CC-chemokine receptor 5 (CCR5) in hepatitis C—at the crossroads of the antiviral immune response?

Abstract

CC-chemokines constitute a structurally related group of chemotactic cytokines which attract and activate specific subsets of inflammatory cells such as monocytes and T lymphocytes to the sites of infection. CC-chemokines bind to specific G-protein coupled receptors to trigger cell activation and migration. In particular, the CC-chemokines CCL3 (macrophagic inflammatory protein 1α, MIP-1α), CCL4 (macrophagic inflammatory protein 1β, MIP-1β) and CCL5 (regulated upon activation, normal T cell expressed and secreted, RANTES) are ligands for the CC-chemokine receptor 5 (CCR5) and attract monocytes and T lymphocytes. Expression of the various different chemokine receptors and tissue specific migration is related to the state of differentiation and activation of inflammatory cells. Whereas naive T lymphocytes express chemokine receptor CXCR4, T helper (Th) cells producing type 1 cytokines such as interferon-γ and tumour necrosis factor as well as a major subset of CD8+ cytotoxic T lymphocytes predominantly express CCR5.1^–3 In contrast, lymphocytes secreting type 2 cytokines are characterized by expression of CCR3, CCR4 and CCR8. Intrahepatic infiltration of mononuclear inflammatory cells is the histological hallmark of hepatitis C virus (HCV) infection. Importantly, lymphocytes infiltrating HCV infected livers express high levels of CCR5 suggesting that Th1 driven immune responses are critically involved in the antiviral immune response of the host.4 Likewise, CCL3, CCL4 and CCL5 are expressed by vascular endothelial cells within portal tracts as well as by CD8+ cytotoxic T lymphocytes in the infiltrates.4

There is accumulating evidence that strong antiviral type 1 immune responses are associated with viral clearance both during the acute phase of primary HCV infection and antiviral therapy of chronic hepatitis C.5^–7 On the other hand, CCR5+ T lymphocytes may also contribute to immune-mediated tissue damage of chronic hepatitis C.8^,9 Unlike lymphocytes in infected livers, CCR5 expression is significantly reduced on CD4+ and CD8+ T lymphocytes in the peripheral blood.4^,10 While this finding may just reflect selective compartmentalization of CCR5+ T cells to the liver, there is recent evidence suggesting that hepatitis C can counteract recruitment of antiviral CCR5+ T lymphocytes by down-regulating CCR5 expression via direct interactions of the HCV E2 envelope protein with the tetraspanin CD81.11 Furthermore HCV proteins such as HCV core and NS5A can modify CCL5 secretion by altering CCL5 promoter activity.12 Increased binding of CCL5 to its receptor CCR5 further decreases CCR5 surface density due to receptor internalization.13

Down-regulation of CCR5 might have important consequences for the host’s immune defence. Early after HCV infection, prominent CD8 cell responses are observed involving transient up-regulation of CCR5 expression.14 In chronic hepatitis C infection, however, CD8+ T lymphocytes show sustained dysfunction with poor type 1 cytokine secretion in response to HCV antigens and reduced expression of CCR5.14^,15 Decreased CCR5 expression in chronic hepatitis C infection might correspond to diminished lymphocellular type 1 cytokine responses reported by several groups.15^,16 Type 2 responses, however, are apparently preserved. Moreover, recruitment of CD8 cells into the liver might become more ineffective the more CCR5 expression is down-regulated. Of interest, sequential liver biopsies in patients with hepatitis C infection demonstrated steadily declining numbers of intrahepatic CD8 cells over the course of chronic infection.17 Thus, interfering with CCR5 mediated signalling may be part of the viral strategy to establish itself as chronic infection.

The CCR5 gene is subject to several mutations affecting its expression. Among these a 32 base pair deletion in the CCR5 open reading frame, CCR5-Δ32, has gained major scientific interest, as it leads to a frameshift and prevents expression of a functional CCR5 protein.18 CCR5-Δ32 has a 9.2% allele frequency in Caucasians but is virtually absent in populations of non-Caucasian descent. Because CCR5 is an essential co-receptor for m-tropic human immunodeficiency virus (HIV) strains,19 subjects homozygous for the CCR5-Δ32 allele, who comprise approximately 1.1% in a Caucasian population,18 are almost completely protected against HIV infection. Furthermore, HIV progression seems to be delayed in CCR5-Δ32 heterozygous patients.20 Beyond that, altered expression due to the CCR5-Δ32 mutation should also affect CCR5-mediated pathways in the immune response against other infectious agents. This hypothesis is supported by studies in CCR5 knockout mice, which demonstrated a shift towards type 2 cytokine activation in a model of experimental colitis.21 Furthermore, such CCR5 knockout animals show subtle impairment of macrophage functions, T cell-dependent immune responses and reduced clearance of the intracellular pathogen Listeria monocytogenes.22 More importantly, CCR5-Δ32 has been proposed to affect the natural course of several human diseases which are putatively associated with a change in the balance between type 1 and type 2 cytokines as part of their pathogenesis (Table 1). Consequently, the concept that type 1 immune responses are critical for the inflammatory reaction and the antiviral immune defence in HCV infection prompts the hypothesis that reduced CCR5 expression due to the CCR5-Δ32 mutation might also affect susceptibility to chronic hepatitis C, its natural course and the outcome of antiviral therapy.

In line with these predictions, the CCR5-Δ32 mutation was found to be more frequent than in the background population in two studies.23^,24 In particular, Woitas et al.24 reported that 7.8% of patients with chronic HCV infection exhibited the homozygous CCR5-Δ32/CCR5-Δ32 genotype compared to 1.0% in a healthy control group. Moreover, CCR5-Δ32 homozygosity in this study was associated with significantly increased HCV viral loads and CD8+ T lymphocyte counts in the peripheral blood.24 This finding would suggest that individuals with the CCR5-Δ32/CCR5-Δ32 genotype have an increased susceptibility to chronic HCV infection. In subsequent studies, however, increased CCR5-Δ32 frequency appeared to be specific to the subgroup of patients with haemophilia who were infected with chronic hepatitis C but remained free of HIV infection.25^–27 Several explanations have been proposed to explain these discrepancies. First, increased CCR5-Δ32 allele frequency in this particular group of patients might reflect selection pressure exerted by a concomitant HIV infection. A detailed statistical analysis of the combined HCV and HIV/HCV infected populations, however, proposes that CCR5-Δ32 homozygous patients were more prevalent in the cohort of Woitas et al., than can be explained by HIV selection pressure alone.28 Alternatively, the strategy of patient selection (haemophilia versus chronic liver disease), might have affected the results. Furthermore, certain individuals may lose anti-HCV and may be left with no markers of HCV infection after spontaneous clearance of HCV. Finally, HCV infected haemophiliacs may represent a group of patients with particularly intense exposure to HCV.

A possible role of the RANTES/CCR5 pathway in the pathogenesis of chronic HCV infection is further supported by histological studies. Although increased susceptibility to chronic HCV infection was also not observed in a large case–control study involving HCV-infected participants recruited from several centres across Europe, it identified the –403 polymorphism in the promoter region of CCL5 (RANTES) gene and CCR5-Δ32 allele as the only mutations among 20 tested polymorphisms of chemokines and chemokine receptors to be significantly associated with reduced hepatic inflammation.29 Moreover, decreased interface hepatitis, lobular necrosis and portal inflammation have been observed in CCR5-Δ32 heterozygous Israeli patients with chronic hepatitis C of Ashkenazi and Sephardim descent.30 Both observations provide further support for the involvement of CCR5 and its ligand CCL5 in the pathogenesis of chronic hepatitis C. However, reports on potential effects of CCR5-Δ32 concerning fibrosis progression remain controversial.

It nevertheless remains a most important issue to clarify whether mutations of the CCR5 gene affect treatment outcomes in chronic hepatitis C. Type 1 interferons and ribavirin are the standard treatment options for chronic hepatitis C. Interferon exerts direct antiviral, anti-proliferative and immunomodulatory effects. Although its precise mechanism of action against hepatitis C virus infection is still poorly understood, Yang et al.31 have reported that interferon mediates immune activation at least partially via up-regulating CCR5 expression on CD4 and CD8 cells. With respect to ribavirin, the mode of action is even less clear. Nevertheless, it has been proposed that this nucleoside analogue can mediate immunomodulation by altering the type 1/type 2 cytokine bias towards more favourable type 1 immune responses.32^,33 In line with the proposed mode of action, HCV-infected carriers of the CCR5-Δ32 mutation showed significantly decreased response rates of viral elimination than wild-type patients in interferon monotherapy.34 In contrast, combination treatment with ribavirin consistently did not reveal any effects of CCR5-Δ32 on treatment outcomes.34^–36 Importantly, CCR5-Δ32 carriers with chronic HCV infection who had not responded to interferon monotherapy showed unusually high response rates when they were retreated with the interferon/ribavirin combination regimen. Thus, the addition of ribavirin seems to compensate for the negative effect of the CCR5-Δ32 mutation on treatment outcome. In this context, it is an intriguing speculation, that ribavirin might counteract the effects of CCR5-Δ32 by strengthening type 1 immune responses. Fortunately, combination treatment has meanwhile become the gold standard of anti-HCV therapy. Thus, CCR5-Δ32 has only little relevance with respect to current clinical practice in chronic HCV infection. However, this mutation may gain considerable importance for further therapeutic developments, because CCR5 inhibitors are currently being developed for the treatment of HIV infection and should be carefully checked in patients with HCV coinfection to avoid undesired side effects.

Taken together, there exist intriguing theoretical concepts as well as first observational evidence to suggest an important role of CCR5 and its ligands with respect to the pathogenesis of HCV infection. However, the available observational data remain controversial at the present stage. Further research in different ethnic populations and consideration of additional genetic markers are needed to understand the role of polymorphic genetic traits in a disease that is of polygenic nature. Furthermore, chronic hepatitis C virus infection may be just a first example for a group of infectious diseases, where alterations in the balance between type 1 and type 2 immune responses may be of importance. Therefore, it should also be worthwhile studying the potential roles of CCR5 and its ligands in other infectious diseases with a putatively altered immune balance such as tuberculosis and leprosy.

References