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HCV Replication is Associated with Mitochondrial Depletion in Coinfected Patients
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"Mitochondrial DNA depletion in HIV-infected patients with chronic hepatitis C and effect of pegylated interferon plus ribavirin therapy"
"....HCV replication is directly associated with mtDNA depletion in PBMC of HCV/HIV-co-infected patients... After the initiation of hepatitis C therapy, there was an overall increase in the mean mtDNA amount in PBMC, which reached statistical significance in patients with baseline serum HCV-RNA levels greater than 6 log IU/ml compared with those with lower HCV-RNA levels (+61 versus -30 mtDNA copies/cell; P = 0.033) and in patients without antiretroviral therapy compared with those under HAART (+133 versus -13 mtDNA copies/cell; P = 0.021). Increases in mtDNA content in PBMC were recognized as soon as week 12 of anti-HCV therapy in patients who suppressed HCV-RNA and were not taking antiretroviral drugs...The recognition of a significant inverse relationship between HCV replication and mtDNA content in PBMC from HIV/HCV-co-infected patients is a remarkable finding. It may help to explain why there is a greater incidence of mitochondrial-related toxicities, such as insulin resistance, dyslipidemia and lipoatrophy, in HIV-infected patients with chronic hepatitis C compared with patients without HCV infection..."
AIDS: Volume 21(5) 12 March 2007 p 583-588
de Mendoza, Carmena; Martin-Carbonero, Luza; Barreiro, Pabloa; de Baar, Michelb; Zahonero, Nataliaa; Rodriguez-Novoa, Soniaa; Benito, Jose Miguela; Gonzalez-Lahoz, Juana; Soriano, Vincenta
From the aDepartment of Infectious Diseases, Hospital Carlos III, Madrid, Spain
bPrimagen, Amsterdam, the Netherlands.
"....This study shows that the level of HCV replication is associated with the extent of mtDNA depletion in PBMC of HIV-infected patients with chronic hepatitis C. Treatment of HCV infection was associated with an improvement in the mtDNA content in this population. This benefit was mainly seen in patients not taking antiretroviral drugs concomitantly, however, suggesting that there is a deleterious effect on mtDNA content when HCV therapy is taken along with antiretroviral drugs..... The use of some nucleoside analogues to treat HIV infection might enhance mitochondral damage, which could be further aggravated by the concomitant prescription of ribavirin in a dose-dependent manner..... Our current results support the belief that the deleterious effect of HCV replication on mtDNA may be reversed with successful anti-HCV therapy. When pegIFN plus ribavirin are provided in HIV-infected individuals along with antiretroviral drugs, however, this benefit is no longer apparent. We postulate that a synergistic detrimental effect of anti-HCV and anti-HIV drugs is the mechanism for this observation.... our data are in favour of a general mechanism of mtDNA depletion in chronic viral diseases, in which high levels of viral particles are produced per day in a given individual. The immunological stress driven by this rapid viral turnover is probably the main cause of this observation [5]. Therefore, our findings may apply not only to HIV and HCV, but also to hepatitis B virus infection. Patients with active chronic hepatitis B may show mtDNA depletion, which could revert somewhat with effective antiviral therapy. Studies are currently ongoing to prove this hypothesis...."
Abstract
Background: The metabolic stress derived from high levels of virus replication in both HIV and hepatitis C virus (HCV) infections results in mitochondrial DNA depletion, which seems to be enhanced in co-infected patients. The use of nucleoside analogues to treat HIV infection may further increase mtDNA depletion by inhibiting gamma DNA polymerase. Information on the impact of therapy with pegylated interferon (pegIFN) plus ribavirin on mtDNA is scarce and conflicting results have been reported.
Patients and methods: Fifty-nine HCV/HIV-co-infected patients (43 on and 16 off antiretroviral therapy) who initiated treatment with pegIFN plus ribavirin were retrospectively analysed. The amount of mtDNA in peripheral blood mononuclear cells (PBMC) was measured at baseline and at the end of HCV therapy.
Results: Mean baseline serum HCV-RNA was 5.8 log IU/ml and 56% of patients were infected by HCV genotype 1. An inverse correlation between serum HCV-RNA levels and PBMC mtDNA content was recognized at baseline (r = -0.370; P = 0.006). HCV-RNA suppression at the end of HCV therapy was associated with a significant increase in mtDNA, particularly in patients with baseline HCV-RNA levels greater than 6 log IU/ml (+61 mtDNA copies/cell) and in subjects not taking antiretroviral therapy (+133 mtDNA copies/cell).
Conclusion: HCV replication correlates with the extent of mtDNA depletion in PBMC, and treatment of chronic hepatitis C is associated with a significant improvement in mtDNA content. This benefit, however, is not recognized when HCV medications are used along with antiretroviral therapy, probably because of a deleterious interaction of these drugs on mitochondria.
Introduction
One third of the HIV-infected population worldwide is chronically infected with hepatitis C virus (HCV) [1,2]. The metabolic stress derived from high levels of virus replication in both HIV and HCV infections results in mitochondrial damage and mitochondrial DNA depletion [3-6], which seems to be further enhanced in co-infected patients [7,8]. As a result, some metabolic complications (i.e. dyslipidemia, insulin resistance, etc.) are more frequent in co-infected individuals [9,10]. After the introduction of HAART, an improvement in the mtDNA content of peripheral blood mononuclear cells (PBMC) is generally seen accompanying HIV-RNA suppression [11,12]. The use of some nucleoside analogues (i.e. stavudine and didanosine) may, however, subsequently reverses this benefit by inhibition of mtDNA polymerase gamma [4,5,13].
The combination of pegylated interferon (pegIFN) plus ribavirin is the current recommended therapy for chronic hepatitis C in HCV-monoinfected as well as in HIV/HCV-co-infected individuals [1,2]. Information on the impact of HCV therapy on the mtDNA content in PBMC or liver tissue is scarce, and conflicting results have been reported in co-infected patients [7,14]. Interactions between ribavirin, which is a guanosine analogue, and some nucleoside reverse transcriptase inhibitors (i.e. didanosine) used as a backbone of HAART regimens are common and may result in high rates of toxicities and treatment discontinuation [15,16]. In this study, we examined longitudinally the mtDNA content in PBMC collected from a group of HCV/HIV-co-infected patients who underwent treatment for chronic hepatitis C.
Patients and methods
Study population
All HCV/HIV-co-infected patients who initiated treatment with pegIFN (Pegasys 180 μg/week) plus ribavirin (1000-1200 mg a day) at one single institution (Hospital Carlos III, Madrid, Spain) during the last semester of 2003 were analysed. To be eligible, patients had to show CD4 cell counts greater than 300 cells/μl, and be abstinent from alcohol and not actively engaged in drug addiction practices for the past year. Only patients with negative serum hepatitis B surface antigen, elevated transaminases and without decompensated liver cirrhosis were considered as candidates for HCV therapy. Following treatment guidelines for HCV/HIV-co-infected patients [1,2], HCV therapy was continued in patients showing early virological response, defined as more than a 2 log reduction in serum HCV-RNA at week 12 of treatment. For the purpose of this study, only patients who had completed the planned course of HCV therapy (6-12 months, depending on HCV genotype) were considered for subsequent analyses.
Quantitation of mitochondrial DNA content in peripheral blood mononuclear cells
Blood was collected in ethylenediamine tetraacetic acid tubes from all individuals. Cryopreservation of PBMC was carried out after maximal depletion of platelets with repeated wash cycles using phosphate buffered saline and RPMI medium solutions. The amount of mtDNA in PBMC was assessed using a duplex real-time nucleic acid sequence-based amplification assay (Retina Mitox; Primagen, Amsterdam, the Netherlands), which allows the amount of mtDNA per cell to be estimated quantitatively. This format provides a direct measurement of mtDNA and nuclear DNA contents, making possible an estimate of a ratio under the same amplification conditions, which avoids the influence of external factors that might interfere when amplifications are made separately. More details about this methodology have been described elsewhere [12,17]. Samples were tested for mtDNA at baseline and at the end of HCV therapy.
Laboratory tests
The measurement of plasma HIV-RNA was performed using a third generation branched-DNA assay (Versant v3.0; Bayer, Barcelona, Spain), following the manufacturer's instructions. The lower limit of detection of the assay is 50 HIV-RNA copies/ml. Serum HCV-RNA was determined using Cobas Taqman (Roche, Barcelona, Spain). The dynamic range of that assay is 10-10 million IU/ml. HCV genotypes were investigated using an hybridization assay (HCV LiPA-II; Bayer). The CD4 T-lymphocyte count was determined by flow cytometry (Coulter, Madrid, Spain), using specific fluorescein-labelled antibodies. Biochemical and haematological parameters were recorded at each visit every 3 months. Serum ribavirin plasma trough concentrations were measured at 12 weeks of HCV therapy using high performance liquid chromatography, as described elsewhere [18].
Statistical analysis
All data were recorded and analysed using SPSS version 11.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Baseline characteristics of the study population were reported as percentages, mean or as median values plus interquartile ranges. Differences between the study groups were compared using chi-square and Fisher's exact tests for categorical variables, and the t-test for quantitative parameters. The correlation between variables was assessed using bivariate analyses for non-parametric tests. Linear regression models were used for multivariate analyses. Differences were considered to be significant when P values were below 0.05.
Results
A total of 59 HCV/HIV-co-infected patients (43 on and 16 off antiretroviral therapy) who initiated treatment with pegIFN plus ribavirin were retrospectively analysed. The mean baseline serum HCV-RNA was 5.8 log IU/ml and 56% of patients were infected by HCV genotype 1. The main characteristics of the study population are recorded in Table 1. Antiretroviral therapy consisted in all instances of two nucleoside analogues plus either a non-nucleoside reverse transcriptase inhibitor (n = 18) or a protease inhibitor (n = 15). The nucleoside backbone included stavudine (n = 7), abacavir (n = 16), lamivudine (n = 32), tenofovir (n = 23) or zidovudine (n = 10). Following international guidelines [1,2], didanosine was not allowed to be taken with ribavirin, given the high risk of mitochondrial toxicity when combining these drugs. Overall, 84% of patients taking antiretroviral therapy had undetectable viraemia and the rest had less than 1000 HIV-RNA copies/ml.
An inverse correlation between serum HCV-RNA levels and mtDNA content in PBMC was recognized at baseline (Spearman's rho -0.370; P = 0.006; Fig. 1). Moreover, individuals infected with HCV genotype 3 and those without antiretroviral therapy showed lower mtDNA content than the rest, although the difference did not reach statistical significance (Table 2). In the multivariate analysis, only serum HCV-RNA levels greater than 6 log IU/ml were significantly associated with a lower mtDNA content in PBMC at baseline in this HCV/HIV-co-infected population.
Overall, 81.6% of patients had undetectable serum HCV-RNA levels at the end of hepatitis C therapy, and 63.4% reached a sustained virological response, defined as negative serum HCV-RNA 6 months after HCV treatment discontinuation. After the initiation of hepatitis C therapy, there was an overall increase in the mean mtDNA amount in PBMC, which reached statistical significance in patients with baseline serum HCV-RNA levels greater than 6 log IU/ml compared with those with lower HCV-RNA levels (+61 versus -30 mtDNA copies/cell; P = 0.033) and in patients without antiretroviral therapy compared with those under HAART (+133 versus -13 mtDNA copies/cell; P = 0.021). Increases in mtDNA content in PBMC were recognized as soon as week 12 of anti-HCV therapy in patients who suppressed HCV-RNA and were not taking antiretroviral drugs.
As expected, patients on stavudine showed significantly lower median mtDNA at baseline than the rest of the patients on HAART (187 versus 338 mtDNA copies/cell; P = 0.004), whereas patients taking zidovudine had a higher baseline mtDNA content than the rest (424 versus 286 mtDNA copies/cell; P = 0.041). Changes in mtDNA during the course of hepatitis C therapy did not, however, differ significantly when comparing patients using distinct nucleoside analogues as part of HAART (data not shown).
The median ribavirin plasma trough concentrations during HCV therapy were 2.11 μg/ml [interquartile range (IQR) 1.56-3.14]. Serum ribavirin plasma levels tended to be inversely associated with the mtDNA content in PBMC at the end of hepatitis C therapy in the subset of patients on HAART (Spearman's rho -0.326; P = 0.074). Moreover, in the multivariate analysis, whatever combination of antiretroviral drugs taken along with HCV therapy was associated with a significant depletion in the mtDNA content in PBMC (Table 3), supporting a potential additive effect on mitochondrial toxicity of ribavirin and antiretroviral drugs.
Discussion
This study shows that the level of HCV replication is associated with the extent of mtDNA depletion in PBMC of HIV-infected patients with chronic hepatitis C. Treatment of HCV infection was associated with an improvement in the mtDNA content in this population. This benefit was mainly seen in patients not taking antiretroviral drugs concomitantly, however, suggesting that there is a deleterious effect on mtDNA content when HCV therapy is taken along with antiretroviral drugs.
The recognition of a significant inverse relationship between HCV replication and mtDNA content in PBMC from HIV/HCV-co-infected patients is a remarkable finding. It may help to explain why there is a greater incidence of mitochondrial-related toxicities, such as insulin resistance, dyslipidemia and lipoatrophy, in HIV-infected patients with chronic hepatitis C compared with patients without HCV infection [9,10]. Interestingly, the mtDNA content (at least in PBMC) improved after the suppression of HCV replication with pegIFN plus ribavirin. This observation may allow us to hypothesize that the eradication of HCV infection with hepatitis C therapy might reduce the chances of developing or the severity of metabolic complications and morphological abnormalities frequently seen in HIV/HCV-co-infected patients. If confirmed by others, this observation could be a further argument to prioritize the treatment of hepatitis C in HIV-positive patients.
It should be noted, however, that this benefit of HCV therapy in the mid-long term must be balanced with its potential short-term complications. In the subset of co-infected patients taking antiretroviral drugs, a more profound mtDNA depletion was seen after the prescription of HCV medications, regardless of HCV suppression. Although the inhibition of mitochondrial function has been reported after the administration of interferon [19], our data favour a more important role for ribavirin, which could interact with some nucleoside analogues, enhancing mitochondrial damage. Accordingly, a trend towards increased mtDNA depletion was noticed in patients on antiretroviral drugs in whom plasma concentrations of ribavirin were greater. Altogether, it seems that a relative mtDNA depletion is associated with active HCV and HIV infections. The use of some nucleoside analogues to treat HIV infection might enhance mitochondral damage, which could be further aggravated by the concomitant prescription of ribavirin in a dose-dependent manner.
Discrepancies reported so far measuring mtDNA in HIV-infected patients with chronic hepatitis C and the effect of anti-HCV therapy with pegIFN plus ribavirin could be explained by the influence of antiretroviral drugs. In an earlier study we recognized an overall detrimental effect, which has not been confirmed in the current larger study; however, most patients in the former study were on antiretroviral therapy [7]. Our current results support the belief that the deleterious effect of HCV replication on mtDNA may be reversed with successful anti-HCV therapy. When pegIFN plus ribavirin are provided in HIV-infected individuals along with antiretroviral drugs, however, this benefit is no longer apparent. We postulate that a synergistic detrimental effect of anti-HCV and anti-HIV drugs is the mechanism for this observation.
Reports of acute pancreatitis, lactic acidosis, hepatic decompensation and severe weight loss have all been described in HCV/HIV-co-infected patients on HAART after the initiation of HCV therapy [15,16,20-23]. Although most of these toxicities have been attributed to an enhancement of mitochondrial toxicity caused by anti-HIV nucleoside analogues, it was unclear whether ribavirin itself might also cause mtDNA damage. Given that the mtDNA content in PBMC uniformly increased during the course of HCV therapy in all our patients not taking antiretroviral drugs, our results suggest that ribavirin does not cause mtDNA depletion by itself. Therefore, HCV therapy should ideally be administered to HCV/HIV-co-infected patients before beginning antiretroviral therapy.
Another interesting observation in this study was that mtDNA depletion at baseline tended to be more pronounced in patients with chronic hepatitis C caused by HCV genotype 3 than in those carrying other HCV genotypes. The relatively low number of patients with HCV genotype 3 probably prevented the results from reaching statistical significance. HCV genotype 3 is particularly prevalent among intravenous drug users in Europe, and responds quite well to HCV therapy [24]. Hepatic steatosis [25,26], liver fibrosis progression [27,28] and episodes of hepatotoxicity after the initiation of antiretroviral therapy [29,30] all seem to be more frequent in individuals infected with HCV genotype 3 than other HCV variants. Therefore, the treatment of chronic hepatitis C caused by HCV genotype 3 should be particularly prioritized in HIV-infected patients. Clearance of infection with this HCV variant in HIV-positive individuals will provide a benefit that is beyond chronic liver disease.
Finally, our data are in favour of a general mechanism of mtDNA depletion in chronic viral diseases, in which high levels of viral particles are produced per day in a given individual. The immunological stress driven by this rapid viral turnover is probably the main cause of this observation [5]. Therefore, our findings may apply not only to HIV and HCV, but also to hepatitis B virus infection. Patients with active chronic hepatitis B may show mtDNA depletion, which could revert somewhat with effective antiviral therapy. Studies are currently ongoing to prove this hypothesis.
In conclusion, HCV replication is directly associated with mtDNA depletion in PBMC of HCV/HIV-co-infected patients, which may explain the higher incidence of mitochondrial-related toxicities seen in this population compared with either HIV or HCV-monoinfected individuals. More importantly, treatment of chronic hepatitis C seems to revert this deleterious effect on the amount of mtDNA. The concomitant administration of antiretroviral drugs during the course of HCV therapy seems to enhance mitochondrial damage further, probably because of a synergistic deleterious interaction between HCV and HIV medications. Long-term follow-up of HIV-infected patients who have cleared HCV with therapy is warranted, and will allow us to confirm whether the incidence of mitochondrial-related toxicities is reduced in this subset of patients.
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