Review the Role of Natural Killer Cells in Hepatitis C Infection

Antiviral Therapy 2013; 18:853–865 (doi: 10.3851/IMP2565)

Review The role of natural killer cells in hepatitis C infection

Jessica Howell1,2,3*, Kumar Visvanathan3

1Liver Transplant Unit, Austin Hospital, Melbourne, Australia 2Department of Medicine, University of Melbourne, Melbourne, Australia 3Innate Immune Laboratory, Monash University, Melbourne, Australia

*Corresponding author e-mail: [email protected]

HCV infection is an exponentially growing health burden viral infection. A recent explosion in studies exploring the worldwide, with an estimated 170 million people infected. role of NK cells in HCV infection has yielded important Although therapies for HCV are continually improving, mechanistic information and intriguing potential thera- there remain a considerable proportion of patients who peutic options for HCV infection. This review provides a do not achieve viral eradication and develop liver disease. general overview of normal NK cell function and outlines Natural killer (NK) cells are crucial for T-cell activation some of the important mechanisms characterizing the and are one of the first-line sentinel cell responders to immune interplay between NK cells and HCV infection.

Introduction

The global impact of HCV infection is rapidly grow- cells in HCV infection has yielded important mecha- ing, with an estimated 170 million people infected [1]. nistic information and intriguing potential therapeu- By 2020, it is predicted that the number of chronically tic options for HCV infection. This review provides a infected individuals who go on to develop cirrhosis general overview of normal NK cell function and their will increase by 30% and the number who develop specific role in HCV infection. hepatocellular carcinoma will increase by 80% [2,3]. These sobering figures occur in the context of improv- Natural killer cells ing HCV treatments; nevertheless, there are still a considerable number of patients who fail to achieve viral NK cells are mononuclear cells that are primary eradication due to either non-response to treatment or responders to microbial infections and tumour cells, failed tolerance of treatment. but also have an important role in hypersensitivity Most research to date regarding HCV immunity reactions, autoimmunity and allogeneic transplanta- has focused on the role of adaptive immunity in tion [4–6]. Their key functions are initiation of antiviral HCV infection. The role of the innate immune sys- pathways and inflammatory cytokine production, and tem has more recently gained interest for two rea- cytolysis of abnormal target cells [7–9]. Importantly, sons: due to its importance in initial viral recognition NK cells do not require priming to recognize infected after inoculation and due to the fact that adaptive cells and thus form part of the innate immune response, immune responses are not able to completely account but their functions are augmented through activation for the observed immune response to HCV. Innate by other cell types [10]. In this way NK cells are key ini- immune responses help initiate subsequent antibody- tiators of the earliest innate antiviral immune responses. mediated specific adaptive immune responses, which NK cells are CD56-positive, CD3-negative and CD16- are impaired in chronic HCV infection, suggesting a positive cells and comprise approximately 5–20% of potential role for innate immunity in impaired adap- peripheral lymphocytes, but up to 30–50% of intra- tive immune responses to HCV. Natural killer (NK) hepatic lymphocytes [11]. NK cells are controlled by cells contribute to T-cell activation and are one of the surface receptors that engage their appropriate ligand first-line sentinel cell responders to viral infection. A on target cells and this initiates a functional response. recent explosion in studies exploring the role of NK These receptors include killer cell immunoglobulin-like

receptors (KIRs), lectin-like receptors (including hyporesponsive, with impaired cytotoxicity, lower NKG2A-F) and natural cytotoxicity receptors (NKp30, inflammatory cytokine production and greater lev- NKp44 and NKp46) [12]. Most KIRs and CD94/ els of immunoregulatory cytokines, such as IL-10 NKG2A are inhibitory to NK cells [13], whereas secreted [22]. However, this has not yet been con- NKG2D, CD94:NKG2C/E, KIR3DS1 and the natural firmed in human intrahepatic NK cells. cytotoxicity receptors activate NK cells [10]. Other activation markers include CD25, CD69, CD80, CD83 and Hepatic natural killer cells and liver fibrosis CD86, and expression reflects the state of underlying activation and maturity of NK cells [14]. Another specific intrahepatic role for NK cells is inhibi- NK cell populations are heterogenous in their function of fibrogenesis via inhibition of hepatic stellate cells tional responses to stimulation and can be subdivided (HSCs), either directly through induction of HSC apop- based upon their relative expression of CD56 into tosis or indirectly via production of IFN-g, which inhib- CD56bright and -dim subsets, with distinct functional its HSC activation by inducing HSC cell cycle arrest and profiles [15]. However, there is considerable overlap signal transducer and activator of transcription (STAT)1- in function between these different subpopulations as mediated apoptosis [23,24]. Down-regulation or blocking they represent cells at different stages of maturation of KIR leads to enhanced NK-mediated HSC apoptosis rather than distinct cell subtypes. Relatively imma- [23]. NK cells only kill early-activated HSCs, not quiescent ture CD56bright NK cells produce large amounts of or fully activated HSCs, which are distinguished by high cytokine with stimulation, including interleukin (IL)-2, levels of retinoic acid early induced transcript (RAE-1) and IL-12, IL-15 and IL-18, but express low levels of per- TRAIL expression, coupled with down-regulated major forin and are less directly cytotoxic than CD56dim cells. histocompatibility complex class I ligands [24]. Greater They constitute approximately 10% of the peripheral peripheral NK cell cytotoxicity has been associated with blood NK cell population and do not express CD16 or less liver fibrosis in HCV infection and likely reflects this KIR, but do express NKG2A [10]. CD56bright cells do mechanism [25]. however express large amounts of tumour necrosis factor-related apoptosis inducing ligand (TRAIL), which Natural killer cell recruitment and activation binds to death ligands DR4 and DR5 on target cells to induce apoptosis [16]. Importantly, CD56bright cells The key initial step in NK cell activation is recruitment to can mature into CD56dim cells [17]. areas of inflammation. NK cells express several chemokine CD56dim cells, by contrast, constitute approximately receptors in order to be attracted to areas of tissue dam- 90% of peripheral circulating NK cells [10]. CD56dim age [26]. Initial recognition between a target cell and NK cells do not produce as much cytokine as CD56bright cell occurs via activating receptors such as CD16, 2B4, cells in response to target cell engagement, but do pro- NKG2D, DNAX accessory molecule 1 and lymphocyte duce significant amounts of antiviral interferon (IFN)-g function associated antigen-1 (LFA-1) that bind to their and have high levels of perforin expression for cytotoxic- respective ligand expressed on target cells and initiate an ity, particularly when they are of the transient CD56dim inside-out signal for activation of LFA-1 and subsequent CD62L-positive CD57-negative subtype [18]. Termi- adhesion [27]. Target cell recognition by NK cells induces nally differentiated CD56dim cells express high levels of pro-inflammatory chemokine and cytokine release early perforin and also produce greater amounts of cytokine after engagement, including macrophage inhibitory pro- than more immature CD56dim cells in response to tar- tein (MIP)-1a, MIP-1b and RANTES (otherwise known get cell recognition [19]. as CCL5), cytokines tumour necrosis factor (TNF)-a and IFN-g [28]. These cytokines and chemokines then recruit Hepatic natural killer cells additional NK cells and other inflammatory cells to the site of inflammation. Inside-out signalling via LFA-1 The liver contains plentiful amounts of NK cells and requires a low threshold for activation, with increasing many more reside here than are circulating periph- strength of activating stimulus required for MIP-1b secre- erally [20]. The intrahepatic NK cell population tion, CD107a surface expression with degranulation, and dwells within the lumen of the sinusoids [21]. An TNF-a and IFN-g secretion, respectively [15]. This likely important concept in liver immunity is that there is explains why viruses such as HCV can affect some NK a tolerant immune environment to minimize the con- cell functions whilst others remain preserved. stant stimulation from bombardment with antigenic stimuli from the portal system draining the gastroin- Accessory cell stimulation of natural killer cells testinal tract [10]. This has implications for immune responses against intrahepatic viruses, such as HCV. NK cells are activated by cytokines released by neigh- Murine intrahepatic NK cells have been shown to be bouring cells, particularly IL-12 and IL-15 [29]. Myeloid

dendritic cells (mDCs) produce IL-12 in response to viral the adaptive immune response [44]. NK cell cytokine stimulation (such as in response to HCV-mediated Toll- secretion matures and activates DCs, enhancing IL-12 like receptor [TLR]-3 signalling) and this induces IFN-g production and antigen presentation, favouring a pre- secretion by NK cells. Type I IFN produced by activated dominantly Th1 response in downstream activated plasmacytoid dendritic cells (pDCs) and to a lesser T-cells [45]. Thus NK cell activation of DCs then fur- degree mDCs is also an important stimulator of NK ther augments NK cell responses in a positive feedback cells, particularly in the context of viral infections such as loop [14]. Depending on the activation level of NK cells HCV [30–33]. IL-2 released by antigen-specific activated and their ratio to DCs, NK cells can either induce matu- T-cells can also further enhance NK cell responses by ration of monocyte-derived DCs or cause lysis of imma- lowering their threshold for activation. Importantly, NK ture DCs [7,8]. NK cells can also induce memory-like cells, particularly NK CD56bright cells, also secrete IL-2, responses similar to T-cells, with some subsets produc- IL-12, IL-15 and IL-18 with stimulation, and this per- ing Th17-cytokine IL-22 [46]. petuates NK cell activation in a positive feedback loop. Similarly, whilst NK cells are stimulated by type I IFN release by pDCs, NK cell activation further increases Natural killer cell receptors and control of type 1 IFN and proinflammatory cytokine production natural killer cell function by pDCs, leading to an enhanced protective secondary antiviral T-cell immune response [7]. NK cell surface receptor expression is an important mod- ulator of NK cell function in response to stimulation, Degranulation and cytotoxicity combining with accessory cell cytokine production to determine NK cell responses. KIRs have a restricted pat- An important function of NK cells is cytotoxicity medi- tern of expression and interact only with a limited number ated through HLA class I molecule expression and their of human leukocyte antigen (HLA)-C ligands [34]. There interactions with NK cell receptors. NK cells share is marked genetic diversity of the KIRs, both at the genetic cytotoxicity with T-cells, releasing lytic granules con- and allelic level, and their HLA molecule counterparts, taining perforin, granzymes and Fas ligand [47]. NK creating considerable genetic diversity [35]. HLA2DL2 cell cytotoxicity is controlled at various levels along the and HLA2DL3 bind to group 1 HLA-C molecules (HLA- signalling pathway through endosomal compartmen- Cw3), whereas KIR2DL1 binds to group 2 HLA-C mol- talization of signalling proteins to prevent inappropri- ecules (HLA-Cw4) [36,37]. The group 1 HLA-C1 epitope ate release of lytic granules and cell damage. NK cell (HLA-C*01/*03/*07/*08/*12/*14/*16) and the group 2 marker expression, particularly natural cytotoxicity HLA-C2 epitope (C*02/*04/*05/*06/*15/*17/*18) are markers and KIR, also control degranulation. classified by a dimorphism at position 80 of the a1 domain Cytolytic activity of NK cells is enhanced by type I [38]. By contrast, NKG2A is able to bind to HLA-E. IFN-producing cells, particularly pDCs [14,30–32]. KIR phenotypes modulate the strength of NK cell This provides an alternate pathway for cytolysis con- immune responses to a given immune stimulus through trol to NK cell marker expression via other signalling the strength of binding between KIR and its corre- receptors such as TLRs, which are expressed by pDCs sponding inherited HLA. The affinity for KIR2DL2 is and activated by viruses such as HCV [48]. lower for HLA-C2 than for HLA-C1 epitopes, hence the interaction between KIR2DL2 and HLA-C2 pro- Antiviral responses duces weaker inhibition [39–41]. NK cell marker expression is also influenced by exter- The other important function of NK cells is secretion nal stimuli. For example, NKG2D expression is reduced of IFN-g in response to viral stimulation. NK cells can by IL-10 and tumour growth factor (TGF)-b secretion by directly inhibit viral replication in infected cells [49] and regulatory T-cells [42]. Some NK cell marker ligands, such also can activate both macrophages [50] and inducible as HLA class I molecules and UL16 binding proteins are nitric oxide synthase-dependent antiviral pathways [51]. stress-inducible [43], allowing alteration of NK cell func- IFN-g secretion is dependent on IL-12 released by acces- tion by the inflammatory microenvironment. The more sory cells [52]. Antiviral products released by NK cells common NK cell receptors are outlined in Figure 1. also increase interferon regulatory factor (IRF)-3 and IRF-7 signalling pathways in hepatocytes, providing an Natural killer cell functions: modulation of important link between peripheral NK cells and liver adaptive immunity cell function in HCV immunity [53]. By contrast with other NK cell functions, which self- NK cells provide an important bridge between innate propagate via positive feedback cytokine loops, IFN-g and adaptive immunity. When activated, they engage secretion is controlled by a negative feedback loop. in a key interplay with DCs leading to activation of IFN-g activates the Janus kinase–STAT pathway in NK

Antiviral Therapy 18.7 855 J Howell & K Visvanathan

Figure 1. Inhibitory and activation marker expression on natural killer cells and their corresponding ligands

PAMPs MIC-A/B,

ULBPs TLR-2, -4 HLA-C2

NKG2D HLA-E HLA-C1 NKG2C KIR2DL1

HLA-E TLR-3, -7, -9 KIR2DL2/3 NKG2 PAMPs HLA-E E NKG2A

BAT-3, B7-H6, CMV, pp65 NKp30 Activating Inhibitory markers markers

Viral NKp44 RIG-1, NOD1 haemagglutinin

PAMPs NKp46 Viral haemagglutinin

CD16

IgG

KIR3DS1

HLA-Bw4

Natural killer (NK) cell function is controlled by the balance between activation and inhibition marker expression. NKp30, NKp44 and NKp46 are activated directly by viral pathogens. CD16 binds to circulating immunoglobulin G (IgG), linking the adaptive immune response. CD94:NKG2A, CD94:NKG2C and CD94:NKG2E all bind to corresponding human leukocyte antigen (HLA)-E, whereas NKG2D binds to MIC-A/B. Killer cell immunoglobulin-like receptors (KIRs) bind to corresponding HLA-C or Bw molecules. NK cells express Toll-like receptors (TLRs)-2, -3, -7, -8 and -9, which are directly activated by pathogen-associated molecular patterns. Retinoid inducible gene-1 (RIG-1) and nucleotide binding oligomerization domain containing protein 1 (NOD1) pathways are also activated in NK cells. PAMPs, pathogen-associated molecular patterns; ULBPs, UL16 binding proteins.

cells, in turn activating suppressor of cytokine signal- amounts of IFN-g when NK cells are primed by prior ling, which then inhibits NK cell IFN-g secretion [54]. exposure to IL-2 or IFN-a [55]. TLR-3 has also been shown to up-regulate both TNF-a and IFN-g Natural killer cells and innate immune expression in NK cells [56,58]. This is important in pathogen recognition receptors HCV infection, as HCV non-structural proteins are pathogen-associated molecular patterns (PAMPs) for NK cell function is also controlled by pathogen recog- TLR-3 and TLR-7/8, allowing both direct and indirect nition receptors, such as TLRs and RNA helicases, both activation of NK cells by HCV. directly through NK cell receptor expression and through Retinoid inducible gene-1 (RIG-1) and nucleotide bind- accessory cell receptor expression, such as by DCs. ing oligomerization domain-containing protein-1 can NK cells express TLR-3, -7, -8 and -9 and these also be activated in NK cells and leads to up-regulation receptors are functional [55,56]. TLR-3 and TLR-7 of NK cell function through the IFNβ promoter stimula- potentiate NK cell cytotoxicity both directly and tor-1–melanoma differentiation-associated gene 5 path- indirectly via accessory cell production of IL-12 [55]. way [56,59]. Similarly, RIG-1 pathway activation by viral The amount of IL-12 produced in this way is usu- PAMPs such as HCV in mDCs also leads to increased NK ally enough to trigger cytotoxicity but not enough to cell expression of activation marker CD69 and enhanced trigger IFN-g production [57]. However, TLR-7 stim- cytotoxicity, mediated by IL-12 [60]. RIG-1 signalling is ulation promotes NK cell production of substantial also targeted and inhibited by HCV infection [61,62].

Thus NK cell function can be activated directly which CD56dim NK cells are particularly adept) dur- through NK cell receptor engagement with corre- ing acute HCV infection correlates with the magni- sponding ligands including viral molecules, or indi- tude of the HCV-specific T-cell response [68]. By con- rectly via accessory cell production of cytokines. The trast, there are greater numbers of CD56bright cells existence of several avenues for modulation of NK cell in patients who become chronically infected rather function, such as via NK cell surface receptor expres- than those who spontaneously clear infection [29]. sion and pathogen recognition receptor pathways, This demonstrates that reduction of the relative num- allows fine control of NK cell immune responses. ber of CD56dim cells to CD56bright cells predisposes They also represent multiple targets for viruses such to abortive viral clearance. Patients who do not clear as HCV infection. virus also have higher NKG2A/C expression and these CD56bright cells (both peripheral and intrahepatic) Natural killer T-cells produce less IFN-g [69]. NKp30 expression is greater in exposed but unin- NK T-cells (NKTs) are distinct from NK cells both in fected subjects [12]. However, reduced frequency function and receptor expression. NKTs do express NK of NKp30+NKp46+CD16+NKG2D+ cells has been cell markers, but also T-cell receptors (TCR) [63]. They reported in those with acute HCV who went on to are a highly heterogenous cell population, classified spontaneously clear infection [70]. as variant or invariant NKTs based upon their diver- NK secretion of IFN-g has been shown to be sity of TCR expression. Invariant NK T-cells (iNKTs) lower in patients with chronic HCV infection com- comprise functionally distinct groups, including CD4+, pared with those who spontaneously clear infection. CD8+ and CD4-CD8- cells. CD4+ and CD4-CD8- iNKTs It is thought that IFN-g leads to heightened NK cell secrete both Th1 (IFN-g) and Th2 cytokines (IL-4, IL-5 cytotoxicity and subsequent increases in STAT1 and and IL-13), although CD4+ iNKTs secrete more Th2- decreased STAT4, which then reduces IFN-g secretion profile cytokines than double negative iNKTs [64]. via negative feedback [12,71]. CD8+ iNKTs secrete Th1 cytokines, such as IFN-g [64]. There have also been KIR:HLA phenotypes asso- The role of NKT-cells in HCV infection is beyond the ciated with spontaneous HCV clearance. Inhibitory scope of this review. However, it is worth noting that KIR2DL3 is associated with spontaneous resolution although NKT-cells do play a role in HCV infection of HCV and homozygosity for KIR2DL3 is more com- that is distinct from NK cells, they also secrete IFN-g mon in seronegative, aviraemic, HCV-exposed indi- and become biased toward secretion of Th2 profile viduals [72]. It has been postulated that this may be cytokines in chronic HCV infection [65]. due to the inherent weak binding of KIR2DL3 to its HLA-C1 ligand, leading to reduced inhibition of NK Natural killer cells in hepatitis C infection cell function and subsequent reduced susceptibility to HCV infection [40,73]. Surprisingly, this KIR-HLA- NK cells have a prime role in HCV infection and there C combination appears to only be protective in those are many changes in NK cells attributable to HCV, but who contract HCV via intravenous drug use and not in it is worth noting that the current literature is not with- those exposed through blood transfusion. Presumably out controversy. in the latter situation the viral load is far greater and overwhelms the NK cell capacity to clear infection [10]. Acute HCV infection and spontaneous HCV clearance KIR2DL1:HLA-C1 and KIR2DL2 are also protec- NK cells have been studied extensively in acute HCV tive against HCV infection [39–41,74]. There is a infection, particularly in those exposed to HCV but progressive effect of KIR2DL2 inheritance, with the who subsequently remain uninfected. greatest protection conferred by KIR2DL2 homozy- In acute HCV, there is higher NKG2D expression gosity. HLA-E R allele homozygosity (ligand for on CD56bright and -dim NK cell subsets, associated CD94:NKG2A) is also protective against infection, with increased IFN-g production and cytotoxicity specifically with HCV genotypes 2 and 3 [56]. This is compared with healthy controls [29,66]. This is in thought to be due to HLA-E having lower affinity for contrast to what happens in chronic HCV infection, peptides, hence it is expressed at lower levels so there where IFN-g production is impaired. is less inhibition of NK cells via CD94:NKG2A [75]. There are several important differences between A summary of the role of NK cells in acute HCV those exposed and infected with HCV and those who infection is outlined in Figure 2. A summary of the key are exposed but remain uninfected. There is greater differences between patients who are exposed to HCV frequency of CD56dim cells in patients who remain and become chronically infected compared with those uninfected post exposure [67]. This fits with further patients exposed to HCV but who clear infection is pre- evidence that increased NK cell degranulation (at sented in Table 1.

Antiviral Therapy 18.7 857 J Howell & K Visvanathan

Figure 2. Natural killer cell function in initial HCV infection TLR-7 mDC pDC IL-12, IL-17, IL-18

IFN-γ IFN-α IFN-γ,TNF-α Th1 TLR-7/8

MHC class II IL-2 NK cell MHC class I CD107a degranulation HCV IFN-γ Hepatocyte TNF-α

Fibroblast

HSC

Fas/FasL

TRAIL R/TRAIL

Plasmacytoid dendritic cells (pDCs) are stimulated by virus via Toll-like receptors (TLRs) and produce interferon (IFN)-α. Myeloid dendritic cells (mDCs) are stimulated by viral proteins to produce interleukin (IL)-12, IL-17 and IL-18. T-cells produce IL-2 with activation. These cytokines lead to activation of natural killer (NK) cell antiviral function. NK cells produce tumour necrosis factor (TNF)-α, which attracts inflammatory cells to the site of infection and up-regulates TNF-related apoptosis inducing ligand (TRAIL)-mediated killing of virally-infected hepatocytes. NK cells also up-regulate cytotoxicity and expression of cytotoxic granule membrane component CD107a. NK cells increase IFN-γ secretion, which has direct antiviral effects and inhibits hepatic stellate cells and subsequent liver fibrogenesis. IFN-γ secretion further activates mDCs and T-cells through a positive feedback loop. NK cells can also be directly activated via NK receptors and Toll-like receptors. MHC, major histocompatibility complex.

Table 1. Associations between natural killer cell marker expression, genotype and function and clinical outcomes of HCV exposure

Exposed to HCV but clear virus Exposed to HCV and develop chronic HCV infection

Increased NK CD56dim cell frequency Increased NK CD56bright cell frequency Increased NKp30 expression Increased NKG2A/C expression on NK CD56bright cells Reduced frequency of NKp30+NKp46+CD16+NKG2D+ cells Impaired NK cell IFN-g secretion Increased NK cell IFN-γ secretion – KIR2DL3 homozygosity – KIR2DL2 heterozygosity or homozygosity – HLA-E R homozygosity –

Based on data from [12,29,39–41,57,67–72,74]. HLA, human leukocyte antigen; IFN, interferon; KIR, killer cell immunoglobulin-like receptors; NK, natural killer.

Chronic HCV: natural killer cell frequency [25,29], as well as increased CD56-CD3-CD16+ NK Overall circulating NK cell frequency is reduced in cell subsets [44]. Reduction in intrahepatic NK cells HCV infection [76]. However, higher numbers of is also described and has been associated with pro- CD56bright cytokine-producing NK cells relative to gression of HCV infection and development of cir- CD56dim cytotoxic NK cells has also been reported rhosis [77].

Chronic HCV: cytotoxicity There have been mixed descriptions of NKG2D It has been widely reported that there is reduced cyto- expression in chronic HCV infection, with some groups toxicity in HCV infection [10,11,78], which reverts to describing down-regulation and others unchanged normal after successful viral eradication with treat- expression [13,87,100,101]. MICA/B expression is also ment [79]. The reported decrease in CD56dim NK reduced in HCV infection, leading to impaired activa- cells relative to NK CD56bright cells is a potential tion of NK cells and NKG2D binds to MICA/B [85]. cause of low perforin expression and impaired cyto- The HCV NS5A protein can stimulate monocytes via toxicity in HCV infection [25,80], as degranulation TLR4 to produce IL-10 and inhibits IL-12 production, marker expression has been reported to be similar to with subsequent downstream impairment of NK cell controls, suggesting preserved NK cell cytotoxicity in NKG2D expression and function [100]. Similarly, the HCV [76]. Some studies have even reported increased HCV NS3/4A protein has been shown to down-regu- cytotoxicity in chronic HCV infection [81,82], and late NKG2D ligand expression [102]. Down-regulation further studies are required to clarify this issue. of NK cell NKG2D may explain reduced NK cell production of IFN-g and impaired cytotoxicity, as NKG2D Chronic HCV: cytokine production expression levels inversely correlate with HCV liver Cytokine production by NK cells and accessory acti- damage and aminotransferase levels [103]. vating cells is also affected by HCV infection. There There is also evidence that activation cytotoxicity is impaired IL-15 production by accessory cells and markers NKp44, NKp46, NKp30 and NKG2C are up- this contributes to lower relative levels of CD56dim regulated on NK cells in chronic HCV [87,98]. Farag et NK cells and impaired function [83]. Supplementa- al. [93] found that activating receptors were increased tion with exogenous IL-15 has been shown to correct on NK cells exposed to HCV infected serum or HCV functional NK deficits [84] and hints at an intriguing pseudoparticles, whereas inhibitory KIR expression was treatment strategy for investigation in HCV. IFN-a reduced. Others have also reported reduced numbers of secretion by pDCs is also reduced in HCV infection, circulating KIR+ NK cells in HCV infection [20,49,50]. resulting in impaired NK cell activation [33,85]. It is Intrahepatic NK cells have high TRAIL expression in also possible that chronic hypersecretion of IFN-a HCV [69,98] and HCV-infected hepatocytes undergo by pDCs leads to suppression of NK cell function TRAIL-mediated apoptosis [10]. through negative feedback [53]. A summary of the role of NK cells in chronic HCV NK cell cytokine production is skewed towards infection is outlined in Figure 3. A summary of the key immunosuppression in chronic HCV, with greater differences between patients with acute and chronic IL-4, IL-10 and TGF-b secretion and impaired IFN-g HCV infection is presented in Table 2. secretion by NK cells, impairing DC activation and prompting Th2 responses from T-cells [29,86,87]. Natural killer cells and HCV treatment This allows an HCV-permissive immune milieu [88]. There have been several studies assessing the relation- Th2-skewed T-cells, regulatory T-cells and dendritic ship between NK cell function and receptor expression cells contribute to this by secreting IL-10 [89,90]. It and response rates to HCV therapy. HCV treatment is postulated that the HCV E2 viral envelope protein results in an initial phase of viral load reduction in the binds to CD81 and has an inhibitory effect on NK first 48 h of therapy, where IFN-a blocks HCV rep- cell function [78,91]. Indeed, treatment of HCV with lication and virion release. The second, slower phase IFN-a leads to down-regulation of CD81 expression of falling viraemia represents elimination of infected and restoration of NK cell function [92], however hepatocytes and NK cells contribute to this process via whether E2-CD81 binding is the direct cause of NK cytotoxic killing of target cells and TRAIL-mediated cell inhibition is disputed [88,93]. apoptosis [10,104]. Patients who achieve sustained virological response Chronic HCV: natural killer cell marker expression (SVR) to treatment have been shown to have greater NKG2A/CD94 expression on CD56bright cells is numbers of NKG2A+, KIR- NK cells than those who increased in chronic HCV infection, which may con- did not achieve SVR [13]. Akin to the findings in acute tribute to impaired cytokine production by NK cells HCV, KIR2DL3 and HLA-C group 1 genotype is associ- in HCV infection [86,94]. NKG2A levels have been ated with SVR, with the KIR2DL3-HLA-Cw*03 alleles observed to return to normal after treatment with IFN particularly associated with SVR [72,105]. Conversely, and ribavirin [13] and NKG2A levels correlate with patients with the KIR2DL2/KIR2DL2-HLA-C1C2 HCV disease activity [69,95–98]. HLA-E also is overex- genotype are less likely to respond to treatment [105]. pressed on HCV-infected cells due to an endogenously KIR2DL5 frequency is greater in HCV non-responders processed HCV core peptide and this also contributes to treatment [106]. Lack of KIR2DS2 and KIR2DL2 to abnormal NKG2A expression [13,99]. are associated with poor SVR rates in the post liver

Antiviral Therapy 18.7 859 J Howell & K Visvanathan

Figure 3. Natural killer cell function in chronic HCV infection TLR-7 mDC pDC TGF-β, IL-10

IFN-α IFN-γ,TNF-α; IL-10 Th2 TLR-7/8

MHC class II IL-10, IL-4, NK cell IL-6, IL-13 T-reg MHC class I CD107a degranulation HCV IFN-γ Hepatocyte

Fibroblast TRAIL

HSC

Fas/FasL

TRAIL R/TRAIL TGF-β, FGF, PDGF, VEGF, ET-1, MMP-2 and -9, TIMP1, 2

Collagen synthesis

Over time, chronic stimulation of natural killer (NK) cells with plasmacytoid dendritic cell (pDC)-derived interferon (IFN)-a leads to a shift in NK cell function, favouring a viral-permissive, Th2 response. NK cells produce interleukin (IL)-10 and tumour growth factor (TGF)-ß, which dampens activation of dendritic cells and promotes Th2 responses in T-cells. Regulatory T-cells (T-reg) also secrete IL-10 and other cytokines which further inhibit NK cell function. Tumour necrosis factor (TNF)-a production and TNF-related apoptosis inducing ligand (TRAIL) expression are preserved, leading to increased hepatocyte damage. Reduced IFN-g production by NK cells leads to loss of inhibition of hepatic stellate cells, with subsequent up-regulation of pro-fibrotic cytokines and activation of fibroblasts culminating in liver fibrosis. ET-1, endothelin-1; FGF, fibroblast growth factor; HSC, hepatic stellate cells; mDC, myeloid dendritic cells; MHC, major histocompatibility complex; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; TIMP, tissue inhibitor of matrix metalloproteinases; TLR, Toll- like receptor; VEGF, vascular endothelial growth factor.

transplant treatment context. Pretreatment expression Degranulation of peripheral NK cells correlated with of KIR2DL1, KIR2DL3 and KIR3DL1 on NK cells is treatment-induced first phase reduction in viral load higher in patients who are non-responders to treatment, and remained higher in virological responders than including those with poor viral decline on treatment by non-responders for weeks [12]. This demonstrates that week 4 [107]. NK cells are strongly activated by IFN-a, particularly

Early virological response (EVR; defined as >2 log10 cytotoxic function, whereas IFN-g secretion is reduced. IU/ml drop in HCV viral load by week 12 of therapy) IFN-g appears less predictive of response to treatment has been associated with a rapid increase in NK cell in HCV than cytotoxicity [12]. There is also a shift expression of activation markers NKG2D, NKp30 towards greater frequency of NK CD56bright cells and CD16 and decreased inhibitory markers NKG2C, later in treatment [12,108]. 2B4, SIGLEC7 and NKG2A [12]. IL-28B genotype TT patients have greater NKG2A expression, suggest- Natural killer cells in HCV recurrence after ing greater NKG2A-mediated NK cell impairment. A liver transplantation summary of associations between NK marker expres- HCV is the leading indication for liver transplanta- sion and genotype and clinical outcomes of HCV tion in Western nations, however HCV recurrence therapy with pegylated IFN and ribavirin are pre- post-transplant is universal. A significant proportion sented in Table 3. of patients develop aggressive HCV recurrence, with

Table 2. Differences in natural killer cell marker expression, genotype and function between patients with acute HCV infection and chronic HCV infection

Acute HCV infection Chronic HCV infection

Increased NKG2D expression on NK CD56bright and CD56dim cells Reduced NK cell frequency Increased NK cell IFN-g secretion Increased NK CD56bright cell frequency Increased NK cell cytotoxicity Reduced intrahepatic NK cell frequency – Reduced NK cell IFN-g secretion – Increased NK CD56bright cell NKG2A/CD94 expression – Increased HLA-E expression on HCV-infected cells – Reduced MICA/B expression on HCV-infected cells and reduced NK cell activation – Reduced NK cell NKG2D expression – Increased NK cell NKp30, NKp44, NKp46 and NKG2C expression – Increased NK cell TRAIL expression – Reduced NK cell KIR expression

Based on data from [20,29,69,76,77,85,86,99,103]. HLA, human leukocyte antigen; IFN, interferon; KIR, killer cell immunoglobulin-like receptors; MICA/B, MHC class I chain related genes A and B; NK, natural killer; TRAIL, tumour necrosis factor-related apoptosis inducing ligand.

Table 3. Associations between natural killer cell marker expression and genotype and clinical outcomes in HCV therapy with pegylated interferon and ribavirin

Early virological responsea Sustained virological responseb Non-virological responsec

Increased NKG2D expression Increased NKG2A+, KIR- NK cell frequency KIR2DL2/HLAC1C2 genotype Increased NKp30 expression KIR2DL3/HLA-C1C1 genotype Non-KIR2S2 genotype Increased CD16 expression KIR2DL3/HLA-cw*03 genotype Non-KIR2DL2 genotype Reduced NKG2C expression – KIR2DL5 genotype Reduced 2B4 expression – – Reduced NKG2A expression – –

a b Based on data from [12,13,72,105]. Early virological response was defined as >2 log10 IU/ml drop in HCV viral load by week 12 of therapy. Sustained virological response was defined as persistent undetectable HCV viral load 24 weeks after cessation of therapy.c Non-virological response was defined as persistent HCV viraemia after cessation of therapy (including patients who were null responders, partial responders who did not achieve sustained virological response and viral relapsers within 6 months of therapy cessation). HLA, human leukocyte antigen; KIR, killer cell immunoglobulin-like receptors; NK, natural killer.

rapid fibrosis progression leading to graft cirrhosis NK cells without achieving viral suppression through and ultimately graft loss [109,110]. For this reason, increased NK cell activation. Whilst NK cells do lyse there has been great interest in the potential role of HCV-infected hepatocytes directly, NK cell release of NK cells in HCV recurrence post transplantation. IFN‑g, which in turn suppresses HCV RNA expres- Host NK cell frequency is related to severity of HCV sion, is the dominant method of NK cell-mediated recurrence post liver transplant [83]. An increase in NK viral control [53,113]. However, NK cells can activate cell NKG2C expression is also noted early post-trans- or lyse hepatic stellate cells that are key modulators of plant and correlates with HCV recurrence and alanine fibrosis progression and this may explain the effect of aminotransferase post-transplant [111]. KIR2DL3 post-transplant [23,114]. Mismatch between donor and recipient KIR-HLA- Good outcomes in liver transplantation are asso- C ligands is associated with more aggressive recur- ciated with HLA-C2 heterozygous or homozygous rence of HCV post-transplant. Recipient KIR2DL3 grafts, with less graft loss and higher survival due to expression correlates with liver fibrosis progression, less chronic rejection and cirrhosis compared with with mismatch between donor and recipient lead- HLA-C1 homozygous grafts [115]. This fits with cur- ing to fibrosis progression only in the presence of rent knowledge, where the HLA-C2-KIR interaction is KIR2DL3. This appears to be in contrast to its pro- stronger than HLA-C1 and therefore results in greater tective role in acute HCV infection [112] and may NK cell inhibition [116]. However, not all groups in perhaps be explained by greater tissue damage by the field have replicated these findings [117].

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Accepted 30 January 2013; published online 4 April 2013

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