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Hepatitis C virus (HCV) is a uniquely successful hepatitis virus in its ability to establish chronic infection in more than two-thirds of those who contract it. The inability of the innate and adaptive immune responses to control HCV invasion and replication contribute to development and persistence of chronic infection. Multiple components have been identified in this process, including pathways by which HCV subverts innate immune recognition and activation, delayed organization of an effective adaptive immune response, the tolerogenic liver environment, and the persistently high levels of viral antigens. We summarize recent findings on the interaction between HCV infection and innate immune responses.

Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References

A critical role for innate and adaptive immunity has been identified in HCV persistence and liver injury. During acute infection, type I interferon (IFN) is induced in the liver; however, HCV viral load does not decrease, suggesting that HCV interferes with antiviral mechanisms. Innate immune cells, including natural killer cells and dendritic cells shape innate immunity and determine adaptive immune activation. Vigorous, multi-epitope–specific, T helper 1-type and sustained CD4+ and CD8+ T cell responses were found in resolved acute HCV infections.1–4 In contrast, in cases that progress to chronic infection, CD4+ T cell responses are weak, short-lived, and targeted to a narrow range of epitopes.1 Many of these defects in adaptive immunity can be linked to deficiencies in innate immune responses to chronic HCV infection. However, viral recognition pathways involved in immunomediated anti-HCV defense are also identified in hepatocytes, the major reservoir and replication site of HCV. The hepatocyte is a specialized cell in a defined tissue content, and the innate immune programs that are specific to hepatocytes and/or to HCV infection are not yet fully defined. Given the central role of the liver in metabolism and detoxification, the hepatocytes encounter both ex vivo and in vivo derived signals that shape their responses to pathogens. Furthermore, the rich surrounding of immune and nonimmune cells in the liver provide a second layer of complexity to the reaction of hepatocytes to pathogens. Thus, one should consider both cell-specific and organism-specific approaches when attempting to understand the complex responses to HCV, especially when dealing with a failure to spontaneously eliminate this pathogen in the majority of infected individuals.

Pattern Recognition Receptors and Modification of Their Function in HCV Infection

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References

As a positive single-stranded RNA (ssRNA) virus, HCV expresses ssRNA and double-stranded RNA (dsRNA) during replication, both of which may act as ligands for different pattern recognition receptors.5 Of the different pattern recognition receptors expressed on the cell surface or intracellularly, some of the Toll-like receptors (TLRs) and retinoid inducible gene 1 (RIG-1)–like RNA helicase receptors play a major role in recognition of pathogen-derived nucleic acid signals such as ssRNA and dsRNA or proteins.6 Activation of these pathogen-sensing receptors leads to induction of inflammatory mediators and type 1 IFNs that can inhibit the replication of the virus; however, HCV has evolved multiple mechanisms to interfere with type 1 IFN pathways.7–11 Although the exact role of TLRs and RNA helicase receptor systems in HCV nucleic acid recognition awaits further clarification, the specificity of these receptors and their distinct cellular distribution may be critical for induction of an appropriate antiviral immunity. For example, TLR3, a receptor for dsRNA,12 and TLR7 and TLR8, receptors for ssRNA,13, 14 are exclusively localized to intracellular compartments, such as endosomes,15 suggesting that these intracellular TLRs recognize nucleic acids following the internalization and lysing of viruses but are unable to sense viruses that directly entered the cytosol and initiated replication to produce dsRNA. Recognition of direct viral invasion and replication occurs via the RIG-I–like helicase receptors, which are freely present in the cellular cytoplasm.6, 16, 17

Toll-Like Receptors in Recognition of RNA Viruses.

TLR3 is expressed in innate immune cells (for example, dendritic cells, macrophages) as well as in hepatocytes.18, 19 TLR3-mediated signaling is triggered by dsRNA in polyinosinic:polycytidylic acid [poly(I:C)] and by purified genomic dsRNA from rheovirus or ssRNA viruses such as respiratory syncytial virus, encephalomyocarditis virus, and West Nile virus.12 TLR3 uses the adapter protein Toll/IL-1 receptor (TIR) domain-containing adapter inducing IFN-β (TRIF), which indirectly activates several transcription factors, including interferon regulatory factor 3 (IRF3), nuclear factor κB (NF-κB), and activator protein (AP) 1. IRF3 coordinates the expression of genes for type I IFN, whereas NF-κB and activator protein 1 transcription factors coordinate inflammatory gene induction. TRIF recruits the kinases IKK-ϵ, and TANK (TRAF family member-associated NF-κB activator)-binding kinase (TBK) to its N-terminal domain, enabling them to phosphorylate IRF3 and allowing the IRF3 homodimerization or heterodimerization.20 The IRF3 complex translocates to the nucleus and triggers type 1 IFN production.21 Increased TLR3 expression in peripheral blood mononuclear cells in chronic HCV infection correlated with sustained virologic response after standard IFN-α plus ribavirin therapy.22

Recent studies demonstrated that HCV can interfere with TLR3 signaling through cleavage of TRIF by the HCV nonstructural3/4A protease23 thereby preventing further activation of the downstream pathway for induction of IFN and proinflammatory cytokines.24 HCV NS3 alone interacts with TBK1, a key protein to the TLR3-mediated IFN induction pathway, disrupting the TBK1/IRF3 relationship.25

TLR7 and TLR8 are expressed in the endosome and recognize a number of ssRNA viruses.26 TLR7 was initially identified as a receptor able to recognize imidazoquinoline derivatives with antiviral activity.27 Subsequently, guanosine-rich or uridine-rich ssRNA derived from human immunodeficiency virus genotype 1 and influenza virus, synthetic polyuridinated RNA, and certain small interfering RNAs were identified as ligands for TLR7.28, 29 TLR7 is expressed in plasmacytoid dendritic cells (PDCs)29 and possibly in hepatocytes, as suggested by productive response to TLR7 ligands in hepatoma cell lines.30 TLR8 is functional in humans but not in mice, and it is expressed in myeloid dendritic cells (MDCs), monocytes, macrophages, and T cells.31, 32 Human TLR8 mediates recognition of human immunodeficiency virus–derived ssRNA and chemical ligand R848,13 and its role in HCV infection is currently unknown. Recent studies revealed functional differences between human TLR7 and TLR8 in which TLR7 agonists primarily activated PDCs, whereas TLR8 agonists activated MDCs, monocytes, and macrophages.33 In addition, the cytokine production profile of TLR7 was dominated by IFN-α induction, while TLR8 triggered predominantly the proinflammatory cytokines and chemokines, such as TNF-α, interleukin (IL)-12, and macrophage inflammatory protein 1α.32 The pattern recognition site for the leucine rich repeats of TLR7/8 molecule is contained within the endosome, while the Toll/IL-1 receptor domain is exposed to the cytoplasm,34 where it transduces intracellular signals by recruitment of the myeloid differentiation primary response gene 88 (MyD88), a common TLR adapter protein. MyD88 further forms complexes with members of the interleukin-1 receptor-associated kinase (IRAK) family (IRAK1 and IRAK4) and TNF receptor-associated factor 6 (TRAF6), which in turn activate TAK1 and result in NF-κB activation. Type I IFN induction after TLR7 activation is independent of IRF3,35 suggesting the possible involvement of other IRF family members in this pathway. IRF7 is structurally similar to IRF3, and whereas its expression is low in most cell types, it is constitutively expressed in PDCs.36 IRF7 is able to form a signaling complex with MyD88, IRAK1, IRAK4, and TRAF6, while TBK1, IKK-ϵ, and IRAK1 are capable of phosphorylating IRF7.29–37 Activated IRF7 homodimerizes and heterodimerizes with other IRFs, allowing this complex to translocate into the nucleus and bind to the IFN-stimulated response element promoter site.38 Type 1 IFNs, composed of IFN-α and IFN-β, are released in response to TLR7 or TLR8 signaling (Fig. 1).

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Figure 1. Toll-like receptor–dependent and RIG-I–like receptor–dependent recognition of HCV-derived products.

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Combined IFN-α and ribavirin is the current treatment for HCV infection,39 therefore activation of TLR7 or TLR8 leading to IFN-α induction would increase host immunity to HCV. Although it remains to be directly determined whether TLR7 or TLR8 recognize HCV, 2 separate groups have illustrated that activation of TLR7 aids in the eradication of the virus.40, 41 Administration of specific TLR7 agonists either to HCV patients or to HCV-replicating cell lines resulted in reduction of HCV virus levels or HCV replication.40, 41

Recognition of Nucleic Acid by RIG-I–like RNA Helicases.

Cytoplasmic receptors of the RNA helicase are a family of pattern recognition receptors that participate in the induction of type 1 IFNs upon stimulation with dsRNA and include the retinoid inducible gene 1 (RIG-I), melanoma differentiation associated gene 5 (Mda5), and RIG-I–like RNA helicase LGP2.42 RIG-I contains a DexDH box RNA helicase and 2 caspase recruiting domain–like domains. The helicase domain interacts with dsRNA, whereas the caspase recruiting domain–like domains are required for activating downstream signaling pathways.43 Mda5 contains 2 caspase recruiting domain–like domains and a helicase domain, while LGP2 lacks the caspase recruiting domain–like domains and is thought to negatively regulate RIG-I and Mda5.42 RIG-I is essential for the recognition of Flaviviruses, Paramyxoviruses, Orthomyxoviruses, and Rhabdoviruses, whereas Mda5 is required for the recognition of a different set of RNA viruses, including Picornaviruses.43 RIG-I–like RNA helicases are expressed in MDCs, macrophages, hepatocytes, and fibroblastic cells but not in PDCs, suggesting that TLR and RIG-I–like RNA helicase pathways are not fully redundant but differentially specialized in antiviral signaling in different cell types.42–45 Sumpter et al. had shown that the helicase domain of RIG-I binds HCV RNA and transduces the activation signal for IRF-3 by its caspase recruiting domain homolog.45 Four different groups independently identified downstream signaling from RIG-I as the mitochondria-associated adapter protein MAVS, also known as Cardif, interferon-beta promoter stimulator 1 (IPS-1), or virus-induced signaling adaptor (VISA).46–49 The mechanisms by which MAVS is regulated by RIG-I and how MAVS signals downstream kinases in the immune cells remain to be fully explored. However, MAVS localization to the mitochondrial outer membrane is critical to the function of the pattern recognition receptor–mediated signaling pathways. Similar to the cell survival proteins of the Bcl family, the mislocalization of MAVS from the mitochondrial membrane to the plasma membrane or endoplasmic reticulum impairs the ability of MAVS to induce IFNs. While the role of intermediate molecules such as TRAF6 has been debated, MAVS activates both the classical IκB kinase (IKK) complex and the downstream NF-κB and TBK-1/IKK-ϵ pathways and leads to IRF3 phosphorylation and type I IFN induction37, 50 (Fig. 1).

Recent studies have shown that HCV NS3/4A protein interrupts IFN-β induction through the RIG-I pathway via proteolytic cleavage of MAVS/Cardif/IPS-1/VISA.8, 51 NS3/4A cleaves MAVS at Cys-508,8, 51 which is located only a few residues before the mitochondrial-targeting domain; as a result, MAVS is dislodged from the mitochondria and becomes inactive. Liver tissues from chronic HCV infection demonstrated altered subcellular distribution of IPS-1 in infected hepatocytes, and IPS-1 cleavage was associated with lack of interferon-stimulated genes 1 (ISG-1) expression.8 Recently it was demonstrated that MAVS is cleaved by NS3/4A during HCV infection and in vitro in liver cell lines infected with the JFH-1 HCV virus.8, 52, 53 HCV NS2 also seems to disrupt promoter activity of RIG-I.53 However, Yoneyama et al.42 suggested that HCV NS3 controls not only RIG-I but also Mda5-mediated pathways. Saito et al.54 recently reported that unlike RIG-I and LGP2, Mda5 does not bind HCV RNA and does not trigger HCV-specific IFN induction. Thus, the role of Mda5 in HCV infection remains to be defined. It is tempting to speculate that because Mda5 shares the IPS adapter with RIG-I and LGP2, HCV infection may hamper Mda5-mediated response to other viruses. (Table 1).

Table 1. Levels of Interference of HCV-Derived Products with Immune Functions
HCV ProteinReceptor/PathwayInterference with Immune ResponseReference
E1BCL-2–dependentInduces production of reactive oxygen species and nitric oxide; lowers the mitochondrial transmembrane potential; induces DNA damage and activation of STAT355
E2CD81Inhibits NK activity via block of tyrosine phosphorylation56, 57
E2CD81Inhibits the non-MHC–restricted cytotoxicity mediated by NK cells but also IFN-γ production by NK cells58
E2CD81Stimulates ERK and p38 MAPK signaling pathways; triggers cell proliferation and secretion of IFN-γ and IL-1059
E2CD81Conditions the failure of dendritic cells to recirculate to lymphoid tissue via modulation of chemokine receptors60
CoreTLR2Stimulates MAPK signaling pathways; triggers secretion of TNF-α and IL-10 in monocytes and dendritic cells61, 62
CoreBCL-2–dependentInduces production of reactive oxygen species and nitric oxide; lowers the mitochondrial transmembrane potential; induces DNA damage and activation of STAT355
CoreJak-STATBinds STAT1; subverts the Jak-STAT kinase by selectively inducing STAT1 degradation63
NS2RIG-IDisrupt promoter activity of RIG-I64
NS3TLR2Stimulates MAPK signaling pathways; triggers secretion of TNF-α and IL-10 in monocytes and dendritic cells61, 65
NS3BCL-2–dependentTriggers mitochondrial permeability transition with production of reactive oxygen species and nitric oxide; lowers the mitochondrial transmembrane potential, induces DNA damage and activation of STAT355
NS3/NS4RIG-IDisrupts RIG-I signaling; prevents the nuclear accumulation of IRF-3; blocks virus-mediated IκBα and the expression of NF-κB target gene IL-610
NS3/NS4TLR3Induces proteolysis of TRIF; reduces its abundance and inhibits polyI:C-activated signaling through the TLR3 pathway before its bifurcation to IRF-3 and NF-κB23
NS4MAPKStimulates IL-10 and inhibits IL-12 production in monocytes66
NS4ACaspase-3–dependent apoptosis pathwayAlters the intracellular distribution of mitochondria; induces mitochondrial damage and induces apoptosis through activation of caspase-3, but not caspase-867
NS4A/B?Inhibits the global ER-to-Golgi traffic, thus reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface68
HCV RNARIG-I, Lgp2Direct binding; triggers IRF-3–dependent and NF-κB–dependent genes42, 45, 50

Surface-Expressed TLRs and Other Receptors Involved in Innate Immune Regulation in HCV.

Increasing evidence suggests that HCV core protein—which is present not only in the infected liver but also in the systemic circulation of HCV-infected patients—stimulates cell activation through common cellular receptors such as TLR262, 65 and complement receptor C1qR.69 TLR2 is activated not only by HCV core but also NS3 protein.65 Upon activation, TLR2 recruits MyD88 and triggers NF-κB leading to production of both anti-inflammatory and proinflammatory cytokines in human monocytes.65, 70 Yakushijin et al.71 reported that TLR2 expression in immature DCs was lower in the chronic HCV-infected group and TLR2-mediated monocyte-derived MDC maturation and antigen-stimulatory capacity was defective. Altered expression of different TLRs has been reported in peripheral blood mononuclear cells and T cells, although the relevance of these findings to HCV-innate immune interactions requires further investigation.22, 72

The complement system in innate immunity is involved in generation of inflammatory cytokine induction and phagocytosis of viruses or bacteria.73 Studies suggest that interaction between complement receptor C1qR and HCV core protein leads to inhibition of T lymphocyte proliferation.69 HCV core protein has multiple immunomodulatory effects,74 thus potential activation of innate immune cells with core protein via gC1qR may be critical in induction of inflammatory cytokines and disruption of T cell functions in chronic HCV infection.

HCV Interferes with IFN-Inducing Pathways

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References

IFNs induced during viral infection include type I (IFN-α, IFN-β), type II (IFN-γ), and type III (IFN-λ, including IL-28 and IL-29).75 The exact role of IFNs in HCV eradication during the natural course of HCV infection is yet to be fully understood. Results from both human and nonhuman infection models have shown that induction of type I IFNs occurs at high levels in both acute and chronic HCV infection in the liver. An increase in type 1 IFN in those who resolve the acute infection and those who progress to chronic infection has also been reported.76 Whereas decreased IFN-γ induction has been correlated with the lack of clearance of the virus and impaired T cell activation,77 the role of IFN-λ has recently received attention. IFN-λ is highly expressed in the livers of HCV-infected patients and exhibits potent anti-HCV activity both in vivo and in vitro.78–80 Thus, it remains to be determined whether the balance and/or the induction pattern of these different IFNs have an impact on viral clearance in the natural course of HCV infection.

HCV not only disrupts TLRs and their pathways, it also disrupts most other type 1 IFN-generating pathways. Protein kinase R was discovered as one of the first IFN-induced pathways disrupted by HCV NS5A and E2 association.80–83 Protein kinase R binds dsRNA and activates a series of substrates that can either inhibit protein synthesis or phosphorylate IκB, ultimately activating NF-κB,84 both of which are detrimental to viral replication within the cell.

HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References

The immune system works as a well-orchestrated interaction of different types of cells whose final goal is to eliminate invading pathogens. Recent research suggests that HCV interacts with and affects the function of different innate immune cell types, including dendritic cells, monocytes, macrophages, natural killer cells, and natural killer T cells. The HCV interference is diverse with regard to cellular levels, targets, and outcomes (Table 1). More importantly, HCV seems to successfully disrupt the coordinated activity of the innate immune cells to result in deficient adaptive immune response and prevent pathogen elimination (Fig. 2).

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Figure 2. The implications of different types of immune cells in recognition and elimination of hepatitis C virus.

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Dendritic Cells in HCV Infection

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References

Dendritic cells are professional antigen-presenting cells with exceptional capacity to stimulate adaptive immune defense. Both MDCs and PDCs circulate in the blood as immature cells and are very effective at antigen capture and processing; however, they all require some form of terminal maturation to become fully immunogenic. Upon antigen uptake, encounter of microbial products, or exposure to inflammatory cytokines such as TNF-α, IL-1, etc, the MDCs and PDCs mature (that is, increase their antigen-loading capacity onto MHC molecules), up-regulate costimulatory molecules, produce cytokines, and ultimately effectively present antigens to T cells and initiate/maintain the adaptive immune response.85

Myeloid Dendritic Cells.

MDCs (CD14, blood dendritic cells antigen (BDCA)-1+, CD11c+, CD83+, CD33+, HLA-DRbright cells) are present at a frequency of 0.5%-1.0% in the peripheral blood and produce cytokines including IL-12, IL-10, and IFN-β but not IFN-α. Decreased frequency of MDCs in peripheral blood of patients with chronic HCV infection has been identified,86, 87 and it has been suggested by some that MDCs are infected with HCV.88 Reports on the function of MDCs in chronic HCV infection are also controversial. Longman et al.89 reported no phenotypic or functional defects in MDCs from peripheral blood in HCV-infected patients. In contrast, findings from Kanto et al.86 and Tsubouchi et al.88 indicate that blood-derived MDCs from HCV patients are functionally impaired. A recent study based on a small group of patients with acute HCV infection indicated that an increase in the frequency of MDCs during acute HCV may be associated with viral clearance, whereas lack of such increase might be an important factor in the development of chronic infection.90 MDCs of patients with chronic HCV infection have impaired abilities to stimulate allogeneic CD4 T cells and to produce IL-12 p70 compared with those from healthy volunteers.61, 86 After exposure to naïve CD4 T cells, MDCs from patients were less able to drive the T helper type 1 response.86 Furthermore, MDCs from the patients primed more IL-10–producing cells than those from healthy volunteers.86 Considering that IL-12 is a key cytokine in the induction of CD4 T cell activation, whereas IL-10 has complex inhibitory effects, HCV-induced modulation of these cytokines may have special importance in altered HCV-specific T cell responses in chronic HCV infection.

Monocyte-Derived MDCs.

The phenotype and cytokine production profile of bone marrow–originated MDCs can be generated from peripheral blood monocytes [monocyte-derived MDCs (Mo-MDCs)] in the presence of IL-4 + granulocytes and monocytes colony stimulating factor (GM-CSF) and serve as a model for MDCs. Data on the function/phenotype of Mo-MDCs during acute HCV infection are scarce, and the opinions on the function of Mo-MDCs in chronic HCV are divided. Although Longman et al.89 reported normal phenotypic characteristics and allogeneic functions in Mo-MDCs in humans and Larsson et al.91 identified similar findings in a nonhuman primate model of chronic HCV infection, the majority of researchers find functional abnormalities in Mo-MDCs of humans with chronic HCV infection.61, 92–94 Decreased expression of the costimulatory molecules CD83 and CD86,92 increased production of IL-10 and low IL-12, impaired capacity to stimulate allogeneic T cells in vitro, and defective maturation were reported in patients with chronic HCV infection regardless of viral genotype or patient age/sex across a wide geographic localization of patients.61, 92–94 Yakushijin et al.71 reported that TLR2 expression in immature dendritic cells was lower in the chronic HCV-infected group and TLR2-mediated Mo-MDC maturation, and antigen-stimulatory capacity was defective. MDCs may be infected with HCV and could possibly act as HCV carriers.95 The frequency of dendritic cells, both MDCs and plasmacytoid dendritic cells (PDCs), is reportedly increased in the livers of HCV-infected patients.60 Furthermore, crosslinking of CD81 with HCV E2 inhibits Mo-MDC migration without interference with maturation/differentiation and surface expression of chemokine receptors.60

PDCs.

PDCs are HLA-DRbright, BDCA-2+, BDCA-4+, and CD123bright cells, all of which lack myeloid markers CD11c and CD33 but are exceptionally potent in IFN-α production. PDCs make up only 0.2%-0.5% of peripheral blood mononuclear cells, but their function is fundamental in the defense against viral infections.29, 85 With few exceptions,96 the majority of researchers reported decreased frequency of PDCs in peripheral blood of patients with chronic HCV infection.87, 88, 92, 97, 98 We recently demonstrated that treatment-naïve patients with chronic HCV infection had reduced frequency of circulating PDCs, which partially may be due to cytokine-induced apoptosis.98 Another feasible explanation is that PDCs may be trapped in the liver, as recently suggested by Spengler's group.99 Our own group indirectly confirmed the findings of Nattermann et al.99 by reporting increased expression of the PDC markers CD123 and BDCA-2 in the livers of patients with chronic HCV compared with normal liver or nonviral hepatitis.98 PDC enrichment in the liver could explain the discrepancy between the elevated expressions of IFN-α genes in the livers of HCV-infected individuals100, 101 that was still associated with their inability to clear the virus. The IFN-α–producing capacity of peripheral blood PDCs from HCV patients appears to be reduced.98, 102, 103 More importantly, while the amounts of IFN-α produced in vivo are insufficient to modulate the antigen-processing and antigen-presenting functions, they are certainly sufficient for transforming the PDCs into potent activators of adaptive immunity during chronic HCV infection. As reported by Murakami et al.,102 PDCs from patients with HCV induced significantly lower numbers of IFN-γ–producing effector T lymphocytes compared with that of controls. Furthermore, PDCs of patients primed more CD4 T cells producing IL-10 than those from normal controls.103 In conclusion, deficient numbers and insufficient IFN-α production by PDCs in the periphery, accompanied by PDC trapping in the liver, may contribute to blunted adaptive immune responses seen in HCV-infected patients.

Monocytes and Macrophages in HCV.

Several studies found functional activation of monocytes, accompanied by changes in expression of mRNA coding for TLR2, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 in patients with chronic HCV infection compared with controls.22, 55, 65, 72, 104 TLR2 and TLR4 protein expression in peripheral blood monocytes are significantly increased in patients with chronic hepatitis C compared with controls, irrespective of HCV genotype or histological stage of liver disease.72, 104 Furthermore, monocyte expression of TLR2, but not of TLR4, correlated significantly with serum TNF-α and IL-6 levels.104 HCV proteins, such as core, NS3 and NS4, activate human monocytes via TLR2 and TLR4, to produce cytokines.61, 62, 65 It is also conceivable that engagement of cellular receptors involved in HCV uptake, such as CD81, also triggers cellular activation.58–60Ex vivo, monocytes of chronic HCV-infected patients show an imbalanced production of cytokines, with excessive production of both the proinflammatory cytokine TNF-α, and the anti-inflammatory cytokine IL-10.61 Thus, monocyte-derived cytokines may influence the cytokine milieu locally or systemically, thereby leading to functional impairment of other immune cells such as PDCs and T cells.98 More importantly, inflammatory cytokines alone and/or together with HCV in the liver may contribute to hepatocyte damage and activation of stellate cells, all leading to progressive liver disease. Monocytes contribute to innate immune response both as inflammatory cells and as immature precursors of dendritic cells and tissue macrophages.105 Interestingly, Sansonno et al.106 reported that CD34+ stem cells may be infected with HCV, thus allowing the speculation that a latent infection may indeed affect the function of the stem cell–derived progeny, including monocytes, macrophages, and dendritic cells.

Tissue-specific macrophages, represented by Kupffer cells (KC) in the liver, contribute to the systemic response to local inflammation, clearance of pathogen-derived soluble molecules and toxins from the circulation, and killing of invading pathogens and neoplastic cells.107 Although we are not aware of ex vivo studies on KCs from HCV-infected patients, the histological examination and immunohistochemistry analysis show markers of KC activation, including increased expression of the classical macrophage markers CD163 and CD68, modified KC morphology, and increased phagocytic capacity and proliferation in HCV patients compared with controls or liver diseases of nonviral etiology.108–110 Burgio et al.110 reported that in patients with chronic HCV infection, most KCs are activated and express high levels of CD80, CD40, and class II MHC molecules, thus acquiring the phenotype of professional antigen presenting cells. Activated KCs demonstrate close contact with CD4+ T lymphocytes; KC–T lymphocyte clusters were found within the sinusoids, across the sinusoid wall, and within the liver parenchyma as well, as a consequence of transendothelial migration.108–110 The positivity of activated KCs for HCV antigens suggests that KC–T cell clusters represent the morphological expression of the functional interaction between KCs, acting as professional antigen-presenting cells, and antigen-experienced CD4+ T lymphocytes within the liver.108–110 Such activation of KCs could be due to their close interaction with HCV-infected hepatocytes, HCV-derived microbial products, or exposure to lipopolysaccharide, because readily detectable HCV core protein and endotoxin in serum were reported in patients with chronic HCV infection.98, 111 Both HCV products, such as core, NS3 and NS4 proteins, and lipopolysaccharide are potent in vitro activators of monocytes/macrophages61–63, 108; thus it is conceivable that they may collectively play a role in the maintenance of chronic inflammation in the liver, because—at least in vitro—the KCs do not appear to be directly susceptible to HCV infection.112

Natural Killer and Natural Killer T Cells

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References

Natural killer (NK) and natural killer T (NKT) cells are uniquely enriched in the liver. In contrast to the peripheral blood that contains approximately 13% NK and 4% NKT cells, the intrahepatic lymphocytes contain 37% NK cells and 26% NKT cells. The percentage of NK cells in the intrahepatic lymphocyte pool may increase to 90% in hepatic diseases.113, 114

NK cells are important antiviral effectors due to their contribution to virus elimination via direct killing of infected cells and cytokine production. Recent studies highlighting the crosstalk between NK cells, dendritic cells, and T cells have prompted re-evaluation of the important role NK cells play in HCV-specific immune responses.114 Reduced frequency of NKs in peripheral blood of patients with chronic HCV infection has been reported,115 which may associate with reduced proportion of NKp46-expressing and NKp30-expressing NK cells116 and reduced or normal cytolytic activity.115–120 In contrast, patients who cleared HCV under antiviral therapy showed normal expression of NKp44, NKp30, and NKp46.117 Furthermore, NK cells may play a role in the progression of liver disease, because higher levels of NK cell cytolysis in HCV-infected patients were associated with less liver fibrosis.115–117 NK cells express CD81, a receptor previously reported as important for virus uptake. Engagement of CD81 with envelope glycoprotein E2 inhibits CD16-mediated or IL-12–mediated NK cell activation, modulates Erk phosphorylation, and inhibits IFN-γ production.116–119 However, despite the apparent beneficial effect of the HCV-mediated NK activation during chronic HCV infection, such activation may act as a double-edged sword by killing hepatocytes and secreting proinflammatory cytokines, thus causing liver damage and contributing to the pathogenesis of liver disease.114 NK cells may also modulate other components of innate immunity. Recently, Jinushi et al.118 reported that negative regulation of NKs by inhibitory receptor CD94/NKG2A leads to altered NK-induced modulation of dendritic cell functions in chronic hepatitis C virus infection. Furthermore, the polymorphism of killer immunoglobulin-like receptors observed in HCV-infected patients may provide an additional layer of regulation of NK function during HCV infection.116, 117 Based on almost 2 decades of research advances, a recent theoretical model for HCV persistence placed the NK cell at the center of HCV immune evasion strategies121; this theory awaits confirmation.

A deficient frequency of natural killer T (NKT) cells in peripheral circulation of HCV-infected patients has been reported.114, 122 The NKT cells appear preactivated in vivo, because the in vitro–stimulated cells from HCV-infected patients gain more ability to secrete IL-13 than those from healthy subjects.123 The frequency of Valpha24/Vbeta11 NKT cells found in the liver is increased, and they also appeared to be activated.124 Furthermore, NKT cells from the livers of HCV-infected individuals express a dysregulated expression of killer immunoglobulin-like receptors125 similar to changes seen in NK cells.119–120 Although such phenotypical change may render the NKT cells autoreactive and promote killing of hepatocytes and other host cells, the role of NKT cells in specific or bystander cellular killing during HCV-induced hepatitis awaits further confirmation.

Innate Immunity As a Target in HCV Therapy

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References

Therapeutic approaches for HCV include IFN-α–based strategies to generally boost antiviral immunity and innate immune responses as well as targeted therapies based on specific defects identified in immune and viral replication pathways. Immune targets for HCV therapy include not only the classical immune cells but also heptocytes that express receptors for type I IFNs and pattern recognition receptors (TLRs and RIG-I–like RNA helicases). In addition, immune cell-derived factors may also modulate hepatocyte responses to HCV infection. Standard therapy for HCV is based on administration of recombinant IFN-α, a pivotal component of innate immune responses to viral infections; however, the full spectrum of IFN-α effects is yet to be fully understood. New strategies to increase production of endogenous type 1 IFN production through administration of TLR9 or TLR7/8 ligands have been promising in early clinical trials and in experimental models.39, 126–129 Activation of these pathways with exogenous ligands during HCV infection may have more complex biological and immune effects than simply induction of type I IFNs. Considering that TLR7/8 and TLR9 are both expressed in PDCs, activation of this cell type appears to have beneficial effects on antiviral immunity; however, TLR7/8 agonists can also impair monocyte-derived DC differentiation and maturation, thereby limiting their overall benefit.130 Currently, clinical trials with synthetic TLR7 agonists are on hold due to toxicity.131 Clinical studies with oligonucleotides containing unmethylated cytosine and guanine dinucleotides in specific base contexts (CpG ODN) to stimulate TLR9-mediated type I IFN production have also ceased due to limited benefit on HCV viral levels,132 suggesting possible exhaustion or limitation in the endogenous IFN-α–producing capacity in chronic HCV infection.

Antiviral therapies targeting selective inhibition of viral enzymes are under intense investigation. The HCV NS3/4A serine protease cleaves the viral glycoprotein into single functional proteins.133 However, NS3/4A also targets multiple innate immune recognition mechanisms, including TLR3, by cleaving its adapter TRIF and the RIG-I pathways by cleaving MAVS from the mitochondria, thereby disabling the key signaling pathways for type I IFN induction.9, 10, 51 Serine protease inhibitors have been suggested to restore host innate immune pathways in hepatocytes by preventing NS3/4A-induced degradation of TRIF and RIG-I.134, 135 Some of the serine proteases, such as BLIN-2061, have been withdrawn from clinical trials due to toxicity, while others, such as VX-950, are potent inhibitors of viral replication; their beneficial influence during HCV infection remains to be investigated in ongoing clinical trials.135 The protease inhibitor SCH 503034 had inhibitory effects in the in vitro replicon system, and emergence of viral resistance was reduced via combination therapy with IFN-α.135 Thus, blockage of NS3/4A protease activity is expected to have dual benefits: first, to directly suppress viral protein production, and second, to restore host immune recognition and responsiveness to IFN-α.136 Other pharmacological inhibitors of viral replication targeting the NS5B RNA-dependent polymerase, nucleoside inhibitors (NM283), and nonnucleoside inhibitors (cyclosporin A and analogues) have antiviral activities with no obvious direct effect on the natural innate immunity of the host,135 thus providing additional therapeutic opportunities to be explored.

Additional approaches include the use of immunomodulators. For example, the use of nonsteroidal anti-inflammatory drugs has been investigated137 and is yet to be fully determined during HCV infection. Nonsteroidal anti-inflammatory drugs may contribute to an attenuation of proinflammatory pathways to benefit ongoing liver damage and/or may lead to inhibition of PGE2, a major inhibitor of T cell activation and a factor contributing to regulatory T cell activation.138 Thus, it is conceivable that nonsteroidal anti-inflammatory drugs could be useful as a symptomatic therapy during HCV infection.

Finally, due to the superb T cell–activating potential of dendritic cells, dendritic cell–induced immunization strategies are under investigation. Recent studies in mice demonstrated that DNA-based immunization can break tolerance in an HCV model.126, 139–142 Future studies targeting the specific sites of HCV–host interactions await evaluation.

Conclusion

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References

A critical role has been established for innate immunity in viral recognition and initiation of virus-specific immune responses in HCV infection. Innate immune pathways including activation of TLRs and RIG-I–like helicase receptors are activated by HCV both in classical immune cells and in hepatocytes to initiate IFN production and inflammatory cell activation. Although there is evidence for activation of these antiviral pathways in acute HCV infection, HCV has evolved multiple mechanisms to subvert innate immune pathways, including destruction of TLR3-induced and RIG-I–induced signaling for IFN induction in hepatocytes, inhibition of PDC IFN-α production, and subversion of MDC functions in T cell activation. These observations collectively suggest that although HCV recognition occurs via innate immune pathways, the host often losses the battle between antiviral immunity and the virus. In this process, there is increasing evidence for nonspecific chronic inflammatory cell activation that manifests as increased proinflammatory cytokine levels and inflammatory cell infiltration in the liver as a result of host and viral factors and contributes to immune dysregulation as well as fibrosis in the liver.

References

  1. Top of page
  2. Hepatitis C Virus, Immune Responses and Hepatocytes—Brief Overview
  3. Pattern Recognition Receptors and Modification of Their Function in HCV Infection
  4. HCV Interferes with IFN-Inducing Pathways
  5. HCV Disrupts the Intracellular and Intercellular Coordination of the Immune System
  6. Dendritic Cells in HCV Infection
  7. Natural Killer and Natural Killer T Cells
  8. Innate Immunity As a Target in HCV Therapy
  9. Conclusion
  10. References
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