HCV Immunopathogenesis: Virus-Induced Strategies against Host Immunity

Clin Liver Dis 10 (2006) 753–771

HCV Immunopathogenesis: Virus-Induced Strategies against Host Immunity Gyongyi Szabo, MD, PhD*, Angela Dolganiuc, MD, PhD Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, LRB 215, Worcester, MA 01605–2324, USA

Worldwide more than 170 million people are chronically infected with the hepatitis C virus (HCV), which is a frequent cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is a small, enveloped virus that belongs to the Hepacivirus in the family of Flaviviridae [1]. Unlike infection with other hepatotropic viruses, only a small percentage of acute HCV infections are cleared and most infected individuals develop lifelong HCV infection in the absence of efficient treatment. It is believed that both viral and host factors contribute to the inability of the host immune system to clear the initial infection and lead to the high propensity of chronic HCV infection.

Overview of the immune response to hepatitis C infection After HCV infects the liver, viral replication continues and viral particles are constantly released into the circulation. It is generally accepted that adaptive immunity plays a critical role during the clinical course of hepatitis. Recent investigations, however, revealed an equally critical role for innate immune activation. Expression of interferon (IFN) and IFN-inducible genes is detectable shortly after the acute HCV infection in the liver [2]. The initial immune response in viral hepatitis involves natural killer (NK) cells, NKT cells, resident macrophages, and dendritic cells (Fig. 1). The proportion of NK and NKT cells is increased even in the normal liver compared with

* Corresponding author. E-mail address: [email protected] (G. Szabo). 1089-3261/06/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2006.08.028 liver.theclinics.com

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Fig. 1. The immune response in viral hepatitis C involves both the innate and adaptive immune system. Innate immunity involves activation of resident liver macrophages (MØ), dendritic cells, NK cells, and NKT cells, whereas CD4 þ T cells, CD8 þ T cells, and B lymphocytes are effectors of adaptive immunity. CTL, cytotoxic T lymphocyte; HCV, hepatitis C virus; IFN, interferon; IL, interleukin; MHC, major histocompatibility complex; NK, natural killer; TNF, tumor necrosis factor.

the peripheral circulation and when activated during viral infection, these cells produce IFN, which inhibits replication of HCV through a noncytolytic mechanism [3,4]. High type I IFN response in the liver is seen during acute infections with HCV; however, it does not correlate with viral clearance and it is unclear whether the IFN source is immune or parenchymal cells [5]. Resident macrophages and immature dendritic cells (DCs) in the HCVinfected liver are capable of taking up viral antigens, processing and presenting them to other immune cells [6]. Encounter with viral antigens can induce migration of immature DCs into regional lymph nodes and their maturation into a highly antigen-presenting phenotype that enables activation of T lymphocytes and influences polarization of helper CD4þ T-cell subsets [7]. CD4þ T cells have immunoregulatory functions mediated by secretion of cytokines that support either Th1 (cytotoxic T lymphocyte, leading to cytotoxic T lymphocyte generation; secretion of cytokines: interleukin [IL]-2, IFN, tumor necrosis factor) or Th2 (B-cell activation and antibody production; cytokines: IL-4, IL-5, IL-10, IL-13) type immune responses (see Fig. 1). The involvement of antigen-specific CD4þ T cells in HCV eradication has been well described during both acute and chronic HCV

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infection [8]. Although CD4þ T cells do not mediate direct liver cell injury in HCV infection, they are critical in facilitating other antiviral immune mechanisms, such as enhancing CD8þ T-cell effector functions. These antigen-primed cytotoxic T lymphocytes recruited to the liver are critical in eradication of virus-infected cells [8,9].

Hepatitis C virus cellular entry mechanisms The HCV genome encodes a single polyprotein precursor of about 3000 amino acids, which is cleaved cotranslationally and posttranslationally by cellular and viral proteases to yield 10 mature products. The structural proteins encoded by HCV include a capsid core protein and two glycoproteins, E1 and E2, and whole nonstructural (NS) proteins including p7 and NS2-5. The exact mechanisms of HCV entry into the cells are not fully understood. In vitro model systems using virus-like particles, pseudoparticles, or the recently described JFH-1 HCV clone allowed identification of potential HCV receptors interacting with the surface glycoprotein complex of HCV [10–12]. CD81, a member of the tetraspanin family, is a putative receptor for HCV. CD81 has been shown as a binding site for E2 [13], but interestingly ectopic expression of the human CD81 in nonhepatic cells does not lead to HCV entry. The scavenger receptor class B type I (the human analogue is CD36) [14], C-type lectins (DC-SIGN, L-SIGN), the low-density lipoprotein receptor, and glycosaminoglycans have also been suggested as potential receptor entry sites for HCV [15]. All these cellular receptors are widely expressed on immune cells and their role in immunopathogenesis of HCV infection is yet to be determined.

Innate immune responses in hepatitis C infection Recognition of an invading pathogen by cells of the innate immune system, particularly by DCs, triggers type I IFN and inflammatory cytokine production and DC maturation [16]. These pathogen-primed DCs provide a critical link between innate and adaptive immune responses by inducing antigen-specific T-cell activation [17]. Induction of the antiviral innate immune response depends on recognition of viral components by host pattern-recognition receptors [16,17]. The key sensors identified to date that recognize viral components, such as genomic DNA and RNA or doublestranded RNA produced in virally infected cells, include members of the Toll-like receptor (TLR) family and various cytoplasmic RNA helicases. These two classes of pattern recognition receptor molecules are expressed in different cell types and different intracellular compartments. On interaction with virus-derived ligands, both classes of pattern-recognition receptors induce type I IFN responses involving distinct signaling pathways (Fig. 2). Although the TLRs are widely expressed and functional in immune cells,

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Fig. 2. Recognition of HCV is mediated both by toll-like receptor (TLR)-dependent and TLR-independent pathways. TLR-dependent viral recognition involves TLR3, a receptor for double-stranded RNA that can be localized in endosomes or on the cell surface, and TLR7/8, the endosomal receptors that recognize virus-derived single-stranded RNA. TLR-independent viral recognition is achieved by two cytoplasmic helicases, RIG-I and MDA-5. Both pathways result in production of type I IFNs. IFN, interferon; IKK, inducible I B kinases; MDA, melanoma-differentiation-associated gene; RIG, retinoic acid–inducible gene; RIP, receptor-interacting protein.

particularly innate immune cells, helicases are also expressed in parenchymal cells and seem to play a key role in HCV recognition in hepatocytes. Toll-like receptors in hepatitis C virus recognition Of the 10 human TLRs, virus-associated nucleic acids are recognized by TLR3 (double-stranded RNA); TLR7 and TLR8 (single-stranded RNA); and TLR9 (CpG DNA motifs) [16,17]. Double-stranded RNA generated during viral infection as a replication intermediate for single-stranded RNA viruses, such as HCV, is recognized by TLR3. Stimulation by TLR3 induces both type I IFN production and proinflammatory signals [16]. Unlike any other TLRs, TLR3 signaling is independent of the common TLR adaptor, MyD88, and uses TRIF for signal transduction [18,19]. TRIF interacts with receptor-interacting protein 1, which is responsible for activation of NF-B [20]. TR receptor-interacting protein IF also activates TRAF-familymember-associated NF-B activator binding kinase 1 (TBK1) and inducible

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I B kinases leading to direct phosphorylation of IRF3 and IRF7 [21]. TLR3 uses TRIF to induce apoptosis through a receptor-interacting protein/ FADD/caspase-8-dependent and mitochondrion-independent pathway [22]. TLR3 is expressed in hepatocytes and a variety of epithelial cells including biliary and intestinal epithelial cells. TLR3 is also highly expressed on CD8þ DCs, facilitates phagocytosis of apoptotic bodies of virus-infected or double-stranded RNA–loaded cells, and triggers DC maturation. Signaling through TLR3 promotes cross-priming of T cells [23]. TLR3 is expressed in conventional DCs that phagocytose dying cells, but not in plasmacytoid DCs [16]. TLR7 and TLR8 show high homology and recognize synthetic antiviral components (R848, imiquimod, and so forth); guanine nucleotide analogues; and uridine-rich or uridine-guanosine–rich single-stranded RNA of both viral and host origin [24–26]. Both TLR7 and TLR8 are expressed within the endosomal membranes with the ligand binding domain facing the lumen of the endosomes and the TIR signal domain positioned in the cytoplasmic side [27]. This is important considering that many enveloped viruses traffic into the cytosol through the endosomal compartment and some of the putative HCV receptors, such as DC-SIGN, follow the endocytic internalization pathway when capturing their ligands [28]. TLR7 and TLR8 activation results in recruitment of the adaptor molecule, MyD88, and the subsequent signaling events by the MyD88-IRAK-TRAF6 signaling module are essential for induction of type I IFNs. It has been shown that MyD88 and TRAF6 can bind to IRF7 directly and recruit IRAK1 to phosphorylate IRF7 [29]. A central component of antiviral immunity is activation of TLR3 and TLR7 and TLR8 that induce phosphorylation of IRF3 and IRF7, formation of IRF dimers, and their translocation into the nucleus, resulting in production of type I IFNs and expression of a set of IFN-inducible genes [20]. Helicases in hepatitis C virus recognition Recognition of viruses can be achieved in TLR-independent pathways involving the cytoplasmic helicase proteins retinoic acid–inducible gene I (RIG-I, also known as Ddx58) and melanoma-differentiation-associated gene 5 (also called Helicard). RIG-I is an RNA helicase with two caspaserecruiting domain-like domains that interact with double-stranded RNA and activate downstream signaling leading to IRF3 and NF-B activation [30,31]. Melanoma-differentiation-associated gene 5 has a structure similar to that of RIG-I and has been linked to the mediation of antiviral responses. The adaptor molecule IPS-1 (also known as MAVS, VISA, and Cardiff), expressed on mitochondria, links RIG-I and melanoma-differentiationassociated gene 5 to downstream signaling mediators including FADD, receptor-interacting protein-1, TBK1, and inducible I B kinases to trigger RIG-I and melanoma-differentiation-associated gene 5–mediated type I

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IFN induction [32–35]. IPS-1 activates NF-B and IPS-1 knockdown blocks IFN responses. Kato and coworkers [30] identified that melanoma-differentiation-associated gene 5 and RIG-I recognize different types of doublestranded RNAs: melanoma-differentiation-associated gene 5 recognizes poly(I:C), whereas RIG-I detects in vitro transcribed double-stranded RNAs. RNA viruses are also differentially recognized by RIG-I and melanoma-differentiation-associated gene 5: RIG-I is essential for the production of IFN in response to RNA viruses including paramyxoviruses, influenza virus, and Japanese encephalitis virus, whereas melanoma-differentiation-associated gene 5 is critical for picornavirus detection. Recent studies suggest that RNA helicases are targets of viral mechanisms for host invasion particularly in HCV infection. Dendritic cells There is a distinct pattern recognition expression profile of various immune and parenchymal cells. Plasmacytoid DCs, also known as IFN-producing cells, are a restricted subset of DCs that are specialized in secreting copious amounts of type I IFNs, particularly IFN, after stimulation with viral nucleic acids [16,36]. Human plasmacytoid DCs express TLR7, TLR8, and TLR9 but not TLR2, TLR3, TLR4, or TLR5. Myeloid DCs express TLR2, TLR3, TLR4, TLR5, and TLR9 and on encountering with pathogen components with these receptors produce inflammatory cytokines, IFN-b, and undergo DC maturation. Genetic experiments demonstrate that TLR7 and TLR8 are essential for IFN induction in plasmacytoid DCs by RNA viruses [37]. In many other cell types, however, including mDCs, macrophages, and fibroblasts, deletion of both MyD88 and TRIF, which abolishes TLR signaling, has no effect on viral induction of type I IFNs likely representing IFN induction by the TLR-independent helicase pathways [31]. In the normal liver both myeloid and plasmacytoid DCs are present. Liver DCs localize near central veins and portal tracts and express low levels of major histocompatibility complex (MHC) and costimulatory molecules, suggesting an immature phenotype and a location favorable for antigen uptake [6,38]. During chronic infection with HCV, the DCs are identified in liver biopsies as components of portal infiltrates located in close proximity to lymphatic vessels; colocalize with CD8þ T cells; and express CD80, CD83, and CD86, suggestive of mature phenotype that establishes close contact with T cells [39]. Further, a relative enrichment of DCs in livers of patients with chronic HCV infection, but not of those with nonviral liver disease, was noted compared with the blood compartment [40]. Natural killer cells The liver is enriched in NK cells compared with the peripheral blood and perhaps it is not unexpected that a close association between the resolution

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of HCV infection and NK cell activity has been demonstrated [41]. Patients homozygous for the NK cell inhibitory receptor, KIR2DL3 with HLA-C1, were more likely to recover from acute HCV infection than those with any other haplotypes suggesting that the diminished inhibitory NK cell response confers protection against HCV. NK cell activation is regulated by DCs by production of IL-12, IL-15, IL-18, or IFN or through expression of NKactivating ligands [42]. Interestingly, DCs from HCV-infected patients are unresponsive to exogenous IFN to enhance MHC class I–related chain A/ B expression and fail to activate NK cells [43]. It has been speculated that the impairment of DCs in NK cell activation is responsible for the failure of HCV control in acute infection where HCV continues to replicate despite high-level IFN expression in the liver [5,44]. Interaction of NK cells with HCV E2 protein by its receptor, CD81, inhibits NK cell activity [45,46], whereas it causes stimulation in T and B lymphocytes. The level of MHC class I expression regulates NK cell activity; it represents a potential target for viral-induced modulation of NK cell responses. HCV core protein has been shown to up-regulate MHC class I levels on hepatocytes resulting in impaired cytolytic activity of NK cells but not of CD8þ T cells [47]. These examples support the contention that virus-induced mechanisms are involved in NK cell dysfunctions in HCV infection. Adaptive immune response in acute infection In acute HCV infection, HCV replication occurs in hepatocytes and HCV genomes appear in the serum within a few days and typically peak at 6 to 10 weeks regardless of the outcome [8]. Parallel to the detection of HCV genome and proteins in hepatocytes, activated HCV-specific T cells enter the liver; however, these immune responses are delayed at least 1 month in both humans and chimpanzees [5,48]. HCV-specific CD4þ and CD8þ T-cell responses and IFN coexpression is associated with a decrease in HCV quantity [5]. Resolution of acute infection is associated with a vigorous, multiepitope-specific, Th1-type and sustained CD4þ T-cell immune response [44,49]. Even transient control of viral replication is temporarily associated with HCV-specific CD4þ and CD8þ T-cell responses, whereas a failure of such responses correlates with high viral replication levels and persistent infection. It is still unclear why the cellular immune response is delayed for several weeks in HCV infection even in humans and chimpanzee who ultimately clear the infection [5,44,50]. The frequency of HCV-specific CD8þ T cells is high during the acute phase of infection (2%–8% of peripheral CD8þ T cells) but it decreases after HCV persistence develops in chronic infection [51,52]. More specifically, the HLA-B27 restricted CD8þ T-cell response seems to be involved in mediation of HCV clearance [53]. Cytotoxic T-lymphocyte responses are ‘‘stunned’’ in the acute phase as demonstrated by the inability to produce IFN and to proliferate in response to HCV antigens [51–54].

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Long-lasting memory of CD4þ and CD8þ T cells against HCV in the absence of viremia and antibody is confirmed in health care workers, spouses, and healthy family members of acute or chronic HCV patients [55–57]. The question whether a protective immunity exists against HCV is yet to be fully answered. Multiple episodes of acute hepatitis C reported in thalassemic children receiving transfusions [58] and reinfection of HCV in chimpanzees that cleared a previous HCV infection suggest that fully protective immunity to HCV does not exist [59]. There is evidence, however, for some protective immunity. Rechallenge of HCV-recovered chimpanzees with HCV infection demonstrates that memory CD4þ and CD8þ T cells play a primary but distinct role in the prevention of persistent HCV infection. Although HCV-immune chimpanzees are susceptible to reinfection, there was a marked reduction in the duration and peak of viremia [60–62]. Depletion of CD8þ T cells in chimpanzees with prolonged viremia after rechallenge with the same HCV strain and viral clearance correlated with the recovery of CD8þ T-cell functions in the liver [60]. Treatment with anti-CD4þ antibodies resulted in HCV persistence, signifying the importance of memory CD4þ T cells in the outcome of infection [63].

Humoral immune response HCV-specific antibodies are not detectable in the serum until the appearance of cellular immune response and occur about 2 months after infection. There is limited evidence from chimpanzees that antibodies specific for the HCV envelope proteins (E1 and E2) may neutralize in vivo HCV infectivity [64] and modulate HCV RNA levels in vaccinated and rechallenged chimpanzees [65]. Longitudinal studies of resolved HCV infection revealed that HCV-specific antibody titers decrease within 10 to 20 years, whereas HCVspecific CD4þ and CD8þ T-cell responses persist even several decades later [66,67].

Chronic hepatitis C virus infection HCV-specific CD8þ T cells can survive for years in the persistently infected liver and likely contribute to the control of viral replication and progressive liver disease. Although virus-specific cytotoxic T lymphocytes are concentrated in the liver in chronic HCV infection, they have not been consistently correlated with disease severity [68–70]. Increased intrahepatic cytokine mRNA levels have been associated with the severity of portal inflammation and liver fibrosis [71]. Recruitment of inflammatory cells, such as macrophages that can mediate tissue injury, also occurs in chronic HCV [72]. Circulating monocytes, precursors of tissue macrophages, are in vivo preactivated in patients with chronic HCV infection and produce increased levels of the proinflammatory cytokines, tumor necrosis factor, and

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IL-8 on ex vivo stimulation compared with monocytes from noninfected individuals [73]. HCV infection that follows a chronic course is usually marked by low frequencies of cytotoxic T lymphocytes targeting few MHC class I–restricted epitopes in the HCV structural and nonstructural proteins [55,74,75]. HCV-driven CD4þ T cell proliferation in individuals who develop persistent infection is usually weak or absent when compared with spontaneously resolving infections [44,76]. Transient CD4þ T cell proliferative responses may occur in both those patients who clear the virus or in patients developing chronic HCV. Loss of this initial robust helper CD4þ T-cell activity during acute HCV infection predicts HCV persistence [77,78].

Mechanisms of hepatitis C virus persistence and subversion of immune responses by the hepatitis C virus Several mechanisms have been postulated to contribute to subversion of the immune responses in support of chronic HCV persistence. In the absence of advanced liver disease, defects in cellular immunity exist that seem to be strictly HCV-specific. There is no evidence in these patients for generalized immune dysfunction. The mechanisms for these HCV-specific cellular immune defects, however, are not well understood. In vitro studies demonstrated immunomodulatory activities of HCV proteins and viral nucleocapsid [79,80]. One of the most commonly proposed mechanisms for cellular immune defects in HCV infection is impaired antigen presentation. Conceptually, this could explain defects in immunity ranging from an apparent absence of HCV-specific T cells in some individuals to a substantially delayed or nonsustained response in others. HCV interferes with the host immune responses at several levels including viral recognition by pattern recognition receptors, DC functions, immunomodulation by viral proteins, and through the inhibitory actions of regulatory T cells (Box 1). Interference with innate immune activation by pattern recognition receptors Recent studies provide evidence for HCV interaction with the evolutionally conserved pattern recognition receptors as a potential strategy to replicate unnoticed by the host cell. HCV achieves this goal by the action of the NS3/4A, the major serine protease, expressed by the HCV that blocks double-stranded RNA–induced IFN production by interfering with IRF3 phosphorylation [79,80]. Studies with subgenomic replicon-harboring Huh cells showed that NS3/4A protein interacts directly with TBK1 to decrease TBK1-IRF3 interaction, whereas NS3 protein alone induces degradation of the TLR3 adaptor TRIF, both leading to down-regulation of IRF3 activity and type I IFN induction [80–83]. In hepatocytes, type I IFN is also induced by activation of PKR or RIG-I, which may occur independently

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Box 1. The mechanisms of immune subversion used by HCV Innate immunity  Inhibition of monocyte-derived DC functions by HCV structural and NS proteins  Reduced antigen-presenting capacity of monocyte-derived DCs  Excessive IL-10 and decreased IL-12 production in monocyte-derived DCs  Loss of circulating plasmacytoid DCs  Relative enrichment of DCs in the liver  Suppression of NK cell function by DCs  Low IFN-g production by NKT cells Adaptive immunity  Suppression of T-cell functions by HCV-derived structural and NS proteins  CD4+ T-cell functional defect  Decreased IL-2 production  Altered T-cell trafficking  Expansion of CD4+CD25+ regulatory T cells  CD8+ T-cell anergy  Selective deletion of HCV-specific CD8+ T cells  Expansion of IL-10 producing CD8+ T cells  Restricted clonotypic CD8+ T cell TCR Viral recognition in liver cells  HCV NS3 protein interferes with type 1 IFN production by degradation of the TLR3 adaptor TRIF and down-regulation of IRF3 activity  NS3/4A protein interacts directly with TBK1 to decrease RIG-I triggered interaction of TBK1 with IRF3, leading to down-regulation of IRF3 activity and impaired type 1 IFN induction The summarized defects of innate and adaptive immunity during HCV infection are based on both in vivo and in vitro findings of multiple research groups that are cited in the references, whereas the interference of HCV proteins with TLR-dependent and TLR-independent recognition of HCV in liver cells and induction of IFNs have been reported in vitro and await in vivo confirmation.

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of TLRs, but involve both IRF3 and IRF7 [84]. NS3/4A cleaves the C-terminal region of IPS-1 adaptor, causing disruption of NF-B and IRF3 activation [34]. Recent studies with the JFH-1 infectious HCV virus demonstrated that acute HCV infection transiently induces RIG-I and IPS-1 dependent IRF-3 activation [85]. This host response initially limits HCV production; however, HCV disrupts this response early in infection through NS3/4A [85]. NS3/4A disrupts the RIG-I pathway through proteolysis of IRF-3 activation and downstream activation of the IFN pathway in hepatocytes [81,86]. These data indicate that HCV subverts both RIG-I recognition and TLR signaling pathways to limit type I IFN induction in hepatocytes. In addition, both TLR9- and TLR7- and TLR8-induced IFN production is diminished in peripheral plasmacytoid DCs of HCV-infected patients [87]. It is unclear to date if HCV proteins may affect TLR7, TLR8, and TLR9 signaling, because, at least in DCs, these TLRs use IRF7 but not IRF3 and are TBK-1 independent [29]. Subversion of dendritic cell functions DCs, the most effective and specialized antigen-presenting cell type, are particularly susceptible to the inhibitory effects of HCV infection. Although viral replication in DCs is yet to be demonstrated, there is mounting evidence that DCs may be infected with HCV [88]. It is currently presumed that DCs may represent the repository for HCV, based on identification of viral RNA in DCs but not in serum of patients who cleared HCV spontaneously or on treatment and had long-term normalization of liver function tests [89]. Further, it seems that DCs host unique quasispecies of HCV with impaired translational activity, which is different from those present in the liver [90]. To date most studies agree that in humans chronic HCV infection affects DC function in the form of impaired antigen presentation and altered cytokine production [73,91–93]. Both DC differentiation and functional maturation seem to be altered in patients with chronic HCV infection and was associated with increased IL-10 and reduced IL-12 production by DCs [73]. These DCs exhibited decreased capacity to induce T-cell proliferation [73,91], were less able to drive the Th1 immune response, and were found to prime more IL-10–producing cells compared with normal DCs [94]. The poor allostimulatory capacity of DCs of HCV-infected patients is possibly caused by the immunomodulatory effects of viral proteins, because DC defects can be reproduced in non–HCV-infected DCs by treatment with recombinant HCV core or NS3 proteins or by adenoviral vector expression of HCV core or NS3 proteins [73,95,96]. These HCV protein-modified DCs induced defective stimulation of CD4þ T cells that were unable to proliferate or to produce IL-2 [95]. The role of virus-induced factors in mediation of DC defects is also supported by the observation that the reduced allostimulatory capacity of DCs from HCV-infected individuals

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occurred in infected patients with persistently normal alanine transaminase levels [94] and returned to normal after treatment-induced clearance of the virus [93]. The antigen-presenting defects of DCs may develop gradually, because some patients with chronic HCV infection have normal functional subsets of circulating myeloid DC [97]. Although there is not a clear understanding of the DC functions during acute infection with HCV in humans, studies from primate models suggest that in early infection HCV sequences are undetectable in DCs of HCV-infected chimpanzees [48,98]. Further, no defects were observed in the maturation process of DCs during the asymptomatic phase of HCV infection. Interestingly, allostimulatory capacity of DCs was decreased in some animals with high viral loads [98], suggesting that RNA levels and the viral protein load may play a role in modulation of DC functions. In addition to monocyte-derived DCs, plasmacytoid DCs are also affected during HCV infection. Reduced frequency of plasmacytoid DCs in the peripheral blood of patients with chronic HCV infection was reported [40,94,99] along with decreased capacity to produce IFN on in vitro stimulation with DNA viruses (HSV), TLR7 and TLR8, or TLR9 ligands [87,99]. Similar to myeloid DCs, the plasmacytoid DCs of patients have defects in antigen-presenting capacity and prime more CD4 T cells producing IL-10 than those from normal controls [94]. The plasmacytoid DC population is re-established in subjects with spontaneously resolved HCV infection, suggesting that viral factors are responsible for plasmacytoid DC depletion [40]. It has been proposed that the liver environment may contribute to suboptimal DC activation in HCV infection. Experimental data from the mouse system suggest that liver resident DCs are less immunogenic compared with splenic DC populations [38]. Liver sinusoidal endothelial cells, which express MHC class I and II and costimulatory molecules, Kupffer cells, stellate cells, and even hepatocytes, may induce T-cell apoptosis and mediate the induction of tolerogenic effects [6]. Immunomodulation by hepatitis C virus proteins The immunosuppressive potential of HCV-derived proteins has been implicated in functional defects of T cells, NK cells, and DCs. T-cell proliferation and IL-2 production is inhibited by HCV core protein interaction with the globular domain of C1q receptor on T cells [100]. Transfection of HCV core and E1 genes into non–HCV-infected DCs reduced their capacity to stimulate allogeneic T cell responses [95]. HCV core, NS3, and NS4 proteins activate monocytes to produce IL-8, IL-6, IL-10, and tumor necrosis factor, and inhibit differentiation and antigen-presenting functions of myeloid DCs [73]. It seems that surface expressed TLR2 is involved in cell activation by HCV core and NS3 proteins resulting in TLR2-mediated downstream activation of the MyD88-dependent proinflammatory pathways in monocytes [101]. Further, presentation of HCV-derived lipopeptides LP22-40 by DCs

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enhances the proliferation and IFN-g secretion in HCV-specific T cells by up-regulation of TLR2 [102]. Marked up-regulation of TLR2 and TLR4 was reported in patients with chronic HCV infection irrespective of HCV genotype and viral load [103] and was detected in hepatocytes, Kupffer cells, and peripheral blood monocytes [104]. TLR2- and TLR4-induced tumor necrosis factor production and circulating tumor necrosis factor levels are increased in HCV-infected patients [73,105]. Interestingly, HCV replication, unlike other RNA viruses, is resistant to tumor necrosis factor [106]. TLR2mediated activation by HCV proteins may contribute to the increased proinflammatory cytokine activation and hepatocyte damage in chronic HCV infection [9]. Regulatory T cells A novel aspect of DC function is induction of immune tolerance by generation of regulatory T cells, as defined by the ability of the CD4þCD25þFoxp3þ cell subset to suppress effector T-cell function in an antigen-specific, cell-contact, and partly cytokine-mediated fashion. DCs that generate regulatory T cells are immature, antigen-stimulated, and express low levels of costimulatory and MHC class II surface molecules [107]. Of the cytokines, IL-10, produced at increased levels by DCs of HCV-infected patients, has been shown to modulate DCs to induce T-cell anergy and decrease IL-12 production [108]. Importantly, myeloid DCs generated from patients with chronic HCV infection show features of this phenotype including increased IL-10, reduced IL-12 production, and decreased expression of costimulatory molecules [73,91]. Furthermore, plasmacytoid DCs can also induce CD4þCD25þ regulatory T cells when stimulated with TLR9 ligands [109]. Studies examining the circulating HCV-specific T-cell frequency, repertoire, and cytokine profiles revealed that HCV persistence was associated with a reversible CD4þ T cell–mediated suppression of HCV-specific CD8þ T cells and with higher frequency of CD4þCD25þ regulatory T cells [110–112]. The mechanisms of CD8þ T-cell suppression involved IL-10 and TFG-b production [110]. Regulatory T cells also suppressed autologous and alloreactive CD4þ responses in an antigennonspecific manner. In the HCV-infected liver, the existence of CD8þ CCR7- regulatory T cells has also been demonstrated. These cells suppress HCV-specific CD8þ T cells by secreting IL-10 and contributing to HCV persistence and liver injury [113]. Summary HCV infection and viral replication in hepatocytes triggers activation of antiviral innate and adaptive immune activation. Determinants of the favorable outcome of acute infection and viral elimination, however, are still only partially understood. HCV recognition by pattern recognition receptors

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including TLRs and helicases is critical for type I IFN production. Coordinated activation of NK cells, DC maturation, and induction of robust HCV-specific Th1-type CD4þ T cell and CD8þ T-cell activation seems to be associated with successful clearance of acute infection. Determinants of chronic infection include low frequency of cytotoxic T lymphocytes directed against few HCV structural and NS proteins and weak CD4þ T-cell proliferative responses. These T-cell defects are associated and possibly related to a defect in functions of both plasmacytoid and myeloid DCs. In addition, the increased proportion of regulatory T cells contributes to the HCV-specific subversion of innate and adaptive immune responses in chronic HCV infection. Increasing evidence suggests that many of these immune defects are related to HCV-induced strategies to undermine immune cell activation. Protease activity of NS3/4A on the different components of pattern recognition receptors and subversion of DC and NK cell functions by HCV proteins create a constantly compromised immune environment. Understanding these new pieces of the puzzle of HCV-induced modulation of host immunity may promise strategies for new, targeted therapeutic approaches. References [1] Lindenbach BD, Rice CM. Molecular biology of flaviviruses. Adv Virus Res 2003;59: 23–61. [2] Cerny A, Chisari F. Pathogenesis of chronic hepatitis C: immunological features of hepatic injury and viral persistence. Hepatology 1999;30:595–601. [3] Guidoti L, Chisari F. Noncytolytic control of viral infections by the innate and adaptive immune response. Annu Rev Immunol 2001;19:65–91. [4] Frese M, Schwarzle V, Barth K, et al. IFN gamma inhibits replication of subgenomic and genomic HCV RNAs. Hepatology 2002;35:694–703. [5] Thimme R, Bukh J, Spangenberg HC, et al. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc Natl Acad Sci U S A 2002;99: 15661–8. [6] Lau AH, Thomson AW. Dendritic cells and immune regulation in the liver. Gut 2003;52: 307–14. [7] Liu Y-J. Dendritic cell subsets and lineages, and their functions in innate and adoptive immunity. Cell 2001;106:259–62. [8] Bowen DG, Walker CM. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature 2005;436:946–52. [9] Nelson DR, Lau JN. Pathogenesis of hepatocellular damage in chronic HCV infection. Clin Liver Dis 1997;1:515–28. [10] Baumert TF, Ito S, Wong DT, et al. HCV structural proteins assemble into viruslike particles in insect cells. J Virol 1998;72:3827–36. [11] Bartosch B, Dubuison J, Coset FL. Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes. J Exp Med 2003;1979:633–42. [12] Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11:791–6. [13] Pileri P, Uematsu Y, Campagnoli S, et al. Binding of hepatitis C virus to CD81. Science 1998;282:938–41. [14] Cocquerel L, Kuo CC, Dubuison J, et al. CD81-dependent binding of hepatitis C virus E 1E2 heterodimers. J Virol 2003;77:10677–83.

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