Conceived and designed the experiments: MWW CS EH SZ AP BK. Performed the experiments: DR SS YM. Analyzed the data: MWW DR CS EH SZ AP BK. Contributed reagents/materials/analysis tools: CS EH SZ AP BK. Wrote the paper: MWW CS SZ AP BK.
The authors have declared that no competing interests exist.
Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity.
Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry.
Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027).
CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis.
Chronic hepatitis C virus (HCV) infection is a major cause of liver cirrhosis, and hepatocellular carcinoma worldwide
HCV is a plus-strand RNA virus without known ability to integrate its genome into the host genome. Chronic hepatitis C is characterized by high turnover of infected cells and persistence of HCV requires continuous
Four essential HCV hepatocyte entry receptors, CD81, the scavenger receptor B1, claudin-1, and occludin have been identified, yet
Yet well known as a cell surface protein, CD81 is also a typical component of exosomes. Exosomes are membrane vesicles secreted by different eukaryotic cells, e.g. hepatocytes and lymphocytes
Aim of the present study was to perform a comprehensive analysis if soluble CD81 is increased in patients with chronic hepatitis C compared to healthy controls, patients with chronic HCV infection but persistently normal alanine transaminase (ALT) levels, and patients with cured hepatitis C. Therefore, a method to quantify soluble CD81 in the exosomal serum fraction was established, and the association of soluble CD81 in the exosomal serum fraction with clinical and virological parameters within the different cohorts was investigated.
The present study is a case control study with 23 healthy controls and 37 patients with chronic hepatitis C and elevated alanine aminotransferase (ALT) level measured at least at 2 time points 14 days or more apart within 6 months before study entry. Furthermore, 24 patients with chronic HCV infection and persistently normal ALT serum levels as documented by three occasional measurements within the last 6 months before study entry and a minimum interval of 4 weeks between measurements, and 7 patients with long-term sustained virologic response 5 years after antiviral therapy with pegylated interferon-alfa 2a and ribavirin were included. All cohorts were independently recruited. Patients with decompensated liver cirrhosis or co-infection with hepatitis B virus or human immunodeficiency virus were excluded. Diagnosis of chronic HCV infection (>6 months) was confirmed by positive anti-HCV antibodies by third-generation enzyme immunoassay and detectable HCV RNA in serum. Grading and staging of chronic hepatitis C was done by a local, experienced pathologist according to the histological activity index described by Knodell et al.
Serum and plasma for ALT and AST levels, CD81 assessment, and HCV-RNA quantification were collected at the same time point and stored at −80°C until use. Enrichment of exosomes was performed according to Masciopinto et al.
Serum derived proteins from different centrifugation steps were separated by dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). As thiol compounds may have a negative impact on anti-CD81 antibody binding
Huh7 cells are characterized by high CD81 expression
Quantitative assessment of HCV RNA in serum was performed by a quantitative reverse transcription polymerase chain reaction assay (Cobas Amplicor HCV MonitorTM 2.0, Roche Diagnostic Systems, Branchburg, NJ; lower detection limit, 600 IU/mL). Samples with HCV RNA concentration above the upper detection limit above 800.000 IU/mL were diluted appropriately. HCV genotyping was performed by reverse hybridization assay (Inno LiPA HCV II, Innogenetics, Gent, Belgium). All contamination prevention measures suggested by Kwok and Higuchi were strictly applied
For comparison with serum HCV RNA and correlation to CD81 concentration within enriched serum fraction p5, HCV RNA was assessed in enriched serum, also. Therefore, differential centrifugation was performed in accordance to the CD81 enrichment protocol. Each pellet from a given centrifugation step was resuspended in 0.25 mL PBS, and HCV RNA was extracted fully automatic (COBAS-Ampliprep, Roche, Mannheim, Germany). Quantification of HCV RNA was performed standardized by real-time polymerase chain reaction (COBAS-Taqman 48 Analyzer Roche, Mannheim, Germany).
Unless indicated otherwise, all tests were two tailed and
Detection of exosomal CD81 in plasma from patients with chronic hepatitis C has been previously described by Masciopinto et al.
(A, B) Immunoblotting of soluble CD81 is shown after enrichment by differential centrifugation of frozen (A) plasma and (B) serum samples. Huh7 cells served as positive control, and HepG2 cells as negative control. While only a marginal fraction was detectable in fraction p4, the main portion was detectable in fraction p5, comparable between plasma and serum samples. (C, D). Soluble CD81 concentration was assessed semi-quantitatively by use of dilutions from a Huh7 cell lysate by densitometric analysis. The lysate dilution immunoblot analysis and the corresponding obtained standard curve are shown in (C) and (D), respectively.
We next investigated whether CD81 concentration in the p5 fraction can be quantified. For this purpose, different dilutions of Huh7cell lysates were detected by Western blot analysis and quantified by densitometry. As shown in
(A) Immunoblot analysis from representative patient samples (P1–P5) and healthy controls (H1–H5) as well as Huh7 dilution series (D1–D3). Enrichment of soluble CD81 had been standardized before sample processing, and a standardized volume of 1 mL serum was used for CD81 enrichment and quantification in all study samples analyzed. (B) Enriched CD81 serum concentrations given in healthy volunteers (n = 23), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with chronic hepatitis C and elevated ALT levels (n = 37), and patients with long-term SVR (n = 7). The CD81 concentration is given in relative units (RU)/mL as box-and-whisker plot showing median, 25 and 75 quartiles as well as the total range. Differences between two groups were calculated by the Mann-Whitney U test. (C) Correlation of CD81 concentration in fraction p5 given in RU/mL and serum ALT level given in U/mL in all patients with chronic HCV infection, including patients with elevated (▵) and persistently normal (○) ALT levels (n = 61). Correlations between two variables were assessed by the Spearman's rank correlation test. (D) Correlation of CD81 concentration in fraction p5 given in RU/mL and serum AST level given in U/mL in all patients with chronic HCV infection, including patients with elevated (▵) and persistently normal (○) ALT levels (n = 61). Correlations between two variables were assessed by the Spearman's rank correlation test.
Aim of this study was to investigate whether soluble CD81 levels are increased in patients with chronic hepatitis C. Therefore, the concentration of soluble CD81 in the exosomal fraction p5 was compared between a cohort of 23 healthy controls and 37 patients with chronic hepatitis C. As shown in
Patients with chronic HCV infection | Patients with SVR | Healthy volunteers | |||
normal | elevated | ||||
ALT | ALT | ||||
(n = 24) | (n = 37) | (n = 7) | (n = 23) | ||
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Age (mean ± SD) | 50.8±11.8 | 53.3±10.2 | 63.6±11.8 | 29.4±5.7 | |
Gender (male/female) | 10/14 | 15/22 | 4/3 | 12/11 | |
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ALT (U/L; mean ± SD) | 41.4±12.8 | 145.3±108.3 | 28.7±10.8 | 26.5±4.9 | |
AST (U/L; mean ± SD) | 39.9±16.9 | 106.7±61.8 | 38.4±4.1 | 29.1±5.5 | |
GGT (U/L; mean ± SD) | 49.3±48.1 | 99.8±71.3 | 24.5±10.6 | 30.0±6.8 | |
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I | (median [min., max.]) | 1 (1, 1) | 1 (1, 3) | n.d. | n.a. |
II | (median [min., max.]) | 1 (1, 1) | 1 (1, 1) | n.d. | n.a. |
III | (median [min., max.]) | 3 (1, 3) | 3 (1,3) | n.d. | n.a. |
IV | (median [min., max.]) | 1 (1, 3) | 3 (1,3) | n.d. | n.a. |
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Genotype distribution (1/2/3) | 17/4/3 | 32/1/4 | n.d. | n.a. | |
HCV RNA (log IU/mL, mean ± SD) | 5.8±0.6 | 6.1±0.6 | n.d. | n.a. |
ALT, alanine transaminase; AST, aspartate transaminase; GGT, gamma-glutamyl transferase (the upper limit of normal was 50 U/L, 50 U/L, 39 U/L for ALT, AST and GGT); max., maximum; min; minimum; n.a., not applicable; n.d., not done NR, nonresponse SD, standard deviation; SVR, sustained virologic response.
Exosomal CD81 may also be associated with inflammatory activity. To investigate a potential association between the CD81 concentration in the p5 fraction and liver inflammation, CD81-p5 was analyzed in a cohort of patients with chronic hepatitis C and persistently normal ALT levels. In the present study, patients with chronic hepatitis C and persistently normal ALT levels showed lower CD81-p5 levels than patients with chronic hepatitis C and elevated ALT levels and comparable levels of CD81-p5 with healthy controls and patients with cured HCV.
Overall, the CD81-p5 level was associated with serum ALT activity in patients with chronic hepatitis C with or without ALT elevation (r = 0.372, p = 0.003,
We also analyzed the association between CD81-p5 and histological liver damage. CD81-p5 was significantly associated with severe vs. non severe fibrosis (area under curve 0.699, p = 0.027,
Higher CD81-p5 levels were associated with sever fibroses (area under curve, AUC, 0.699; p = 0.027).
As HCV binds to CD81, the CD81 concentration in fraction p5 may be associated with the HCV RNA level. The HCV RNA concentration was quantified in unfractionated serum and in the enriched serum fraction p5 in all patients with chronic HCV infection (
Correlations between two variables were assessed by the Spearman's rank correlation test. (A) Serum HCV RNA and CD81 concentration in fraction p5 were not correlated. (B) Quantification of HCV RNA in the different serum fractions showing the highest HCV RNA concentration in fraction p5. (C) Correlation between HCV RNA concentration in fraction p5 and serum showing high correlation between HCV RNA concentration in unfractionated and enriched serum. (D) Concentrations of HCV RNA in fraction p5 and CD81 fraction p5 showed no correlation. P values and correlation coefficient r are given for each correlation.
The tetraspanin CD81 is an essential HCV hepatocyte cell entry receptor
CD81 containing exosomes have not been studied in larger cohorts of patients with chronic hepatitis C, so far. Exosomes are small vesicles secreted by vital cells only, amongst others considered to modify immune response in viral and malignant diseases
Enrichment of exosomes from plasma or serum, as performed within the current study, can be performed by differential centrifugation
In the present study, soluble CD81 levels were increased in the exosomal serum fraction p5 in patients with chronic hepatitis C compared to healthy volunteers. Moreover, in patients cured from hepatitis C, CD81 levels normalized to levels found in healthy subjects. Overall these results suggest that infection with the hepatitis C virus is associated with increase of soluble CD81.
Soluble CD81 could be associated with HCV replication. Therefore, we assessed whether soluble CD81 correlates with the HCV RNA level. In the present study, however, HCV RNA serum levels were not correlated with the CD81 concentration in the exosomal serum fraction p5. HCV RNA may also bind to exosomal CD81 which is suggested by data from Masciopinto et al., who have shown an association of HCV envelope proteins and HCV RNA with exosomal CD81
Recently, proinflammatory exosomes have been described to contribute to inflammation e.g. in sarcoidosis
Patients with HCV infection and persistently normal ALT levels may show a more benign course than patients with elevated ALT levels. Nevertheless, several studies described significant histological liver lesions and increased ALT levels over time in these patients
To further investigate the observed correlation between soluble CD81 levels and necroinflammatory activity, we compared CD81 levels to ALT and AST levels. Here, a significant correlation between serum ALT as well as AST levels and soluble CD81 was observed. This may indicate an association between hepatocyte cell death and higher CD81 serum levels. The association between soluble CD81 and surrogate markers of cell death could be due to release of CD81 containing exosomes or cell detritus from hepatocytes during the necroinflammatory process. However, the correlation coefficients between soluble CD81 levels and biochemical markers of liver damage, even though statistically significant, were weak. A final conclusion that soluble CD81 levels are mostly derived from inflammatory processes in patients with hepatitis C cannot be drawn from the results of the current study.
Another explanation for the increase of soluble CD81 levels in patients with chronic hepatitis C is secretion of CD81 containing exosomes by lymphocytes. Interaction of HCV particles with CD81 expressed on PBMC as well as alteration of CD81 expression on PBMC during HCV infection is well known and of clinical importance
In summary, the results of the present study show that chronic HCV infection is associated with increase of soluble CD81 in the exosomal serum fraction. CD81 in the exosomal fraction in patients with chronic hepatitis C appears to be associated with inflammatory activity. This is a new finding with potential implications in the understanding of HCV persistence and HCV-associated necroinflammation. In concordance, soluble CD81 levels were associated with higher stage liver fibrosis.