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Increased Rates of Liver Cell Death in HCV/HIV Coinfection
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Increased Hepatocyte Fas Expression and Apoptosis in HIV and Hepatitis C Virus Coinfection
....HCV/HIV-coinfected patients show higher levels of hepatocytes expressing Fas and undergoing irreversible apoptosis (cell death) than do HCV-infected patients ......Antiapoptotic therapy needs further investigation for the growing number of patients failing regimens of interferon plus ribavirin, particularly in the setting of rapidly progressive liver disease related to HCV/HIV coinfection..... A loss of regulatory CD4+ cell responses against HCV in patients with lower CD4+ cell nadirs could be involved in hepatocyte apoptosis.... inflammatory cytokines can induce Fas expression and are involved in hepatocyte apoptosis.... aberrant immune activation associated with HIV infection. Immune deactivation is less intense in patients with HCV/HIV coinfection than in patients without HCV...
The Journal of Infectious Diseases Nov 1 2005;192:1566-1576
Juan Macias,1 Miguel A. Japon,3 Carmen Saez,3 Rosa B. Palacios,1 José A. Mira,1 José A. Garcia-Garcia,1 Nicolas Merchante,1 Salvador Vergara,1 Fernando Lozano,2 Jesús Gomez-Mateos,2 and Juan A. Pineda1,2
1Servicio de Medicina Interna, 2Unidad de Enfermedades Infecciosas, Hospital Universitario de Valme, and 3Departamento de Anatomia Patologica, Hospital Universitario Virgen del Rocio, Seville, Spain
ABSTRACT
Background. Chronic hepatitis C disease (CHC) follows an accelerated course in human immunodeficiency virus (HIV) coinfection. The reasons for this are unclear. Fas-mediated hepatocyte apoptosis is involved in the pathogenesis of hepatitis C virus (HCV) infection. We sought to compare the expression of Fas on hepatocytes and irreversible apoptosis of hepatocytes among patients with CHC with and without HCV/HIV coinfection.
Methods. Fas-immunostained hepatocytes were semiquantified, and apoptotic hepatocytes were detected by staining caspase-cleaved cytokeratin 18 filaments and counted across the entire section of liver-biopsy specimens from HCV-infected patients with and without HCV/HIV coinfection.
Results.
One hundred thirty-four HCV/HIV-coinfected and 100 HCV-infected patients were included.
HCV/HIV coinfection was associated with both diffuse distribution of Fas-stained hepatocytes (adjusted odds ratio [AOR], 7.4 [95% confidence interval {CI}, 3.8-14.4]) and with apoptotic hepatocyte counts greater than the median (AOR, 2.5 [95% CI, 1.5-4.5]). In HCV/HIV-coinfected patients, CD4+ cell nadir <200 cells/mL was associated with both Fas expression (AOR, 2.9 [95% CI, 1.3-6.8]) and hepatocyte apoptosis (AOR, 2.3 [95% CI, 1.1-4.9]).
Conclusion. HCV/HIV-coinfected patients show higher levels of hepatocytes expressing Fas and undergoing irreversible apoptosis than do HCV-infected patients. However, low CD4+ cell nadirs in coinfected patients are associated with hepatocyte Fas expression and apoptosis.
DISCUSSION
In the present study, we report, for the first time (to our knowledge), that HCV/HIV-coinfected patients show higher levels of hepatocytes expressing Fas and undergoing irreversible apoptosis than do HCV-infected patients. Also, low CD4+ cell nadirs are associated with hepatocyte Fas expression and apoptosis in HCV/HIV-coinfected patients.
Fas system-mediated apoptosis is involved in the pathogenesis of CHC [6, 7]. However, the role of the Fas system in the pathogenesis of CHC in HIV coinfection is not known. We assessed this issue in a large number of liver-biopsy specimens from patients with CHC both with and without HIV coinfection. This large sample size enabled us to control for confounding factors in a multivariate analysis. We found that HIV coinfection was the factor with the strongest independent association with Fas expression on hepatocytes. In addition, the irreversible apoptosis of hepatocytes, as assessed by staining of caspase-cleaved cytokeratin 18 filaments, was independently related to the expression of Fas on hepatocytes. Finally, HIV coinfection was also associated with hepatocyte apoptosis. For these reasons, Fas-mediated hepatocyte apoptosis seems to be enhanced in HCV/HIV coinfection. This increased hepatocyte apoptosis could drive the accelerated course of CHC in HIV-coinfected patients.
The present study had a few limitations. First, we examined hepatocyte apoptosis during CHC in a cross-sectional survey. However, the rate of hepatocyte apoptosis is not likely to be fixed. Thus, host, viral, and environmental factors that may influence apoptosis change over time. The design of the study did not allow for an analysis of the evolution of hepatocyte apoptosis in HCV infection; therefore, no definitive conclusion can be drawn about the causal effect of hepatocyte apoptosis on the progression of liver fibrosis. To assess this, a study of sequential liver-biopsy specimens is needed. However, such a study design would pose ethical problems and would probably result in poor patient compliance. Moreover, should a longitudinal study of serial liver-biopsy samples be undertaken, acute variations in hepatocyte apoptosis would be missed between biopsies. Therefore, the effect of antiretroviral agents on hepatocyte apoptosis has been examined only for drugs that were administered on the date of the liver biopsy. The influence of antiretroviral drugs received previously on hepatocyte apoptosis cannot be evaluated with the current study design. Second, patients included in the study group were not probably representative of the general population with CHC. Patients with HCV infection were proposed to undergo liver biopsy because they were compliant with clinical visits and were more likely to adhere to anti-HCV therapy. Moreover, patients with decompensated cirrhosis do not routinely undergo biopsies. In this way, patients with the most advanced end-stage liver disease had no chance of inclusion in the study.
Alcohol was unexpectedly not related to hepatocyte apoptosis. Ethanol exerts a dose-dependent induction of hepatocyte apoptosis in acute animal models [16]. However, the levels of hepatocyte apoptosis observed in liver disease caused by chronic alcohol abuse are far lower than those observed in chronic viral hepatitis in human studies [17]. Because of this, the contribution of alcohol consumption to hepatocyte apoptosis could have been outweighed by virus-related apoptosis in the study. In addition, we cannot exclude the misclassification of alcohol intake. In our survey, alcohol consumption was estimated from the clinical interview. This method of calculating daily ethanol intake is unreliable in alcohol drinkers [18].
Hepatocyte apoptosis can be identified by morphologic characteristics on hematoxylin-eosin-stained samples. However, this approach is subjective and can detect only very late apoptosis. In addition, different hepatocytes could be misclassified as being apoptotic. Because of this, we needed a more objective and specific method to detect apoptotic hepatocytes. DNA fragmentation assays have been used for this purpose [9]. However, DNA fragmentation takes place during late stages of apoptosis. By contrast, caspase-cleaved cytokeratin 18 immunostaining allows the detection of early, but not reversible, and late apoptosis of parenchymal cells [19]. Very late apoptotic bodies and blood cells that commonly infiltrate and die in the diseased liver are not reactive to M30 MAbs [19]. Therefore, higher numbers of apoptotic hepatocytes are detected by M30 immunostaining than by a DNA fragmentation assay in CHC [20]. In the present study, we assessed hepatocyte apoptosis by M30 immunostaining, which strengthened our results.
A possible link between hepatocyte apoptosis and liver fibrosis has been hypothesized [7]. Thus, macrophages that have ingested apoptotic cells in vitro secrete transforming growth factor-B [21, 22], a profibrogenic cytokine. In the present study, we found that hepatocyte apoptosis was associated with the presence of liver fibrosis in HCV/HIV-coinfected patients. This relationship was not observed in patients without HIV coinfection. The latter finding agrees with previous surveys of hepatocyte apoptosis in CHC performed in non-HIV-infected patients [20, 23, 24]. Apoptosis is a rapid and fluctuating phenomenon, and it is sometimes difficult to detect [6]. To the contrary, liver fibrosis accumulates more or less constantly in response to continued damage. Thus, a liver-biopsy specimen could give a more approximate picture of liver fibrosis than of the history of hepatocyte apoptosis. We speculate that sustained and increased levels of hepatocyte apoptosis result in faster fibrosis progression in HIV-coinfected patients with CHC.
A recently reported in vitro study suggested that "innocent bystander" cell death could be implicated in hepatocyte apoptosis in HCV/HIV coinfection [12]. Cultures of primary hepatocytes or HepG2 cells exposed to surface proteins of both viruses showed higher levels of apoptosis [12]. In our study, we observed a trend toward lower apoptosis in those patients with more prolonged HIV suppression. The number of subjects with undetectable HIV viral loads was relatively low, and this could have precluded the detection of small differences.
Low CD4+ cell nadirs were associated with hepatocyte apoptosis in the present study. Lower CD4+ cell nadirs have been associated with a persistent loss of CD4+ cell responses against HCV [25]. A loss of regulatory CD4+ cell responses against HCV in patients with lower CD4+ cell nadirs could be involved in hepatocyte apoptosis. However, inflammatory cytokines can induce Fas expression [7] and are involved in hepatocyte apoptosis [26]. In CHC, many hepatocytes express Fas, but very few undergo final apoptosis [27]. This down-regulation of the Fas pathway could be overcome by aberrant immune activation associated with HIV infection. Immune deactivation is less intense in patients with HCV/HIV coinfection than in patients without HCV infection [28].
We and others have reported a slower progression of liver fibrosis in HCV/HIV-coinfected patients who receive PIs as part of their HAART [29, 30]. However, we did not observe any association between hepatocyte apoptosis and HAART in the present study. The accumulation and degradation of liver scar matrix are complex processes. Therefore, from hepatic stellate cell activation and fibrogenesis to reversion of liver fibrosis, there are multiple potential sites of antifibrotic activity. The results reported here do not suggest any specific effect of PIs on hepatocyte apoptosis, nor do they exclude other possible effects on liver fibrosis.
Therapy for HCV infection is far from satisfactory. A sustained virologic response is achieved in <50% of the patients infected with HCV genotype 1 [31, 32]. HCV/HIV coinfection is an even worse scenario, with lower response rates [33]. The present results support enhanced Fas-mediated hepatocyte apoptosis in HCV/HIV coinfection. There have been preliminary results regarding the positive effect of anti-caspase treatment of HCV infection [34]. Antiapoptotic therapy needs further investigation for the growing number of patients failing regimens of interferon plus ribavirin, particularly in the setting of rapidly progressive liver disease related to HCV/HIV coinfection.
BACKGROUND
Chronic hepatitis C disease (CHC) is a clinical problem of increasing importance in HIV-coinfected patients. Coinfection with HIV and hepatitis C virus (HCV) is highly prevalent among intravenous drug users (IDUs) and hemophiliacs [1]. Additionally, CHC follows an accelerated course in HCV/HIV coinfection [2]. Before the introduction of highly active antiretroviral therapy (HAART) in clinical practice, HCV/HIV-coinfected patients did not have this rapid progression to liver failure, because they died overwhelmingly of AIDS [1, 3, 4]. HAART effectively prevents death due to AIDS. Because of this, liver failure is becoming a leading cause of morbidity and mortality in HCV/HIV-coinfected patients, because HAART is used extensively [5].
The reasons for the accelerated evolution of CHC in HCV/HIV coinfection are not known. One possible explanation is enhanced hepatocyte apoptosis. In this regard, Fas system-mediated apoptosis is involved in the pathogenesis of CHC [6, 7]. Thus, an up-regulation of Fas expression on hepatocytes in patients singly infected with HCV has been reported by immunohistochemical studies [8, 9]. Increased Fas mRNA transcripts have been observed in HCV-infected livers [10]. Enhanced expression of Fas has been associated with increased DNA fragmentation in the liver [9, 11]. Recently, it has been shown that apoptosis of primary hepatocytes and HepG2 cells was enhanced by exposition to surface proteins of both HCV and HIV [12]. However, it is not known whether this mechanism has a role in vivo. Indeed, there are no available data on Fas expression and apoptosis of hepatocytes in HCV/HIV-coinfected patients. Because of this, we compared the expression of Fas on hepatocytes and irreversible apoptosis of hepatocytes in patients with CHC and who were either HIV infected or noninfected.
The following variables were considered in the statistical analysis: age at infection, age at liver biopsy, duration of HCV infection, stage of liver fibrosis, FPR, HIV infection status, sex, alcohol intake, alanine aminotransferase (ALT) level at liver biopsy, HCV genotype, and serum RNA level. For HCV/HIV-coinfected patients, the following variables were also considered: CD4+ cell count at liver biopsy; CD4+ cell nadir; presence of clinical AIDS; persistent, undetectable HIV viral load; use of individual antiretroviral drugs at liver biopsy; and the duration of exposure to each of the drugs considered.
RESULTS
Characteristics of the study population.
One hundred thirty-four HCV/HIV-coinfected patients (84%) and 100 HCV-infected patients (41%) were included. The characteristics of the patients according to HIV coinfection status are shown in table 1. There was a higher proportion of men, IDUs, and patients with an alcohol intake >50 g/day in the group with HCV/HIV coinfection. Age at liver biopsy, duration of HCV infection, and percentage of infection with HCV genotype 1 were higher in the group without HIV coinfection. The median FPR of the study population was 0.109 fibrosis units/year (range, 0.056-0.177 fibrosis units/year). The median FPR of the HCV/HIV-coinfected group was 0.138 fibrosis units/year (range, 0.059-0.25 fibrosis units/year), and that of the HCV-infected group was 0.091 fibrosis units/year (range, 0.043-0.124 fibrosis units/year) (P < .001).
Detection of Fas-expressing and apoptotic hepatocytes.
Hepatocytes expressing Fas could be clearly identified. In most cases, Fas-stained hepatocytes did not show morphologic signs of apoptosis. However, hepatocytes with more Fas immunostaining occasionally showed early nuclear changes suggestive of initial apoptosis. Liver-biopsy specimens were classified according to the extent and distribution of hepatocytes showing Fas expression. Nine liver biopsies (4%) needed reevaluation because of discrepant scores between observers. The staining of caspase-cleaved cytokeratin 18 by the M30 MAb allowed the detection of early and late irreversible apoptosis of hepatocytes. M30-stained hepatocytes could be found dispersed across the liver-biopsy specimens.
Factors associated with hepatocyte Fas expression.
In the univariate analysis, Fas expression of hepatocytes was associated with hepatocyte apoptosis. The higher the Fas score, the higher the AI (apoptotic index). The expression of Fas on hepatocytes was related to HIV coinfection status. Therefore, a higher proportion of HCV/HIV-coinfected patients showed diffuse distribution of Fas. Conversely, no Fas expression or focal expression of Fas was found more frequently in HCV-infected patients. The proportion of Fas-immunoreactive hepatocytes was associated with liver fibrosis stage. A higher hepatocyte expression of Fas in HCV/HIV-coinfected patients was observed during stages F0-F3 of liver fibrosis. Age at liver biopsy, duration of HCV infection, ALT level at liver biopsy, and FPR were also associated with Fas expression in the univariate analysis.
HCV/HIV coinfection, liver fibrosis stage (F0 vs. F1-F4), duration of HCV infection (below vs. above the median, 16 years), age at liver biopsy (below vs. above the median, 38 years), ALT level (<100 vs. >100 UI/mL), and alcohol intake (⩽50 vs. >50 g/day) were entered into a logistic regression model. The following factors were independently associated with diffuse Fas expression on hepatocytes: HCV/HIV coinfection (AOR, 7.4 [95% CI, 3.8-14.4]; P < .001), the presence of liver fibrosis (AOR, 4.3 [95% CI, 1.9-9.7]; P = .001), and older age at liver biopsy (AOR, 2.1 [95% CI, 1.1-4.0]; P = .02).
Factors associated with hepatocyte apoptosis.
In the univariate analysis, HCV/HIV-coinfected patients had higher AIs than did patients without HIV coinfection. The median AI was associated with the stage of liver fibrosis. The median AI of HCV/HIV-coinfected patients was significantly higher at liver fibrosis stages F2 and F3 than that of patients without HIV coinfection. For fibrosis stage F4, a borderline association was found. AI was associated with liver fibrosis in HCV/HIV-coinfected patients, but it was not associated in patients without HIV coinfection. Additionally, risk group, ALT level at liver biopsy, and FPR were also associated with the AI.
HCV/HIV coinfection, liver fibrosis stage (F0 vs. F1-F4), risk group (IDU vs. non-IDU), ALT level (<100 vs. >100 UI/mL), and alcohol intake (<50 vs. >50 g/day) were entered into the multivariate analysis. The variables independently associated with an AI above the median were HIV coinfection (AOR, 2.5 [95% CI, 1.5-4.5]; P = .001) and ALT level at liver biopsy (AOR, 2.5 [95% CI, 1.5-4.5]; P = .001).
Fas expression and hepatocyte apoptosis in HCV/HIV-coinfected patients.
Fas-stained hepatocytes distributed diffusely were more frequently found in older patients, those with a longer duration of HCV infection, those with lower CD4+ cell nadirs, and those with liver fibrosis. Duration of HCV infection, liver fibrosis, and CD4+ cell nadirs were independently associated with diffuse distribution of Fas on hepatocytes.
An AI above the median was more frequent in patients with a longer duration of HCV infection, higher ALT levels, liver fibrosis, and lower CD4+ cell nadirs. Liver fibrosis and CD4+ cell nadirs were independently associated with hepatocyte apoptosis.
In HCV/HIV-coinfected patients, 53 (40%) had never received HAART. Forty-one (77%) of them were antiretroviral naive. Eighty-one patients (60%) had been prescribed HAART before liver biopsy. Fifty-two patients (64%) were receiving protease inhibitors (PIs) on the date of liver biopsy. Twenty-nine patients (36%) receiving HAART were receiving nonnucleoside reverse-transcriptase inhibitors on the date of liver biopsy-15 patients were receiving efavirenz, and 14 were receiving nevirapine.
The use of antiretroviral drugs on the date of liver biopsy, the duration of exposure to them, and the presence of undetectable HIV viral loads were not associated with Fas expression on hepatocytes or the AI. In 54 patients with persistent, undetectable HIV viral loads, the median duration of suppressed HIV viral load was 26 months (range, 16-41 months). Eighteen patients with absent or focal Fas immunostaining and 36 patients with diffuse Fas immunostaining had a median duration of suppressed HIV viral load of 31 months (range, 24-41 months) and 23 months (range, 15-42 months), respectively (P = .3). The median duration of undetectable HIV viral loads was 30 months (range, 21-44 months) for 29 patients with an AI below the median, and it was 23 months (range, 13-39 months) for 25 patients with an AI above the median (P = .1).
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