs AND IFN-BASED TREATMENT

Until recently, IFN-α comprised an integral component of treatment for HCV infection. NK cells are one of the primary cell populations responding to IFN-α; therefore, it is logical to assume that NKs will be intimately involved in the response to IFN-based antiviral therapy for chronic HCV (Mondelli 2015). NK cells recognize HCV-infected hepatoma cells after IFN-α stimulation in a DNAM-1-dependent manner. Furthermore, interaction of IFN-α-stimulated NK cells with HCV-infected hepatoma cells efficiently reduces HCV replication (Stegmann et al. 2012). IFN-α also induces TRAIL expression on NK cells and increased expression of TRAIL on NK cells has been associated with control of HCV infection; these observations might account for the second-phase decline in HCV-RNA levels during pegylated-IFN-α therapy (Stegmann et al. 2010). Successful treatment with IFN-based therapies is associated with decreased inhibitory NKG2A expression (Golden-Mason et al. 2011), higher natural and antibody-dependent NK cytotoxicity (Oliviero et al. 2013), and normalization of NK cell levels as well as IFN-γ production (Dessouki et al. 2010). Successful antiviral therapy restores NK cell levels in the liver. Analysis of paired liver biopsy samples has shown that sustained virological response (SVR) is associated with an increase in the total number of intrahepatic NK cells, following treatment with IFN-α alone or combined with ribavirin (Van Thiel et al. 1994; Yamagiwa et al. 2008).

NKs AND DAA THERAPY

The development of drugs directly acting on specific HCV target structures, DAAs, has revolutionized the treatment of chronic HCV and successful treatment can now be offered to virtually all patients irrespective of their comorbidity (Spengler 2018).

IFN-α is a potent activator of NK cells; therefore, it is not surprising that NK cell activation has been identified as a key factor associated with SVR to IFN-α-based therapies (Mondelli 2015). In contrast, DAAs would not be expected to have a direct effect on NK cell phenotype and function; however, rapid control of virus could result in decreased endogenous IFN-α, which may result in decreased activation. Indeed, pilot studies on first-line DAA therapy regimes (daclatasvir/asunaprevir) suggest that NK cells may contribute to clearance of HCV during DAA therapy (Burchill et al. 2015; Serti et al. 2015, 2017; Spaan et al. 2016; Childs et al. 2017). Early studies focused mainly on differences between responders and nonresponders or comparisons with external healthy control groups without liver disease. Together, these studies showed down-regulation of activation markers, in particular, cytotoxicity receptors, on NK cells and normalization of NK cell function after successful DAA therapy. In a more recent study, a Chinese cohort treated with ledipasvir/sofosbuvir, a current mainstream treatment for HCV, expression levels of cytotoxicity receptors NKp30 and NKp46 and the inhibitory receptor NKG2A were down-regulated to yield an NK phenotype resembling that observed in healthy controls, suggesting that NK cell function might be normalized with DAA therapy (Li et al. 2019). In another study, Wang and colleagues showed similar phenotypic changes in NK cells with respect to NKG2A expression and normalization of IFN-γ production 12 weeks after treatment (Wang et al. 2018). In our own study, we evaluated the levels, phenotype, and function of peripheral NK cells during and after successful ledipasvir/sofosbuvir treatment in a homogeneous cohort of all-male HCV genotype 1–infected subjects (Golden-Mason et al. 2018). Our phenotype data suggested early transient activation of NK cells and a reduction in cytotoxic activity 12 weeks after the end of treatment. However, we found no evidence to support improvement in IFN-γ production by NK cells or a reduction in cytotoxicity in our cohort at an early time point (week 2 of treatment) or 12 weeks after the end of treatment, suggesting that NK cell immune function is not fully restored after successful DAA therapy. It remains unclear whether NK restoration is needed for DAA-mediated HCV cure, and reported changes in this population may simply result from the disappearance of virus. Disappearance of the virus correlates with phenotypic changes in the NK cell population. However, several changes are not evident until several weeks after clearance. In addition, the sparse functional data available suggest that NK cell restoration (increased IFN-γ production and reduced cytotoxicity) is not essential for DAA-mediated cure. However, changes seen in the peripheral NK cell populations may not reflect hepatic NK cell populations; therefore, we cannot preclude an essential role for NKs at the site of viral replication.

MACROPHAGES IN HCV INFECTION

Monocytes and macrophages play vital roles in the innate immune defense against pathogens. They produce cytokines and other factors that shape both innate and adaptive immunity by regulating NK cell function and T-cell differentiation, which influences the outcome of HCV infection. Liver-resident KCs are critical for maintaining a tolerogenic environment and homeostasis. KCs can act as accessory cells that sense viral RNA and activate or inhibit NK cells by secreting cytokines. Conversely, activated NK cells produce cytokines that regulate macrophage polarization and function. KCs sense tissue injury by recognition of danger-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) derived from damaged hepatocytes. Activation of KCs induces inflammatory cytokines and chemokines promoting infiltration of bone marrow–derived monocytes, which differentiate into macrophages in response to the hepatic environment (Krenkel and Tacke 2017). The distinction of KCs from monocyte-derived macrophages is not well established in humans (Tacke 2017). Liver macrophages show great plasticity and can adopt a classically activated (M1-like) proinflammatory, an alternatively activated (M2-like) anti-inflammatory, or a tissue repair phenotype (Wynn and Vannella 2016). The polarized M1/M2 model is an oversimplification of the heterogeneity of tissue macrophages, and liver macrophages often express both M1- and M2-like markers simultaneously and are likely involved in tissue repair in response to danger signals (Wynn and Vannella 2016; Krenkel and Tacke 2017). Several studies suggest that polarization toward an M2-like or suppression of the proinflammatory M1-like macrophage phenotype contributes significantly to HCV persistence and chronic inflammation in HCV-infected liver (Ohtsuki et al. 2016; Saha et al. 2016). Chronic hepatitis C is associated with immune infiltration and the infected liver shows a significant increase in total macrophage numbers (Khakoo et al. 1997). Similar to NK cells, HCV-core protein can regulate the activity of monocytes and macrophages. HCV core protein engages TLR2 on peripheral blood monocytes and induces production of multiple inflammatory cytokines. One of the key intrahepatic inflammatory soluble factors produced by KCs in response to DAMP or PAMP interaction is IL-1β, and HCV interaction with macrophages triggers IL-1β production and release through the activation of the NLRP3 inflammasome (Shrivastava et al. 2013). Serum levels of IL-1β are increased in HCV-infected patients (Negash et al. 2013). HCV-core protein is the major specific NLRP3 inflammasome agonist that drives inflammasome assembly leading to the production/release of bioactive IL-1β from macrophages (Negash et al. 2019). The interaction of HCV-core protein with TLR2 on KCs, induces secretion of IL-1β, TNF-α, and IL-10 and up-regulation cell surface PD-L1, which is involved in suppression of HCV-specific T-cell responses promoting viral persistence (Tu et al. 2010). TNF-α produced by activated macrophages induces relocalization of the tight junction protein occludin and increases the permissiveness of hepatocytes to HCV infection (Fletcher et al. 2014). In addition, HCV-core blocks TLR3-mediated secretion of antiviral cytokines IFN-α and IFN-β from KCs (Tu et al. 2010). HCV entry into macrophages mainly depends on phagocytosis, which results in increased expression of IL-1β and IL-6 and promotes apoptosis of macrophages (Liu et al. 2019). Peripheral monocyte phagocytosis and differentiation into M1 or M2 macrophages is impaired in chronic HCV-infected patients compared with healthy controls and can be partially rescued by DAAs (Fan et al. 2015; Zhang et al. 2016). In the setting of HCV infection, impaired macrophage phagocytosis may contribute to chronic infection and subsequent uncontrolled inflammation promotes liver disease.

Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Galectin-9 is highly expressed in several organs including the liver and is the natural ligand for the T-cell immunoglobulin domain and mucin domain protein 3 (Tim-3) (Zhu et al. 2005). Tim-3 is significantly up-regulated on virus-specific T cells of HCV-infected patients, and Tim-3 blockade reverses exhaustion and restores CD4⁺ and CD8⁺ T-cell function in chronic infection (Golden-Mason et al. 2009; McMahan et al. 2010). Galectin-9 production from monocytes and macrophages is induced by IFN-γ (the principal cytokine secreted by activated NK cells), circulates at very high levels in the serum, and its hepatic expression is significantly increased in patients with chronic HCV (particularly on KCs) compared with normal controls (Fig. 2; Mengshol et al. 2010). Exosomes released from HCV-infected hepatocytes induce galectin-9 in cultured monocytes, and circulating nonclassical CD16^posCD14^posHLA-DR^pos monocytes have the highest Gal-9 protein levels in chronically infected patients (Harwood et al. 2016). Galectin-9 induces expansion of regulatory T cells, contraction of CD4⁺ effector T cells, and apoptosis of HCV-specific cytotoxic T lymphocytes (CTLs) (Mengshol et al. 2010). Functional impairment of NK cells, including cytotoxicity and cytokine production, results from galectin-9 engagement (Golden-Mason et al. 2013). Galectin-9 production by hepatic macrophages may help establish and maintain chronic HCV infection through regulation of NK- and T-cell responses.

Figure 2.

Hepatitis C virus (HCV) patients have increased galectin-9 expression in Kupffer cells. Immunohistochemistry was used to stain for galectin-9 protein in paraffin-embedded liver biopsy samples from HCV-infected patients and normal control subjects. Kupffer cells can be identified by their characteristic cigar-shaped morphology. (A) Galectin-9 staining in normal liver (brown). (B) Galectin-9 staining in an HCV patient showing clear up-regulation (Mengshol et al. 2010).

NK AND MACROPHAGE CROSS TALK

NK cells have been shown to play important roles in every stage of HCV infection (Fig. 3). The cross talk between NK cells and macrophages at the site of infection is likely critical for effective control of viral infection. Soluble mediators such as IL-12 produced by TLR-stimulated macrophages induces NK cell IFN-γ production, which leads to further activation of KCs and release of proinflammatory cytokines, including TNF-α, IL-18, and IL-1β. The importance of NK and macrophage function in control of HCV infection is reflected in the diverse strategies adopted by HCV to dysregulate NK cell and macrophage coordinated responses. As discussed above, HCV proteins have been shown to have a direct effect on both NK cells and macrophages. Different viral proteins have been implicated in various effects. For example, HCV core protein inhibits the maturation of NK cells, whereas the HCV NS3 protein down-regulates NCRs important for viral control. The HCV NS5A protein down-regulates NKG2D expression and impairs NK cell functions. NS5A triggers IL-10 production and inhibits IL-12 production by macrophages, which then inhibits NK cell degranulation and IFN-γ production. Insufficient IFN-γ production by NK cells likely favors the polarization of macrophages toward an M2 phenotype. HCV targets NK cells and macrophages indirectly through their interaction with hepatocytes. Direct contact of NKs with HCV-infected hepatocytes impairs both IFN-γ production and cytolytic activity of NK cells. HCV up-regulates MHC class I and HLA-E on hepatocytes, which act as ligands for inhibitory KIR and NKG2A. The HCV protease NS3/4A reduces the expression of NKG2D ligands on hepatocytes further impairing their function. Exosomes released from HCV-infected hepatocytes induce galectin-9 in macrophages, which has an inhibitory effect on NK cells. The many strategies HCV uses to perturb both NK cell and macrophage function and the obvious dysregulation of these populations in the setting of HCV infection highlights the important role these innate cell populations play in control of HCV infection.

Figure 3.

Natural killer (NK) cells contribute to control of hepatitis C virus (HCV) infection. NK cells have been implicated at every stage of HCV infection; the association of NK cells with natural history, acute and chronic infection, and treatment outcome is shown. (KIR) killer immunoglobulin-like receptor, (MHC I) major histocompatibility complex class I, (NCRs) natural cytotoxicity receptors, (TRAIL) TNF-related apoptosis-inducing ligand, (IFN-γ) interferon γ, (NKG2) natural killer group 2 D/A, (SVR) sustained virological response.

FUTURE DIRECTIONS

HCV has evolved with evasive strategies that lead to viral persistence in the majority of cases. The current advances in DAA treatment for HCV makes the eradication of this disease a realistic goal, although there is a residual risk of disease progression (including development of hepatocellular carcinoma). Understanding the role of NK cells, macrophages, and other innate immune cells post-SVR remains paramount for prevention of disease pathogenesis and progression. Novel strategies to treat liver disease may be aimed at targeting these cells.