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ORIGINAL ARTICLE
Year : 2015  |  Volume : 28  |  Issue : 3  |  Page : 774-779

Low-density lipoprotein receptor on peripheral lymphocytes as a candidate receptor for hepatitis C virus


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
3 Department of Clinical Pathology, Benha Teaching Hospital, Benha, Egypt

Date of Submission04-Jan-2015
Date of Acceptance03-Mar-2015
Date of Web Publication22-Oct-2015

Correspondence Address:
Samar M Emara
1 Bazar Alexandria ST, Shebin El-Kom, 32512 Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.167922

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  Abstract 

Objective
The aim of this work was to assess the role of low-density lipoprotein receptor (LDLR) on peripheral lymphocytes as a candidate receptor for hepatitis C virus (HCV) in patients with chronic HCV both without interferon treatment and after interferon treatment in comparison with a control group.
Background
HCV is one of the most prevalent infectious diseases in Egypt. It is considered the main cause of hepatic morbidity in Egypt, with progression to chronic liver disease, cirrhosis, and hepatocellular carcinoma. Pioneering studies have reported the involvement of the LDLR in the cellular entry of HCV. LDLR was found to play a role in the pathogenesis of HCV infection.
Materials and methods
Peripheral blood lymphocytes were isolated from 40 patients with chronic HCV without interferon treatment and 20 patients with HCV after interferon treatment. Quantification of LDLR expression was performed by flow cytometry and the results were compared with those of 20 normal volunteers as a control group.
Results
There was a significant difference between the mean ± SD of LDLR% between HCV cases (89.08 ± 4.89), post-interferon treatment cases (57.45 ± 4.72), and the control group (54.69 ± 8.48). There was a significant positive correlation between LDLR%, LDLR mean fluorescence intensity, and viral load.
Conclusion
There was a significant positive correlation between the amount of LDLR expression on peripheral lymphocytes and viral load as quantified by PCR. This indicates the involvement of LDLR in the pathogenesis of HCV infections.

Keywords: Flow cytometry, hepatitis C virus, low-density lipoprotein receptor


How to cite this article:
Montaser LM, Sonbol AA, Anees SE, Emara SM. Low-density lipoprotein receptor on peripheral lymphocytes as a candidate receptor for hepatitis C virus. Menoufia Med J 2015;28:774-9

How to cite this URL:
Montaser LM, Sonbol AA, Anees SE, Emara SM. Low-density lipoprotein receptor on peripheral lymphocytes as a candidate receptor for hepatitis C virus. Menoufia Med J [serial online] 2015 [cited 2020 Feb 24];28:774-9. Available from: http://www.mmj.eg.net/text.asp?2015/28/3/774/167922


  Introduction Top


Hepatitis C virus (HCV) has been a major global concern since its discovery more than 20 years ago. Approximately 10-20% of individuals who develop chronic hepatitis C will develop chronic liver disease complications such as cirrhosis and hepatocellular carcinoma [1]. It is most prevalent in central and east Asia, North Africa, and the Middle East [2]. In high-endemic areas such as Egypt, the local rate of prevalence may reach 30% [3]. Residents of Nile Delta, in particular, have the highest seroprevalence of HCV infection worldwide [4]. The HCV is a small (~55-65 nm) enveloped single-stranded positive-sense RNA virus [5]. HCV shows marked sequence heterogeneity; six genotypes and different subtypes have been described with different geographical distributions [6]. This genetic variability causes variable response to interferon treatment [7]. Various modes can cause infection by the virus including blood transfusions before 1991 such as injection drug users, occupational (needlestick) exposure, sexual transmission, hemodialysis, organ transplantation, and perinatal transmission [8].

HCV cell entry involves many viral and cellular factors in a multistep process essential for virus uptake into the hepatocyte [9]. Antibody blocking and low-density lipoprotein receptor (LDLR) receptor knockdown experiments have recently shown the involvement of the LDLRs in the entry of viral particles into hepatocytes [10]. LDLR is composed of 839 amino acids (after removal of 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich LDL [11]. It is a cell surface receptor that recognizes the apoprotein B100, apoE protein, and VLDL remnants (IDL). In humans, the LDLR protein is encoded by the LDLR gene, which is a member of the LDLR gene family [12],[13]. LDLR has been implicated in HCV nonproductive entry of HCV particles, in addition to its function for optimal replication of the HCV genome [14]. The aim of this work was to determine the relationship between LDLRs on peripheral lymphocytes by flow cytometry and hepatitis C-RNA viral load in chronic hepatitis C patients.


  Materials and methods Top


Materials

The protocol for this study followed the ethical standards and was approved by the ethical committee of our institution, and all participants provided informed consent to participate in this study. Sixty patients with chronic HCV were selected from the liver institute outpatient clinic in the period from November 2012 to February 2014 in addition to 20 age-matched and sex-matched healthy controls on the basis of the following inclusion criteria:

(a) Chronic hepatitis C patients confirmed by consistent detection of HCV-RNA (6 months), positive anti-HCV antibodies by third-generation enzyme immunoassay and histological examination performed by an experienced pathologist.

(b) Negative for hepatitis B surface antigen.

(c) Negative for antibodies to human immunodeficiency virus type 1 and type 2. Exclusion criteria were as follows: patients with chronic liver diseases of origin other than hepatitis C viral etiology, past and present substance abuse, and patients with decompensated liver diseases.

The participants in this study were classified into three groups: group 1: 40 chronic HCV patients with no history of interferon treatment, group 2: 20 chronic HCV patients treated with a combination of pegylated IFN-a-2a 180 μg subcutaneously once weekly and ribavirin tablets 800-1200 mg/day for 48 weeks in the outpatient clinic of National Liver Institute, and group 3: 20 apparently healthy individuals, age and sex matched, as a control group.


  Methods Top


All individuals were subjected to the following laboratory investigations: Complete blood count (CBC), liver function tests, serum lipid profile, quantification of HCV-RNA by RT-PCR, and flow cytometric assessment of LDLR.

Sampling

A 12-14 h fasting venous blood sample (10 ml) was withdrawn from each individual under aseptic conditions and then dispensed into four tubes. Three milliliters of blood was collected in an EDTA-containing tube for CBC and LDLR analysis. 1.8 ml of blood was collected in a tube containing 0.2 ml trisodium citrate for measurement of prothrombin time. Three milliliters of blood was collected in a plain tube in which serum was separated and used for the assessment of liver function testes and hepatitis viral markers. Two milliliters of blood was collected for measurement of the lipid profile.

Flow cytometric analysis of LDLR expression was carried out on peripheral lymphocytes.

Reagents

Phosphate buffered saline (PBS) provided by Sigma and stored at 4°C was used.

Monoclonal antibodies: phycoerythrin-conjugated mouse monoclonal anti-human antibodies against LDLR (clone 472413, mouse anti-human IgG; R&D Systems, 614 Mckinley Place NE Minneapolis, MN 55413, USA) were used.

Principle of the test

Washed cells were incubated with labeled monoclonal antibodies that bound to cells expressing the antibody of interest; unbound antibody was then washed from the cells and cells expressing monoclone were fluorescently stained, with the intensity of staining directly proportional to the density of expression of the monoclone.

Sample preparation

(1) Two milliliters of ficoll was placed in a centrifuge tube and layered with 1 ml of blood-containing EDTA sample to ensure that the blood and ficoll do not mix. Centrifugation was performed at 1800 rpm for 20 min.

(2) The mononuclear layer was placed in a separate tube. The cell suspension was washed three times with PBS and centrifuged at 3000 rpm for 5 min.

Sample staining

(1) For each sample, two tubes were prepared: one for the test and the other for the unstained auto control.

(2) One hundred microliters of cells suspension in PBS was mixed with 10 μl phycoerythrin-conjugated mouse monoclonal LDLR, the mixture was incubated at 2-8°C for 30 min. and the cells were washed twice in 2 ml PBS.

(3) Finally, the cells were resuspended in 200-400 μl of PBS and then measured by flow cytometry.

All samples were analyzed using a (Becton Dickinson immune cytometry systems, San Jose, CA95131-1807, USA) flow cytometer.

Statistical analysis

The collected data were statistically analyzed using the statistical package for social science program version 17 (SPSS Statistics for windows, SPSS Inc., Chicago). Quantitative data were expressed as mean ± SD. The Student t-test was used to compare the same variables between two different groups. The analysis of variance test was used for comparison of three or more groups with quantitative variables and the Pearson correlation (r) was used to study the correlation between two normally distributed quantitative variables. The Mann-Whitney U-test was performed for assessment of non-normally distributed quantitative variables. The χ2 -test was used to study the association between two qualitative variables. P value less than 0.05 was considered significant.


  Results Top


The study included 60 patients, 40 patients with chronic liver diseases) 23 men and 17 women), 20 post-interferon treatment cases (12 men and eight women), and 20 volunteers as a control group (13 men and seven women). In terms of sociodemographic data, there was no significant difference in age or sex between the three groups studied. The age of HCV patients ranged from 21 to 48 years, with a mean of 38.02 ± 7.78. The age of post-interferon treatment patients ranged from 26 to 48 years, with a mean of 36.1 ± 6.33. The present study also showed a significant difference between the HCV patients and the post-interferon group in the three parameters of CBC, namely, hemoglobin, WBCs, and platelets, and no significant difference between HCV patients and the control group [Table 1].
Table 1: Comparison between hepatitis C virus cases, post-interferon treatment cases, and the control group in CBC parameters

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In terms of liver function tests in this study, there was a statistically significant difference between HCV patients, the post-interferon group, and the control group for alanine transaminase (ALT) and aspartate transaminase (AST) results. The liver function tests were in general better in the control group than post-interferon treatment and HCV cases [Table 2]. In terms of lipid profile results, the present study showed no significant difference in the results between HCV cases, post-interferon treatment cases, and the control group.
Table 2: Comparison between hepatitis C virus cases, post-interferon treatment cases, and the control group in liver functions

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In the present study, there was a significant difference between the mean ± SD of LDLR% between HCV cases (89.08 ± 4.89), post-interferon treatment cases (57.45 ± 4.72), and the control group (54.69 ± 8.48). Also, there was a significant difference between the mean ± SD of LDLR mean fluorescence intensity (MFI) between HCV cases (98.11 ± 12.45), post-interferon treatment cases (57.23 ± 8.11), and the control group (48.25 ± 7.18) [Table 3]. There was a significant positive correlation between the degree of LDLR expression, MFI, and viral load; as quantified by PCR [Table 4] and [Figure 1] and [Figure 2].
Figure 1: Correlation betwee n LDLR % and viral load among hepatitis C virus cases. LDLR, low-density lipop rotein receptor.

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Figure 2: Correlation between LDLR MFI and viral load among hepatitis C virus cases. LDLR, low-density lipoprotein receptor; MFI, mean fluores cence intensity.

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Table 3: Comparison between hepatitis C virus cases, post-interferon treatment cases, and the control group in low-density lipoprotein receptor parameters

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Table 4: Relationship between the viral load and low-density lipoprotein receptor parameters

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  Discussion Top


Worldwide, the HCV has infected about 130-170 million individuals, mostly as a chronic infection [15]. HCV particlesare known to exist in conjunction with lipoproteins [14]. The virus has been shown to increase the lipid production of infected cells [16]. As a result of this interaction, the LDLR has been proposed as a channel for HCV entry into hepatocytes; however, its role in virus entry remains unclear [14].

In this study, the sociodemographic data of HCV patients without treatment, HCV patients after interferon treatment, and the control group did not show significant differences in age and sex. The age of HCV patients ranged from 21 to 48 years, mean 38.02 ± 7.78. The age of post-interferon treatment patients ranged from 26 to 48 years, mean 36.1 ± 6.33; this is in contrast to a recently published survey on HCV infection in the civilian population of the USA. In this survey, the prevalence was higher in men than women (1.9 vs. 1.1% and P < 0.001), and greater than two-thirds (70.1%) of those infected were in the 45-65 years age group [17].

The survey of Ditah et al. [17] reported the prevalence rather than the incidence of the disease and this explains the age group of the already infected cases rather than new cases, which is not the case in Egypt. In western countries, the most important mode of HCV infection is intravenous drug abuse,which can justify the higher spreadamong males than females.

This predominance of prevalence among men and in the age group older than 40 years old was also found in the research of Mohamoud et al. [18], who reported that the prevalence and incidence of HCV across diverse population groups in Egypt were found to be much higher than those in other countries worldwide. This makes HCV and its complications one of the leading public health problems that Egypt faces today [18].

The present study also showed a significant difference between the HCV cases and post-interferon group in the three parameters of CBC, namely, hemoglobin, WBCs, and platelets, and no significant difference between HCV cases and the control group. The decrease in these parameters after interferon treatment may be attributed to the bone marrow-suppressive effect of interferon. The results of Bruin et al. [19] are in agreement with those of the present study.

In terms of liver function tests in this study, there was a statistically significant difference between HCV cases, the post-interferon group, and the control group in ALT and AST results. The liver function tests were in general better in the control group than the post-interferon treatment and HCV cases. This is in agreement with a Saudi study on the effect of combined pegylated interferon-ribavirin therapy in chronic hepatitis C infected patients, which showed a statistically significant reduction in the mean values of AST and ALT after 24 weeks of antiviral treatment [20].

In terms of lipid profile results, the present study showed no significant difference in the results between HCV cases, post-interferon treatment, and the control group. This is in contrast to the study of Ghadir et al. [21], who showed that four variables studied (HDL, LDL, total cholesterol, and TG) were significantly lower in cirrhotic patients than in the comparison group. Furthermore, the degree of decrease in serum HDL, LDL, and total cholesterol (but not TG) showed a positive correlation with the severity of liver damage, which is reasonably expected as liver biosynthesis is reduced [21]. This is in agreement with Dai et al. [22], who reported the viral effect on patients' serum lipid profile and concluded that HCV viremia is statistically associated with lower serum cholesterol and TG levels, which is not the case in the present study because HCV cases are early chronic cases and there was neither liver damage nor cirrhosis.

In the present study, there was a significant difference between the mean ± SD of LDLR% between HCV cases (89.08 ± 4.89), post-interferon treatment cases (57.45 ± 4.72), and the control group (54.69 ± 8.48). Also, there was a significant difference between the mean ± SD of LDLR MFI between HCV cases (98.11 ± 12.45), post-interferon treatment cases (57.23 ± 8.11), and the control group (48.25 ± 7.18). These results showed a significant correlation between the degree of LDLR expression and viral load.

This is in agreement with Albecka et al. [14], who investigated the role of the LDLR in the HCV life cycle by comparing entry of the virus in the mechanism of lipoprotein uptake. They confirmed that this receptor plays a role in the life cycle of HCV generated in cell culture. This specific interaction was speculated on observing a decrease in HCV-RNA replication by blocking the LDLR with a specific antibody. Nevertheless, a soluble form of the LDLR inhibited both HCV entry into the hepatocytes and its binding to the LDLR expressed on Chinese hamster ovary cells. They further investigated this role in the HCV life cycle and found that it played a significant role in viral replication rather than entry into hepatocytes [12]. The study of Von Hahn et al. [23] showed that natural ligands to oxidized LDL are inhibitors of HCV cell entry.

HCV virions are reported to exist in serum in a complex form with b-lipoproteins and/or immunoglobulins, and infect cells through LDLR. The increase in b-lipoproteins interferes with the rate of HCV infection of liver cells, and results in decreased HCV replication in the cells and decreased HCV Ag in sera [24].

Also, Syed et al. [16] concluded that HCV modulates host lipid metabolism to promote enrichment of lipids in the intracellular environment, which are essential in multiple aspects of the HCV life cycle. Their observations suggest that HCV upregulates LDLR expression at both the protein and the transcript level. Furthermore, they found that this upregulation likely contributes toward the uptake of serum lipids by infected hepatocytes. Abrogation of HCV-mediated upregulation of LDLR inhibits serum lipid uptake and thereby perturbs HCV replication. Siagris et al. [25] found this finding to be more pronounced in patients infected with HCV genotype 3a and concluded that further studies are necessary to define the pathophysiology of the relationship between lipid metabolism and HCV infection.

Almost the same conclusion was reported by Hsu et al. [26] on the correlation of the lipid profile with various genotypes of HCV, which suggests that further studies should be carried out to investigate the correlation of lipid profile and each HCV genotype. The close relationship between host lipids and the HCV life cycle generates opportunities for new therapeutic options that target lipid regulation. Indeed, host factor targeting overcomes viral resistance because of emerging escape variants and genotype variability [27].


  Conclusion Top


The present study has clearly shown that LDLR plays a significant role in HCV entry into hepatocytes as well as virus replication. The laboratory monitoring of this receptor can reflect the severity of infection and can be used to predict the response to interferon treatment. The association between LDLR and viral load is in agreement with the concept that this receptor plays an important role in the life cycle of HCV.


  Acknowledgements Top


Conflicts of interest

There are no conflicts interest.

 
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