Chemokines in the immunopathogenesis of hepatitis C infection

Authors

  • Mathis Heydtmann,

    1. NIHR Biomedical Research Unit for Liver Disease, MRC Centre for Immune Regulation, Institute of Biomedical Research, University of Birmingham, Birmingham, United Kingdom
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  • David H. Adams

    Corresponding author
    1. NIHR Biomedical Research Unit for Liver Disease, MRC Centre for Immune Regulation, Institute of Biomedical Research, University of Birmingham, Birmingham, United Kingdom
    • NIHR Biomedical Research Unit for Liver Disease, MRC Centre for Immune Regulation, Institute for Biomedical Research, 5th Floor, University of Birmingham, Birmingham, B15 2TH, United Kingdom
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    • fax: 0121 4158701


  • Potential conflict of interest: Nothing to report.

Abstract

Chronic infection with the hepatitis C virus, a noncytopathic hepatotropic RNA virus, affects over 170 million people worldwide. In the majority of cases, neither the early innate immune response nor the later adaptive immune response succeeds in clearing the virus, and the infection becomes chronic. Furthermore, in many patients, the ineffective inflammatory response drives fibrogenesis and the development of cirrhosis. It is critical to understand this immune pathology if preventative and curative therapies are to be developed. Chemokines are a superfamily of small proteins that promote leukocyte migration and orchestrate the immune response to viruses, including hepatitis C virus. Chemokines are crucial for viral elimination, but inappropriate persistence of expression in chronic hepatitis C infection can drive tissue damage and inflammation. Here we review the role of chemokines and their receptors in hepatitis C virus infection. (HEPATOLOGY 2009;49:676–688.)

Hepatitis C Virus (HCV) Immunopathology

HCV is a hepatotropic virus consisting of a polyprotein processed into structural proteins (core and envelope proteins 1 and 2), nonstructural proteins (NS2 to NS5), and a protein of unknown function (p7).1, 2 The viral polymerase lacks proof-reading capability, and this results in the generation of sequence diversity and quasispecies that contribute to evasion of the host immune response and chronic infection.2–4 Although hepatocytes are the primary target of HCV, the virus can interact with monocyte, lymphocyte, and endothelial cells.5, 6 The recent description of in vitro models of HCV replication is elucidating HCV biology,7 but the lack of small-animal models hinders studies of disease course and outcome.3

The liver is a unique immunological environment with a dual blood supply and distinct rheological requirements for leukocyte recruitment. Although hepatic immune tolerance prevents exuberant responses to food antigen,8 the liver can generate strong immune responses to infections, including hepatitis A and E viruses.9 In general, a vigorous intrahepatic immune response requires activation of T cells by activated dendritic cells (DCs) within secondary lymphoid tissues, whereas activation within the liver by hepatocytes or endothelial cells results in tolerance.10 This allows the liver to tolerate food antigens captured by endothelial cells and self-antigens on hepatocytes while responding appropriately to infections that cause inflammation and activation of DCs. The ability of HCV to infect hepatocytes without causing marked inflammatory damage may prevent the activation of a full immune response.2, 11

Whether HCV infection is acute and self-limiting or persistent is determined early.3, 4 The first line of defense is provided by infected hepatocytes that secrete interferon (IFN) and down-regulate RNA translation in response to infection before innate immune cells, including macrophages, DCs, natural killer (NK) cells, and natural killer T (NKT) cells, are activated to amplify secretion of type I IFNs and IFN-response genes. Activation of pattern recognition receptors, particularly Toll-like receptors, triggers chemokine secretion, which amplifies leukocyte recruitment.2, 3, 10 Innate immune activation facilitates the development of adaptive immunity through DCs, which take up and process viral antigens and migrate to lymph nodes to activate naïve T cells.12 Two functionally distinct subsets of DCs, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs), have been implicated in HCV persistence.13 pDCs secrete type-1 IFN, prime T helper 1 (Th1) responses, and activate cascades of chemokine secretion in HCV infection.14 mDCs prime Th1 responses via interleukin 12 (IL-12) but produce little type-1 IFN. The frequency of IFN-α–secreting intrahepatic pDCs is reduced in chronic HCV infection,15 but it is unclear whether mDC function is impaired.13, 14 Viral clearance is associated with a vigorous, multispecific CD4+ and CD8+ T cell response, with Th1 responses dominating.3, 4 Regulatory T cells (Tregs) are found in the HCV-infected liver, where they have a dualistic effect: on the one hand, they suppress HCV-specific CD8 T cells and promote viral replication; on the other hand, they suppress collateral inflammatory damage to reduce liver injury in chronic infection.16, 17

It is unclear how HCV evades immune responses and why persistence in the liver leads to hepatitis and fibrogenesis.2, 11 Although HCV does not cause general immunosuppression, it compromises innate and adaptive immunity in many ways, including effects on chemokines and their receptors.

Abbreviations

CCL, chemokine (C-C motif) ligand; CCR, chemokine (C-C motif) receptor; CXCL, chemokine (C-X-C motif) ligand; CXCR, chemokine (C-X-C motif) receptor; CX3CL, chemokine (C-X3-C motif) ligand; CX3CR, chemokine (C-X3-C motif) receptor; DC, dendritic cell; HCV, hepatitis C virus; HSC, hepatic stellate cell; ICAM-1, intercellular cell adhesion molecule 1; IFN, interferon; IL, interleukin; KC, Kupffer cell; LFA-1, lymphocyte function-associated antigen 1; MCMV, murine cytomegalovirus; mDC, myeloid dendritic cell; Mo, monocyte; NK, natural killer; NKG2A, natural killer group 2A; NKT, natural killer T; pDC, plasmacytoid dendritic cell; PMN, polymorphs; PRR, pattern recognition receptor; Th1, T helper 1; Th2, T helper 2; Th17, T helper 17; TNFα, tumor necrosis factor α; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; Treg, regulatory T cell; VAP-1, vascular adhesion protein 1; VCAM-1, vascular cell adhesion molecule 1; VLA-4, very late antigen 4; XCL, chemokine (C motif) ligand; XCR, chemokine (C motif) receptor.

Chemokines and Chemokine Receptors

Chemokines are 8- to 12-kDa heparin-binding cytokines that coordinate the homeostatic trafficking of leukocytes and their recruitment to inflammatory sites (Figs. 1 and 2). Dysregulation of chemokine expression or function underlies many inflammatory diseases.18 They can be divided functionally into homeostatic and inflammatory chemokines. Although most chemokines are inducible with inflammation, homeostatic chemokines are constitutively expressed and bring cells together within primary, secondary, and tertiary lymphoid organs to form functional microenvironments.19 However, several chemokines do not fall clearly into one category, and others previously thought to be homeostatic are implicated in inflammation.18 Chemokines mediate their effects through seven-transmembrane spanning receptors that signal via heterotrimeric guanosine triphosphate–binding proteins. Homeostatic chemokine receptors bind only one or two chemokines, for example, a monogamous pair—chemokine (C-X-C motif) receptor 5 (CXCR5) and chemokine (C-X-C motif) ligand 13 (CXCL13)—recruits B cells to follicles in lymph nodes, whereas receptors that recruit cells to inflammatory sites often have several ligands. Chemokines and their receptors also undergo posttranslational modifications that alter their function, providing almost limitless options that bring exquisite specificity to the control of leukocyte homing and positioning in tissues.19

Figure 1.

Important major human chemokines that act on cells of the lymphocyte and Mo lineages are shown. Fifty human chemokines and 18 chemokine receptors have been identified, and they can be divided into subfamilies on the basis of the position of conserved cysteine residues within a conserved tetracysteine motif. In CC chemokines, the first two consensus cysteines are next to each other; in CXC chemokines, they are separated by a nonconserved amino acid. These two subfamilies account for all but three of the known chemokines, the others being CX3CL1 (three intervening amino acids between the first cysteines) and XCL1 and XCL2, which lack two of four canonical cysteines. Ligands and their receptors are connected by lines, and the main leukocytes expressing receptors are shown by crosses. Receptors that are important for leukocyte recruitment to the liver are highlighted in yellow, and the intrahepatic cell types expressing the receptors are shown in the far right-hand column. Data have been drawn from many sources, including Viola and Luster,18 Rot and von Andrian,19 Boisvert et al.,43 and our own unpublished data on liver-infiltrating lymphocytes. Abbreviations: CCL, chemokine (C-C motif) ligand; CCR, chemokine (C-C motif) receptor; CXCL, chemokine (C-X-C motif) ligand; CXCR, chemokine (C-X-C motif) receptor; CX3CL, chemokine (C-X3-C motif) ligand; CX3CR, chemokine (C-X3-C motif) receptor; imm, immature; mat, mature; mDC, myeloid dendritic cell; Mo, monocyte; NK, natural killer; NKT, natural killer T; pDC, plasmacytoid dendritic cell; PMN, polymorphs; T mem, memory T cell; T naïve, naïve T cell; Th1, T helper 1; Th2, T helper 2; Th17, T helper 17; T reg, regulatory T cell; XCL, chemokine (C motif) ligand; XCR, chemokine (C motif) receptor.

Figure 2.

Chemokines are critical factors in regulating lymphocyte recruitment from the blood into the liver. (A) The process of leukocyte extravasation into tissue involves at least four stages.20 Cells are captured by carbohydrate-dependent tethering, which brings the flowing cell into contact with the vessel wall, allowing interactions with the endothelium. In the subsequent triggering stage, chemokines (immobilized on endothelial proteoglycans) activate chemokine receptors on leukocytes, resulting in activation of integrin binding to endothelial ligands such as ICAM-1 and VCAM-1. During this stage, the leukocyte stops on the vessel wall and then migrates on the endothelium, looking for the signals that drive transendothelial migration into tissue. Chemokines are also involved in transmigration, during which leukocytes migrate across the endothelium and enter tissue. Once in tissue, the cell follows chemokine gradients to sites of infection, using chemokine-mediated changes in the actin cytoskeleton to propel migration.23 (B) Chemokines are immobilized on the endothelium by proteoglycans in the glycocalyx that present chemokines to flowing leukocytes. The endothelium can itself secrete chemokines or present chemokines secreted by underlying cells in tissue, including hepatocytes, stellate cells, Kupffer cells, and infiltrating leukocytes, or capture chemokines secreted upstream by other structures such as cholangiocytes. (C) The CXCR3 ligands CXCL9 to CXCL11 dominate in the recruitment of CXCR3-expressing effector lymphocytes via the sinusoidal endothelium, whereas CCR5 ligands are more important for recruitment via the portal vascular endothelium. The critical trigger for CXCR3 ligand induction in the infected liver is IFNγ acting synergistically with proinflammatory cytokines such as TNFα secreted by multiple cells in response to viral infection and injury. Abbreviations: CCR, chemokine (C-C motif) receptor; CXCL, chemokine (C-X-C motif) ligand; CXCR, chemokine (C-X-C motif) receptor; ICAM-1, intercellular cell adhesion molecule 1; IFN, interferon; TNFα, tumor necrosis factor α; VCAM-1, vascular cell adhesion molecule 1.

Chemokines and Leukocyte Trafficking

The process of leukocyte extravasation into tissue involves at least four stages (shown later in Figs. 3-5).20 An initial carbohydrate-dependent tether brings the flowing cell into contact with the vessel wall, and a second rolling step mediated by selectins slows the leukocyte, allowing interactions with the endothelium. In the liver, rolling is attenuated as a result of low levels of shear stress in hepatic sinusoids, and there is little role for selectins.3 In the subsequent triggering step, signaling from leukocyte chemokine receptors triggers conformational activation of integrins and binding to endothelial ligands such as intercellular cell adhesion molecule 1. During this stage, the leukocyte stops on the vessel wall and then migrates on the endothelium, looking for the signals that drive transendothelial migration into tissue. Integrin-mediated arrest depends on a rapid in situ increase in integrin affinity in response to chemokines bound to the endothelial glycocalyx. The signaling complexes activated by chemokines are found in pre-assembled complexes that differ between leukocytes, thereby providing a degree of cell specificity.22

Figure 3.

Complex networks of autocrine and paracrine interactions involving many cell types determine the chemokine milieu within the hepatitis C virus–infected liver and thereby determine which leukocytes are recruited. Early events are a result of viral infection of hepatocytes and activation of PRRs on resident liver cells. This triggers the secretion of chemokines, including CCL2, CXCL10, CCL3, and CCL5, which recruit the first wave in innate immune cells, including NK cells, NKT cells, monocytes, and pDCs. These infiltrating cells amplify and broaden chemokine secretion by secreting interferons. Infiltrating monocytes and NKT cells secrete large amounts of IFNγ, which further stimulates resident liver cells to secrete chemokines, including CXCL10, and provide autocrine stimulation of monocytes to secrete CXCL10 and CCL5. pDCs secrete CCL3 and also large amounts of IFNα, which acts on monocytes to induce them to secrete CCL2, CCL4, and CXCL10. Effector T cells including Th1 cells are recruited in response to CXCL10 and secrete more IFNγ, helping to sustain the Th1 polarized local environment. Abbreviations: CCL, chemokine (C-C motif) ligand; CCR, chemokine (C-C motif) receptor; CXCL, chemokine (C-X-C motif) ligand; CXCR, chemokine (C-X-C motif) receptor; IFN, interferon; mono, monocytes; NK, natural killer; NKT, natural killer T; pDC, plasmacytoid dendritic cell; PRR, pattern recognition receptor; Th1, T helper 1.

Figure 4.

Distinct chemokines are involved in the recruitment and positioning of effector cells in the hepatitis C virus–infected liver. Recruitment via the portal vascular endothelium involves CCR5 ligands and the adhesion molecules ICAM-1, VCAM-1, and VAP-1 on the liver endothelium, which together promote adhesion and transmigration into tissue. Recruitment via the sinusoids appears to be dependent on CXCR3 rather than CCR5. The chemokine signals are displayed on the endothelial glycocalyx as described previously. The mechanisms of migration through the subendothelial tissues are poorly understood but probably involve interactions with the underlying matrix and fibroblasts. Localization at infected hepatocytes involves integrin-mediated adhesion to the hepatocytes triggered by chemokines up-regulated on infected cells, including the transmembrane chemokine CXCL16, shown in this example, and fractalkine. Abbreviations: CCL, chemokine (C-C motif) ligand; CCR, chemokine (C-C motif) receptor; CXCL, chemokine (C-X-C motif) ligand; CXCR, chemokine (C-X-C motif) receptor; ICAM-1, intercellular cell adhesion molecule 1; VAP-1, vascular adhesion protein 1; VCAM-1, vascular cell adhesion molecule 1.

Figure 5.

A sketch of the liver microarchitecture from the sinusoidal space (top) to the matrix between hepatocytes (bottom) is shown. Direct effects of viral infection of hepatocytes or interactions between viral particles or proteins and liver cells may trigger chemokine secretion (yellow and green stars) and leukocyte recruitment. Lymphocytes are seen crossing the sinusoidal endothelium using the adhesion molecules VAP-1, ICAM-1, and VCAM-1 and then interacting with hepatic stellate cells (purple) in the space of Disse using VCAM-1 to promote motility into the underlying tissue. Multiple chemokines are involved in each stage, secreted as part of local paracrine and autocrine networks induced by viral infection. Chemokines are retained in the infected liver by binding to the endothelial glycocalyx and cell matrix (pink filaments). Viral proteins and the effects of pathogen-associated molecular patterns during viral infection can activate endothelium and induce local chemokine secretion; viral proteins also activate stellate cells to secrete chemokines, which can then be retained in the matrix or transcytosed to the endothelial surface to promote recruitment. Infection of hepatocytes themselves leads to activation of chemokine transcription and secretion; finally, viral proteins or particles can interact with infiltrating leukocytes to modulate the secretion of and response to chemokines. Abbreviations: ICAM-1, intercellular cell adhesion molecule 1; LFA-1, lymphocyte function-associated antigen 1; VAP-1, vascular adhesion protein 1; VCAM-1, vascular cell adhesion molecule 1; VLA-4, very late antigen 4.

During diapedesis, leukocytes migrate across the endothelium and basement membrane to enter tissue.20 Hepatic sinusoids lack tight junctions and a basement membrane, and little is known about how leukocytes migrate through sinusoidal endothelium or how they cross the space of Disse.21 Once in tissue, the cell follows chemokine gradients to sites of infection, using chemokine-mediated changes in the actin cytoskeleton to propel migration.23

Transcytosis and presentation on the endothelial glycocalyx localize chemokines to specific sites in the vasculature, where they undergo posttranslational modification and activation/deactivation by ectoenzymes such as CD26.19, 24 Furthermore, a group of promiscuous, nonsignaling, chemokine receptors, termed interceptors, act as a sump to remove chemokines or promote their disposal via lymphatics25 (Fig. 2).

Chemokines and the Immune Response

Chemokines are secreted early after infection in response to activation of pattern recognition receptors on epithelial, stromal, and immune cells. These chemokines recruit the first wave of immune cells, including neutrophils, monocytes, NK cells, and NKT cells, all of which express inflammatory chemokine receptors. Chemokines also recruit DCs, which provide the link between innate and adaptive immunity. In response to pathogens, DCs take up antigen and increase expression of chemokine (C-C motif) receptor 7 (CCR7), which promotes migration via lymphatics into lymph nodes.26, 27 CCR7 is also expressed on naïve T and B cells, and the presence of its ligands, chemokine (C-C motif) ligand 21 (CCL21; on high endothelial venules and lymphatics) and CCL19 (within lymph node stroma), brings naïve lymphocytes and DCs together in the T-cell zone to allow immune activation. B lymphocytes also express CXCR5, and this allows them to respond to another chemokine, CXCL13, the expression of which is restricted to follicles.28 A subset of T cells up-regulates CXCR5, which promotes their migration to follicles, where they provide help to activated B cells.

T-cell activation in lymph nodes expands antigen-specific effector cells, which are imprinted with receptors that direct their homing back to tissue. In the gut and skin, lymphocytes are imprinted with tissue-specific homing receptors.29 It is not clear whether liver-specific homing receptors exist, although the inflammatory chemokine receptors CXCR3, CCR5, and CXCR6 are strongly associated with infiltration into the inflamed liver.30–32 The activation state of the DC and the local cytokine milieu determine whether Th1, T helper 2 (Th2), or T helper 17 (Th17) effector cells or Tregs are generated. Differential expression of chemokine receptors between lymphocyte subsets determines where and when they are recruited to tissue. Thus, Th2 cells express CCR4 and CCR8, whereas Th1 cells preferentially express CCR5 and CXCR3, and Th17 cells express CCR6 and CXCR6.33, 34 Tregs in lymphoid tissues and blood express CCR4, CCR5, and CCR6, whereas those in the liver express CXCR3 and in some situations CXCR6 and CCR10.35 All subsets display shared receptors consistent with the requirement for cells to be recruited to many tissues under different conditions.36

T-cell priming also generates long-lived CCR7+ central memory T cells that circulate through lymphoid tissues and CCR7 effector memory cells that home to inflamed tissues.37 CCR7 has regulatory properties, being required for the activation of Tregs and for the emigration of CCR7+ effector cells out of tissue via lymphatics during resolution of inflammation.27, 38 Thus, chemokines are critical for the initiation, maintenance, and resolution of immune responses, and this implies that they are central to disease pathogenesis in HCV infection.

Chemokines in HCV Pathogenesis

Given the lack of animal models of HCV infection, our understanding of disease pathogenesis relies on observational studies in infected humans and in vitro experiments. Expression studies using infected liver tissue have implicated specific chemokines in HCV pathogenesis,39–44 and these observations are supported by reports linking chemokine gene polymorphisms to altered susceptibility and progression of disease.45, 46 Here we review the role of chemokines and their cognate receptors in the different stages of HCV infection.

Chemokines in the Innate Immune Response Against HCV.

Viral infection of hepatocytes activates chemokine secretion,40, 41 which results in the recruitment of innate immune cells, including NK and NKT cells, which sustain local IFNγ production.47 HCV core protein,48 NS4A, NS4B, and NS5A49, 50 can all induce chemokine secretion in vitro, and some of these chemokines subvert the antiviral immune response. For example, CXCL8 secretion increases as a consequence of transactivation of the CXCL8 promoter by HCV proteins in hepatic stellate cells (HSCs),51 hepatocytes,49 and infected macrophages or endothelial cells.52, 53 The resulting increased CXCL8 levels promote immune evasion by inhibiting IFN antiviral activity.54 However, IL-8 increases expression of the death-inducing receptor tumor necrosis factor–related apoptosis-inducing ligand R2 (TRAIL-R2) on hepatocytes, which may sensitize them to cytotoxicity mediated by TRAIL-expressing cytotoxic T cells.55 HCV proteins and full-length virus can also inhibit CCL3, CCL5, CXCL8, and CXCL10 induction by other viruses such as the Sendai virus.56

NK and NKT cells are present at high frequencies in the liver57 and express chemokine receptors associated with tissue infiltration.43 CXCR6 and its ligand CXCL16 support the recruitment and survival of NKT cells,58 allowing them to sustain high local levels of IFNγ and thereby promoting the recruitment of Th1 cells.59 The existence of intrahepatic chemokine cascades has been demonstrated in murine cytomegalovirus (MCMV),60 and it is likely that similar mechanisms shape the immune/inflammatory response in HCV. MCMV stimulates local IFN secretion, which triggers CCL2 release by Kupffer cells (KCs). This leads to the recruitment of CCL3-secreting monocytes that recruit NK cells, which in turn secrete IFNγ, thereby triggering macrophage secretion of CXCL9 and CCL3 and the recruitment of CD4+ T cells. The secretion of IL-12 and IL-18 by macrophages and KCs results in a local Th1 response, and because Th1 cells secrete IFNγ, they create a feedback loop that promotes further T cell recruitment. Such a cascade is difficult to demonstrate in humans, but in chimpanzees, the early IFNα response and up-regulation of chemokines correlate with viral elimination; this suggests that it is crucial in determining outcome61 (Fig. 3). Thus, in the early phase of HCV infection, chemokines, particularly CXCL8, CXCL16, CCL2, and CCL3, promote recruitment of innate immune cells to the liver, including DCs, which then initiate the adaptive immune response.

Chemokines and the Trafficking of DCs: The Link Between Innate and Adaptive Immunity.

Secretion of chemokines by KCs and infected hepatocytes enhances the recruitment of DC precursors. In the inflamed liver, DCs become activated, take up antigen, and migrate via the space of Disse and portal tracts to draining lymph nodes, where they activate adaptive immunity.62 The signals that recruit mDC precursors into the liver include chemokines, particularly CX3CL1, displayed on sinusoidal endothelium61 (Aspinall and Adams, unpublished data, 2008). pDCs express the inflammatory chemokine receptors CCR2, CCR5, and CXCR3, which direct them to the inflamed HCV-infected liver. There, they secrete type-1 IFNs, tumor necrosis factor α (TNFα), CCL3, CCL4, and CXCL10 and induce CCL2 secretion by other cell types as part of a cascade that amplifies leukocyte recruitment.14

HCV can interfere with DC trafficking to prevent efficient antigen presentation. Nattermann et al.63 reported that HCV-induced secretion of CCL5 attracts CCR5+ immature DCs to the liver, where they are rendered unresponsive to CCR7 ligands as a result of HCV-E2 binding to CD81. The inability to use CCR7 to migrate into lymphatics traps DCs within the liver and thereby delays the establishment of an effective immune response in the crucial initial phase of infection.

Adaptive Immune Response and T Cell Recruitment in HCV

Th1 immune responses dominate in the HCV-infected liver,30, 64 and intrahepatic T cells express chemokine receptors associated with Th1 responses, including CXCR3, CXCR6, CCR1, and CCR5.30, 32, 42, 43 The distribution of chemokines within the liver compartmentalizes recruitment to different anatomical sites; CCR5 recruits lymphocytes to portal tracts, whereas CXCR3 is essential for recruitment into the parenchyma via sinusoids, and CXCR6 localizes cells to infected hepatocytes31, 65–67 (Figs. 2 and 4). In other viral infections, the CXCL8 receptor CXCR1 has been found on populations of virus-specific cytolytic CD8 T cells,68 and we have detected CXCR1+ cells within the HCV-infected liver; this suggests that the CXCL8/CXCR1 axis may be important for recruitment of HCV-specific cytotoxic T cells.

CXCR3 and Lymphocyte Recruitment.

Expression of CXCR3 is closely linked to Th1 function, and its ligands CXCL9, CXCL10, and CXCL11 are induced by the Th1 cytokines IFNγ and TNFα. Many studies using different approaches have shown that CXCR3 ligands are increased in the blood and liver of HCV-infected patients.69, 70 Successful antiviral therapy is associated with an increase in circulating CXCR3+CD8+ T cells and a reduction in CXCL10 and CXCL9 levels in blood. Furthermore, high levels of CXCL10 reduce the probability of sustained virological response to therapy.69–71 A polymorphism that results in a deletion in the CXCL11 promoter and reduced CXCL11 expression is more frequent in HCV patients than controls,72 and this is consistent with a role for CXCR3 in viral clearance as well as liver injury. The sources of CXCR3 ligands in HCV infection include hepatocytes, HSCs, and sinusoidal endothelium,30, 73 which express CXCL9-11 on stimulation with IFNγ and TNFα. These and other proinflammatory cytokines are released by KCs in response to infection and are amplified by the initial wave of infiltrating pDCs, NK cells, and NKT cells. An additional contribution comes from activated CD4+ T cells, which release CXCR3 ligands after interacting with HCV antigens in hepatocytes.73 This provides a feedback loop in which antigen-specific cells maintain the expression of the chemokines required for effector cell recruitment (Figs. 3 and 5).

Hepatic CXCR3 ligands are increased in many liver diseases, and this suggests that they play a generic role in effector cell recruitment to the inflamed liver.66 Endothelial CXCR3 ligands drive transendothelial migration into tissue and can be secreted by the endothelium itself, by neighboring cells (and transcytosed to the endothelium), or by upstream cells (and captured from the slow-flowing sinusoidal blood by proteoglycans within the endothelial glycocalyx).67 Hepatocytes promote lymphocyte recruitment by increasing endothelial chemokine secretion and adhesion molecule expression.75 Thus, hepatocyte stimulation by HCV infection together with IFNγ and TNFα derived from KCs may provide a paracrine amplification signal to increase lymphocyte recruitment via sinusoidal endothelium in chronic HCV.76

In MCMV, CXCR3 ligands recruit antigen-specific T lymphocytes to the liver,77 but it is difficult to ascertain what proportion of lymphocytes recruited to the liver in HCV are viral-specific cells as opposed to bystander cells. Bystander cells contribute to tissue injury in several ways. They express CD40 ligand, which allows them to activate CD40 on hepatocytes leading to nuclear factor kappa B–dependent chemokine secretion.78 Both antigen-specific and bystander cells express CXCR3 and use this receptor to enter the liver.79, 80 Tregs also use CXCR3 to enter tissue, although other signals may determine to where they migrate within the inflamed liver and hence where they mediate their anti-inflammatory effects.35 Thus, complex networks have evolved to induce CXCR3 ligands in the infected liver. These recruit antiviral effector cells, which could promote viral clearance in early disease, but in chronic infection, their persistence results in continuing effector cell recruitment and collateral liver injury. The fact that CXCR3+ effector cells drive damage makes CXCR3 a potential therapeutic target in chronic infection. Whether the anti-inflammatory effects would be outweighed by enhanced viral proliferation remains to be seen (Fig. 5).

Leukocyte Recruitment Mediated by CCR5 and CCR1 in HCV.

CCR2 and CCR5 are characteristic of memory T cells,18 and CD8 T cells expressing these receptors are enriched in the liver in HCV.30, 42, 43 The receptors share chemokine ligands; CCR5 interacts with CCL3, CCL4, CCL5, and CCL8, and CCR2 interacts with CCL2, CCL13, CCL7, and CCL8, all of which have been detected in the liver.30, 81 CCR5 ligands are strongly expressed on portal endothelium82 and in murine models of graft versus host disease; CCR5 and CCL3 support effector cell recruitment to portal tracts.64 The complexity of chemokine networks is illustrated by the finding that mice lacking CCR5 are more susceptible to concanavalin A–induced hepatitis and exhibit extensive inflammation mediated by CCR1+ effectors.65 Thus, under some conditions, CCR5 recruits anti-inflammatory and effector cells.

In HCV infection, a subset of CD8 T cells co-express CCR5 and the inhibitory natural killer group 2A (NKG2A) receptor. These T cells are attracted to the liver in response to CCR5 ligands induced by HCV-E2 antigen. However, engagement of the NKG2A receptor results in their inactivation, and this demonstrates another mechanism by which HCV can manipulate chemokine networks to subvert effective immune responses.81 The consequences of HCV on T cells can be unpredictable; for example, although HCV-E2 binding to CD81 on CD8 T cells increases CCL5 secretion, this causes autocrine desensitization of CCR5 and a loss of migratory capacity.83 These chemokine-mediated effects may be enhanced by the ability of the virus to interfere with cell motility directly,84 and this suggests that disabling cell migration is an important mechanism of immune subversion for HCV.

Gene association studies are cited to support the importance of CCR5 in HCV pathogenesis. However, the evidence is not clear-cut, and although some studies have reported that polymorphisms in CCL5 or CCR5 influence HCV pathogenesis, others have not confirmed these findings.45, 85 For example, Woitas et al.86 reported an increased frequency of the CCR5δ32 polymorphism (associated with reduced CCR5 function) in patients with HCV, but another study showed no association and concluded that the original findings could be explained by an overrepresentation of this mutation among human immunodeficiency virus–seronegative individuals with HCV.87 The same study did detect an association between hepatic inflammation and the CCL5 promoter polymorphism 403-A, which results in overexpression of CCL5. However, patients with the polymorphism had less inflammation than controls, and this finding was replicated in a second independent study.88 An analysis of an Irish cohort of women infected with HCV-contaminated anti-D after childbirth found no significant relationship between CCR2 or CCL5 polymorphisms and disease outcome or severity, although heterozygosity for the CCR5δ32 mutation was associated with spontaneous viral clearance and reduced hepatic inflammation in patients with specific human leukocyte antigen types.89 CCR5/CCL5 may affect the response to treatment because haplotypes carrying mutations associated with reduced CCL5 secretion are more frequent in nonresponders than sustained responders.90

CXCR6, CXCL16, and Leukocyte Localization at Epithelial Surfaces in the Liver.

CXCR6 is expressed on CD4 and CD8 T cells and NK and NKT cells in the HCV-infected liver, and its ligand CXCL16 is up-regulated as a transmembrane protein on inflamed bile ducts and hepatocytes.31 The engagement of CXCR6 on T cells by cholangiocyte CXCL16 promotes β1 integrin–dependent adhesion, which may position and retain effector cells in the HCV-infected liver. A recent study reported a unique subset of HCV-specific CXCR6+ liver-infiltrating CD8 T cells in HCV. These cells may have distinct effector functions because they express the C-type lectin CD161, which is expressed by NKT cells and Th17 cells.91, 92

Other chemokines may also be involved in retaining T cells within the liver. These include CXCL1293, 94 and the transmembrane chemokine CX3CL1 (fractalkine), both of which are expressed on inflamed bile ducts.95 The fractalkine receptor CX3CR1 is expressed by Th1 cells and NK cells and may help to retain these cells at sites of epithelial inflammation or infection. We detected another epithelial chemokine, CCL28, on cholangiocytes in several liver diseases, including HCV.35 However, a high proportion of the liver-infiltrating cells that expressed CCR10, the CCL28 receptor, were functional FoxP3+CD4+ Tregs. In comparison with blood Tregs, the CCR10+ liver-derived Tregs express high levels of CXCR3 and low levels of CCR7 consistent with a tissue-infiltrating phenotype. This led us to propose that these cells use CXCR3 to enter the liver and then localize to inflamed bile ducts using CCR10. The role of Tregs in HCV infection is controversial. High numbers of Tregs in the HCV-infected liver suggest an active role in suppressing antiviral immune responses, although in established infection with chronic hepatitis, they may suppress collateral damage.16, 96

In summary, the chemokine receptors CCR1, CCR5, CXCR1, CXCR3, and CXCR6 may all contribute to the recruitment of an effective antiviral immune response. However, in chronic infection, they recruit damaging effector cells that perpetuate liver injury without effectively clearing the virus.

Homeostatic Chemokines, Lymphoid Follicles, and Lymphocyte Egress from the Liver.

Homeostatic chemokines are up-regulated at sites of chronic inflammation, where they promote the formation of lymphoid follicles that have features of secondary lymphoid tissues.97 Such structures express CCL19, CCL21, and CXCL13, resulting in the recruitment of CCR7+ T cells and CXCR5+ B cells and their compartmentalization into T and B cell areas. Hepatic lymphoid follicles are seen in some patients with chronic HCV infection and can be sites for aberrant antibody production, which in some cases drives the development of type II mixed cryoglobulinemia.98 The vasculitic lesions of cryoglobulinemia in nerves and skin are characterized by up-regulation of CCL3, CCL4, and CXCL10 and infiltration by CXCR3+ Th1 cells and CCR5+ monocytes. Thus, the same proinflammatory chemokines are implicated in the vasculitic complications of cryoglobulinemia and hepatitis.99

CCR7+ T cells have been detected in livers from patients with HCV,98 but most if not all are CD62Llow and LFA-1high, characteristic of memory cells rather than naïve cells.32 Because CCL19 and CCL21 are expressed on sinusoids and lymphatic vessels in portal tracts,100 we suggest that CCR7 promotes the exit of T cells from the liver via lymphatics to draining lymph nodes, where they are restimulated by antigen. The reduced numbers of intrahepatic CCR7+ memory T cells in chronic HCV infection may reflect a defect in this pathway.32

Chemokines and Fibrosis

Chemokines are involved in fibrosis both indirectly by recruiting inflammatory cells that drive fibrogenesis and also by direct effects on HSCs.101 HSCs, which play a central role in fibrogenesis following their transition to myofibroblasts, express chemokines and chemokine receptors, allowing them to both contribute to the local chemokine milieu and respond to it.102, 103 CCL2 secreted by HSCs recruits CCR2+ macrophages and T cells, and levels are strongly associated with fibrogenesis. Inhibiting CCL2 reduces progression of fibrosis in vivo.102 CCL3, which binds CCR1 and CCR5, is secreted by macrophages and epithelial cells and is profibrotic in several animal models. Studies using CCR1- and CCR5-deficient mice show how chemokines can have a broader influence on inflammation and fibrogenesis. A lack of CCR1 or CCR5 is associated with decreased IL-4 and IL-13 expression, and this suggests a link between chemokine signaling and the secretion of profibrotic cytokines. Because IL-4 and IL-13 can induce CCL3 and CCL2 expression, a positive feedback loop may promote and sustain expression of both profibrotic Th2 cytokines and chemokines during fibrogenesis.104

HSCs also respond to activation by chemokines. CXCR3-binding chemokines stimulate phosphoinositide 3-kinase–dependent migration and proliferation of HSCs, and this demonstrates how chemokines can directly promote myofibroblast activation and scar formation.105 Thus, chemokines in the liver modulate the progression of fibrosis through several interlinked mechanisms. The involvement of such mechanisms in HCV infection is supported by the association of the CCR5δ32 mutation and a polymorphism in CCL8 (Q46K) with the severity of fibrosis in HCV infection.88

Conclusions

Chemokines are critical regulators of immunity and inflammation in all phases of HCV infection. They function within cytokine cascades that regulate the immune response to the virus. However, in chronic infection, their persistent expression can drive chronic inflammation in the absence of effective antiviral immunity, leading to liver injury and cirrhosis. The complex roles played by different chemokines during distinct stages of HCV infection may explain some of the conflicting findings from studies analyzing the impact of chemokine gene mutations on HCV pathogenesis. This also means that targeting chemokines therapeutically is complex. Although blocking chemokines that drive inflammation and fibrogenesis may be beneficial, such approaches run the risk of inhibiting the antiviral immune response, allowing unchecked viral replication. Future research will need to focus on the precise role of specific chemokines at each stage of HCV infection. In particular, we need to know the intrahepatic source of chemokines, what triggers their production, and which cells are responding at different stages of HCV infection. This will elucidate which chemokine networks promote immune cell recruitment and viral elimination as opposed to those that drive collateral liver damage through inflammation and scar formation. In addition, we need to understand better how HCV dysregulates chemokine expression and function to subvert antiviral immune responses. Such information is critical if chemokines are to be targeted therapeutically in HCV infection.

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