Liver damage in hepatitis C virus (HCV) infection results from cellular immune responses against HCV-infected hepatocytes rather than direct cytopathic effects of the virus itself [1,2]. It is therefore surprising that despite immunodeficiency HIV/HCV-co-infected patients display enhanced liver disease with accelerated fibrosis progression compared with immunocompetent HCV-mono-infected patients . Possible explanations may lie in the local expression of regulatory cytokines or the cellular composition of inflammatory infiltrates.
In both HCV and HIV mono-infection the chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein 1 alpha (MIP-1α), interferon-inducible protein 10 (IP-10) and IFNγ regulate local inflammation [4–16]. The cytokines macrophage chemoattractant protein 1 (MCP-1), secondary lymphochemokine (SLC) and stroma-derived factor 1 (SDF-1) were linked to hepatic fibrosis in hepatitis C [17–19]. In HIV/HCV co-infection cytokine production is altered by HIV and its immunodeficiency, and also by HAART [20,21].
Using quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR) to amplify gene-specific messenger RNA in liver biopsies from HCV-mono-infected patients, Leroy et al.  found increased expression of IFNγ, RANTES and TNFα, as well as major changes in intrahepatic T-lymphocyte subsets. As this approach closely reflects the in-vivo intrahepatic inflammatory situation, we applied this qRT–PCR to compare mRNA expression patterns of cellular markers and cytokine genes in liver biopsies of HIV/HCV-co-infected and HCV-mono-infected patients.
Materials and methods
Liver biopsies from 33 HCV-mono-infected and 40 HIV/HCV-co-infected patients (Table 1a) naive for anti-HCV treatment were obtained at a single timepoint before anti-HCV treatment. Liver diseases other than hepatitis C were excluded. Among the HIV/HCV-co-infected patients 26 were on HAART (Table 1b). Of note is the fact that immunity was well preserved in co-infected patients: only nine had CD4 T-cell counts below 400 cells/μl. This more severely immunocompromised subgroup did not differ significantly in alanine aminotransferase (ALT) levels, viral loads, histological inflammation and fibrosis scores from the co-infected patients with CD4 T-cell counts greater than 400 cells/μl. The study protocol conformed to the Declaration of Helsinki with a priori approval by the local Institutional Review Board. Written informed consent was obtained from each study participant.
Complimentary DNA synthesis and quantitative real-time reverse transcriptase–polymerase chain reaction
Biopsy specimens were washed in 0.9% sodium chloride to eliminate contamination by blood lymphocytes and then divided into two parts. One part was scored for inflammatory activity and fibrosis by a single histopathologist at each site (Ishak score ). The other part was snap-frozen in liquid nitrogen and stored at −80°C until liver tissue was homogenized and total RNA was extracted including DNAse digestion (RNeasy mini-kit; Qiagen, Hilden, Germany). cDNA was synthesized using the Omniscript RT-Kit (Qiagen) and Oligo-dT-Primer (Promega, Madison, Wisconsin, USA). Quantitative PCR was performed on cDNA transcripts using LightCycler-FastStart-DNAMaster ‘Plus’ SYBRGreen-I (Roche, Mannheim, Germany) according to the manufacturer's instructions on a LightCycler (Roche). Sequences and PCR conditions for CD3ε, TCRα, CD4, CD8α, CD8β, CD56 and CD69 were chosen as described  and are summarized for all other markers in supplementary Table 1. Target-gene mRNA concentrations were determined relative to a standard curve using the ΔCt method and were normalized with respect to β-Actin mRNA levels. As a result of the small sample size with limited RNA yields, MCP-1 mRNA levels could not be determined in seven HCV-mono-infected patients, and analysis of CD3ε, CD4, CD8α, CD8β, CD69, CD56 and TCRα was limited to 23 and 28 patients with HCV and HIV/HCV infection, respectively.
Quantitative results between different patient groups were compared by the Mann–Whitney-U-test. PCR reactions that failed to amplify a specific product were excluded from the analysis. Pearson's correlations between mRNA levels were calculated using SPSS 12.0 (SPSS Inc., Chicago, Illinois, USA) after logarithmic transformation and subsequent normalization to a mean of 0 to account for non-Gaussian distribution. For ordinal parameters Spearman's rank correlations were used. Bonferroni-type corrections for multiple comparisons were applied based on permutated datasets as described by Moore et al. . All comparisons were two-tailed. P values below 0.05 were considered to indicate statistical significance.
To characterize intrahepatic differences in the composition of inflammatory infiltrates and in cytokine expression patterns we compared mRNA levels of the cellular markers CD3ε, TCRα, CD8α, CD8β, CD4, CD68, CD56 and CD69, and of the cytokines IFNγ, MIP-1α, RANTES, IP-10, MCP-1, SDF-1 and SLC in liver biopsies of HCV-mono-infected and HIV/HCV-co-infected patients.
Correlations between cellular surface markers correspond to distinct clusters of inflammatory cells
We first validated and cross-checked the internal consistency of our data by calculating clusters of correlations for cellular markers as described by Leroy et al. . In both patient groups we confirmed highly significant (P < 0.01) correlations between mRNA levels of CD3ε and TCRα (HCV r = 0.881 and HIV/HCV r = 0.846), CD8α (r = 0.744 and r = 0.765), and CD8β (r = 0.852 and r = 0.748). We also found a significant relationship between CD3ε and CD4 (r = 0.603 and r = 0.651), whereas CD4 was not correlated with CD8α and CD8β. These findings confirmed that clusters of CD3+TCRα+CD8+ and CD3+TCRα+CD4+ T cells could be differentiated reliably. We did not, however, find any correlations between CD3ε and CD56.
Significant differences in intrahepatic inflammatory infiltrates and cytokine expression
In comparing median mRNA levels of leukocyte surface markers between the two patient groups, CD3ε, TCRα, CD8α and CD8β were increased more than twofold in HIV/HCV-co-infected patients, whereas CD4 mRNA levels were markedly reduced. In contrast, there was no significant difference in CD68, CD56 and CD69 (Fig. 1). To illustrate changes in the relative proportions of cellular subsets among infiltrating CD3-positive cells we calculated mRNA ratios of CD4 and CD8α/β relative to CD3ε levels. Both median CD8α/CD3ε (HCV 0.74 versus HIV/HCV 1.0; P = 0.023) and CD8β/CD3ε ratios (0.14 versus 0.18; P = 0.042) were approximately 1.3-fold elevated in HIV/HCV-co-infected patients, whereas CD4/CD3ε ratios were reduced to approximately 25% (4.8 versus 1.3; P < 0.001).
In examining intrahepatic mRNA levels of inflammatory cytokines, IFNγ, RANTES, MIP-1α and IP-10 were increased 1.8–3.1-fold in HIV/HCV-co-infected patients compared with HCV-mono-infected patients. In contrast, mRNA levels of the profibrogenic chemokines MCP-1, SDF-1 and SLC were not significantly different between the two patient groups (Fig. 1).
Previous studies reporting reduced or maintained intrahepatic cytokine mRNA levels  or HCV-specific T-cell functions in HIV/HCV co-infection [25–29] were mostly carried out on cohorts displaying equal ALT levels between groups. Moreover, HCV genotype 3 infection may be associated with enhanced liver disease in HIV/HCV co-infection . We therefore repeated the above analyses in a subgroup of ALT matched patients (21 HCV mono and 21 HIV/HCV-co-infected patients, mean ALT 2.65 × upper limit of normal each), and in non-GT3-infected patients. Here we found no evidence that our observations were significantly confounded by different ALT levels or HCV genotypes between patient groups (data not shown).
Attenuated inflammatory reactions in HIV-co-infected patients on HAART
When HIV/HCV-co-infected patients on HAART were analysed separately, significantly lower TCRα, CD8α, CD8β and CD68 mRNA levels were detected than in untreated co-infected patients. There was also a trend towards the reduced expression of the activation marker CD69, whereas CD4 and CD56 did not reveal major changes between treated and untreated patients (Fig. 1). Average intrahepatic mRNA levels of IFNγ, MIP-1α and RANTES were markedly lower in the subgroup of HIV/HCV-co-infected patients on HAART than in untreated patients, whereas there were no significant differences in MCP-1, SDF-1 and SLC (Fig. 1).
Correlations between cytokines, cellular subsets, transaminases and fibrosis scores
To disclose functional associations between cellular infiltrates, cytokines and clinical markers of liver damage and fibrosis, we analysed correlations between intrahepatic mRNA levels and aspartate aminotransferase (AST), ALT and histological inflammation and fibrosis scores. The inflammatory cytokines IFNγ, RANTES, MIP-1α and IP-10 were closely correlated with TCRα and CD8α in both patient groups (P < 0.01, r > 0.55 for all). IP-10 was correlated with AST (P = 0.018, r = 0.498) and ALT (P = 0.001, r = 0.594) serum levels in HCV-mono-infected but not in HIV/HCV-co-infected patients, and CD8α showed a weak correlation with AST (P = 0.061) in both patient groups. Beyond that there were no additional correlations between cytokine mRNA and aminotransferase levels. Finally, SLC mRNA was correlated with the histological fibrosis score in HCV-mono-infected patients (r = 0.526; P = 0.003), and considerably less so in HIV/HCV-co-infected patients (r = 0.314; P = 0.055).
In the present study we determined differences in gene expression levels for a large panel of leukocyte markers and cytokines in liver biopsies from HCV-mono-infected and HIV/HCV-co-infected patients. As a result of limited sample sizes, immunohistochemistry studies to evaluate directly the amount and localization of expressed proteins could not be performed. It is, however, well documented that mRNA levels enable an indirect assessment of the expression of cellular surface molecules [31–33] and cytokine secretion . Here, increased CD8 and reduced CD4 mRNA levels in HIV/HCV-co-infected patients probably reflect HIV-induced CD8 T-cell expansion and CD4 T-cell depletion, whereas CD3ε, TCRα, CD68 and CD56 mRNA levels also suggested a general intrahepatic enrichment of T cells, but not macrophages or natural killer cells in HIV/HCV-co-infected patients. Enhanced expression of MIP-1α, RANTES, IFNγ, and IP-10 was probably related to effector functions of CD8 T cells as suggested by correlations between these cytokines and CD8. Importantly, IP-10, which is induced in hepatocytes by IFNγ and Fas-ligand [10,11,13] during HCV infection, was also correlated with aminotransferase levels in HCV-mono-infected patients, indicating a role of CD8 T cells and their effector cytokines in the pathogenesis of HCV-related tissue damage. Here, MIP-1α and RANTES are also of interest as they may be released together with granzymes from cytolytic granules of CD8 T cells [5,12]. These observations are in line with findings by Leroy et al. , who identified CD8 T cells rather than natural killer cells as the predominant inflammatory cell type in HCV mono-infection.
A previous publication hypothesized a defect in intrahepatic cytokine activation , and functional studies found equivalent or reduced HCV-specific T-cell responses in HIV/HCV-co-infected compared with mono-infected patients [25–29]. As HCV-specific cytokine production is influenced by progressive immunodeficiency [26,29], the relatively well-preserved CD4 cell counts in our HIV-positive patients may explain contrasting results. Moreover, our method measures the collective intrahepatic cytokine expression. As chemotactic stimuli from the HCV-infected liver attract lymphocytes regardless of their antigen specificity, local accumulation of HIV-specific, activated lymphocytes probably also contributes to increased cytokine levels in our study. As a result of the lack of liver tissue from a control group of HIV-mono-infected patients we could not, however, quantitate this effect. It remains to be determined whether bystander cytotoxicity directly caused by inflammatory cytokines [35–38] from HIV-specific T cells could be involved in the pathogenesis of enhanced liver disease in HIV/HCV-co-infected patients.
Recent studies reported beneficial effects of HAART on HCV disease progression in HIV/HCV-co-infected patients [21,39–42]. In line with a recent publication , inflammatory cytokine expression and the composition of cellular infiltrates in our HAART-treated patients appeared to match more closely our findings in HCV-mono-infected individuals. Although longitudinal samples before and after treatment initiation are needed to address this point adequately, our cross-sectional data support the idea that enhanced intrahepatic inflammatory processes in co-infected patients might be reversible under HAART.
MCP-1 and SLC may be involved in hepatic fibrogenesis by the attraction and activation of hepatic stellate cells in hepatitis C [17,18]. Profibrogenic properties of SDF-1 were deduced from its expression in neo-blood vessels at inflammatory foci . Positive correlations between SLC and fibrosis scores in our study support a functional role for SLC in HCV-induced fibrogenesis. Considering the lack of differential regulation observed here it appears unlikely, however, that SLC, MCP-1 and SDF-1 directly influence the enhanced fibrosis progression in HIV/HCV co-infection.
In summary, our results indicate an accumulation of cytotoxic CD8 T cells with an increase in inflammatory mediators, possibly leading to enhanced tissue damage in HIV/HCV-co-infected patients, whereas we found no differential regulation of profibrogenic cytokines. Reduced inflammatory reactions in HIV-positive patients receiving HAART may explain the protective effect of HIV-specific treatment on liver disease in HIV/HCV co-infection.
The authors would like to thank Martin Wolff for sample donation, and Georg Lauer, Victoria Kasprowicz and Galit Alter for reviewing this manuscript.
Authors' contributions: T.K. collected sample material, designed and performed research, analysed and interpreted data and drafted the manuscript; C.T., B.C., B.K. provided samples and clinical data; B.L. performed the statistical analysis; G.F. and H.D.N. provided samples and primer sequences and assisted with the method; T.S. and J.K.R. provided samples, interpreted data and reviewed the manuscript; U.S. designed research, analysed and interpreted data and drafted the manuscript.
Sponsorship: This work was supported by the Heinz-Ansmann-Foundation.
Conflicts of interest: J.K.R. declared relations (consulting, speaker's bureau) with Roche, Schering, Gilead, GSK, BI and Abbott. U.S. received travel grants from Roche and Essex. C.T. declared relations (speakers bureau, advisory board) with Roche. All other authors have no conflicts of interest.
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