Association of cytokine genetic polymorphism with hepatites B infection evolution in adult patients

ARTICLES

Association of cytokine genetic polymorphism with hepatites B infection evolution in adult patients

Cátia Silene Sversuti Ribeiro; Jeane Eliete Laguila Visentainer; Ricardo Alberto Moliterno ⁺

Departamento de Análises Clínicas, Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900 Maringá, PR, Brasil

ABSTRACT

The infection by the hepatitis B virus (HBV) has different forms of evolution, ranging from self-limited infection to chronic hepatic disease. The objective of this study was to evaluate the influence of cytokine genetic polymorphisms in the disease evolution. The patients were divided into two groups, one with chronic HBV (n = 30), and the other with self-limited infection (n = 41). The genotyping for TNF (-308), TGFB1 (+869, +915), IL-10 (1082, -819, and -592), IL-6 (-174), and IFNG (+874) was accomplished by the PCR-SSP (polymerase chain reaction with sequence specific primers technique using the One Lambda kit. Although no statistically significant differences were found between the groups, the combination of TNF -308GG and IFNG +874TA was found in a lower frequency in chronic patients than in individuals with self-limited infection (26.7 versus 46.3%; P = 0.079; OR = 0.40; IC95% = 0.14-1.11). In chronic patients with histological alterations it was not observed the genotype TGFB1+869 C/C, against 24.4% in the self limited infection group (100 versus 75.6%; P = 0.096; OR = 7.67; IC95% = 0.42-141.63). Further studies in other populations, and evaluation of a greater number of individuals could contribute for a better understanding of the cytokine genetic polymorphism influence in HBV infection evolution.

Key words: cytokine gene polymorphism - hepatitis B - genetic association

The infection by the hepatitis B virus (HBV) appears under different forms of evolution, ranging from the asymptomatic and self-limited infection to the chronic state, which can develop into chronic hepatitis, cirrhosis, and hepatic-cellular carcinoma (Thomas & Strickland 2000). It is estimated that the virus infected 350 million people in the whole world, forming a reservoir which facilitates the spreading of the disease (Kane 1995). Furthermore, in spite of the transmission reduction after the vaccination advent (Shih et al. 1999, Berlioz-Arthaud et al. 2003), the infection remains as an important cause of chronic hepatic disease (Thomas & Strickland 2000).

The Health Ministry estimates that 1% of the Brazilian population has chronic diseases related to HBV (Ministério da Saúde 2003, Ferreira 2004). The factors that lead a patient with acute infection by HBV to become persistently infected are not totally established. Environmental factors as well as factors innate to the virus do not completely explain the different forms of the HBV infection evolution (Chu & Lok 2002, Kao et al. 2002), bringing up a discussion of the implication of genetic factors in disease evolution (Thio et al. 1999, Thursz 2001).

The initial anti-HBV immune response promotes the killing of infected cells by the virus and the secretion of antiviral cytokines by cells of the innate immunity (Ferrari et al. 2003). However, the infection control is not reached at this stage of HBV response. A vigorous and polyclonal immune response of T cells is pointed out as fundamental for the virus elimination and disease resolution (Guidotti et al. 1999). The well-successful response to HBV is a result of both specific anti-virus cytotoxic T-cells activity, which eliminates the infected cells, and non-cytolitic mechanisms exerted through cytokines released by T and non-T cell infiltrates. The cytokines are able to suppress the viral expression and replication, mediating the infection control without causing death of the infected cell. The evolution of the infection to chronicity is associated to a weak or undetectable humoral immune response, and is identified by the persistency of HBV's surface antigen (HBsAg) in the circulation (Jung & Pape 2002, Ferrari et al. 2003).

Several studies have evaluated the influence of genetic factors in the cytokine production by cells of the immune system. The genetic polymorphism was pointed out because it correlates to both the transcription level and in vitro production of tumor necrosis factor alfa (TNF-a) (Kroeger et al. 1997, Westendorp et al. 1997, Hajeer & Hutchinson 2001). The genotypic variation of interferon gama (IFN)(Pravica et al. 1999), interleukin 6 (IL-6) (Fishman et al. 1998), T cell growth factor beta (TGFB1) (Awad et al. 1998) and interleukin 10 (IL-10) (Crawley et al. 1999) were also shown to influence its production.

As cytokines play a fundamental role in the immune response to HBV, and HBV infection may present different forms of evolution (self-limited or persistent and progressive), we decided to evaluate the correlation of the INFG, TNF, IL-6, IL-10, and TGFB1 genetic polymorphisms with the disease evolution. These cytokines can affect the infection control either by suppressing viral replication (INF-g, TNF-a and IL-6), or inhibiting the T-cell activation (IL-10, TGF-b).

PATIENTS AND METHODS

Patients - Thirty white Brazilian patients with chronic infection by HBV seen at Ambulatório de Doenças Infecciosas-Hepatite from Hospital Universitário Regional de Maringá (HURM) and at Ambulatório de Hepatite da Policlínica Zona Sul from Maringá municipality, Paraná, were enrolled in this study. All patients were serum positive for the HBV surface antigen (HBsAg) and 26 of them were serum positive in two or more tests performed in a time interval higher than six months. From the other four patients, in one of them the hepatic biopsy accomplished 14 months after the HBsAg test showed the presence of active chronic hepatitis in cirrhosis phase. The other three patients were negative for anti-HBs antibodies with intervals higher than six months after the HBsAg test and presented normal transaminase values in two or more evaluations.

A second group comprising 41 white Brazilian candidates for blood donation that showed positive results for the antibody against both the HBV core (anti-HBc) and the surface antigen of HBV (anti-HBs) was also enrolled. In this study, this group was named as self-limited infection group (Table I). All of the patients and blood donors gave the informed consent to participate in the study, which was approved by the Ethics Committee in Research with Human Beings of Maringá State University. All the participants of this study are white-Brazilians from Paraná, Brazil. Paraná's white population is predominantly of European origin (80.6%), with a small but significant contribution of African (12.5%) and Amerindians (7%) genes (Probst et al. 2000).

DNA extraction and genotyping of the cytokine genes - Genomic DNAs were extracted and purified from whole blood collected with ACD (acid citric dextrose) using EZ-DNA kit (Biological Industries, Israel), according to the manufacture's instructions.

The single nucleotide polymorphisms (SNPs) were analyzed for TNF (-308 G or A), TGFB1 (+869 T or C, and +915 C or G), IL-10 (-1082 A or G, -819 T or C, and -592 A or C), IL-6 (-174 C or G), and IFNG (+874 T or A) by polymerase chain reaction (PCR) with sequence specific primers through the Cytokine Genotyping Primers Kit (One Lambda^Ò, CA, US). All amplifications were performed according to the manufacturer's recommendations. The PCR products were then visualized by electrophoresis in 2% agarose gel stained with ethidium bromide.

Statistical analysis - The allelic and genotypic frequencies of each SNP were obtained by direct counting. The cytokine genetic polymorphism distribution of both HBV chronic patient group (CP) and self-limited infection group (SLI) were compared by Qui-square test and/or Fisher exact test with the aid of the Statistica 6.0 program. The P values smaller or equal to 0.05 were considered significant. For each comparison, it was calculated the value of OR (odds ratio), using Haldane's modifications of the Wolf method when necessary.

RESULTS

Tables II and ^III summarize, respectively, the cytokine genotype distribution and allelic frequencies of patients with either self-limited infection (n = 41), or with chronic hepatitis (n = 30).

The genotype and the allele frequencies between these groups showed no significant statistical differences for both TNF and IFNG polymorphisms. However, the genotypic combination of TNF -308 G/G with IFNG +874 T/A showed a lower frequency in chronic patients (25 versus 47.5%; P = 0.079; OR = 0.37; IC 95% = 0.13-1.01).

The TGFB1 +869 and +915 genotypic and allelic frequencies between the groups with self-limited infection and chronic patients were not statistically significant. However, 11 of the 30 chronic hepatitis patients showed histological alterations confirmed by biopsy, and it was not observed the genotype TGFB1+869 C/C in this group, against 24.4% in the self limited infection group (Table IV).

The genotypic and allelic distribution related to the three SNPs of IL-10 (-1082 A or G, -819 T or C, and -592 A or C) did not show any statistical difference among the studied groups.

Considering the genotype combinations, it was also verified a higher frequency of TGFB1 +869 T/C with IL-10 -819 C/C in chronic patients with histological alterations, as compared to the self-limited infection group (54.5% versus 24.4%; P = 0.073; OR = 3.72; IC 95% = 0.93-14.85).

The polymorphism of IL-6 -174 did not show significant differences in relation to alleles, genotypes or phenotypic expression. Only 8.7% of individuals showed the C/C genotype.

DISCUSSION

Cytokines are soluble polypeptides that act in defense against viral infections, either directing the Th1/Th2 host's immune response predominant pattern, or directly inhibiting viral replication. Nucleotide variations in regulatory gene regions can result in two or more alleles for each cytokine locus, which in some cases manifest by the increase or decrease of cytokine gene transcription (Bidwell et al. 1998).

Studies on the cytokine gene polymorphisms points out to its role on the evolution of HBV infection, ranging from the pathogenesis of fulminating hepatitis (Leifeld et al. 2002) until its action in viral replication inhibition (Chen et al. 2005). Studies in HBV infected patients point out to the role of IFN-g in the infection control. Suri et al. (2001), investigating the non-cytolitic activity over the patients' hepatocites with acute and chronic infection observed the action of IFN-g as a viral replication inhibitor. Recently, Szkaradkiewicz et al. (2003) related the Th1 activity to self-limited hepatitis B recuperation. Nevertheless, the predominantly cytokine pattern found in chronic patients was related to an increase of IL-10 production. Hyodo et al. (2004) observed an increased production of this cytokine in response to the core antigen of HBV (AgHBc) in patients with chronic infection, suggesting that the excessive production of IL-10 may contribute to HBV persistency in these patients.

Considering the findings that the level of TNF-a was decreased in animal models that developed into chronicity of the infection by HBV (Hodgson & Michalak 2001), various studies have been carried out seeking to demonstrate the association of TNF genetic polymorphism and the infection evolution.

In a Korean population with chronic disease patients and individuals promptly recovered, Kim et al. (2003a) observed an association between the allele A in position –308 (G/A or A/A) with resolution of HBV infection. In accordance with these findings, a study with HBeAg positive mothers was carried out demonstrating a significantly higher frequency of both TNF 308GG and allele G in mothers with chronic infection (Kim et al. 2003b). Cheong et al. (2006) did not find associations of the TNF 308 polymorphism with either resolution or persistence of HBV infection. Nonetheless, he found that the haplotype -308 G/ -238 G was associated with HBV persistence. On the other hand, Xu et al. (2005) verified a higher frequency of both, TNF –308 GA and the allele A in chronic patients with severe disease in relation to controls, asymptomatic patients and patients with mild disease. Nevertheless, they did not see any difference between chronic patients, as a single group, and controls. They also found that the TNF-a serum level was significantly higher in G/A genotype patients with severe disease in relation to those with G/G genotype. These results point out to the contribution of TNF-a to the severity of HBV infection. The outcome of HBV infection was also evaluated in relation to the polymorphism of other positions at the TNF-a promoting region, where it was observed an association between –238 G/G SNP and chronic infection (Lu at al. 2004, Du et al. 2006).

We could not find the same relation when evaluating the TNF polymorphism by itself. However, it was observed a higher frequency of patients with self-limited infection with the combination that has been shown as lower production potential of TNF-a (TNF –308 G/G) (Kroeger et al. 1997), and intermediate production potential of (INF-g +874 T/A) (Pravica et al. 1999).

In the present study, the susceptibility to chronic infection by HBV was not associated to the genetic polymorphism of IL-10 or IFN-g as for alleles or genotypes. Accordingly, a study conducted by Miyazoe et al. (2002) did not find a significant difference between HBV infected patients and health volunteers. However, the frequencies of the IL -10 alleles T–819 and A–592 were significantly higher in asymptomatic patients when compared to patients with chronic progressive disease. Another study investigating the genetic capability of cytokine's production and susceptibility to chronic infection (Ben-Ari et al. 2003) did not show any association with IL-10 production potential as determined by polymorphisms at 1–082, –819, and –592 positions. However, the majority of patients with chronic infection (65.2%) in this last study presented a genotype the authors related to INF-g low level production potential in relation to control group (37.5%).

We also did not find any relation to TGFB1 polymorphism. Nonetheless, 11 patients of the chronic patients' group had histological alterations characterized by hepatic biopsy. The number of patients in this group was small, and a definite association with genetic polymorphism of cytokines could not be established. However, all HBV chronic patients with histological alterations in the present study showed the allele T at position +869 of TGFB1. According to Perrey et al. (1998) and Letterio et al. (1998), the allele T at position +869 and G at position +915 are associated with higher production of TGF-b1.

One consequence of chronic hepatic injury is the recurrence of tissue repair process, leading to fibrosis of the liver. TGF-b1 is a cytokine with immunosupressant properties that include regulation of T-cell proliferation and differentiation (Perrey et al. 1998). Moreover, its activity is related to tissue repair and to hepatic fibrosis' formation, since it affects the transcription of genes linked to fibrosis and can retard the hepatocites proliferation (Perrey et al. 1998). As observed by Bissel et al. (2001), there is an inter-individual variation in the response to hepatic injury; and the genetic polymorphism of TGFB1 seems to affect the progression timing to fibrosis. Awad et al. (1998) observed that patients with both cystic and pulmonary fibrosis showed a greater frequency of the allele +869 T of this cytokine. It was also observed an association of patients with pre-transplant pulmonary fibrosis and the TGFB1 +915 GG genotype.

The participants of this study were also compared as for the genetic polymorphism of TNF, TGFB1, IL-10, IL-6, and IFNG with a healthy white Brazilian population of bone marrow donors from the South and Southeast regions of Brazil (Visentainer et al. 2005). It was verified that both the allelic and genotypic frequencies of this population were intermediary as compared with the self-limited infection group and the chronic patient group. Nonetheless, we did not find statistically significant differences between the populations of the two studies.

In this study, we could not characterize definitive evidences pointing to the involvement of the cytokine polymorphisms in the evolution of HBV infection. A greater number of chronic patients with and without histological alterations could allow a more accurate definition of the influence of the genetic polymorphism on infection evolution.

ACKNOWLEDGMENTS

To Maringá State University Hemocenter, Maringá State University Hospital, and South Zone Maringá Health Care Unit for allowing patients sample harvesting.

Received 27 July 2006

Accepted 7 March 2007

• Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV 1998. Genotypic variation in the transforming growth factor-1 gene. Transplantation 66: 1014-1020.
• Ben-Ari Z, Mor E, Papo O, Kfir B, Sulkes J, Tambur AR, Tur-Kaspa R, Klein T 2003. Cytokine gene polymorphisms in patients infected with hepatitis B virus. Am J Gastroenterol 98: 144-150.
• Berlioz-Arthaud A, Perolat P, Buisson Y 2003. 10 year assessment of infant hepatitis B vaccination program, in the Loyalty Islands (New Caledonia). Vaccine 21: 2737-2742.
• Bidwell JL, Wood NAP, Morse HR, Olomolaiye OO, Laundy GJ 1998. Human cytokine gene nucleotide sequence alignments. Eur J Immunogen 25: 83-265.
• Bissel DM, Roulot D, George J 2001. Transforming growth factor b and liver. Hepatology 34: 859-867.
• Chen Y, Wei H, Gao B, Hu Z, Zheng S, Tian Z 2005. Activation and function of hepatic NK cells in hepatitis B infection: an underinvestigated innate immune response. J Viral Hepatol 12: 38-45.
• Cheong JY, Cho SW, Hwang IL, Yoon SK, Lee JH, Park CS, Lee JE, Hahm KB, Kim JH 2006. Association between chronic hepatitis B virus infection and interleukin-10, tumor necrosis factor-a gene promoter polymorphisms. J Gastroenterol Hepatol 21: 1163-1169.
• Chu CJ, Lok ASF 2002. Clinical significance of hepatitis B virus genotypes. Hepatology 35: 922-929.
• Crawley E, Kay R, Sillibourne J, Patel P, Hutchinson IV, Woo P 1999. Polymorphic haplotypes of the interleukin-10 5 flanking region determine variable interleukin-10 transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis. Arthr Reumatol 42: 1101-1108.
• Du T, Guo XH, Zhu XL, LI JH, Lu LP, Gao JR, Gou CY, Li Z, Liu Y, Li H 2006. Association of TNF-alpha promoter polymorphisms with the outcomes of hepatitis B virus infection in Chinese Han population. J Viral Hepatol 13: 618-624.
• Ferrari C, Missale G, Boni C, Urbani S 2003. Immunopathogenesis of hepatitis B. J Hepatol 39: S36-S42.
• Ferreira CT 2004. Hepatites virais: aspectos da epidemiologia e da prevenção. Rev Bras Epidemiol 7: 473-487.
• Fishman D, Faulds G, Jeffry R, Mohamed-Alli V, Yudkin JS, Humphries S, Woo P 1998. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 102: 1369-1376.
• Guidotti LG, Rochford R, Chung J, Shapiro M, Purcell R, Chisari FV 1999. Viral clearance without destruction of infected cells during acute HBV infection. Science 284: 825-828.
• Hajeer AH, Hutchinson IV 2001. Influence of TNF-a gene polymorphisms on TNF-a production and disease. Hum Immunol 62: 1191-1199.
• Hodgson PD, Michalak TI 2001. Augmented hepatic interferon gamma expression and T-cell influx characterize acute hepatitis progressing to recovery and residual lifelong virus persistence in experimental adult woodchuck hepatitis virus infection. Hepatology 34: 1049-1058.
• Hyodo N, Nakamura I, Imawari M 2004. Hepatitis B core antigen stimulates interleukin-10 secretion by both T cells and monocytes from peripheral blood of patients with chronic hepatitis B virus infection. Clin Exp Immunol 135: 462-466.
• Jung MC, Pape GR 2002. Immunology of hepatitis B infection. Lancet Inf Dis 2: 43-50.
• Kane M 1995. Global program for control of hepatitis B infection. Vaccine 13 (Suppl. 1): 47-49.
• Kao JH, Chen PJ, Lai MY, Chen DS 2002. Genotypes and clinical phenotypes of hepatitis B virus in patients with chronic hepatitis B virus infection. J Clin Microbool 40: 1207-1209.
• Kim JH, Pyo CW, Hur SS, Kim YK, Kim TG 2003b. Influences of IL-1B, IL-1RN, TNF-a and TNF-b alleles on chronic HBV infection and perinatal infections of hepatitis B virus after immunoprophylaxis. Hum Immunol 64 (Suppl. 1): 5143.
• Kim YJ, Lee HS, Yoon JH, Kim CY, Park MH, Kim LH, Park BL, Shin HD 2003a. Association of TNF-a promoter polymorphisms with clearance of hepatitis B virus infection. Hum Mol Gen 12: 2541-2546.
• Kroeger KM, Carville KS, Abraham LJ 1997. The -308 tumor necrosis factor- á promoter polymorphism effects transcription. Mol Immunol 34: 391-399.
• Leifeld L, Cheng S, Ramakers J, Dumoulin FL, Trautwein C, Sauerbruch T, Spengler U 2002. Imbalanced intrahepatic expression of interleukin 12, interferon gamma, and interleukin 10 in fulminant hepatitis B. Hepatology 36: 1001-1008.
• Letterio JJ, Roberts AB 1998. Regulation of immune response by TGF-b. Ann Rev Immunol 16: 137-161.
• Lu LP, Li XW, Liu Y, Sun GC, Wang XP, Zhu XL, Hu QY, Li H 2004. Association of 238 G/A polymorphism of tumor necrosis factor-alpha gene promoter region with outcomes of hepatitis B virus infection in Chinese Han population. World J Gastroenterol 10: 1810-1814.
• Ministério da Saúde 2003. Hepatites Virais. O Brasil Está Atento, Programa Nacional de Hepatites Virais, Brasília, p.1-20.
• Miyazoe S, Hamasaki K, Nakata K, Kajiya Y, Kitajima K, Nakao K, Daikoku M, Yatsuhashi H, Koga M, Yano M, Eguchi K 2002. Influence of interleukin-10 gene promoter polymorphisms on disease progression in patients chronically infected with hepatitis B vírus. Am J Gastroenterol 97: 2086-2092.
• Perrey C, Pravica V, Sinnott PJ, Hutchinson IV 1998. Genotyping for polymorphisms in interferon-g, interleukin-10, transforming growth factor-b1 and tumor necrosis factor-a genes: a technical report. Transpl Immunol 6: 193-197.
• Pravica V, Asderakis A, Perrey C, Hajeer A, Sinnott PJ., Hutchinson IV 1999. In vitro production of IFN-g correlates with CA repeat polymorphism in the human IFN-g gene. Eur J Immunogen 26: 1-3.
• Probst CM, Bompeixe EP, Pereira NF, Dalalio MMO, Visentainer JEL, Tsuneto LT, Petzl-Erler ML 2000. HLA polymorphism and evaluation of European, African, and Amerindian contribution to the white and mulatto populations from Paraná, Brazil. Human Biol 72: 597-617.
• Shih HH, Chang MH, Hsu HY, Lee PI, Ni YH, Chen DS 1999. Long term immune response of universal hepatitis B vaccination in infancy: a community-based study in Taiwan. Ped Inf Dis J 18: 427-432.
• Suri D, Schilling R, Lopes AR, Mullerova I, Colucci G, Williams R, Naoumov NV 2001. Non-cytolitic inhibition of B virus replication in human hepatocytes. J Hepatol 35: 790-797.
• Szkaradkiewicz A, Jopek A, Wysocki J, Grzymislawski M, Malecka I, Wozniak A 2003. HBcAg-specific cytokine production by CD4 T lymphocytes of children with acute and chronic hepatitis B. Virus Res 97: 127-133.
• Thio CL, Carrington M, Marti D, O'Brien SJ, Vlahov D, Nelson KE, Astermborski J, Thomas DL 1999. Class II HLA alleles and hepatitis B virus persistence in African Americans. J Inf Dis 179: 1004-1006.
• Thomas D, Strickland GT 2000. Viral Hepatitis. In TG Strickland, Hunter's Tropical Medicine and Emerging Infections Diseases, 8th ed., Saunders, Philadelphia. p. 231-235.
• Thursz M 2001. Genetic susceptibility in chronic viral hepatitis. Ant Res 52: 113-116.
• Visentainer JEL, Cavichioli AD, Sell AM, Lieber SR, Persoli LBL, Marques SBD, Vigorito AC, Aranha FJ, de Souza CA 2005. TNF-a, IL-6, IFN-g, IL-10, and TGF-b1 genotypes among Brazilian caucasoids. Rev Bras Hematol Hemot 27 (Supl. 1): 61-65.
• Xu XW, Lu MH, Tan DM 2005. Association between tumor necrosis factor gene polymorphisms and the clinical types of patients with chronic hepatitis B virus infection. Clin Microbiol Infect 11: 52-56.
• Westendorp RGJ, Langermans JAM, Huizinga TWJ, Verweij CL, Sturk A 1997. Genetic influence on cytokine production in meningococcal disease. Lancet 349: 1912-1913.

Publication Dates

• Publication in this collection
  17 May 2007
• Date of issue
  June 2007

History

• Accepted
  07 Mar 2007
• Received
  27 July 2006