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Article first published online: 6 MAY 2009
Copyright © 2009 American Association for the Study of Liver Diseases
Volume 50, Issue 1, pages 34–45, July 2009
How to Cite
Kottilil, S., Yan, M. Y., Reitano, K. N., Zhang, X., Lempicki, R., Roby, G., Daucher, M., Yang, J., Cortez, K. J., Ghany, M., Polis, M. A. and Fauci, A. S. (2009), Human immunodeficiency virus and hepatitis C infections induce distinct immunologic imprints in peripheral mononuclear cells. Hepatology, 50: 34–45. doi: 10.1002/hep.23055
Disclaimer: The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organization imply endorsement by the U.S. Government.
Potential conflict of interest: Nothing to report.
- Issue published online: 23 JUN 2009
- Article first published online: 6 MAY 2009
- Accepted manuscript online: 6 MAY 2009 12:00AM EST
- Manuscript Accepted: 22 JAN 2009
- Manuscript Received: 13 NOV 2008
- Intramural Research Program of the NIH (NIAID and NIDDK)
Coinfection with hepatitis C virus (HCV) is present in one-third of all human immunodeficiency virus (HIV)-infected individuals in the United States and is associated with rapid progression of liver fibrosis and poor response to pegylated interferon (IFN) and ribavirin. In this study we examined gene expression profiles in peripheral blood mononuclear cells (PBMCs) from different groups of individuals who are monoinfected or coinfected with HIV and HCV. Data showed that HIV and HCV viremia up-regulate genes associated with immune activation and immunoregulatory pathways. HCV viremia is also associated with abnormalities in all peripheral immune cells, suggesting a global effect of HCV on the immune system. Interferon-α-induced genes were expressed at a higher level in PBMCs from HIV-infected individuals. HCV and HIV infections leave distinct profiles or gene expression of immune activation in PBMCs. HIV viremia induces an immune activated state; by comparison, HCV infection induces immunoregulatory and proinflammatory pathways that may contribute to progression of liver fibrosis. Conclusion: An aberrant type-I IFN response seen exclusively in HIV-infected individuals could be responsible for the poor therapeutic response experienced by HIV/HCV coinfected individuals receiving interferon-α-based current standard of care. (HEPATOLOGY 2009;50:34–45.)
Chronic coinfection with hepatitis C virus (HCV) is documented in one-third of all human immunodeficiency virus (HIV)-infected persons in the United States, and is associated with increased morbidity and mortality relative to monoinfection with either virus.1, 2 Since the advent of antiretroviral therapy (ART) for controlling HIV replication in vivo, acquired immune deficiency syndrome (AIDS)-associated opportunistic infections have declined considerably.3 However, recent data suggest an increasing number of HIV-infected individuals are now dying from liver disease.3–5 Moreover, HCV/HIV coinfected individuals see a rapid progression of liver fibrosis to cirrhosis when compared to HCV monoinfected individuals.6 Several adverse effects associated with ART are exacerbated in HCV/HIV coinfected individuals, making it difficult to accomplish adequate virologic control of HIV infection among such individuals.7–12 Additionally, HCV/HIV coinfected individuals have a higher HCV RNA viral load than do HCV monoinfected individuals.13–17 Finally, coinfection with HIV decreases the rates of sustained virologic response (SVR) of HCV and increases the rate of relapses after discontinuation of anti-HCV therapy among those who have achieved an end-of-treatment response (ETR) to combination therapy.14–17
Chronicity of infection with HCV monoinfected individuals is associated with an inconspicuous immune response against the virus18; in contrast, in HIV monoinfected individuals the resultant immune response is readily detectable, but is unable to contain HIV replication, leading to establishment of chronic infection.19 The characterization of and relationships between the immune responses against HCV and HIV in coinfected individuals are not completely understood.
To determine the differential host immune responses to each virus, we employed a DNA microarray study using peripheral blood mononuclear cells (PBMCs) from five different groups of individuals. DNA microarrays have been used previously to study the pathogenesis of HCV20 and HIV in monoinfected individuals.21 However, such studies failed to define gene expression imprints and/or adequately compare the differentiated gene profiles induced by each of these viruses alone and in coinfection because the studies did not involve direct comparison of the gene expression profiles of HCV and HIV coinfected individuals. In this study, we performed DNA microarray analysis on PBMCs from HIV-negative, HIV-viremic, HIV-aviremic, HCV-viremic, and HCV/HIV-coinfected individuals to determine the differential gene expression among these groups. To our knowledge, this study is the most comprehensive DNA microarray study that involves cross-sectional analysis of all five control groups. These genetic imprints provide insights into the pathophysiology of chronic HCV infection in those subjects who are coinfected with HIV.
Subjects and Methods
PBMCs were obtained by venipuncture from 33 subjects belonging to the following clinical categories: HIV-negative (n = 7), HCV-monoinfected viremic (n = 7), HIV-monoinfected viremic (n = 8), HCV/HIV-coinfected (n = 5), and HIV-aviremic (n = 6) (Table 1). All donors signed informed consents approved by the Institutional Review Board (IRB) of the National Institute of Allergy and Infectious Diseases (Bethesda, MD).
|Group||Start Date||Age||Gender||Race||Risk||HCV Genotype||TCD4||CD4 (%)||HIV VL||HCV VL||HCV Treatment Response|
Isolation of PBMCs and RNA.
PBMCs were isolated from white blood cells by the standard Ficoll-Hypaque Plus (Amersham Biosciences, Uppsala, Sweden) density gradient separation technique. RNA was isolated using Qiagen messenger RNA isolation kits (Qiagen, Germantown, MD) following the manufacturer's protocol.
Each patient's PBMCs (2 × 106) were incubated in 12-well plates with complete Roswell Park Memorial Institute-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), penicillin-streptomycin (Invitrogen), and L-glutamine (Invitrogen). Cultures were incubated at 37°C in a 5% CO2 incubator for up to 48 hours and supernatants from each culture were collected.
DNA Microarray Analysis.
PBMCs were analyzed using Affymetrix U133A 2.0 oligonucleotide arrays according to the protocols specified by the manufacturer (Affymetrix, Santa Clara, CA). A significant analysis of microarray (SAM) algorithm was used to determine the genes that were differentially expressed after an extensive filtering processes.22 Genes with low variability or undetectable expression levels (for the majority of samples) were eliminated from analysis if the Guanosine-Cytosine Robust Multi Array values for these genes were within the interquartile range of <0.263 or a 75th percentile of <5.
Branched DNA (bDNA) Multiplex Assay.
Validation of DNA microarray data was performed using a novel customized bDNA multiplex assay capable of detecting the expression of 35 genes. The RNA transcripts are released from cells in the presence of lysis mixture and hybridized to the probe sets. The RNA-probe set complexes are captured to their respective capture beads through the cooperative hybridization of multiple capture extenders (CE) with the capture probes on the capture beads during an overnight incubation. Signal amplification is performed by sequential hybridization of the bDNA amplifier and biotinylated label probe. The streptavidin-conjugated R-phycoerythrin (SAPE) binds to the biotinylated label probe. The capture beads are analyzed using a Luminex instrument. The amount of each target RNA present in a sample is quantified by determining the amount of SAPE fluorescence signal and the identity of the beads.
For flow-cytometric analyses, the following combinations of fluorochrome-conjugated antibodies were used: CD3 (allophycocyanin-conjugated [APC]) with CD54 (phosphatidylethanolamine [PE]), CC chemokine receptor 2 (CCR2) (PE), CCR7 (PE), CD10 (PE), CD80 (PE), CD86 (PE), CD274 (PE), CX3CR1 (PE), IGF-1R (PE), and NCR3 (PE). All antibodies and appropriate isotype controls were obtained from BD Biosciences (San Jose, CA). After washing, PBMCs were incubated with appropriate antibodies for 30 minutes at 4°C. The cells were washed, fixed, and suspended in 1% paraformaldehyde in phosphate-buffered saline (PBS) and flow-cytometric analysis was performed on a fluorescent-activated cell sorting (FACS) Array (BD Biosciences). For subset analysis, a lymphocyte gate and gates uniquely identifying CD3+ cells were applied, ≈100,000 events were collected, and the frequency of CD3+ and CD3− cells expressing each receptor was analyzed with FlowJo software (TreeStar, Ashland, OR).
Enzyme-Linked Immunosorbent Assay (ELISA).
Culture supernatants from PBMCs at 48 hours were tested for levels of interleukin-23A (IL-23A), β2 microglobulin, tumor necrosis factor (TNF), CC chemokine ligand-7 (CCL-7), CCL-20, IL-8, and chemokine ligand (CXCL1) by ELISA (R&D Systems, Minneapolis, MN).
Analysis of variance (ANOVA) with Tukey's multiple comparison test was used to compare means of the independent groups. The paired t test with Bonferroni adjustment for multiple testing was used to compare paired responses.
Differential Gene Expression Profiles in PBMCs of HCV-Infected and HIV-Infected Individuals.
In order to identify the gene expression profiles induced by both HCV and HIV, we performed DNA microarray analyses using total RNA isolated from fresh PBMCs from the aforementioned five patient groups. There were no significant differences in the five groups based on the major demographic characteristics such as sex, age, treatment responses, etc. (Table 1). Using Affymetrix human genome U133A oligonucleotide arrays consisting of probes encompassing over 22,000 genes and a SAM algorithm,22 we identified 1,813 differentially expressed genes (Fig. 1). The corresponding genes and samples from the individuals were grouped by using hierarchical clustering. Differences in relative levels of gene expression (Z-score) are indicated in color, where red indicates up-regulation and green indicates down-regulation relative to that of corresponding gene expression in controls (Fig. 1). The hierarchical analyses classified the genes into six distinct clusters based on differential expression between the five groups. Of these, cluster 1 consists of 174 genes down-regulated both in HCV-infected and HIV-infected individuals (groups B and C). Cluster 2 consists of 369 genes up-regulated in HCV-infected individuals (groups B and D). Cluster 3 consists of genes up-regulated in HIV-monoinfected individuals (groups C and E). Cluster 4 consists of genes up-regulated in only HIV-viremic individuals (group C). Cluster 5 consists of 302 genes down-regulated in only HCV-monoinfected individuals (group B). Cluster 6 consists of 599 genes up-regulated in only HCV-monoinfected individuals (group B). To identify HCV-induced changes in gene expression in PBMCs, we chose to focus our analyses on clusters 2, 5, and 6. In this regard, cluster 2 distinctively showed genes up-regulated by HCV viremia as observed in both HIV-negative and HIV-positive individuals. Meanwhile, clusters 5 and 6 showed genes that are either down-regulated or up-regulated by HCV viremia alone as observed in HCV-monoinfected individuals. Hierarchical clustering analyses indicated that there was a similarity in the transcriptional profile of genes that were differentially expressed in PBMCs of HCV-infected individuals (seen in cluster 2 genes) and also distinct gene expression profiles in HCV-monoinfected and HCV/HIV-coinfected individuals (seen in cluster 5 and 6 genes). Representative genes that belong to each cluster identified using rigorous literature-mining algorithms and statistical analyses are shown in Fig. 1.
Selection of Genes for Validation of DNA Microarray by Amplification.
To validate our DNA microarray data, we selected genes based on an extensive literature-mining algorithm, significance of microarray analysis data, and biology of the disease process (both HIV and HCV). This analytical approach following the biology of response rather than the mere expression levels of genes will help us ascertain that our results are driven by the biology (disease process, infection status, etc.) rather than race, age, sex, etc. Although this approach reduces the influence of these variables in interpretation of results, it will certainly not eliminate the influence completely, warranting validation of these results in a larger study. We performed gene amplification from RNA, estimated surface expression of receptors of freshly isolated PBMCs, and the levels of secreted proteins in culture supernatants. Gene amplification performed by bDNA analysis of selected genes that were differentially regulated using a custom-designed bDNA array is shown in Fig. 2. Two candidate genes OAS1 (Fig. 2A,B) and MX1 (Fig. 2C,D) were selected for validation of gene expression and were consistently reproduced by bDNA assay.
Selection of Genes/Gene Products for Validation by Microarray Flow Cytometry and ELISA.
For further validation of our DNA microarray analyses at the level of surface expression of proteins, we selected the most biologically relevant gene products based on biological relevance (Fig. 3, Table 2). From cluster 2, we chose IGF-1R, a cell surface receptor that increases insulin secretion upon stimulation23; CCR7, which is a chemokine homing receptor of immune cells,24 and natural killer (NK)p30, which is one of the NK activating receptors facilitating killing of infected targets.25 From cluster 5, we examined CCR2 and CX3CR1, two chemokine receptors involved in inflammatory response and lymphocyte activation.26 From cluster 6, we studied the expression of CD10, CD54, CD80, and CD274. CD10 is a cell surface marker of immature B cells27; CD54 is involved with cell-cell adhesion and formation of immunological synapses28; CD80 is a major T-cell costimulatory factor29; and CD274 is a cell surface molecule with a T-cell regulatory function.30 To elucidate the effect of HIV and HCV on the secretory function of PBMCs, we tested the ability of PBMCs to secrete the following proteins: CCL-7, CCL-20, CX3CL1, IL-8, and TNF-α. CCL-7 (monocyte chemotactic protein [MCP]-3) is a chemokine that attracts monocytes to sites of inflammation and regulates macrophage function.24 CCL-20, otherwise called liver activation regulated chemokine (LARC) or macrophage inflammatory protein-3 A (MIP-3α), is a chemokine that attracts lymphocytes, but is a weaker target for monocytes.31 CX3CL-1 also known as growth regulated oncogene (GRO)-α or fractalkine, is a cytokine belonging to the CX3C chemokine family and is a neutrophil chemoattractant, which is also involved in angiogenesis, inflammation, and tissue healing.32 TNF-α and IL-8 are proinflammatory cytokines secreted by T cells that mediate chemotaxis and inflammatory responses.33 All these genes formed part of cluster 6, which are up-regulated in HCV-monoinfected individuals.
|Name of Gene||Function||Relationship with Disease||Relevance in This Study|
|CD10||Surface marker for immature B cells||Up-regulated in HCV||Global effect of HCV replication on B cells|
|NKp30||NK activating receptor||Up-regulated in HCV||Increased in chronic HCV and HCV/HIV|
|CD80||Major T-cell costimulatory factor||Up-regulated in HCV||Global effect of HCV replication on T cells|
|CX3CL1||Neutrophil chemoattractant involved in angiogenesis, inflammation, and tissue healing||Up-regulated in HCV||Increased in chronic HCV|
|IGF-1R||Cell surface receptor that increases insulin secretion upon stimulation||Up-regulated in HCV||Increased in chronic HCV and HCV/HIV|
|CCL-7||Monocyte chemotaxis||Up-regulated in HCV||Increased in chronic HCV|
|CCL-20||Lymphocyte chemotaxis||Up-regulated in HCV||Increased in chronic HCV|
|APOBEC3A||mRNA editing enzyme||Up-regulated in HIV||HIV-induced type-I IFN response|
|APOBEC3G||Interferes with replication of retroviruses||Up-regulated in HIV||HIV-induced type-I IFN response|
|TRIM5||Iinnate immune defense against retroviruses||Up-regulated in HIV||HIV-induced type-I IFN response|
|EIF2AK2||Viral defense, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|G1P3||Regulation of apoptosis, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFI27||Impacts cellular apoptosis, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFI44||Impacts cellular apoptosis, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFIT1||IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFIT3||IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFITM1||IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFITM3||IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFNA2||IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFNB||IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IFNG||IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|IRF7||Transcriptional activation of virus-inducible cellular genes, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|ISG15||Ubiquitin like modifier for innate defense, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|ISG20||Ubiquitin like modifier for innate defense, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|LY6E||IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|MX1||Responsible for antiviral state against influenza virus infection, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|MX2||Responsible for antiviral state against influenza virus infection, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|OAS1||Viral RNA degradation and the inhibition of viral replication, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|OAS2||Viral RNA degradation and the inhibition of viral replication, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|PLSCR1||Responsible for the translocation of phospholipids, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|PPIA||Involved in T cell activation, IFN-inducible||Up-regulated in HIV||HIV-induced type-I IFN response|
|SP110||Transcriptional coactivator, IFN-inducible gene||Up-regulated in HIV||HIV-induced type-I IFN response|
|STAT1||IFN signaling||Up-regulated in HIV||HIV-induced type-I IFN response|
HCV Infection Has a Global Effect on Peripheral Blood Immune Competent Cells.
To investigate the effect of ongoing HCV replication on various peripheral blood immune competent cells, we performed flow cytometry analysis for the expression of surface molecules whose genes were differentially regulated in the DNA microarray analysis. As show in Fig. 4, HCV replication has profound effects on B cells, NK cells, and on antigen-presenting cells. B cells from HCV-monoinfected individuals expressed a significantly higher percentage of CD10+ immature B cells in their peripheral blood when compared to that seen in the other groups (14.2 ± 0.8% [group B] versus 7.1 ± 0.3% [group A], 8.2 ± 1.1% [group C], 3.2 ± 0.1% [group D], and 3.9 ± 0.3%; P < 0.03 for group B versus the others). A significantly higher proportion of NK cells from HCV-monoinfected and HIV/HCV-coinfected individuals expressed the natural cytotoxicity receptor 3 (NCR3 or NKp30) on their surface compared to the other three groups (3 ± 0.4% [group A], 9.8 ± 0.3% [group B], 2.6 ± 0.4% [group C], 8.7 ± 0.7% [group D], and 1.8 ±0.2% [group E]; P < 0.04 for groups B and D versus groups A and C and P < 0.03 for groups B and D versus group E). The percentage of cells expressing CD80 in the peripheral blood of HCV-monoinfected subjects was significantly higher compared to that seen with the other four groups (5.1 ± 0.6% [group A], 14.3 ± 0.9% [group B], 4.8 ± 0.8% [group C], 5.1± 0.7% [group D], and 4.4 ± 0.5% group E; P < 0.03 for group B versus the others). The levels of expression of CD274 (PD-L1) were not significantly different among the groups (data not shown).
Higher Levels of Markers of Hepatic Injury Were Secreted by PBMCs of Individuals with Chronic Hepatitis C Infection.
Several noninvasive markers of liver fibrosis have been identified in individuals with chronic hepatitis B and C infection.34 We selected markers of liver fibrosis CX3CL1 and IGF-R1 from cluster 6 and two whose gene expressions were also up-regulated in groups B and D (HCV-monoinfected and HCV/HIV-coinfected subjects) (Fig. 3). CX3CL1 and its receptor CX3CR1 have been found to be associated with liver injury and have been suggested to be a marker of liver disease.35 When we measured the levels of CX3CL1 expression in the supernatants of PBMCs, the cells from HCV-monoinfected subjects produced significantly higher levels of CX3CL1 than did cells from the other four groups (Fig. 5A; 276 ± 18 pg/mL [group A], 2870 ± 420 pg/mL [group B], 1020 ± 180 pg/mL [group C], 950 ± 110 pg/mL [group D], and 180 ± 30 pg/mL [group E]; P < 0.02 for group B versus groups C and D and P < 0.01 for group B versus groups A and E). IGF-R1 expression (mean fluorescent intensity) was significantly higher among HCV-monoinfected and HCV/HIV-coinfected subjects than that of the other groups (72 ± 3% [group A], 198 ± 9% [group B], 82 ± 8% [group C], 202 ± 12% [group D], and 87 ± 5% [group E]; P value < 0.03 for groups B and D versus others). These results also validate the respective gene expression pattern observed with DNA microarray analysis.
PBMCs from HCV-Monoinfected Subjects Secrete Higher Levels of Proinflammatory Cytokines.
Because both HCV and HIV are chronic viral infections that could result in immune activation and proinflammatory responses, we measured the levels of proinflammatory cytokines whose genes were differentially expressed among the groups in the DNA microarray analysis (cluster 6, Fig. 3). The levels of CCL-7 (MCP-3) in the culture supernatants of PBMCs from HCV-monoinfected subjects were significantly increased compared to those of the other four groups (Fig. 6A; 90 ± 9 pg/mL [group A], 2190 ± 650 pg/mL [group B], 150 ± 15 pg/mL [group C], 240 ± 22 pg/mL [group D], 75 ± 5 pg/mL [group E]; P < 0.001 between group B versus the others). The levels of CCL-20 (MIP-3α) were also significantly elevated in the culture supernatants of PBMCs from HCV monoinfected subjects compared to those of the other 4 groups (Fig. 6B; 10 ± 1 pg/mL [group A], 51 ± 8 pg/mL [groups B], 2 ± 0.1 pg/mL [group C], 48 ± 9 pg/mL [group D], 1.8 ± 0.1 pg/mL [group E]; P < 0.01 between group B versus groups A and D and P < 0.005 for group B versus groups C and E). These results consistently validate our DNA microarray data.
HIV-Infected Subjects Express Higher Levels of IFIG Than Do HCV-Monoinfected or Normal Subjects.
We had observed previously that interferon inducible gene (IFIG) expression in PBMCs is significantly higher in HCV/HIV-coinfected individuals who fail to respond to treatment.36 Furthermore, we demonstrated that non-responders to IFN-α therapy failed to induce IFIG expression even after exogenous IFN-α treatment (article in prep.). It is unclear whether HCV/HIV-coinfected individuals have a higher level of IFIG expression when compared to HCV-monoinfected individuals. Therefore, we examined the microarray data for IFIG expression in all five groups (Fig. 7A -F) and measured the expression of 25 IFIG by bDNA multiplex. Our results showed that HIV-monoinfected individuals had significantly higher expression of IFIG genes than did the other groups (P < 0.05).
In this study we demonstrate that HCV and HIV infections induce distinct immunological profiles in PBMCs as determined by gene expression. Although HCV infection leads to the expression of genes that reflect a proinflammatory immune response, mainly on non-T cells. HIV infection induces an immune activation profile involving CD4+ and CD8+ T cells. Liver activation-specific inflammatory markers were induced in PBMCs of both HCV mono and HIV/HCV-coinfected individuals. HCV infection induces activation of all peripheral immune cells, reflecting a global effect of chronic HCV replication on the immune system. Moreover, the IFIG expression, which is highly predictive of the therapeutic response to IFN-α-based therapy, is expressed at a higher level in HIV-infected individuals, reiterating a mechanistic role for type-I IFN baseline hyperexpression in vivo in HCV/HIV-coinfected individuals who fail to clear HCV with IFN-α therapy.
Several studies have demonstrated that HIV infection induces a state of immune activation, which is responsible in part for the immunopathogenesis of the disease.37 HIV viremia has been shown to induce cellular activation as demonstrated by serum markers,38 activation of B cells,39 NK cells,25 pDCs,40 CD4+,41 and CD8+ T cells.42 Although analysis of HIV-induced gene expression profiles in PBMCs was not the primary objective of this study, our results largely confirmed the activation of peripheral T cells in HIV-infected viremic subjects when compared to those who were aviremic or HIV-seronegative. These changes were most remarkable among CD8+ T cells, which showed overexpression of genes coding for surface markers, such as CD38, HLA-DR, CD25, as well as granzyme and perforin genes, which represent an activated peripheral CD8 response to ongoing HIV replication (data not shown).
Unlike HIV, HCV primarily infects and replicates in human hepatocytes.43 Although several studies have shown that HCV can be detected in nonhepatic cells and tissues, maximal replication is sustained only in primary hepatocytes.44 Recent studies also have shown that chronic HCV infection does not lead to the extent of immune activation of T cells seen in HIV-infected subjects.43 Our results also show increased expression of certain genes and expression of various receptors on NK cells (NKp30), B cells (CD10), and pDCs (CD80) in PBMCs from HCV-infected subjects, when compared with HIV-infected or HIV-seronegative subjects. This increased expression of NKp30 on NK cells suggests a level of activation of NK cells in chronically viremic HCV monoinfected subjects. Future functional studies should confirm whether this increased expression of a natural cytotoxicity receptor may result in enhanced cytotoxic capacity of NK cells from HCV viremic individuals or whether the overexpression of this receptor merely represents a state of aberrant activation or anergy of NK cells from HCV viremic subjects. HCV-infected subjects had a significantly increased proportion of CD10+ immature B cells when compared to normal controls. Expansion of CD10+ B cells have been described in other chronic persistent viral infections such as HIV and is thought to represent an accelerated release of immature B cells as a result of IL-7 that is responding to the CD4+ T-cell lymphopenia.39 In this regard, it is possible that chronic HCV infection also results in B cell dysfunction and might explain to some extent the defective humoral immune responses previously described in chronic HCV-infected subjects.45 Finally, PBMCs from HCV-monoinfected subjects express higher levels of the T-cell costimulatory molecule, CD80, when compared to controls. The functional significance of the overexpression of this molecule on antigen-presenting cells is not completely understood, yet it may represent chronic activation of antigen-presenting cells as a result of the persistence of HCV antigens.
Several inflammatory cytokines have been linked to liver damage and the recruitment of effector cells to the liver. There are several markers of liver injury and dysfunction that are elevated in peripheral blood. Expression of CX3CL1 was increased in individuals infected with HCV when compared to those who do not have underlying liver disease. Recent studies have suggested that fractalkine (CX3CL1) recruits CX3CR1-expressing monocytes to the liver that may participate in the process of hepatic inflammation, in agreement with the proinflammatory role of fractalkine in other conditions of inflammation.35 These findings reiterate that CX3CL1/CX3CR1 interactions do play a significant role in inducing the hepatic inflammation seen in chronic HCV infection. Our results indicate that fractalkine expression is elevated in the peripheral blood cells of individuals with chronic HCV infection and suggest that this parameter be useful as a marker of liver fibrosis/injury when validated in larger studies. Furthermore, metabolic abnormalities, such as insulin resistance, diabetes, and hyperlipidemia are much more common in HCV-infected individuals than in the other groups.37, 40 The expression of IGF-1R on cells from HCV-infected individuals was higher than that in the other patient groups. As demonstrated in our study, PBMCs of HCV-monoinfected individuals produce increased levels of several proinflammatory cytokines. These results suggest a role for these cytokines in recruiting effector inflammatory cells to the liver and also serve as a noninvasive marker of liver fibrosis. Both HCV-monoinfected and HIV/HCV-coinfected individuals have increased serum levels of markers of liver fibrosis compared to seronegative HIV individuals and HIV-infected individuals without liver disease. These findings, when validated in larger studies, will definitively reiterate their role as possible noninvasive markers of liver fibrosis.
In summary, some immune markers such as NKp30 and IGF-R1 seem to be up-regulated among patients with HCV with or without HIV coinfection, whereas some others seem to be selectively up-regulated in HCV-monoinfected subjects (CD10, CD80, CCL-7 CCL20, and CX3CL1). It is plausible that HIV infection may have resulted in regulating the expression of some of these receptors. Additionally, it is also possible that the liver disease staging might have influenced the levels of some of these cytokines as well. Further studies are warranted to address whether HIV infection specifically interferes with the effects of chronic HCV infection on immune cells.
Our previous studies have described that high IFIG expression is the single most important predictor of therapeutic nonresponse to IFN-based treatment for HCV.36 This study demonstrates the higher expression of IFIG expression among HIV-infected individuals than HCV-monoinfected individuals, suggesting that HIV infection is the major driving factor in turning on a type-I IFN signature gene expression. Although our study does not involve follow-up analysis of treatment responses of all HCV-infected patients, this is the only study that has compared HCV-monoinfected subjects to HIV/HCV-coinfected subjects with respect to IFIG expression. The results are highly suggestive that HIV-coinfection drives type-I IFN signature gene expression, which could result in blunting of responsiveness to exogenous IFN therapy. A recent study has suggested that increased IFIG expression was seen in the liver of HCV-monoinfected patients who were IFN-nonresponders than those who achieved SVR.46 However, our study did not examine the hepatic IFIG expression, nor select a patient population based on treatment response. Therefore, a comparative study to look at hepatic and peripheral IFIG expression among HCV-monoinfected patients is warranted to see if there is a strong correlation between the two compartments.
In summary, our study offers a comprehensive analysis of the differential regulation of host immune responses in HCV-infected and HIV-infected subjects. The results show that both chronic viral infections have distinct immunological profiles that are consistent with the pathogenesis of the disease process. Future studies will be focused on identifying the specific mechanisms involved in the interactions of HIV and HCV that contribute to the establishment of chronicity and the accelerated progression of liver disease seen in coinfected individuals.
- 9Hepatitis C virus and HIV co-infection: a sleeping giant wakes. Hopkins HIV Rep 1999; 11: 10–12..