Up-regulation of Plasma Hexosylceramide (d18:1/18:1) Contributes to Genotype 2 Virus Replication in Chronic Hepatitis C

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INTRODUCTION H epatitis C virus (HCV) infection has been recognized as a leading cause of chronic liver disease, since it was discovered in 1989. 1 Approximately 3% of the population worldwide-130 to 170 million people-has an HCV infection. 2 Approximately 10% to 20% of patients with chronic HCV infection progress to cirrhosis in a period of 20 to 30 years. 3 Although many factors are associated with disease progression, persistent HCV replication has been recognized as one of the main determinants of hepatic deterioration. 4 Suppression of viral replication is an important strategy for preventing the progression of chronic liver disease. Although the combination of pegylated interferon-a and ribavirin or direct-acting antiviral agents could remove the viruses, a few patients suffer from treatment failure, and they require treatment in the future. Therefore, further searches for novel targets that are a closely related to the HCV replication process are urgently needed.
Sphingolipids interact with cholesterol and glycosphingolipid during the formation of lipid rafts. These provide a platform for signal transduction and pathogen infection, 5 and also play crucial roles in the viral life cycle. 6 For instance, lipid rafts may be the sites for HCV-RNA synthesis. 7 Recent studies have demonstrated that sphingolipids are closely related to liver disease, especially ceramide metabolites. 8 Ceramides play an essential role in inhibiting HCV entry. 9 During HCV infection, the suppression of sphingolipid biosynthesis with a serine palmitoyltransferase inhibitor, which is the first-step enzyme in the ceramide biosynthetic pathway, could suppress viral replication in HCV-infected chimeric mice and HCV replicon cells. [10][11][12] Additionally, HCV infection regulates various aspects of lipid metabolism within infected hepatocytes. [13][14][15] For example, HCV upregulates the sphingomyelin and ceramide levels, promoting viral replication in the hepatocytes of humanized chimeric mice. 16 Although multiple reports have indicated that dysregulation of sphingolipid synthesis affects HCV replication in animal models and cells, the relationship of plasma sphingolipids with HCV replication in chronic hepatitis C (CHC) patients remains poorly understood.
Previously, we established mature high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) techniques to quantify sphingolipids. 17,18 On the basis of a platform that improved quantitative high-throughput lipidomic, we initially confirmed a relationship between the plasma sphingolipids and hepatic inflammation in CHC. 19 Subsequently, we demonstrated that glycosphingolipid and sphingomyelin were closely related to hepatic steatosis and fibrosis in CHC patients, respectively. 20,21 On the basis of the previous reports and studies, we hypothesized that one or a subset of sphingolipids might be involved in HCV replication.
Therefore, in the present study, with the help of HPLC-MS/MS, liver biopsy, and HCV-RNA quantification, we aimed to evaluate the relationship between plasma sphingolipids and HCV replication among treatment-naïve patients with CHC, and also the subgroup of patients without significant necroinflammation (grade 2) or with HCV genotype 2.

METHODS Patients
A cohort of 122 treatment-naïve CHC patients with a clear history of paid plasma donation from 1992 to 1995, from Dingxi City (Gansu Province, China), who were followed between July 2010 to June 2011, were enrolled in the present study. 22 The CHC diagnosis was made in accordance with established criteria. 23,24 Patients were excluded if they were coinfected with HBV, human immunodeficiency virus (HIV), or hepatitis delta virus (HDV), or if they had a malignant disease, including hepatocellular carcinoma, or there were no biopsy results. Two cases were excluded; one was for ascites, and the other one was because the liver biopsy specimen volume was too small. One hundred twenty patients were eligible for the study. Informed consents were obtained. The study was performed according to the provisions of the Declaration of Helsinki and was approved by the Institutional Review Board of Beijing YouAn Hospital, Capital Medical University, Beijing, China.

Clinical Date Collection
The blood biochemical indicators, HCV-RNA load, and immunological markers were collected as previously described. 22 Fasting blood samples were collected at the time of biopsy and stored at À808C. Routine serological indications were extracted in all patients.

Liver Biopsy
Liver biopsies were performed as described. 22 The Scheuer scoring system was used to assess hepatic inflammatory activity. 25 We selected patients without significant necroinflammation (grade 2) for the subgroup statistical analysis.

HPLC-MS/MS
Serum sphingolipids were detected by HPLC-MS/MS as previously described. 17 Sphingolipidomic assays were performed at the Institute of Materia Medica, Peking Union Medical College (Beijing, China).

Statistical Analysis
Data were expressed as mean AE standard deviation (SD), unless otherwise specified. Statistical comparisons for continuous variables were performed using the independent-samples t test or Mann-Whitney U test. Categorical variables were analyzed using the Pearson chi-square test. Correlation analysis between the plasma sphingolipids and HCV-RNA load was made using Spearman rank correlation. After univariate analysis, stepwise logistic regression analysis with a forward selection, and the P values of entry and removal were set to 0.05 and 0.1, respectively. Statistical analysis was performed using SPSS version 19.0 (Chicago, IL). A P value <0.05 was considered statistically significant.

Clinical Characteristics of Patients
In our cohort, there were 57 (47.5%) male and 63 (52.5%) female patients who had a mean age of 51.33 years. The mean serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values were 60.42 and 47.94 U/L, respectively; they were mildly elevated at the time of biopsy. Data are presented as mean AE standard deviation (SD) or number (%). ALT ¼ alanine aminotransferase, AST ¼ aspartate aminotransferase, GGT ¼ gamma-glutamyl transferase, HCV ¼ hepatitis C virus, SD ¼ standard deviation.

Relationship Between Plasma Sphingolipids and HCV-RNA Load in CHC Patients
Plasma sphingolipids in the 120 patients were detected by HPLC-MS/MS. Forty-four sphingolipids were identified and quantified. Twenty-two plasma sphingolipids showed a significant difference between patients with a low viral load (LVL, HCV-RNA load <10 6 IU/mL) and a high viral load (HVL, HCV-RNA load !10 6 IU/mL) (P < 0.05) ( Table 2). To further analyze the correlation between these differentially expressed sphingolipids and HCV-RNA load, Spearman correlation analysis was performed. The results of the correlation analysis revealed that 9 sphingolipids were correlated with the HCV-RNA load (Table 3).

DISCUSSION
In this study, a cohort of antiviral treatment naïve, nondiabetic, and nonobese CHC patients were enrolled. After eliminating the influence of antiviral treatment, we revealed the true relationship between plasma sphingolipids and HCV replication. For the first time, we found that the altered plasma sphingolipids were closely associated with HCV replication in chronic HCV infection, especially for CHC patients with genotype 2.
Sphingolipids have recently been found to be involved in liver disease; for example, they play an important role in hepatitis C virus entry, 9 HCV infection, virion maturation, 30 liver fibrosis progression, and antiviral therapy responsiveness. 31 To the best of our knowledge, the role of plasma sphingolipids in HCV replication of CHC patients is currently unknown. In our study, although several sphingolipids correlate with HCV replication, only HexCer (d18:1/18:1) always performed outstandingly in the subsequent analysis and subgroup analysis.
Ã P values are acquired by univariate analyses.
y P values are acquired by multivariate analyses. Our previous studies indicated that plasma sphingolipids are related to hepatic inflammation in CHC, especially hepatic necroinflammation (grade >2). 18,19 To eliminate the impact of necroinflammation on plasma sphingolipids and the possibly accompanying cytotoxic T-lymphocyte response on HCV replication, we analyzed the relationship between plasma sphingolipids and HCV replication in CHC patients without hepatic necroinflammation (grade 2). Remarkably, the plasma HexCer (d18:1/18:1) level was also independently related to HCV replication in this subgroup analysis. However, another sphingolipid,  HexCer (d18:1/18:0), as an independent factor for HCV replication in CHC patients, was not associated with viral replication in CHC patients who had hepatic inflammation grade 2. In our cohort, HCV genotypes 1b and 2 were the major types, which were consistent with the HCV genotype prevalent in China, where genotypes 1b and 2 account for nearly 80% of HCV infection patients. 33,34 In CHC patients with genotype 2, the plasma HexCer (d18:1/18:1) level was also independently related to HCV replication after adjusting for the confounding factor in the multivariate analysis. However, Cer (d18:1/24:0), which was an independent factor for viral replication in genotype 2 CHC patients, was not associated with HCV replication in any of the 120 CHC cohort patients. We also explored the role of sphingolipids in CHC patients with genotype 1b. Regrettably, no association was found between these sphingolipids and HCV replication in CHC patients with genotype 1b (data not shown).
Although our study did not reveal the mechanisms for plasma HexCer (d18:1/18:1) in HCV replication on the basis of the present data, we speculated that elevated HexCer (d18:1/ 18:1) might contribute to the pathogenesis of viral replication in CHC patients, especially for genotype 2 CHC patients. The sample size in this cohort was not sufficiently large, and patients with other genotypes were not analyzed in this study. Therefore, it would make sense to verify the role of HexCer (d18:1/18:1) in large-scale clinical studies in the future.
In conclusion, our results suggest, for the first time, the crucial role of plasma HexCer (d18:1/18:1) in HCV replication. Additionally, HexCer has potential as a novel therapeutic target for antiviral therapy in CHC, especially in patients with genotype 2. These findings will provide new insights into the molecular mechanism of HCV replication in CHC. Further studies are necessary to determine the detailed mechanisms responsible for these associations.