Impact of the IL-18 gene polymorphism in response to antiviral therapy in chronic HCV genotype 4 patients

Introduction: Interleukin (IL)-18 is a well-known major proinfl ammatory cytokine with broad biological effects. The major immunomodulatory functions of IL-18 include enhancing T cell and natural killer cell cytotoxicity. Serum levels of this cytokine were shown to increase in chronic hepatitis C patients compared to non-infected healthy people. An association between IL-18 gene promoter polymorphisms and pegylated interferon (PEG-IFN) and ribavirin treatment outcomes has been reported for individuals with chronic hepatitis C virus genotype 1 (HCV-1). In this study, HCV genotype 4 (HCV-4) patients were assessed for IL-18 gene polymorphisms and treatment outcomes or severity of liver disease because data concerning the impact of IL-18 gene polymorphisms on patients with HCV-4 infections are limited. Methods: This study included 123 chronic HCV-4 Egyptian patients and 123 apparently healthy volunteer blood donors who served as a control group. HCV genotyping was performed using the line probe assay. IL-18 genotyping was performed using the TaqMan Real-Time PCR method in all 246 patient and control samples. Results: In our study, all patients had HCV-4. IL-18 gene single nucleotide polymorphism (SNP) (-607C/A) genotype distributions and allele frequencies did not differ between HCV patients and normal healthy subjects or between patient groups when compared according to the therapeutic response. Moreover, the presence of an IL-18 SNP was not associated with histological disease severity. We conclude that the presence of the IL-18 SNP rs1946518 does not affect the outcome of chronic HCV-4 treatment in Egyptian patients. Conclusions: The IL-18 SNP rs1946518 does not affect response to treatment in chronic HCV-4 patients.

Hepatitis C virus (HCV), a member of the Flaviviridae family, is a main cause of morbidity and mortality, with more than 170 million infected people worldwide.The main cause of chronic HCV in the Middle East and Egypt is HCV genotype 4 (HCV-4).Recently, HCV infection has been reported in southern Europe among immigrants, mostly from Africa, and among intravenous drug users 1,2 .
With 15% of the population infected, Egypt has the greatest burden of HCV infection in the Middle East, and HCV-4 represents 90% of the infected cases.The standard of care therapy (SOC) for HCV-4 consists of a 48-week combination therapy of pegylated interferon (PEG-IFN) α and ribavirin.However, only 50% of treated patients achieve a sustained virological response (SVR) [3][4][5] .The response to therapy and the progression of the disease to more severe liver injury are determined by many factors, including host (immunologic and genetic) and viral factors 6 .Genetic analysis of different cytokines, chemokines and receptors that are implicated in HCV pathogenesis has revealed many HCV-related polymorphisms 7 .
Interleukin (IL)-18, which was previously known as the interferon (IFN)-γ inducing factor, belongs to the IL-1 cytokine family and is a proinfl ammatory cytokine with broad biological effects [8][9][10] .IL-18 is produced mainly by monocytes and macrophages.Together with IL-12, IL-18 enhances IFN-γ production.Other major immunomodulatory functions for IL-18 include the stimulation of naïve T cells to differentiate into T helper 1 cells, thereby enhancing T cell and natural killer cell cytotoxicity (Fas/Fas ligand and perforin-mediated cytotoxicity) and modulating immunoglobulin production by B cells.IL-18 activity results in the production of a wide range of infl ammatory mediators, such as nitric oxide (NO) and cytokines (e.g., TNFα), that mediate the proinfl ammatory actions of IL-18.This activity can also be attributed to the upregulation of macrophage recruitment chemokines, e.g., IL-8, macrophage infl ammatory protein (MIP-1α), MIP-1β and monocyte chemoattractant protein (MCP-1) 8,11,12 .IL-18 serum levels were shown to increase in chronic hepatitis C patients compared to non-infected healthy people, and the serum levels were strongly correlated with the alanineaminotransferase (ALT) serum activity 13,14 .Three main genetic

METHODS
variants in the promoter region of the IL-18 gene have been described by Giedraitis and colleagues 15 .Two of these IL-18 promoter variants (-137 and -607) showed evidence of a functional relevance.A change in the HATF1 transcription factor binding site to a binding site for another factor found in the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter resulted from a -137G>C conversion in this gene.Similarly, the -607C>A promoter variant disrupts the binding site for a cAMP-responsive protein.IL-18 variants have been associated with various chronic infl ammatory conditions and viral diseases, such as hepatitis B 16 and Alzheimer's disease 17,18 .
The Egyptian population is a unique population to study HCV infection due to the high prevalence and predominance of HCV-4.Recently, several studies have focused on the role of the IL-28B polymorphism as a predictor of antiviral treatment in chronic HCV genotype 1 and 4 (HCV-1,4) patients.
The objectives of the present study were the following: I) To determine and compare the IL-18 (rs1946518) genotype distributions between chronic HCV-4 patients and healthy individuals in Egypt.II) To investigate the impact of IL-18 (rs1946518) gene polymorphisms in HCV-4 patients in response to SOC combination therapy or fi brosis progression.

Patients
A total of 123 chronic HCV patients were enrolled in the present study.Patients were eligible for antiviral treatment according to the Egyptian treatment guidelines for HCV.To collect demographic data, the patients and controls were interviewed using a standardized questionnaire.Demographic data included age at the start of therapy, gender and residence.

Controls
A total of 123 apparently healthy adult volunteer blood donors from the same geographical regions who had no previous complaints of liver disease and were serologically negative for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodefi ciency virus (HIV) served as controls.All patients and healthy subjects provided written informed consent before being enrolled in the study.

Serology
Venous blood was collected from patients, and serum was separated and stored at -80°C.A third-generation enzyme-linked immunosorbent assay(ELISA) kit (Diasorin, Italy) was used to detect antibodies against HCV.Elevated liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for at least 6 months with positive serology for HCV were the main diagnostic criteria.Both hepatitis B surface antigen and HIV-Ab were negative in all patients by enzyme immunoassay (Diasorin, Italy).Screening for antischistosomal antibodies was performed using the indirect hemaglutination (IHA) test according to the manufacturer's instructions (Fumouze Diagnostics, France).

HCV hepatitis C virus-polymerase chain reaction and genotyping
Hepatitis C virus (HCV) ribonucleic acid (RNA) was detected using a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay (Artus HCV RG RT-PCR, Qiagen, Hilden, Germany).A line probe assay (INNO-LiPA HCV II Kit, InnogeneticsQ2, Ghent, Belgium) was used to determine the genotype of the amplifi ed HCV-complementary DNA (cDNA).

Histological examination
A percutaneous liver biopsy was performed on all patients before treatment.The modified Ishak scoring system was used to determine the stage of hepatic fi brosis and grade of infl ammation.

Dose of PEG-IFN and RBV
A combination of Pegylated interferon (PEG-IFN) α and ribavirin (RBV) administered for 48 weeks was used as the SOC therapy for all patients.PEG-IFN was subcutaneously injected near the umbilicus in a single weekly dose of 180µg for IFN α-2a and 1.5ug/kg body weight for interferon gamma (IFN-α)-2b together with RBV (either 1,000-1,200mg/day with IFN α-2a or 800-1,400mg/day with IFN α-2b, depending on body weight).

Treatment outcomes
Patient outcomes following combination therapy were classifi ed according to the clearance or recurrence of hepatitis C virus-ribonucleic acid (HCV-RNA) into the following groups: sustained virological response (SVR) (negative for HCV-RNA 6 months after the end of treatment), transient virological response (TVR) (negative for HCV-RNA at the end of treatment with recurrence afterwards) or null virological response (NVR) (less than a 2-log unit decline in the serum level of HCV RNA from the pretreatment baseline value within the fi rst 12 weeks and detectable viremia at week 24 after the initiation of treatment).Patients with TVR and NVR were grouped together (non-SVR) for further analysis.

Genomic DNA extraction for IL-18 genotyping
Blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes.QIAamp DNA Blood Mini Kits were used to extract genomic DNA from 200µl of whole peripheral blood according to the manufacturer's instructions (QIAGEN, Inc., Hilden, Germany).DNA quality and quantity were assessed by calculating the absorbance ratio OD 260nm/280nm using a NanoDrop spectrophotometer (ThermoFisher, Asheville, NC).

IL-18 genotyping
IL-18 gene variant (rs1946518) 607C>A was genotyped in all samples using TaqMan SNP genotyping assays (Applied Biosystems "AB" 7500 Real-Time "PCR" system).Allelespecifi c labeled TaqMan MGB probes were used (FAM-and VIC-labeled).Each reaction contained 1X assay mix, 1X TaqMan Genotyping MasterMix (AB) and 20ng of template DNA in a total volume of 25µL.Cycling conditions were as follows: 95°C for 10min and 40 cycles of amplifi cation  consisting of 15 sec each and 1min at 60°C.Plate reading and genotyping (by clustering) were performed using 7500 system software version 1.2.3 in the AB 7500 Real-Time PCR system.

Statistical analysis
Single nucleotide polymorphism occurrence was in accordance with Hardy-Weinberg equilibrium in both cases and controls.Data were expressed as means ± SDs (standard deviation) for quantitative data and as numbers and percentages for qualitative data.Categorical variables such as treatment responses were compared using chi-square (χ2) tests.Data were analyzed using Statistical Package for the Social Sciences (SPSS) for Windows version 15 (SPSS Inc., Chicago, IL, USA).Comparisons of genotype frequencies and associations of polymorphisms with liver histological severity were examined for statistical signifi cance using χ2.Odds ratios (ORs) with 95% confi dence intervals (CIs) were calculated to assess the odds conferred by a specifi c allele.A result was considered statistically signifi cant when the p-value was less than 0.05.

Ethical considerations
All experiments were performed according to the ethical principles of the Declaration of Helsinki.

Population characteristics
This study was conducted on 123 patients with chronic HCV-4; the patient age range was 18-59 years (mean=42.3).Most (83.4%) patients were older than 30 years old, and the had a male predominance (67.5%).Table 1 shows the baseline characteristics of these patients and the healthy controls.

Relationships between treatment response and the IL-18 gene (SNP rs1946518) and clinical variables (univariate analysis)
Among the treated patients (n=123), the distribution of rs1946518 genotypes was as follows: CC 32.5%, CA 51.2% and AA 16.3%.Similar frequencies of the -607C>A variant were found in both the patient and control groups (Table 2).No relationships were found in univariate analyses among response type and gender, age at start of therapy, fi brosis stage, infl ammation grade or HCV RNA viral load (Table 3).In contrast, the relationship between the response type and the baseline ALT was signifi cant (Tables 3 and 4).IL-18 SNP (rs1946518) genotypes showed no relationship with the mean baseline viral loads (Table 3).The genotype distributions for the IL-18 SNP (rs1946518) were similar in both the responder and non-responder patients (p=0.02).
The genotype distributions for the IL-18 polymorphism (rs1946518) were not significantly different among the responder, relapsed and non-responder patients (p=0.269).The distribution of rs1946518 genotypes according to different treatment outcomes is shown in Table 5.
This study is the fi rst in Egypt to characterize the impact of functional IL-18 promoter variants on treatment outcomes in chronic HCV patients.The -607C/A IL-18 promoter variant was not associated with the treatment response in chronic HCV-4 patients.However, Haas et al. 19 showed a signifi cant association between both the -137 and -607 IL-18 promoter variants and the treatment response in patients infected with HCV-1.Together with IL-12, IL-18 is an obligatory cytokine for IFN-γ production 20 .IL-18 plays a signifi cant role in the immune response by enhancing T cell responses, regulating IFN-γ production and promoting the development of Th1 immune responses.The treatment outcome and resolution of HCV infection have been strongly attributed to genetic host factors.HCV elimination requires a strong Th1 antiviral immune response 19 .This proinflammatory cytokine has a complex biological role in hepatic diseases with unclear characterization.Similar to the serum sources of IL-18, hepatic Kupffer cells, activated macrophages, Natural Killer (NK) cells and NK T cells contribute for hepatic IL-18 production 21 .Liver injury mediated by IL-18 can be attributed to IFN-γ induction with the aid of IL-12.In mice primed with Propionibacterium acnes and subsequent lipopolysaccharide (LPS) challenge, the protective role of anti-IL-18 antibodies against liver damage was previusely illustrated 10 .In IL-18 transgenic mice, marked hepatic damage was associated with the upregulation of IL-18 expression 21 .However, the inhibitory effect on the replication of hepatitis B virus indicated a protective role for IL-18 during viral infections, which was explained by IFN-γ production by hepatic NK cells and NK T cells stimulated by IL-18 22 .Similar effects were observed in other viral mouse models 23,24 .Interestingly, in a monocytic cell line, IL-18 stimulated HIV replication 25 .
In our study, the distributions of -607C/A genotypes were not different between infected patients and healthy controls.The CA genotype was the most common genotype among the patients and the controls.Accordingly, the C allele was the most common allele in both patients and controls.These fi ndings are consistent with previous studies that reported no differences in -607C/A genotype distributions and allele frequencies between patients and controls 26,27 .
Recently, Manohar et al. investigated the impact of the -607 and -137 polymorphisms on the severity of HCV infection.The -607 and -137 allelic distributions were similar in patients and controls.However, when patients were classifi ed into 2 groups according to the severity of liver disease based on both the histological activity index (HAI ≤5 or >5) and the hepatic fi brosis score (≤2 or >2), a protective role for the IL-18 promoter  variant -607AA was evident.This fi nding was attributed to an association with milder forms of liver disease in chronic HCV 27 .
Zhang PA and coworkers studied the role of IL-18 variants -137 and -607 in HBV infection 28 .The authors found that a lower viral load was linked to the -607AA genotype.The same genotype was more frequently observed in HCV patients when compared with matched controls in a Thai population 16 .
In PEG-IFN and ribavirin-treated patients, serum IL-18 was highly related to the HCV-RNA titers 29 , serum ALT and AST activities 30 .
An et al. 31 showed an association between the -137C and -607A alleles and spontaneous clearance of HCV in African American intravenous drug users compared with those with chronic unresolved infection.Bouzgarrou et al. and colleagues 26 studied the correlations among serum IL-18, HCV infection outcomes and IL-18 gene promoter SNPs (-137G/C and -607C/A).A comparison of different stages and severities of HCV infection and IL-18 levels revealed a positive correlation with disease progression.Additionally, complicated cases with cirrhosis and hepatocellular carcinoma (HCC) were associated with the -607C allele.Manohar et al. excluded the effe ct of IL-18 gene polymorphisms on susceptibility to chronic HCV infection 27 .
In the present study, no association was found between the IL-18 promoter variant -607 and hepatic fibrosis or infl ammation.Similarly, no association could be determined between this polymorphism and levels of AST or ALT.This fi nding is consistent with Haas et al. 20 fi ndings that the severity of hepatic infl ammatory lesions, fi brosis grades and transaminase serum levels were not associated with either IL-18 promoter variant.
In conclusion, the IL-18 SNP rs1946518 does not affect treatment response in Egyptian HCV-4 patients included in our study.Furthermore, no correlation was identifi ed between this SNP and the severity of liver disease.Future studies should include more patients for more reliable assessment.

TABLE 1 -
Baseline characteristics of HCV-4 patients and healthy controls.

TABLE 2 -
Genotyping distributions for the IL-18 polymorphism rs1946518 in HCV-4 patients and healthy controls.

TABLE 3 -
Association between baseline characteristics and sustained viral response/Non-sustained viral response of hepatitis C virus treatment (univariate analysis).

TABLE 4 -
Logistic regression analysis for predictors of the treatment outcome (SVR).This model correctly predicted 64.8% of treatment outcomes.Model fi t (p=0.048*);-2Log likelihood=148.5.The signifi cant predictor for was ALT <40 IU; those patients had a 3-fold greater probability of developing SVR compared to patients with higher ALT levels.The AA genotype was associated with a 2.3-fold greater susceptibility to the development of SVR compared to the CC genotype, but this difference was not statistically signifi cant.Rural residence and infl ammation grades (0-8) also conferred a greater probability for SVR (2.2-and 2.0-fold, respectively), but these differences were not statistically signifi cant.SVR: sustained viral response; B: coeffi cient of regression; SE: standard error of the coeffi cient; OR: odds ratio; 95%CI: 95% confi dence interval; AST: aspartate aminotransferase; ALT: alanine aminotransferase; HCV-RNA: hepatitis C virus-ribonucleic acid viral load; CC, CA, AA: IL 18 promotor genotypes.

TABLE 5 -
Distribution of the rs1946518 genotype according to different responses to SOC therapy in HCV gt-4 patients.
SOC: standard of care; HCV: hepatitis C virus; AA, CA, CC: IL 18 promotor genotypes; NVR: null virological response SVR: sustained virological response TVR: transient virological response.NS: no statistically signifi cant difference.a Linear-by-linear association chi-square test.