High Risk Injection Drug Users and HCV Infection
High Risk Injection Drug Users and HCV Infection
This study has characterised serum cytokine levels in a specific group of individuals who are likely to be repeatedly exposed to HCV through high risk drug injecting yet remain uninfected. We demonstrate that these EU cases have a serum cytokine profile that clearly distinguishes them from other HCV exposed groups and controls, with high levels of pro-inflammatory cytokines and chemokines associated with an innate immune response. Cytokine pathways and cascades are complex, with the assigning of cytokines to specific arms of the immune system simplifying the source and function of several cytokines (e.g. IL-12 and TNF-α are released by both adaptive and innate immune cells). However, overall the pattern shown here is distinct in EU and thus suggests an association with apparent resistance to HCV.
The cytokines that principally differentiate EU from spontaneous resolvers and those who have chronic hepatitis C are IL-6 and IL-8. IL-8 belongs to the CXC family of chemokines and is a chemo-attractant for neutrophils and lymphocytes. IL-8 is known to be released by hepatocytes, endothelial cells and monocytes at an early stage in response to HCV infection promoting recruitment of innate immune cells, including monocytes, dendritic cells and NK cells, with the resultant initiation of early intrahepatic antiviral immune responses. IL-6 is a potent pleiotropic cytokine released by a number of cells including monocytes, dendritic cells and endothelial cells. Amongst its roles, it activates innate immune cells as well as inducing an acute phase response in the liver, which has been shown to promote hepatocyte survival. In other infectious hepatitis models, IL-6 production has been shown to reduce early hepatitis B viral replication independent of the effect of IFN-α or -γ. It is therefore possible that this combination of cytokines allows rapid recruitment and activation of innate immune cells including monocytes, NK cells and NK-T cells to prevent HCV infection from becoming established. Early clearance of the HCV viraemia can then occur before readily detectable downstream adaptive immune responses have developed.
Th1 cellular immune responses are important for effective clearance of HCV viraemia and IL-12 is considered a key cytokine in promoting a Th1 response. A functional single nucleotide polymorphism in IL12B gene has previously been found to be associated with this EU cohort. However in this study IL-12 levels were low in EU, which demonstrates the challenges with extrapolation of functional correlates from single nucleotide genetic associations. Weak HCV-specific Th1 cellular responses in 58% of EU have been previously described. These responses appear to diminish within months of cessation of IDU, suggesting that they may not be key to preventing HCV infection. Rather, they can be regarded as surrogate markers of recent HCV exposure in EU, requiring priming by repeated low dose exposure to HCV in order to be maintained.
Natural killer (NK) cells are important innate immune cells with potent anti-viral functions. Their activity is governed by interactions between activating and inhibitory receptors and class 1 human leucocyte antigen (HLA) molecules. A compound homozygous state for the NK cell inhibitory killer immunoglobulin-like receptor (KIR), KIR2DL3, and it cognate ligand HLA-C1 has been demonstrated to be associated with spontaneous clearance of HCV. This genotype has also been shown to be over-represented in our EU cohort and is thought to provide less inhibition to NK cell function, permitting a more responsive NK cell phenotype. Of particular interest to the findings of raised innate cytokines, the protective effect of this genotype is strongest in those with low dose exposure to HCV, such as through intravenous drug use, suggesting that at this low level, effective innate immune responses are mounted but not overwhelmed. Relating this to the observed cytokine profiles in EU, IL-6 can activate as well as potentiate the cytotoxic potential of NK cells and IL-8 has been shown to be a strong chemo-attractant factor for NK cells in vitro.
The importance of innate immune cytokines in HCV clearance has been brought to the fore recently by the multiple studies that have shown an association between spontaneous and treatment induced clearance of HCV and polymorphisms in or near the IL-28B gene (reviewed in). The IL-28B gene encodes a type III interferon, interferon-λ3 (or IL28B), which has been shown to have similar antiviral properties to type I interferons, such as IFN-α. However, at present the functional significance of these IL-28B gene polymorphisms is poorly understood and it is unclear whether they result in higher levels of IFN-λ3 or not. Presently there is no available assay to quantify serum levels of IFN-λ3 in serum. However, in relation to EU, we have recently shown that the rs12979860 SNP CC genotype associated with spontaneous HCV resolution is not over-represented in this cohort. This implies that the functional correlates of this genotype on spontaneous and treatment induced clearance do not necessarily apply to protection from HCV.
Bearing in mind that most cytokines have a short half-life in serum, raised levels of the distinguishing cytokines may have been missed in some of the EU as longitudinal serum sampling during continued injecting was not done. Correlation with intrahepatic cytokine expression would have helped to determine whether these cytokine levels are related to an immune response specific to hepatic C virus. However, these subjects are healthy without signs of liver disease so obtaining liver tissue for study is not possible.
It is possible that the actual act of injection drug use could be resulting in raised levels of IL-6 and IL-8 as a result of drug inoculation, local trauma or even microbial contamination. Against this is our finding that levels of IL-6 and IL-8 were not significantly raised in the IDU who were currently injecting in the two HCV antibody positive groups. Furthermore, studies in active IDU have shown that the injecting of opiates, such as heroin, has an immunosuppressive effect and results in a dampened proinflammatory cytokine response, making this a less likely explanation for the raised cytokines in EU.
Understanding the factors that can protect from HCV infection is important in the context of developing drug targets and vaccines. EU IDU possess markers of activated innate immunity that may well be a result of repeated exposure to HCV. As they remain uninfected, these cytokine profiles may provide some further clues into the mechanisms of resistance to HCV infection. Further study of the cellular components of innate immunity, particularly NK cells, is clearly important in these high risk intravenous drug users.
Discussion
This study has characterised serum cytokine levels in a specific group of individuals who are likely to be repeatedly exposed to HCV through high risk drug injecting yet remain uninfected. We demonstrate that these EU cases have a serum cytokine profile that clearly distinguishes them from other HCV exposed groups and controls, with high levels of pro-inflammatory cytokines and chemokines associated with an innate immune response. Cytokine pathways and cascades are complex, with the assigning of cytokines to specific arms of the immune system simplifying the source and function of several cytokines (e.g. IL-12 and TNF-α are released by both adaptive and innate immune cells). However, overall the pattern shown here is distinct in EU and thus suggests an association with apparent resistance to HCV.
The cytokines that principally differentiate EU from spontaneous resolvers and those who have chronic hepatitis C are IL-6 and IL-8. IL-8 belongs to the CXC family of chemokines and is a chemo-attractant for neutrophils and lymphocytes. IL-8 is known to be released by hepatocytes, endothelial cells and monocytes at an early stage in response to HCV infection promoting recruitment of innate immune cells, including monocytes, dendritic cells and NK cells, with the resultant initiation of early intrahepatic antiviral immune responses. IL-6 is a potent pleiotropic cytokine released by a number of cells including monocytes, dendritic cells and endothelial cells. Amongst its roles, it activates innate immune cells as well as inducing an acute phase response in the liver, which has been shown to promote hepatocyte survival. In other infectious hepatitis models, IL-6 production has been shown to reduce early hepatitis B viral replication independent of the effect of IFN-α or -γ. It is therefore possible that this combination of cytokines allows rapid recruitment and activation of innate immune cells including monocytes, NK cells and NK-T cells to prevent HCV infection from becoming established. Early clearance of the HCV viraemia can then occur before readily detectable downstream adaptive immune responses have developed.
Th1 cellular immune responses are important for effective clearance of HCV viraemia and IL-12 is considered a key cytokine in promoting a Th1 response. A functional single nucleotide polymorphism in IL12B gene has previously been found to be associated with this EU cohort. However in this study IL-12 levels were low in EU, which demonstrates the challenges with extrapolation of functional correlates from single nucleotide genetic associations. Weak HCV-specific Th1 cellular responses in 58% of EU have been previously described. These responses appear to diminish within months of cessation of IDU, suggesting that they may not be key to preventing HCV infection. Rather, they can be regarded as surrogate markers of recent HCV exposure in EU, requiring priming by repeated low dose exposure to HCV in order to be maintained.
Natural killer (NK) cells are important innate immune cells with potent anti-viral functions. Their activity is governed by interactions between activating and inhibitory receptors and class 1 human leucocyte antigen (HLA) molecules. A compound homozygous state for the NK cell inhibitory killer immunoglobulin-like receptor (KIR), KIR2DL3, and it cognate ligand HLA-C1 has been demonstrated to be associated with spontaneous clearance of HCV. This genotype has also been shown to be over-represented in our EU cohort and is thought to provide less inhibition to NK cell function, permitting a more responsive NK cell phenotype. Of particular interest to the findings of raised innate cytokines, the protective effect of this genotype is strongest in those with low dose exposure to HCV, such as through intravenous drug use, suggesting that at this low level, effective innate immune responses are mounted but not overwhelmed. Relating this to the observed cytokine profiles in EU, IL-6 can activate as well as potentiate the cytotoxic potential of NK cells and IL-8 has been shown to be a strong chemo-attractant factor for NK cells in vitro.
The importance of innate immune cytokines in HCV clearance has been brought to the fore recently by the multiple studies that have shown an association between spontaneous and treatment induced clearance of HCV and polymorphisms in or near the IL-28B gene (reviewed in). The IL-28B gene encodes a type III interferon, interferon-λ3 (or IL28B), which has been shown to have similar antiviral properties to type I interferons, such as IFN-α. However, at present the functional significance of these IL-28B gene polymorphisms is poorly understood and it is unclear whether they result in higher levels of IFN-λ3 or not. Presently there is no available assay to quantify serum levels of IFN-λ3 in serum. However, in relation to EU, we have recently shown that the rs12979860 SNP CC genotype associated with spontaneous HCV resolution is not over-represented in this cohort. This implies that the functional correlates of this genotype on spontaneous and treatment induced clearance do not necessarily apply to protection from HCV.
Bearing in mind that most cytokines have a short half-life in serum, raised levels of the distinguishing cytokines may have been missed in some of the EU as longitudinal serum sampling during continued injecting was not done. Correlation with intrahepatic cytokine expression would have helped to determine whether these cytokine levels are related to an immune response specific to hepatic C virus. However, these subjects are healthy without signs of liver disease so obtaining liver tissue for study is not possible.
It is possible that the actual act of injection drug use could be resulting in raised levels of IL-6 and IL-8 as a result of drug inoculation, local trauma or even microbial contamination. Against this is our finding that levels of IL-6 and IL-8 were not significantly raised in the IDU who were currently injecting in the two HCV antibody positive groups. Furthermore, studies in active IDU have shown that the injecting of opiates, such as heroin, has an immunosuppressive effect and results in a dampened proinflammatory cytokine response, making this a less likely explanation for the raised cytokines in EU.
Understanding the factors that can protect from HCV infection is important in the context of developing drug targets and vaccines. EU IDU possess markers of activated innate immunity that may well be a result of repeated exposure to HCV. As they remain uninfected, these cytokine profiles may provide some further clues into the mechanisms of resistance to HCV infection. Further study of the cellular components of innate immunity, particularly NK cells, is clearly important in these high risk intravenous drug users.