SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. References

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

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. References

Study Subjects.

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).

Table 1. Demographics and Immune Profiles of Study Participants
GroupStart DateAgeGenderRaceRiskHCV GenotypeTCD4CD4 (%)HIV VLHCV VLHCV Treatment Response
  1. HCV, hepatitis C virus; Hetero, heterosexual; HIV, human immunodeficiency virus; IDU, injection drug user; MSM, men who have sex with men; N/A, not applicable; NR, nonresponder; SVR, sustained viral response; VL, viral level.

A10/27/0541FWhiteN/AN/A73755N/AN/AN/A
A10/18/0534MWhiteN/AN/A122543N/AN/AN/A
A10/26/0556FWhiteN/AN/A60659N/AN/AN/A
A10/26/0537FBlackN/AN/A49044N/AN/AN/A
A10/25/0542MWhiteN/AN/A86346N/AN/AN/A
A10/17/0546FWhiteN/AN/A72647N/AN/AN/A
A10/20/0539MHispanicN/AN/A35237N/AN/AN/A
B10/31/0551MWhiteIDU1b  N/A2,500,000NR
B11/9/0553FWhiteIDU1a  N/A473000Relapser
B10/24/0551MBlackIDU1  N/A441,000SVR
B10/26/0545MWhiteIDU1a  N/A3,820,000Relapser
B11/9/0542MWhiteIDU2  N/A10,900,000SVR
B11/2/0559FBlackIDU2b  N/A7,810,000SVR
B10/26/0570MWhiteIDU1b  N/A3,830,000SVR
C12/13/0541MWhiteMSMN/A2902229576N/AN/A
C12/19/0520MBlackHETERON/A58827168629N/AN/A
C12/2/0542MWhiteMSMN/A3373632602N/AN/A
C12/13/0536FBlackHETERON/A415171964N/AN/A
C11/10/0534MHispanicMSMN/A3562123457N/AN/A
C12/1/0533MHispanicMSMN/A31814112697N/AN/A
C12/1/0559FBlackHETERON/A5072712648N/AN/A
C12/19/0523MHispanicHETERON/A152108507N/AN/A
D12/22/0549MBlackIDU1a123345121>7,692,310Naïve
D11/28/0540MBlackMSM1a146044494,976,400Viral breakthrough
D11/16/0551MBlackIDU1b72730493945420NR
D12/8/0549FBlackIDU1a79425491054510Naïve
D12/8/0555MBlackMSM1b100843499504730NR
E11/16/0544MWhiteMSMN/A7453149N/AN/A
E11/28/0541MBlackHETERON/A2581649N/AN/A
E6/20/0545FHispanicHETERON/A2021049N/AN/A
E11/16/0536FHispanicHETERON/A4622276N/AN/A
E11/22/0551MWhiteMSMN/A3623249N/AN/A
E12/20/0544MHispanicMSMN/A46433296N/AN/A

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.

Cell Cultures.

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.

Flow Cytometry.

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).

Statistical Analysis.

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.

Results

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. References

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.

thumbnail image

Figure 1. Clustering of differentially expressed genes in PBMCs from five groups (see Subjects and Methods). Levels of gene expression were assayed using Affymetrix Human Genome U133A chips. A total of 1,813 differentially expressed genes were identified. Genes were grouped using K-means clustering, and samples were grouped by 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. The numbers in parentheses indicate the number of genes in each cluster. Some genes are listed twice because certain probes recognize multiple regions of a single gene. Cluster 1 consists of genes down-regulated both in group B and C individuals. Cluster 2 consists of genes up-regulated in groups B and D individuals. Cluster 3 consists of genes up-regulated in groups C and E. Cluster 4 consists of genes up-regulated only in group C individuals. Cluster 5 consists of genes down-regulated only in group B individuals. Cluster 6 consists of genes up-regulated only in group B individuals. Select biologically relevant genes of clusters 2, 5, and 6 are shown on the right.

Download figure to PowerPoint

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.

thumbnail image

Figure 2. Validation of DNA microarray data by bDNA analysis. DNA microarray expression of IFIG genes was validated using a customized bDNA assay. The expression of two representative IFIGs by DNA microarray (left) and bDNA assay (right) for the two gene candidates OAS1 (A,B) and MX1 (C,D) indicate that HIV-infected individuals have a higher level of IFIG expression overall than the other groups of individuals. Fold change of gene expression is calculated as the change within a group compared to that observed in normal volunteers. The expression of both OAS1 and MX1 were significantly different between group C and others by microarray (P < 0.03 and P < 0.02, respectively) and bDNA (P < 0.04 and P < 0.01, respectively).

Download figure to PowerPoint

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.

thumbnail image

Figure 3. Average differential gene expression profiles of the five groups of individuals. Using either flow cytometry (surface expression) or ELISA (secreted proteins) selected genes were validated based on their biological relevance (following literature-mining algorithms). Clusters 2, 5, and 6 were selected based on the identification of cell surface receptors and secreted proteins most relevant to HCV infection status.

Download figure to PowerPoint

Table 2. Functional Characteristics of All the Genes Selected for Validation in the Study
Name of GeneFunctionRelationship with DiseaseRelevance in This Study
  1. Immune profiling was performed by flow cytometry for all individuals except HCV monoinfected individuals. HIV and HCV viral load had a lower limit of detection of 50 copies/mL and 615 IU/mL respectively. Group A consisted of healthy individuals seronegative for HIV and HCV; group B included HCV monoinfected viremic individuals; group C constituted HIV monoinfected viremic individuals; group D contained HCV/HIV coinfected individuals that were HCV viremic and HIV aviremic, and group E consisted of HIV monoinfected, but aviremic individuals. For Group E individuals, antiretroviral therapy (ART) consisted of at least one HIV protease inhibitor and/or one non-nucleoside reverse transcriptase inhibitor and two reverse transcriptase inhibitors. HCV, hepatitis C virus; HIV, human immunodeficiency virus; IFN, interferon.

CD10Surface marker for immature B cellsUp-regulated in HCVGlobal effect of HCV replication on B cells
NKp30NK activating receptorUp-regulated in HCVIncreased in chronic HCV and HCV/HIV
CD80Major T-cell costimulatory factorUp-regulated in HCVGlobal effect of HCV replication on T cells
CX3CL1Neutrophil chemoattractant involved in angiogenesis, inflammation, and tissue healingUp-regulated in HCVIncreased in chronic HCV
IGF-1RCell surface receptor that increases insulin secretion upon stimulationUp-regulated in HCVIncreased in chronic HCV and HCV/HIV
CCL-7Monocyte chemotaxisUp-regulated in HCVIncreased in chronic HCV
CCL-20Lymphocyte chemotaxisUp-regulated in HCVIncreased in chronic HCV
APOBEC3AmRNA editing enzymeUp-regulated in HIVHIV-induced type-I IFN response
APOBEC3GInterferes with replication of retrovirusesUp-regulated in HIVHIV-induced type-I IFN response
TRIM5Iinnate immune defense against retrovirusesUp-regulated in HIVHIV-induced type-I IFN response
EIF2AK2Viral defense, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
G1P3Regulation of apoptosis, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFI27Impacts cellular apoptosis, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFI44Impacts cellular apoptosis, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFIT1IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFIT3IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFITM1IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFITM3IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFNA2IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFNBIFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IFNGIFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
IRF7Transcriptional activation of virus-inducible cellular genes, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
ISG15Ubiquitin like modifier for innate defense, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
ISG20Ubiquitin like modifier for innate defense, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
LY6EIFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
MX1Responsible for antiviral state against influenza virus infection, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
MX2Responsible for antiviral state against influenza virus infection, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
OAS1Viral RNA degradation and the inhibition of viral replication, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
OAS2Viral RNA degradation and the inhibition of viral replication, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
PLSCR1Responsible for the translocation of phospholipids, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
PPIAInvolved in T cell activation, IFN-inducibleUp-regulated in HIVHIV-induced type-I IFN response
SP110Transcriptional coactivator, IFN-inducible geneUp-regulated in HIVHIV-induced type-I IFN response
STAT1IFN signalingUp-regulated in HIVHIV-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).

thumbnail image

Figure 4. HCV infection has a global effect on immune cells. (A) The percentage of CD10+ immature B cells in the peripheral blood measured by flow cytometry from five individuals in the HCV-monoinfected group was significantly higher than that of the other four groups (P < 0.03). (B) The percentage of NKp30+ cells among NK cells in the peripheral blood was measured by flow cytometry from the five groups. HCV-infected (both monoinfected and HIV-coinfected) individuals had a significantly higher percentage of NKp30+ NK cells than that of the other groups (P < 0.04). (C) The percentage of CD80+ cells among CD3− in HCV-monoinfected individuals was significantly higher than that of the other four groups (P < 0.03).

Download figure to PowerPoint

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.

thumbnail image

Figure 5. Chronic HCV infection is associated with markers of liver injury. (A) The levels of CX3CL1 present in the supernatants of PBMCs from the five groups were determined by ELISA. PBMCs from HCV-monoinfected individuals secreted significantly higher levels of CX3CL1 than did the other four groups (P < 0.02). (B) Mean fluorescent intensity (MFI) of IGF-1R expression on PBMCs showed that HCV-infected (both monoinfected and HIV-coinfected) individuals had significantly higher expression of IGF-1R than did the other three groups (P < 0.02).

Download figure to PowerPoint

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.

thumbnail image

Figure 6. Chronic HCV infection is associated with increased levels of proinflammatory chemokines. (A) The levels of CCL-7 (MCP-3) present in the supernatants of PBMCs from the five groups were quantified by ELISA. PBMCs from HCV-monoinfected individuals secreted significantly higher levels of CCL-7 than did the other four groups (P < 0.001). (B) The levels of CCL-20 (MIP-3 α) present in the supernatants of PBMCs from HCV-monoinfected individuals were significantly higher than those of the other four groups (P < 0.01).

Download figure to PowerPoint

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).

thumbnail image

Figure 7. HIV infection results in increased expression of IFIG that is not seen in normal individuals and HCV-infected individuals. The gene expression profile of several differentially regulated genes was measured using a customized bDNA assay. IFIG genes were found to be up-regulated in HIV viremic individuals compared to other groups of individuals (P < 0.05). (A) IFI27 gene expression was significantly higher in PBMCs from group C compared to group A (P < 0.04), group B (P < 0.035), group D (P < 0.048), and group E (P < 0.034). (B) LY6E gene expression was significantly higher in PBMCs from group C compared to group A (P < 0.02), group B (P < 0.01), group D (P < 0.04), and group E (P < 0.015). (C) IFI44 gene expression was significantly higher in PBMCs from group C compared to group A (P < 0.038), group B (P < 0.02), group D (P < 0.04), and group E (P < 0.038). (D) OAS2 gene expression was significantly higher in PBMCs from group C compared to group A (P < 0.02), group B (P < 0.005), group D (P < 0.03), and group E (P < 0.028). (E) IRF7 gene expression was significantly higher in PBMCs from group C compared to group A (P < 0.042), group B (P < 0.039), group D (P < 0.04), and group E (P < 0.044). (F) MX2 gene expression was significantly higher in PBMCs from group C compared to group A (P < 0.02), group B (P < 0.018), group D (P < 0.022), and group E (P < 0.02).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. References

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.

References

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. References
  • 1
    Alter MJ, Kruszon-Moran D, Nainan OV, McQuillan GM, Gao F, Moyer LA, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 1999; 341: 556562.
  • 2
    Sherman KE, Rouster SD, Chung RT, Rajicic N. Hepatitis C virus prevalence among patients infected with human immunodeficiency virus: a cross-sectional analysis of the US adult AIDS Clinical Trials Group. Clin Infect Dis 2002; 34: 831837.
  • 3
    Selik RM, Byers RH Jr, Dworkin MS. Trends in diseases reported on U.S. death certificates that mentioned HIV infection, 1987-1999. J Acquir Immune Defic Syndr 2002; 29: 378387.
  • 4
    Jain MK, Skiest DJ, Cloud JW, Jain CL, Burns D, Berggren RE. Changes in mortality related to human immunodeficiency virus infection: comparative analysis of inpatient deaths in 1995 and in 1999-2000. Clin Infect Dis 2003; 36: 10301038.
  • 5
    Selik RM, Lindegren ML. Changes in deaths reported with human immunodeficiency virus infection among United States children less than thirteen years old, 1987 through 1999. Pediatr Infect Dis J 2003; 22: 635641.
  • 6
    Benhamou Y, Bochet M, Di Martino V, Charlotte F, Azria F, Coutellier A, et al. Liver fibrosis progression in human immunodeficiency virus and hepatitis C virus coinfected patients. The Multivirc Group. HEPATOLOGY 1999; 30: 10541058.
  • 7
    Aceti A, Mangoni ML, Pasquazzi C, Fiocco D, Marangi M, Miele R, et al. Alpha-defensin increase in peripheral blood mononuclear cells from patients with hepatitis C virus chronic infection. J Viral Hepat 2006; 13: 821827.
  • 8
    Sulkowski M. HIV and hepatitis C virus co-infection. Hopkins HIV Rep 1998; 10: 812.
  • 9
    Sulkowski MS. Hepatitis C virus and HIV co-infection: a sleeping giant wakes. Hopkins HIV Rep 1999; 11: 1012.
  • 10
    Sulkowski MS. Hepatitis C virus infection in HIV-infected patients. Curr Infect Dis Rep 2001; 3: 469476.
  • 11
    Sulkowski MS, Mast EE, Seeff LB, Thomas DL. Hepatitis C virus infection as an opportunistic disease in persons infected with human immunodeficiency virus. Clin Infect Dis 2000; 30( Suppl 1): S77S84.
  • 12
    Sulkowski MS, Moore RD, Mehta SH, Chaisson RE, Thomas DL. Hepatitis C and progression of HIV disease. JAMA 2002; 288: 199206.
  • 13
    Eyster ME. Concurrent HIV and HCV infections hasten liver failure. Am Fam Physician 1992; 46: 536.
  • 14
    Carrat F, Bani-Sadr F, Pol S, Rosenthal E, Lunel-Fabiani F, Benzekri A, et al. Pegylated interferon alfa-2b vs standard interferon alfa-2b, plus ribavirin, for chronic hepatitis C in HIV-infected patients: a randomized controlled trial. JAMA 2004; 292: 28392848.
  • 15
    Laguno M, Murillas J, Blanco JL, Martinez E, Miquel R, Sanchez-Tapias JM, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for treatment of HIV/HCV co-infected patients. Aids 2004; 18: F2736.
  • 16
    Soriano V, Perez-Olmeda M, Rios P, Nunez M, Garcia-Samaniego J, Gonzalez-Lahoz J. Hepatitis C virus (HCV) relapses after anti-HCV therapy are more frequent in HIV-infected patients. AIDS Res Hum Retroviruses 2004; 20: 351353.
  • 17
    Torriani FJ, Ribeiro RM, Gilbert TL, Schrenk UM, Clauson M, Pacheco DM, et al. Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) dynamics during HCV treatment in HCV/HIV coinfection. J Infect Dis 2003; 188: 14981507.
  • 18
    Rehermann B. Interaction between the hepatitis C virus and the immune system. Semin Liver Dis 2000; 20: 127141.
  • 19
    Fauci AS, Pantaleo G, Stanley S, Weissman D. Immunopathogenic mechanisms of HIV infection. Ann Intern Med 1996; 124: 654663.
  • 20
    Bukh J, Miller RH, Purcell RH. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Semin Liver Dis 1995; 15: 4163.
  • 21
    Chun TW, Justement JS, Lempicki RA, Yang J, Dennis G Jr, Hallahan CW, et al. Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals. Proc Natl Acad Sci U S A 2003; 100: 19081913.
  • 22
    Tibshirani R, Hastie T, Narasimhan B, Chu G. Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc Natl Acad Sci U S A 2002; 99: 65676572.
  • 23
    Kulkarni RN. Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function. Biochem Soc Trans 2002; 30: 317322.
  • 24
    Forster R, Davalos-Misslitz AC, Rot A. CCR7 and its ligands: balancing immunity and tolerance. Nat Rev 2008; 8: 362371.
  • 25
    Byrd A, Hoffmann SC, Jarahian M, Momburg F, Watzl C. Expression analysis of the ligands for the natural killer cell receptors NKp30 and NKp44. PLoS ONE 2007; 2: e1339.
  • 26
    Saederup N, Chan L, Lira SA, Charo IF. Fractalkine deficiency markedly reduces macrophage accumulation and atherosclerotic lesion formation in CCR2-/- mice: evidence for independent chemokine functions in atherogenesis. Circulation 2008; 117: 16421648.
  • 27
    Loken MR, Shah VO, Hollander Z, Civin CI. Flow cytometric analysis of normal B lymphoid development. Pathol Immunopathol Res 1988; 7: 357370.
  • 28
    Lebedeva T, Dustin ML, Sykulev Y. ICAM-1 co-stimulates target cells to facilitate antigen presentation. Curr Opin Immunol 2005; 17: 251258.
  • 29
    Slavik JM, Hutchcroft JE, Bierer BE. CD28/CTLA-4 and CD80/CD86 families: signaling and function. Immunol Res 1999; 19: 124.
  • 30
    Dong H, Chen X. Immunoregulatory role of B7–H1 in chronicity of inflammatory responses. Cell Mol Immunol 2006; 3: 179187.
  • 31
    Zeremski M, Petrovic LM, Talal AH. The role of chemokines as inflammatory mediators in chronic hepatitis C virus infection. J Viral Hepat 2007; 14: 675687.
  • 32
    Sager R, Haskill S, Anisowicz A, Trask D, Pike MC. GRO: a novel chemotactic cytokine. Adv Exp Med Biol 1991; 305: 7377.
  • 33
    Mahalingam S, Karupiah G. Chemokines and chemokine receptors in infectious diseases. Immunol Cell Biol 1999; 77: 469475.
  • 34
    Rockey DC, Bissell DM. Noninvasive measures of liver fibrosis. HEPATOLOGY 2006; 43: S113S120.
  • 35
    Wasmuth HE, Zaldivar MM, Berres ML, Werth A, Scholten D, Hillebrandt S, et al. The fractalkine receptor CX3CR1 is involved in liver fibrosis due to chronic hepatitis C infection. J Hepatol 2008; 48: 208215.
  • 36
    Lempicki RA, Polis MA, Yang J, McLaughlin M, Koratich C, Huang DW, et al. Gene expression profiles in hepatitis C virus (HCV) and HIV coinfection: class prediction analyses before treatment predict the outcome of anti-HCV therapy among HIV-coinfected persons. J Infect Dis 2006; 193: 11721177.
  • 37
    Appay V, Sauce D. Immune activation and inflammation in HIV-1 infection: causes and consequences. J Pathol 2008; 214: 231241.
  • 38
    Sipsas NV, Sfikakis PP, Touloumi G, Pantazis N, Choremi H, Kordossis T. Elevated serum levels of soluble immune activation markers are associated with increased risk for death in HAART-naive HIV-1-infected patients. AIDS Patient Care STDs 2003; 17: 147153.
  • 39
    Malaspina A, Moir S, Ho J, Wang W, Howell ML, O'Shea MA, et al. Appearance of immature/transitional B cells in HIV-infected individuals with advanced disease: correlation with increased IL-7. Proc Natl Acad Sci U S A 2006; 103: 22622267.
  • 40
    Boasso A, Shearer GM. Chronic innate immune activation as a cause of HIV-1 immunopathogenesis. Clin Immunol 2008; 126: 235242.
  • 41
    Kestens L, Vanham G, Vereecken C, Vandenbruaene M, Vercauteren G, Colebunders RL, et al. Selective increase of activation antigens HLA-DR and CD38 on CD4+ CD45RO+ T lymphocytes during HIV-1 infection. Clin Exp Immunol 1994; 95: 436441.
  • 42
    Kestens L, Vanham G, Gigase P, Young G, Hannet I, Vanlangendonck F, et al. Expression of activation antigens, HLA-DR and CD38, on CD8 lymphocytes during HIV-1 infection. AIDS (Lond) 1992; 6: 793797.
  • 43
    Kanto T, Hayashi N. Immunopathogenesis of hepatitis C virus infection: multifaceted strategies subverting innate and adaptive immunity. Intern Med 2006; 45: 183191.
  • 44
    Dahari H, Major M, Zhang X, Mihalik K, Rice CM, Perelson AS, et al. Mathematical modeling of primary hepatitis C infection: noncytolytic clearance and early blockage of virion production. Gastroenterology 2005; 128: 10561066.
  • 45
    Chen M, Sallberg M, Sonnerborg A, Weiland O, Mattsson L, Jin L, et al. Limited humoral immunity in hepatitis C virus infection. Gastroenterology 1999; 116: 135143.
  • 46
    Chen L, Borozan I, Feld J, Sun J, Tannis LL, Coltescu C, et al. Hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis C viral infection. Gastroenterology 2005; 128: 14371444.