Sei sulla pagina 1di 7

Human Immunology 70 (2009) 230-236

Contents lists available at ScienceDirect

Combined analysis of interferon- and interleukin-10 gene polymorphisms and chronic hepatitis C severity
Nadia Bouzgarrou a, Elham Hassen a,*, Karim Farhat a, Olfa Bahri b, Sallouha Gabbouj a, Nadia Maamouri c, Nabil Ben Mami c, Hammouda Saffar d, Abdelhalim Trabelsi e, Henda Triki b, Lot Chouchane a,f
a

Molecular Immuno-oncology Laboratory, Faculty of Medicine, Monastir, Tunisia Clinical Virology Laboratory, Pasteur Institute, Tunis, Tunisia c Gastroenterology B Unit, La Rabta Hospital, Tunis, Tunisia d Gastroenterology Unit, University Hospital Fattouma Bourguiba, Monastir, Tunisia e Research Unit UR06SP20, Bacteriology-Virology Laboratory, University Hospital Sahloul, Sousse, Tunisia f Genetic Medicine Department, Weill Cornell Medical College in Qatar, Qatar
b

A R T I C L E

I N F O

A B S T R A C T

Article history: Received 30 June 2008 Accepted 26 January 2009 Available online 29 January 2009

Keywords: Interleukin-10 Interferon-gamma Polymorphisms HCV Chronic infection

Today there is increasing evidence concerning the contribution of pro-/anti-inammatory cytokine balance and genetic factors in hepatitis C pathogenesis and interindividual heterogeneity of disease outcome. In the current study, we investigated the inuence of functionally described single nucleotide polymorphisms (SNPs) present in interferon- (IFN ) and interleukin-10 (IL-10) genes, on chronic hepatitis C severity. IFN ( 874T/A) and IL-10 ( 1082G/A) genotypes were determined in 100 hepatitis C patients with different disease severities (chronic hepatitis, n 42, liver cirrhosis [LC], and hepatocellular carcinoma in liver cirrhosis [HCC], n 58) and 103 healthy controls using allele-specic polymerase chain reaction. No statistical differences in allele or genotype distributions of IFN and IL-10 genes were observed between patients and controls. However, some signicant differences in IFN genotype frequencies were observed between the two groups of patients. IFN high producer genotypes TT and TA were signicantly more common in patients with LC and HCC (odds ratio 2.65; p 0.019). Although IL-10 genotypic frequencies were comparable between the different clinical forms of the disease, the combination of IFN low producer and IL-10high producer genotypes was signicantly associated with a lower risk of LC and HCC (odds ratio 0.21; p 0.015). In conclusion, our ndings suggest that the imbalance between the pro-inammatory and antiinammatory responses mediated by polymorphisms in the IFN and IL-10 genes may inuence the outcome of chronic HCV infection. 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Infection with hepatitis C virus (HCV) is the major culprit for chronic liver disease, characterized by the persistence of necroinammatory damage that can progress to cirrhosis and hepatocellular carcinoma (HCC) [1,2]. A number of factors, including age at infection, gender, alcohol consumption, viral genotype, human immunodeciency virus (HIV), or hepatitis B virus (HBV) co-infection and duration of infection, affect disease outcomes. In Tunisia, the prevalence of HCV infection ranges from 0.4% to 0.7%, with a predominance of the HCV genotype 1b [3,4]. This genotype is assumed to resist antiviral treatment but does not appear to inuence the development of HCV-associated HCC [5]. Today there is increasing evidence that immunologic and host genetic factors contribute to
Nadia Bouzgarrou and Elham Hassen contributed equally to the present study. * Corresponding author. E-mail address: elham.hassen@isbm.rnu.tn (E. Hassen).
0198-8859/09/$32.00 - see front matter doi:10.1016/j.humimm.2009.01.019

the natural history of HCV infection [6,7]. Immune response against HCV, in particular the cell-mediated response by the action of the cytokine system, is reported to contribute to both viral clearance [8,9] and treatment response [10,11]. Clinical research has also shown the prominent role of T helper cell type 1 (Th1) and type 2 (Th2) cytokines in the pathogenesis of chronic hepatitis C. Indeed, the percentage of Th1-type cells found in peripheral blood was higher in cirrhotic patients than in asymptomatic HCV carriers [12]. On the contrary, Th2 cytokines modulate self-inicted injury by suppressing the Th1 response, leading to the milder forms of chronic hepatitis C [1315]. Accordingly, we supposed that the genetic variation affecting cytokine expression might inuence chronic hepatitis C outcomes. In fact, cytokine genes are polymorphic at specic sites, and some of these mutations within the coding and regulatory regions (i.e., promoter sequences), have been associated with a different expression level of the specic cytokines [16].

2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

N. Bouzgarrou et al. / Human Immunology 70 (2009) 230-236

231

One of the most important Th1 cytokines is interferon- (IFN ), which plays a major role in defense against viruses and intracellular pathogens and induces immune-mediated inammatory response. Furthermore, its implication in hepatic pathology has already been suggested [14,17,18]. A signicant correlation was observed between IFN mRNA expression levels and stage of brosis or progressive liver injury in chronic hepatitis C [13,19]. It has been established that the T-to-A polymorphism at position 874 of the rst intron of IFN gene could directly inuence IFN production level [20]. The IFN 874 T/A SNP lies within a putative nuclear factor B (NF- B) binding site, and the presence of the T allele may be important in the induction of a genetically higher IFN production. Pravicia et al. have demonstrated the positive correlation between the occurrence of the 874T allele and the presence of the high-producing microsatellite allele number 2. Indeed, the 874 T/A SNP is in strict linkage disequilibrium with the IFN microsatellite polymorphism (CA repeat) within the rst intron [20]. Allele number 2, with a (CA)12 repeat, has been associated with high IFN production in stimulated lymphocytes in vitro [21]. Subsequently, some studies reinforced these ndings. In Biolo et al. study, the IFN 874 A/A, low-producer genotype was associated with signicant decreased mRNA levels of IFN [22]. Through in vitro lymphoproliferation, Anuradha et al. found that individuals with TT genotype showed signicantly higher antigen-induced IFN as compared with those with TA/AA genotypes [23]. Interleukin-10 (IL-10) is an immunoregulatory cytokine produced by Th2 cells, monocytes/macrophages, and regulatory T cells. IL-10 is also produced by various cell types in the liver, including hepatocytes, sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and liver-associated lymphocytes. It plays an anti-inammatory action by inhibiting the synthesis of cytokines such as IL-1 , IL-1 , IL-6, IL-8, IL-12 and tumor necrosis factor (TNF- ) in activated macrophage and IFN by T cells [24], and it also has some antibrotic properties [25]. Chronically HCV-infected patients who received either short- or long-term therapy with recombinant IL-10 showed a decrease in hepatic inammation and brosis [26,27]. The IL-10 promoter is highly polymorphic, and three SNPs 1082G/A, 819C/T, 592C/A were associated with differential IL-10 expression [28]. The G-to-A polymorphism located at position 1082 might directly inuence IL-10 production level [28]. Turner et al. [28] demonstrated that the 1082A allele was associated with lower in vitro IL-10 production by concanavalin Astimulated PBMC from normal subjects and reported that the observed correlation was independent of the polymorphisms at positions 819 and 592. Other investigators also demonstrated a positive correlation between the 1082G allele and high IL-10 production, alone or in haplotypes [29 32]. Several reports were interested in the study of the implication of IFN and IL-10 gene polymorphisms in the pathogenesis of HCV infection, particularly in viral clearance and treatment response [3335]. However, little is known about IFN and IL-10 gene polymorphisms according to chronic hepatitis C severity. In the present study we investigated whether single nucleotide polymorphisms in IFN and IL-10 genes are associated with susceptibility and/or are markers of prognosis to chronic hepatitis C outcomes. 2. Subjects and methods 2.1. Patients and controls This study included 100 Tunisian subjects with chronic hepatitis C patients who attended either the Gastroenterology Unit of La Rabta or that of Fattouma Bourguiba hospital from May 2005 to March 2008 (28 men and 72 women; mean age, 56 years; range, 37 81 years). Diagnosis in all patients was made by biochemical and molecular assays, including the detection of anti-HCV antibod-

ies using the third-generation commercial enzyme immunoassays (INNOTEST HCV Ab III, Innogenetics-Belgium and Murex anti-HCV, Murex Diagnostics, Chatillon, France) and the detection of HCV RNA in serum (Amplicor HCV assay, Roche Diagnostics, Mannheim, Germany). Patients presenting with other causes of chronic hepatitis (i.e., alcoholic or autoimmune hepatitis), testing positive for other hepatotropic viral antigens, or treated with antiviral drugs before liver biopsy or echography were excluded. The liver histological severity was assessed according to the histologic analysis of liver biopsy samples by two blinded anatomopathologists or according to clinical and ultrasonographic studies. The patients were divided into two groups: the rst group included 42 noncirrhotic patients with chronic hepatitis, and the second group comprised cirrhotic patients with liver cirrhosis (LC; n 35) and hepatic carcinoma patients with liver cirrhosis (HCC; n 23). A total of 103 healthy control subjects who tested negative for HCV infection were selected randomly from blood donors (42 men and 61 women; mean age, 46 years; range, 30 75 years). Patients and controls were gender and age matched and were from the same geographical area. Blood donors were healthy individuals without any illness. Approval for the study was given by the National Ethical Committee, and informed consent was obtained from each patient and control subject. 2.2. DNA extraction Peripheral blood (5 ml) was collected in ethylenediaminetetraacetic acid (EDTA) tubes. Genomic DNA was extracted from peripheral leukocytes using the standard method (salting out procedure) [36] and stored at 20 C. Briey, blood cells were mixed with Triton lysis buffer (0.32 mol/l sucrose, 1% Triton X-100, 5 mmol/l MgCl2, 10 mmol/l Tris-HCl, pH 7.5). Leukocytes were spun down and washed with sterilized H2O. The pellet was incubated with proteinase K at 56 C and subsequently salted out at 4 C using a 5-mol/l NaCl solution. Precipitated proteins were removed by centrifugation. DNA in the supernatant uid was precipated with ethanol. The DNA pellet was dissolved in 400 l sterilized H2O and stored at 20 C until use. 2.3. IFN (+874 T/A) and IL-10 ( 1082 G/A) genotyping Genotyping for polymorphism in IFN ( 874T/A) and IL-10 ( 1082G/A) was carried out by the allele-specic PCR (AS-PCR) assay (IFN and IL-10 gene, GenBank Accession nos. J00219 and X78437, respectively). For the genotyping of IFN ( 874T/A), three primers described by Pravica et al. were used [20]: 5= primers specic for IFN 874T (5=-TTCTTACAACACAAAATCAAATCT-3=) or IFN 874A (5=-TTCTTACAACACAAAATCAAATCA-3=), were separately mixed to a 3= primer (5=-TCAACAAAGCTGATACTCCA-3=) under the following conditions: 10 l reaction mixture containing 25100 ng genomic DNA, 200 mol/l dNTPs, 2 mmol/l MgCl2, 1 Taq polymerase buffer, 0.5 unit of Taq DNA polymerase (Amersham, Paris, France), 1 mol/l of specic primer mix, and 0.3 mol/l of internal control primers mix, which amplify a human growth hormone sequence to check for successful PCR amplication (forward: 5=-GCCTTCCCAACCATTCCCTTA-3=; reverse: 5=-TCACGGATTTCTGTTGTGTTTC-3=). The reaction conditions used with the thermal cycler (Biometra, Gttingem, Germany) were as follows: one cycle of 96 C for 130 seconds and of 63 C for 60 seconds; nine cycles of 96 C for 10 seconds and 63 C for 60 seconds; and 20 cycles of 96 C for 10 seconds, 60 C for 50 seconds, and 72 C for 30 seconds. Similarly, primers described by Mullighan et al. were used to detect the G-to-A transition polymorphism at position 1082 of IL-10 gene [37]. The specic primer sequences used were as follows: the 3= primer 5=-CAGTGCCAACTGAGAATTTGG-3=; the IL-10 1082G: 5= CTACTAAGGCTTCTTTGGGAG-3=; the IL-10 1082A: 5=ACTACTA-

232

N. Bouzgarrou et al. / Human Immunology 70 (2009) 230-236

Table 1 Demographic, virologic, and clinical factors associated with chronic hepatitis severity Characteristic Chronic hepatitis (n 42) 51.0 7.6 LC/HCC (n 58) 61.6 9.8 p Value

Age (y) Gender Male Female HCV genotype HCV-1 HCV non-1 Not determined Pretreatment ALT levels UI/ml Pretreatment AST levels UI/ml

0.0001 NS

cies of allele, genotype, and combined genotypes were compared among the groups by multivariate logistic regression analysis. Data were analyzed using the Epi-Info statistical program (version 5.01a-1991; Centers for Disease Control and Epidemiology Program ofce, Atlanta, GA). Multivariate analysis was made using SPSS 13.0 software (SPSS Inc., Chicago, IL). 3. Results 3.1. Study populations Table 1 summarizes the main features of the patients studied. Patients were divided according to the presence or absence of cirrhosis. Some statistical differences were observed between cirrhotic and noncirrhotic patients in regard to age and HCV genotype using univariate analysis, and only in regard to age by multivariate analysis. Patients and controls were matched for age and gender, and differences were not statistically signicant (p 0.581 and p 0.06, respectively). 3.2. IFN (+874T/A) and IL-10 ( 1082G/A) allelic and genotypic distribution IFN and IL-10 allele and genotype frequencies in all collected samples are shown in Table 2. Genotype distributions in control subjects were consistent with Hardy-Weinberg equilibrium. No statistically signicant differences were observed between patients and controls for both polymorphisms. Genotype frequency analysis of IFN at position 874 between chronic hepatitis patients in regard to the disease severity (chronic hepatitis C or LC/HCC) showed a signicantly higher rate of TT and TA genotypes in patients with cirrhosis (0.7 vs 0.48; p 0.019). These genotypes were associated with an approximately 2.5-fold risk of progression to the above-mentioned severe forms of the disease (OR 2.65, 95% CI 1.07 6.63). In addition, the IFN 874T allele was found to be approximately two times more frequent in patients with end-stage hepatitis C than in patients with chronic hepatitis (OR 2; 95% CI 1.08 3.70, p 0.018). Multivariate analysis of IFN genotypes did not reveal the 874T carriage to be an independent positive predictor for disease severity. When comparing allele and genotype frequencies of the IL-10 1082, no signicant differences between the two groups of patients were observed.

8 (0.19) 34 (0.81) 32 (0.82) 7 (0.18) 3 99.8 80.8 78.3 54.6

20 (0.34) 38 (0.66) 34 (1) 0 (0) 24 83.0 48.2 89.2 43.0

0.012

NS NS

HCC, hepatocellular carcinoma in liver cirrhosis; LC, liver cirrhosis; NS, not significant.

AGGCTTCTTTGGGAA-3=. All reactions were performed in 10- l reaction volumes containing 25 100 ng genomic DNA, 200 mol/l dNTPs, 2 mmol/l MgCl2, 1 Taq polymerase buffer, 0.5 unit of Taq DNA polymerase (Paris, France, Amersham), 0.5 mol/l of specic primer mix, and 0.3 mol/l of internal control primer mix. Reaction conditions were as follows: 1 minute at 96 C followed by ve cycles of 96 C for 25 seconds, 70 C for 45 seconds, and 72 C for 45 seconds, followed by 21 cycles of 96 C for 25 seconds, 65 C for 40 seconds, and 72 C for 45 seconds, followed by four cycles of 96 C for 25 seconds, 55 C for 60 seconds, and 72 C for 120 seconds. The amplied products were separated by ethidium bromide stained 2% agarose gel electrophoresis and visualized with ultraviolet light. The interpretation of the polymerase chain reaction results was based on the presence of the internal control band together with the presence or absence of a specic amplied fragment. 2.4. Statistical analysis The allele frequencies were tested for the Hardy-Weinberg equilibrium using the 2 test. The same test was used to evaluate any signicant differences in the IFN and IL-10 allele/genotype frequencies between patients and controls and among subgroups of patients (chronic hepatitis vs LC/HCC). Values of p 0.05 were considered statistically signicant. Odds ratios (ORs) and 95% condence intervals (CIs) were calculated to assess the relative disease risk conferred by a specic allele or genotype. Genotype frequen-

Table 2 Differential distribution of the IFN- ( 874T/A) and IL-10 ( 1082G/A) genotypes in healthy controls and hepatitis C virus (HCV) patients at different stages of chronic HCV infection Controls (n 103) HCV patients (n 100) OR (95% CI) pa Chronic hepatitis (n 42) LC/HCC (n 58) OR (95% CI) pb

Genotype 874T/A IFNTT TA AA TT TA Allele T A Genotype IL-10 -1082G/A GG GA AA GG GA Allele G A

23 (0.22) 47 (0.46) 33 (0.32) 70 (0.68) 93 (0.45) 113 (0.55)

30 (0.30) 31 (0.31) 39 (0.39) 61 (0.61) 91 (0.45) 109 (0.55)

1.1 (0.512.40) 0.56 (0.281.12) 1 0.74 (0.401.37) (0.671.53) 1

0.786 0.076 0.229 0.942

10 (0.24) 10 (0.24) 22 (0.52) 20 (0.48) 30 (0.36) 54 (0.64)

20 (0.34) 21 (0.36) 17 (0.30) 41 (0.70) 61 (0.53) 55 (0.47)

2.59 (0.877.88) 2.72 (0.918.23) 1 2.65 (1.076.63) 2.00 (1.083.70) 1

0.056 0.043 0.019 0.018

12 (0.11) 49 (0.48) 42 (0.41) 61 (0.59) 73 (0.35) 133 (0.65)

19 (0.19) 43 (0.43) 38 (0.38) 62 (0.62) 81 (0.4) 119 (0.6)

1.75 (0.694.45) 0.97 (0.511.85) 1 1.12 (0.622.05) 1.24 (0.811.89) 1

0.192 0.920 0.685 0.293

9 (0.22) 19 (0.45) 14 (0.33) 28 (0.67) 37 (0.44) 47 (0.56)

10 (0.18) 24 (0.41) 24 (0.41) 34 (0.59) 44 (0.38) 72 (0.62)

0.65 (0.182.28) 0.74 (0.271.97) 1 0.71 (0.281.75) 0.78 (0.421.43) 1

0.445 0.502 0.413 0.384

CI, confidence interval; HCC, hepatocellular carcinoma in liver cirrhosis; OR, odds ratio.
a b

Patients vs controls. Liver cirrhosis/hepatocellular carcinoma (LC/HCC) vs chronic hepatitis.

N. Bouzgarrou et al. / Human Immunology 70 (2009) 230-236

233

Table 3 Combined effects of the IFN- and IL-10 genotypes on chronic hepatitis C virus (HCV) infection severity Genotype Controls (n 103) HCV patients (n 100) OR (95% CI) pa Chronic hepatitis (n 42) 15 (0.36) 7 (0.17) 14 (0.33) 6 (0.14) LC/HCC (n 58) OR (95% CI) pb

IFNIFNIFNIFN-

low/IL-10high low/IL-10low high/IL-10high high/IL-10low

19 (0.18) 14 (0.14) 42 (0.41) 28 (0.27)

23 (0.23) 16 (0.16) 40 (0.40) 21 (0.21)

1.61 (0.654.04) 1.52 (0.554.21) 1.27 (0.592.76) 1

0.257 0.365 0.510

8 (0.14) 9 (0.15) 26 (0.45) 15 (0.26)

0.21 (0.050.9) 0.51 (0.102.47) 0.74 (0.202.68) 1

0.015 0.337 0.611

CI, confidence interval; HCC, hepatocellular carcinoma in liver cirrhosis; LC, liver cirrhosis; OR, odds ratio.
a b

Patients vs controls. Liver cirrhosis/hepatocellular carcinoma (LC/HCC) vs chronic hepatitis.

3.3. Combined effect of IFN (+874T/A) and IL-10 ( 1082G/A) gene polymorphisms Considering the inhibitory effect of IL-10 on IFN synthesis, the effect of IFN and IL-10 combined genotypes on chronic hepatitis C outcome was analyzed. Genotypes were grouped into high or low producer genotypes. For IFN , TT and TA genotypes at position 874 were considered as high producer genotypes (IFN high producer) and AA genotype as a low producer genotype (IFN low producer). For IL-10, GG and GA at position 1082 were grouped into high producer genotypes (IL-10high producer) and AA genotype was regarded as a low producer genotype (IL-10low producer). No signicant differences in IFN and IL-10 combined genotype distributions between patients and controls were observed (Table 3). IFN low producer and IL-10high producer combined genotypes were signicantly more common in patients with chronic hepatitis C compared with those with LC or HCC (0.36 vs 0.14; OR 0.21, 95% CI 0.05 0.9, p 0.015). In contrast, IFN low producer and IL-10low producer combined genotypes frequency distribution in chronic hepatitis and LC/HCC patients was comparatively similar (0.17 vs 0.15). The simultaneous occurrence of IFN low producer and IL-10high producer genotypes seems to have a protective effect against disease aggressiveness. 4. Discussion Hepatitis C virus infection is the leading cause of chronic liver disease worldwide and may, in some patients, develop into LC and HCC. There is increasing evidence that the immune response and the host genetic background play a prominent role in hepatitis C pathogenesis and interindividual heterogeneity of disease outcome. A shift to Th1-type cytokines has been associated with an increased necroinammatory activity and liver brosis [13,15,19,38]. It has been widely proposed that the necrosis of hepatocytes resulting from chronic inammation and subsequent regeneration enhances mutagenesis and proto-oncogene activation in the host cells, leading to HCC [39,40]. Cytokine production varies among individuals and is associated with certain mutations within the coding and the regulatory regions [16]. In a previous study we found that the 607 polymorphism of the pro-inammatory IL-18 cytokine was associated with a higher risk for LC and HCC [41]. In this study, we investigated the inuence of single nucleotide polymorphisms of IFN , a pro-inammatory cytokine, and IL-10, an anti-inammatory cytokine, on chronic hepatitis C severity. Despite the similarities of genotype and allele distributions of IFN ( 874 T/A) and IL-10 ( 1082 G/A) in HCV patients and controls, we found an interesting and signicant association between the inheritance of the T allele at position 874 of IFN gene and the worst outcomes of chronic hepatitis infection. Our results demonstrated that, in chronic HCV infection, the IFN TT and TA genotypes were associated with an approximately 2.5-fold risk of progression

to LC and possibly to HCC. Concordant results have been reported by a recent large-scale study conducted by Dai et al. on Taiwanese HCV-infected patients [42]. A higher incidence of liver LC was found in patients with the T allele at position 874 of the IFN gene compared with those homozygotic for A allele [42]. Some associations between the IFN ( 874 T/A) polymorphism and inammation or brosis were provided by other studies [22,43]. A 2-year follow-up of Italian hemodialysis patients with chronic renal failure showed a preventive effect of AA genotype on C-reactive protein (CRP) elevation in comparison to patients with TT or TA genotypes [22]. In addition, the 12 CA repeat microsatellite allele, in complete linkage disequilibrium with the T allele at position 874, has been previously associated with lung allograft brosis [43]. 874 However, the ndings about the associations between IFN T/A polymorphism and brosis in hepatitis C are conicting. Barrett et al. showed no signicant association between this polymorphism and viral clearance or the severity of liver disease in HCVinfected women [44]. In a study conducted in 40 chronic hepatitis patients, Abbas et al. found no signicant association with brosis severity [45]. The relationship between IFN and brogenesis is still unclear. Despite its anti-brogenic properties, IFN participates in the immune response involved in brogenesis. Several studies provide evidence that IFN plays a prominent role in Th1-type cellular response leading to inammation and liver brosis in HBV- or HCV-related chronic liver disease. Tang et al. reported an increase in IFN level in patients with HBV-related active LC, whereas IL-10 was higher in patients with stationary liver LC [46]. Gigi et al. showed a predominance of IFN mRNA expression in PBMCs isolated from patients with chronic hepatitis C and stimulated with NS3 nonstructural HCV antigen [13]. These investigators also reported a signicant correlation between IFN expression and the stage of brosis. The inefcient eradication of HCV in chronic hepatitis C might be responsible for the continuous stimulation of cellular immune response leading to nonspecic tissue damage. In addition, the long-lasting inammation of the liver was reported to enhance hepatocellular carcinogenesis. Indeed, in the case of chronic hepatitis C, an anti-inammatory treatment has been shown to decrease the rate of hepatocellular carcinogenesis [47]. We also previously reported a higher level of IL-18, initially known as IFN -inducing factor, in HCC patients [41]. A study conducted by Qiu et al. [48] demonstrated a predominance of IFN in primary hepatic cancer tissues. The implication of IFN in carcinogenesis was further demonstrated by Hanada et al., who reported a benecial effect of anti-IFN antibody on colon tumor development [49]. In chronic hepatitis C, an enzyme-catalyzing nitric oxide expression, the inducible nitric oxide synthase (iNOS), was shown to be responsible for disease severity [50], DNA damage, and progression to HCC [51]. One of the mechanisms explaining the link between chronic inammation and tumor genesis is the iNOS induction. There is increasing evidence that iNOS overexpression is involved

234

N. Bouzgarrou et al. / Human Immunology 70 (2009) 230-236

in carcinogenesis in several human gastrointestinal neoplasms including HCC [52]. Considering the fact that iNOS expression is upregulated in an IFN manner, it may be speculated that the implication of IFN in hepatitis C severity is related to iNOS expression level. However, further studies investigating the relationship between IFN ( 874T/A) polymorphisms and iNOS and/or NO expression should be carried out to conrm this hypothesis. IFN synthesis is downregulated by IL-10, a potent antiinammatory cytokine that also has a modulatory effect on hepatic brogenesis [53]. Louis et al. have reported the antibrogenic property of IL-10 in carbon tetrachloride (CCl4)treated mice [54]. More recently, chronic hepatitis C patients treated with recombined IL-10 showed a decrease in liver brosis [26, 27]. IL-10 production is thought to be genetically controlled [28]. Thus we wanted to nd out whether the 1082G/A promoter polymorphism affecting IL-10 expression could inuence chronic hepatitis severity. A previous study conducted on Argentinean HCV-infected patients has found an association between the inheritance of G allele at position 1082 and a lower risk of progression to LC [55]. In addition, a Japanese population study has reported an association between the IL-10 (GCC), a high-producer haplotype, and a reduction in liver brosis [56]. However, in the current study, we have not found any association between IL-10 ( 1082G/A) polymorphism and hepatitis C susceptibility or aggressiveness. The frequency of G allele was comparable in the two groups of patients (0.44 for chronic hepatitis and 0.38 for LC/HCC). So far, no studies have reported the combined effect of IFN / IL-10 gene polymorphisms on chronic hepatitis C severity. Interestingly, we observed a signicantly higher frequency of an antiinammatory IFN low producer/IL-10high producer combined genotype in patients with chronic hepatitis C in comparison to those with liver LC or HCC (0.36 vs 0.14; OR 0.21, 95% CI 0.05 0.9, p 0.015). It appears that the pro-/anti-inammatory response imbalance mediated by polymorphisms in IFN and IL-10 genes may inuence the outcome of chronic HCV infection. However, a recent study of Falleti et al. suggested that IFN high producer/IL-10low producer genotypes were signicantly associated with a slower brosis progression in comparison to IL-10 high producer/IFN low producer genotypes. This inconsistency with our results could be explained by patient characteristics. Indeed, in the Falleti et al. study, the study group included patients who underwent liver transplantation for end-stage liver disease and who were receiving immunosuppressive treatment. In addition, both donor age and transplanted liver may inuence brosis progression [57]. Throughout the study of the functional polymorphisms of TNF- and IL-10, Lio et al. established a positive association of the combined TNF- low high producer producer and IL-10 genotypes and the recovery from HCV infection compared with patients with persistent HCV infection [58]. Furthermore, the combined analysis of pro-/anti-inammatory cytokine polymorphisms, IL-6high producer and IL-4low producer combined genotypes, was studied as well and was found to be implicated in a higher disease risk among end-stage renal disease patients [59]. Moreover, if we take into account the implication of Th1 and Th2 imbalance in the aggressiveness of hepatitis C outcomes, our results will be in accordance with previous studies that described a shift to Th1 cytokine prole in patients with advanced liver injury [1315,19]. In general, the present ndings, along with others, suggest an apparent role of SNPs in the progression of liver brosis and or LC in patients with chronic hepatitis C. Recently, Sermasathanasawadi et al. reported that two SNPs of the interferon regulatory factor 7 (IRF-7) gene, 1047A/G SNP and 2157A/G SNP genotypes, were strongly associated with LC. They suggested the 1047G and 2157G alleles should be used as markers of host factors associated with a higher risk of LC in Japanese patients with chronic HCV infection [60]. In a large-scale population study, Huang et al. per-

formed a gene-centric disease association study of 24,832 putative functional SNPs [61]. They found that patients with chronic hepatitis C carrying DDX5 minor allele or DDX5-POLG2 haplotypes were at increased risk for developing advanced brosis, whereas those carrying the CPT1A minor allele were at decreased risk [61]. Subsequently, Huang et al. completed the previous study and tried to develop a LC risk score (CRS) based on genetic markers for Caucasian patients [62]. Seven SNPs, one SNP in the antizyme-inhibitor1 gene (AZIN1), one SNP in the Toll-like receptor-4 gene (TLR4) and ve SNPs in ve genes of unclear function were used to dene the CRS signature [62]. Although these associations between SNPs and the presence of LC are strong, they may differ from one patient population to another. The role of SNPs should also be evaluated and validated in other chronic HCV patient groups [63]. In fact, some controversial ndings have been reported in different studies estimating polymorphism association. For instance, the 1082 position of the IL-10 gene was found to be associated with LC in HCV patients from the United Kingdom [64]. This is not the case in the current study. The differences in IFN ( 874T/A) as well as in IL10(1082G/A) allele frequencies between racial or ethnic groups were observed [65,66]. Compared with Caucasians, Asians demonstrated some marked differences in IFN and IL-10 genotype frequencies. They have a signicantly higher proportion of genotypes that result in low IL-10 and low IFN production. These ethnicitybased differences could at least partly explain the discrepancy observed in the association of alleles and genotypes of the cytokine genes with the disease. Further studies on a large multiethnic cohort are required to conrm the associations reported. In chronic hepatitis C, brosis progression results from the complex and multifactorial interaction between exogenous (viral) factors and genetic (host) determinants. This study indicates 874T allele that in Tunisian HCV-infected patients, the IFN may be a susceptibility factor for end-stage chronic hepatitis C. We also observed that patients with simultaneous inheritance of IFN low producer and IL-10high producer genotypes have a lower risk for developing LC and possibly later HCC. These ndings can provide more evidence concerning the role of interindividual genetic variations in cytokine genes in hepatitis C pathogenesis. Candidate genetic markers of brosis progression or of LC development should be studied in several large cohorts of patients together with exogenous factors that may inuence the natural course of chronic HCV infection [63]. Because pro-/anti-inammatory cytokines play a key role in the development of liver injury, genomic scanning for SNPs in the genes of several important inammatory mediators needs to be further investigated. These investigations could help to identify patients with markedly increased risks of disease progression and could guide the design of individualized treatment strategies for chronic hepatitis C. Acknowledgments This work was supported by the Ministre de lEnseignement Suprieur de la Recherche Scientique et de la Technologie (LR/05 Hpatites et maladies virales pidmiques), and by the Ministre de la Sant Publique de la Rpublique Tunisienne. We thank Adel Rdissi for English revision. References
[1] Boyer N, Marcellin P. Pathogenesis, diagnosis and management of hepatitis C. J Hepatol 2000;32:98 112. [2] Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol 2005;5:21529. [3] Triki H, Said N, Ben Salah A, Arrouji A, Ben Ahmed F, Bouguerra A, et al. Seroepidemiology of hepatitis B, C and delta viruses in Tunisia. Trans R Soc Trop Med Hyg 1997;91:11 4. [4] Mejri S, Ben Salah A, Triki H, Ben Alaya N, Djebbi A, Dellagi K. Contrasting patterns of hepatitis C virus infection in two regions from Tunisia. J Med Virol 2005;76:18593.

N. Bouzgarrou et al. / Human Immunology 70 (2009) 230-236

235

[5] Reid AE, Koziel MJ, Aiza I, Jeffers L, Reddy R, Schiff E, et al. Hepatitis C virus genotypes and viremia and hepatocellular carcinoma in the United States. Am J Gastroenterol 1999;94:1619 26. [6] Thio CL, Thomas DL, Carrington M. Chronic viral hepatitis and the human genome. Hepatology 2000;31:819 27. [7] Bertoletti A, Ferrari C. Kinetics of the immune response during HBV and HCV infection. Hepatology 2003;38:4 13. [8] Cramp ME, Carucci P, Rossol S, Chokshi S, Maertens G, Williams R, et al. Hepatitis C virus (HCV) specic immune responses in anti-HCV positive patients without hepatitis C viraemia. Gut 1999;44:424 9. [9] Pape GR, Gerlach TJ, Diepolder HM, Gruner N, Jung M, Santantonio T. Role of the specic T-cell response for clearance and control of hepatitis C virus. J Viral Hepat 1999;6:36 40. [10] Marinho RT, Pinto R, Santos ML, Lobos IV, Moura MC. Effects of interferon and ribavirin combination therapy on CD4 proliferation, lymphocyte activation, and Th1 and Th2 cytokine proles in chronic hepatitis C. J Viral Hepat 2004; 11:206 16. [11] Trapero-Marugn M, Garca-Buey L, Muoz C, Quintana NE, MorenoMonteagudo JA, Borque MJ, et al. Sustained virological response to peginterferon plus ribavirin in chronic hepatitis C genotype 1 patients is associated with a persistent Th1 immune response. Aliment Pharmacol Ther 2006;24: 11728. [12] Kawakami Y, Nabeshima S, Furusyo N, Sawayama Y, Hayashi J, Kashiwagi S. Increased frequency of interferon-gamma-producing peripheral blood CD4 T cells in chronic hepatitis C virus infection. Am J Gastroenterol 2000; 95:3670 3. [13] Gigi E, Raptopoulou-Gigi M, Kalogeridis A, Masiou S, Orphanou E, Vrettou E, et al. Cytokine mRNA expression in hepatitis C virus infection: Th1 predominance in patients with chronic hepatitis C and Th1-Th2 cytokine prole in subjects with self-limited disease. J Viral Hepat 2008;15:14554. [14] Sobue S, Nomura T, Ishikawa T, Ito S, Saso K, Ohara H, et al. Th1/Th2 cytokine proles and their relationship to clinical features in patients with chronic hepatitis C virus infection. J Gastroenterol 2001;36:544 51. [15] Falasca K, Ucciferri C, Dalessandro M, Zingariello P, Mancino P, Petrarca C, et al. Cytokine patterns correlate with liver damage in patients with chronic hepatitis B and C. Ann Clin Lab Sci 2006;36:144 50. [16] Ollier WE. Cytokine genes and disease susceptibility. Cytokine 2004;28: 174 78. [17] Mihm S, Hutschenreiter A, Fayyazi A, Pingel S, Ramadori G. High inammatory activity is associated with an increased amount of IFNgamma transcripts in peripheral blood cells of patients with chronic hepatitis C virus infection. Med Microbiol Immunol 1996;185:95102. [18] Bertoletti A, DElios MM, Boni C, De Carli M, Zignego AL, Durazzo M, et al. Different cytokine proles of intraphepatic T cells in chronic hepatitis B and hepatitis C virus infections. Gastroenterology 1997;112:1939. [19] Napoli J, Bishop GA, McGuinness PH, Painter DM, McCaughan GW. Progressive liver injury in chronic hepatitis C infection correlates with increased intrahepatic expression of Th1-associated cytokines. Hepatology 1996;24:759 65. [20] Pravica V, Perrey C, Stevens A, Lee JH, Hutchinson IV. A single nucleotide polymorphism in the rst intron of the human IFNgamma gene: Absolute correlation with a polymorphic CA microsatellite marker of high IFNgamma production. Hum Immunol 2000;61:863 6. [21] Pravica V, Asderakis A, Perrey C, Hajeer A, Sinnott PJ, Hutchinson IV. In vitro production of IFNgamma correlates with CA repeat polymorphism in the human IFNgamma gene. Eur J Immunogenet 1999;26:13. [22] Biolo G, Amoroso A, Savoldi S, Bosutti A, Martone M, Pirulli D, et al. Association of interferon-gamma 874A polymorphism with reduced long-term inammatory response in haemodialysis patients. Nephrol Dial Transplant 2006;21: 131722. [23] Anuradha B, Rakh SS, Ishaq M, Murthy KJ, Valluri VL. Interferon-gamma low producer genotype 874 overrepresented in Bacillus Calmette-Guerin nonresponding children. Pediatr Infect Dis J 2008;27:3259. [24] DAndrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon-gammaproduction by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med 1993;178:1041 8. [25] Tsukamoto H. Is interleukin-10 antibrogenic in chronic liver injury? Hepatology 1998;28:17079. [26] Nelson DR, Lauwers GY, Lau JY, Davis GL. Interleukin 10 treatment reduces brosis in patients with chronic hepatitis C: A pilot trial of interferon nonresponders. Gastroenterology 2000;118:655 60. [27] Nelson DR, Tu Z, Soldevila-Pico C, Abdelmalek M, Zhu H, Xu YL, et al. Long-term interleukin 10 therapy in chronic hepatitis C patients has a proviral and anti-inammatory effect. Hepatology 2003;38:859 68. [28] Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV. An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet 1997;24:1 8. [29] Crawley E, Kay R, Sillibourne J, Patel P, Hutchinson I, Woo P. Polymorphic haplotypes of the interleukin-10 5= anking region determine variable interleukin-10 transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis. Arthritis Rheum 1999;42:1101 8. [30] Yilmaz V, Yentur SP, Saruhan-Direskeneli G. IL-12 and IL-10 polymorphisms and their effects on cytokine production. Cytokine 2005;30:188 94. [31] Stanilova SA, Miteva LD, Karakolev ZT, Stefanov CS. Interleukin-10-1082 promoter polymorphism in association with cytokine production and sepsis susceptibility. Intensive Care Med 2006;32:260 6.

[32] Miteva L, Stanilova S. The combined effect of interleukin (IL)-10 and IL-12 polymorphisms on induced cytokine production. Hum Immunol 2008;69: 562 6. [33] Knapp S, Hennig BJ, Frodsham AJ, Zhang L, Hellier S, Wright M, et al. Interleukin-10 promoter polymorphisms and the outcome of hepatitis C virus infection. Immunogenetics 2003;55:3629. [34] Minton EJ, Smillie D, Smith P, Shipley S, McKendrick MW, Gleeson DC, et al. Trent Hepatitis C Study Group. Clearance of hepatitis C virus is not associated with single nucleotide polymorphisms in the IL-1, -6, or -10 genes. Hum Immunol 2005;66:12732. [35] Huang Y, Yang H, Borg BB, Su X, Rhodes SL, Yang K, et al. A functional SNP of interferon-gamma gene is important for interferon-alpha-induced and spontaneous recovery from hepatitis C virus infection. Proc Natl Acad Sci U S A 2007;104:98590. [36] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. [37] Mullighan CG, Marshall SE, Bunce M, Welsh KI. Variation in immunoregulatory genes determines the clinical phenotype of common variable immunodeciency. Genes Immun 1999;1:137 48. [38] Baroni GS, Pastorelli A, Manzin A, Benedetti A, Marucci L, Solforosi L, et al. Hepatic stellate cell activation and liver brosis are associated with necroinammatory injury and Th1-like response in chronic hepatitis C. Liver 1999;19: 2129. [39] Farinati F, Cardin R, Bortolami M, Burra P, Russo FP, Rugge M, et al. Hepatitis C virus: From oxygen free radicals to hepatocellular carcinoma. J Viral Hepat 2007;14:8219. [40] Matsuzaki K, Murata M, Yoshida K, Sekimoto G, Uemura Y, Sakaida N, et al. Chronic inammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma. Hepatology 2007;46:48 57. [41] Bouzgarrou N, Hassen E, Schvoerer E, Stoll-Keller F, Bahri O, Gabbouj S, et al. Association of interleukin-18 polymorphisms and plasma level with the outcome of chronic HCV infection. J Med Virol 2008;80:60714. [42] Dai CY, Chuang WL, Hsieh MY, Lee LP, Hou NJ, Chen SC, et al. Polymorphism of interferon-gamma gene at position 874 and clinical characteristics of chronic hepatitis C. Transl Res 2006;148:128 33. [43] Awad M, Pravica V, Perrey C, El Gamel A, Yonan N, Sinnott PJ, et al. CA repeat allele polymorphism in the rst intron of the human interferon-gamma gene is associated with lung allograft brosis. Hum Immunol 1999;60:343 6. [44] Barrett S, Collins M, Kenny C, Ryan E, Keane CO, Crowe J. Polymorphisms in tumour necrosis factor-alpha, transforming growth factor-beta, interleukin10, interleukin-6, interferon-gamma, and outcome of hepatitis C virus infection. J Med Virol 2003;71:212 8. [45] Abbas Z, Moatter T, Hussainy A, Jafri W. Effect of cytokine gene polymorphism on histological activity index, viral load and response to treatment in patients with chronic hepatitis C genotype 3. World J Gastroenterol 2005;11:6656 61. [46] Tang JT, Fang JY, Gu WQ, Li EL. T cell immune response is correlated with brosis and inammatory activity in hepatitis B cirrhotics. World J Gastroenterol 2006;12:30159. [47] Ikeda K, Saitoh S, Arase Y, Chayama K, Suzuki Y, Kobayashi M, et al. Effect of interferon therapy on hepatocellular carcinogenesis in patients with chronic hepatitis type C: A long-term observation study of 1,643 patients using statistical bias correction with proportional hazard analysis. Hepatology 1999;29: 1124 30. [48] Qiu FB, Wu LQ, Lu Y, Zhang S, Zhang BY. Predominant expression of Th1-type cytokines in primary hepatic cancer and adjacent liver tissues. Hepatobiliary Pancreat Dis Int 2007;6:63 6. [49] Hanada T, Kobayashi T, Chinen T, Saeki K, Takaki H, Koga K, et al. IFNgammadependent, spontaneous development of colorectal carcinomas in SOCS1decient mice. J Exp Med 2006;203:13917. [50] Atik E, Onlen Y, Savas L, Doran F. Inducible nitric oxide synthase and histopathological correlation in chronic viral hepatitis. Int J Infect Dis 2008;12: 125. [51] Machida K, Cheng KT, Sung VM, Lee KJ, Levine AM, Lai MM. Hepatitis C virus infection activates the immunologic (type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes. J Virol 2004;78:8835 43. [52] Sun MH, Han XC, Jia MK, Jiang WD, Wang M, Zhang H, et al. Expressions of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma. World J Gastroenterol 2005;11:59317. [53] Zhang LJ, Wang XZ. Interleukin-10 and chronic liver disease. World J Gastroenterol 2006;12:16815. [54] Louis H, Van Laethem JL, Wu W, Quertinmont E, Degraef C, Van den Berg K, et al. Interleukin-10 controls neutrophilic inltration, hepatocyte proliferation, and liver brosis induced by carbon tetrachloride in mice. Hepatology 1998; 28:160715. [55] Paladino N, Fainboim H, Theiler G, Schroder T, Muoz AE, Flores AC, et al. Gender susceptibility to chronic hepatitis C virus infection associated with interleukin 10 promoter polymorphism. J Virol 2006;80:9144 50. [56] Hamada H, Yatsuhashi H, Yano K, Arisawa K, Nakao K, Yano M. Interleukin-10 promoter polymorphisms and liver brosis progression in patients with chronic hepatitis C in Japan. J Hepatol 2003;39:457 8. [57] Falleti E, Fabris C, Toniutto P, Fontanini E, Cussigh A, Caldato M, Rossi E, Bitetto D, Minisini R, Smirne C, Pirisi M. Genetic polymorphisms of inammatory

236

N. Bouzgarrou et al. / Human Immunology 70 (2009) 230-236

[58]

[59]

[60]

[61]

cytokines and liver brosis progression due to recurrent hepatitis C. J Interferon Cytokine Res 2007;27:239 46. Lio D, Caruso C, Di Stefano R, Colonna Romano G, Ferraro D, Scola L, et al. IL-10 and TNF-alpha polymorphisms and the recovery from HCV infection. Hum Immunol 2003;64:674 80. Mittal RD, Manchanda PK. Association of (IL)-4 intron 3 and IL-6 -174 G/C gene polymorphism with susceptibility to end-stage renal disease. Immunogenetics 2007;59:159 65. Sermasathanasawadi R, Kato N, Muroyama R, Dharel N, Shao RX, Chang JH, et al. Association of interferon regulatory factor-7 gene polymorphism with liver cirrhosis in chronic hepatitis C patients. Liver Int 2008;28:798 806. Huang H, Shiffman ML, Cheung RC, Layden TJ, Friedman S, Abar OT, et al. Identication of two gene variants associated with risk of advanced brosis in patients with chronic hepatitis C. Gastroenterology 2006;130: 1679 87.

[62] Huang H, Shiffman ML, Friedman S, Venkatesh R, Bzowej N, Abar OT, et al. A 7 gene signature identies the risk of developing cirrhosis in patients with chronic hepatitis C. Hepatology 2007;46:297306. [63] Papatheodoridis GV, Paraskevis D. Role of genetic polymorphisms in the progression of liver brosis in chronic hepatitis C virus infection. Liver Int 2008; 28:764 6. [64] Knapp S, Hennig BJ, Frodsham AJ, Zhang L, Hellier S, Wright M, et al. Interleukin-10 promoter polymorphisms and the outcome of hepatitis C virus infection. Immunogenetics 2003;55:3629. [65] Hoffmann SC, Stanley EM, Cox ED, DiMercurio BS, Koziol DE, Harlan DM, et al. Ethnicity greatly inuences cytokine gene polymorphism distribution. Am J Transplant 2002;2:560 7. [66] Farhat K, Hassen E, Gabbouj S, Bouaouina N, Chouchane L. Interleukin-10 and interferon-gamma gene polymorphisms in patients with nasopharyngeal carcinoma. Int J Immunogenet 2008;35:197205.