Interleukin-2 and Interferon-Gamma Single Nucleotide Polymorphisms in Iranian Patients with Chronic Heart Failure


PDF - Export to EndNote - PubMed Central XML format - PubMed Central XML format



  • - Division of Cardiology, Department of Internal Medicine, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

  • - Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran

  • - Department of Hematology, Faculty of Allied Medical Science, Iran University of Medical Sciences, Tehran, Iran

  • - Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA

  • - Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran

  • - Faculty of Nutrition and Dietetics, Tehran University of Medical Sciences, Tehran, Iran

  • - Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran

  • - Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran

  • - Department of Epidemiology and Biostatistics, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran

  • - Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran
    - Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Corresponding author Children’s Medical Centre Hospital, Tehran, Iran, Tel: +98 21 66929234, Fax: +98 21 66929235, E-mail: rezaei_nima@tums.ac.ir
    - Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran
    - Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
    - Research Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
    - Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN) , Tehran, Iran

Abstract:

Background: Inflammatory cytokines have been known to be associated with Chronic Heart Failure (CHF). Given the importance of cytokines in the context of the failing heart, the prevalence of Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ) polymorphisms was studied in patients with CHF due to ischemic heart disease in a case-control study.
Methods: Fifty-six Iranian patients with CHF were enrolled in this study as the case group and compared with 139 healthy subjects, using polymerase chain reaction with sequence-specific primers method, so as to determine the frequency of alleles, genotypes and haplotypes of IFN-γ (+874 A/T) and IL-2 (-330 G/T, +166 G/T) SNPs. 
Results: The GG genotype at IL-2 -330 in patients with CHF was significantly overrepresented in comparison with the control group (p=0.013). Such a positive genotypic association was also observed for IL-2 +166/TT (p=0.022). Meanwhile, the GT genotype frequency at IL-2 -330/GT in the patient group was significantly lower than the one in healthy controls (p=0.049). No significant association was detected between the IFN-γ gene polymorphisms and individuals’ susceptibility to CHF.
Conclusion: Certain genotypes in IL-2 gene were overrepresented in patients with CHF, which could render individuals more vulnerable to this disease.


 

 


Introduction :

Chronic Heart Failure (CHF) is a serious clinical condition, characterized by impaired contractile function and progressive ventricular dilation 1. As with any other major health issue, CHF greatly influences the quality of life of patients with this condition; therefore, it stands to reason that introduction of promising future genetic markers which could affect individual proneness to this disease, seems essential to initiate therapy in advance.
Various etiologies such as hypertension, coronary artery disease and infection, result in heart failure. Different heart failure models indicate the roles of cytokines, as major actors in different immune mechanisms, in the aforementioned etiologies leading to CHF 2-4. Interleukin-2 (IL-2) is one such proinflammatory cytokine, which induces proliferation of T cells 5. The other cytokine of this category is Interferon-gamma (IFN-γ), which is mainly produced by Natural Killer (NK) cells and T cells. This cytokine is known to be associated with T helper 1 (Th1) responses 6. Previous investigations have indicated the importance of these two inflammatory cytokines, IL-2 and IFN-γ, in the etiopathogenesis of various conditions such as atherosclerosis and ischemic and non-ischemic dilated cardiomyopathy, which could stand as the underlying cause of CHF development 7-9.
It has been postulated that Single Nucleotide Polymorphisms (SNPs) within coding and promoter sequences of cytokine genes could affect their secretion pattern 10,11. Numerous studies have been performed on cytokine gene polymorphisms in the context of various immunological disorders 12-20. However, due to the paucity of data regarding the contribution of cytokines’ gene polymorphisms in CHF susceptibility, achieving consensus seems impossible so far. To the best of our knowledge, this is the first study to explore certain IL-2 and IFN-γ gene polymorphisms in Iranian patients with CHF.
This study was conducted in a group of Iranian patients with CHF in order to assess the associations of SNPs in IL-2 at positions -330 and +166 as well as IFN-γ at position +874 with the disease.

 


Materials and Methods :

Subjects: Fifty-six Iranian patients with CHF (42 males, 14 females) with the mean age of 57.96±12.24 years were enrolled in this study. Diagnosis of CHF in patients was based on intensive history taking, thorough physical examination, electrocardiography and impaired Left Ven¬tricular (LV) systolic function (LV ejection fraction ≤40%) and LV dilation (LV end-diastolic diameter >5.5 cm) on echocardiography. Subjects with recent myocardial infarction, malignancies, chronic lung disease and acute decompensated HF within 3 months before enrollment, were excluded from the study. Only those patients in stable clinical condition, who had received conventio¬nal medical therapy for at least 3 months, were enrolled in this study. Baseline demographic and clinical characteristics of patients with CHF, included in the current study, are depicted in table 1. One hundred and thirty nine unrelated healthy subjects (mean age 45.63±10.84; 100 men, 39 women) who were randomly selected from blood donors at Iranian blood transfusion organizations, were also selected as the control group 21. This study was approved by the Ethics Committee of Tehran University of Medical Sciences. Written informed consent was obtained from all participants prior to sampling.
Genotyping: After DNA extraction from the peripheral blood leukocytes using the "salting out" technique 22, the polymerase chain reaction, with sequence-specific primers (PCR-SSP assay kit; Heidelberg University, Germany) was employed for cytokine gene typing 21. Briefly, amplification of the extracted gene was performed by a Techne Flexigene thermal cycler (Roche) under the following conditions: initial denaturation at 94°C for 2 min; denaturation at 94°C for 10 s; annealing+extension at 65°C for 1 min (10 cycles); denaturation at 94°C for 10 s; annealing at 61°C for 50 s; and extension at 72°C for 30 s (20 cycles). Subsequently, the availability of the Polymerase Chain Reaction (PCR) products was assessed using 2% agarose gel electrophoresis. Thereafter, the gel was placed on an Ultraviolet (UV) transilluminator, and a picture was taken for analysis and documentation. The frequencies of alleles, genotypes and haplotypes of IL-2 (G/T at -330 and +166) and IFN-γ (A/T at -+874) were counted.
Statistical analysis: Allele, genotype and haplotype frequencies were calculated by direct gene counting. In order to test the Hardy-Weinberg equilibrium, the frequencies of various genotypes were compared using the chi square test. The odds ratios and 95% Confidence Intervals (CI) were estimated for each allele, genotype and haplotype. A p-value of less than 0.05 was considered significant.

 


Results :

Alleles, genotype and haplotype frequencies: The allelic and genotype frequencies in patients with CHF and healthy controls are depicted in table 2. The GG genotype at IL-2 -330 in patients with CHF was significantly increased in comparison with the control group [p=0.013, OR=3.56, 95%CI: 1.32-9.57]. Such a positive genotypic association was also observed for IL-2 +166/TT [p=0.022, OR=6.72, 95% CI: 1.26-35.71]. Meanwhile, the GT genotype frequency at IL-2 -330 in the patient group was significantly lower than the one in healthy controls [p=0.049, OR=0.51, 95%CI: 0.27-0.97]. On the other hand, no significant association was found between the IFN-γ gene polymorphisms at +874 position and individuals’ vulnerability to CHF.
No significant differences were found between the two groups for GG, TG, TT and GT haplotypes at positions -330 and +166 of IL-2 gene (Table 3).

 


Discussion :

Several pieces of evidence have shown that inflammation is an important actor in cardiovascular diseases, including Left Ventricular Dysfunction (LVD) and subsequent heart failure, which constitutes an ultimate common pathway for a multitude of cardiac disorders 23,24. Recently, the potential role of circulating inflammatory markers, such as IL-2 and IFN-γ, as risk predictors of cardiovascular events has been a topic of intensive research 25. It has long been speculated that cytokine gene polymorphisms could affect their serum level, as discussed in advance. Therefore, considering the significance of inflammation in cardiac diseases, the present study was designed to examine a sample of Iranian patients with CHF for the SNPs in IFN-γ gene at position +874 as well as IL-2 polymorphisms at positions -330 and +166.
IL-2 is a secretory cytokine generated by activated T lymphocytes, which induces T cells, B cells, and NK cells to proliferate and produce other cytokines 26. Reports have suggested IL-2 as a predictor of vascular disease 7. Recent studies have proposed that elevated levels of IL-2 indicate intensified T cell response to different antigens, which are assumed to be critical in the promotion of atherosclerosis 7,8. Previous studies have suggested the IL-2 G allele at position -330 is associated with enhanced IL-2 expression. IL-2 (-330) GG genotype is recognized as a polymorphism with an increased level of cytokine production following anti-CD3/CD28 stimulation of lymphocytes. However, the GT genotype at the same position is acknowledged as a genotype with an intermediate level of IL-2 gene expression. IL-2 (-330) TT genotype is also known to cause low IL-2 levels 27. Our statistical analysis of IL-2 gene polymorphisms disclosed increased frequency of IL-2 -330 GG genotype as well as IL-2 +166 TT genotype in patient group, compared with control category, while IL-2 -330 GT genotype was shown to be more frequent in healthy controls. Our results are consistent with the findings of a recent study conducted by Ding et al 28, which revealed the association of IL-2 -330 GG genotype with increased risk of coronary artery disease. Their results also showed that subjects carrying IL-2 -330 GG genotype had increased serum level of IL-2 in comparison with those with TG or TT genotypes 28.
IFN-γ is a proinflammatory cytokine produced by Th1 cells, which enhances the expression of MHC class I and class II molecules. An increment of IFN-γ-positive CD4 (+) T cells has been previously reported in patients with CHF 6. On the contrary, diminishment in IFN-γ serum levels has been described 29, in a group of patients with CHF secondary to ischemic and non-ischemic dilated cardiomyopathy. Several investigations have demonstrated the expression of IFN-γ in the immunological activation of atherosclerotic lesions from both clinical samples together within preclinical mouse atherosclerosis models 9,30. Among multitude of SNPs reported in IFN-γ gene, IFN-γ +874 A/T polymorphism, which maps to the putative Nuclear Factor-kB (NF-kB) binding site is known to enhance the expression of IFN-γ gene, where A mutant allele is present, while the presence of T allele is known to be involved in underexpression of IFN-γ 31. Multiple studies carried out in both humans and rodent models have investigated a proatherogenic role of IFN-γ 32-35. Garg et al 36 suggested a significant role of IFN-γ +874 T allele in the occurrence of coronary heart disease. In the current investigation, no association was found between polymorphisms in IFN-γ at position +874 and individual susceptibility to CHF.
This study has certain limitations that should be acknowledged. Firstly, our limitations to measure the serum levels of IL-2 and IFN-γ hindered evaluation of the relevance of gene variants in terms of cytokine levels in patients with CHF. Additionally, previous studies performed in this field have not resulted in a consensus regarding the aforementioned cytokines’ serum levels in patients with CHF, as some have reported elevated levels of IL-2 and IFN-γ in patients with CHF, while some others have suggested decreased levels of these cytokines in such patients. These contradictory results warrant further analysis of Il-2 and IFN-γ levels in CHF. Moreover, the relatively small number of our subjects could diminish the statistical power of our analysis.

 


Conclusion :

To conclude, it is suggested that certain single nucleotide polymorphisms in IL-2 gene can affect the risk of developing CHF. These associations may help us define both predisposing and protective genetic markers with regard to CHF. However, in order to delineate the role of IFN-γ and IL-2 genotypes in the pathogenesis of CHF and influence on IFN-γ and IL-2 production, further studies on cytokine gene polymorphisms in other populations, using larger sample size, are required.

 


Acknowledgement :

This study was funded by Tehran University of Medical Sciences and Health Services (grant number: 87-04-93-9584).

 


Conflict of Interest :

Authors declare no conflicts of interest.

 



<p>Table 1. Baseline demographic and clinical characteristics of patients with CHF</p>
<p>COPD, Chronic Obstructive Pulmonary Disease; CABG, Coronary Rrtery Bypass Grafting; PCI, Percutaneous Coronary Intervention.</p>

Table 1. Baseline demographic and clinical characteristics of patients with CHF

COPD, Chronic Obstructive Pulmonary Disease; CABG, Coronary Rrtery Bypass Grafting; PCI, Percutaneous Coronary Intervention.





<p>Table 2. <em>IL-2</em> and <em>IFN-&gamma;</em> allele and genotype polymorphisms in Iranian patients with CHF and controls</p>

Table 2. IL-2 and IFN-γ allele and genotype polymorphisms in Iranian patients with CHF and controls





<p>Table 3. <em>IL-2</em> haplotype polymorphism in Iranian patients with CHF and controls</p>

Table 3. IL-2 haplotype polymorphism in Iranian patients with CHF and controls






References :
  1. Lloyd-Jones DM, Larson MG, Leip EP, Beiser A, D’Agostino RB, Kannel WB, et al. Lifetime risk for developing congestive heart failure the Framingham Heart Study. Circulation 2002;106(24):3068-3072.   [PubMed]
  2. Hansson GK, Robertson AK, Söderberg-Nauclér C. Inflammation and atherosclerosis. Annu Rev Pathol 2006;1:297-329.   [PubMed]
  3. Kleemann R, Zadelaar S, Kooistra T. Cytokines and atherosclerosis: a comprehensive review of studies in mice. Cardiovasc Res 2008;79(3):360-376.   [PubMed]
  4. Lesnik P, Haskell CA, Charo IF. Decreased atherosclerosis in CX3CR1–/–mice reveals a role for fractalkine in atherogenesis. J Clin Invest 2003;111(3):333-340.   [PubMed]
  5. Ding R, Gao W, Ostrodci DH, He Z, Song Y, Ma L, et al. Effect of interleukin-2 level and genetic variants on coronary artery disease. Inflammation 2013;36(6):1225-1231.   [PubMed]
  6. Fukunaga T, Soejima H, Irie A, Sugamura K, Oe Y, Tanaka T, et al. Expression of interferon-gamma and interleukin-4 production in CD4+ T cells in patients with chronic heart failure. Heart Vessels 2007;22(3):178-183.   [PubMed]
  7. Elkind MS, Rundek T, Sciacca RR, Ramas R, Chen HJ, Boden-Albala B, et al. Interleukin-2 levels are associated with carotid artery intima-media thickness. Atherosclerosis 2005;180(1):181-187.   [PubMed]
  8. Simon AD, Yazdani S, Wang W, Schwartz A, Rabbani LE. Elevated plasma levels of interleukin‐2 and soluble il‐2 receptor in ischemic heart disease. Clin Cardiol 2001;24(3):253-256.   [PubMed]
  9. Frostegård J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, et al. Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis 1999;145(1):33-43.   [PubMed]
  10. Miyake K, Nakashima H, Akahoshi M, Inoue Y, Nagano S, Tanaka Y, et al. Genetically determined interferon‐γ production influences the histological phenotype of lupus nephritis. Rheumatology (Oxford) 2002;41(5):518-524.   [PubMed]
  11. Ohtsuka K, Gray JD, Stimmler MM, Horwitz DA. The relationship between defects in lymphocyte production of transforming growth factor-beta1 in systemic lupus erythematosus and disease activity or severity. Lupus 1999;8(2):90-94.   [PubMed]
  12. Amirzargar A, Shahram F, Nikoopour E, Rezaei N, Saeedfar K, Ziaei N, et al. Proinflammatory cytokine gene polymorphisms in Behcet's disease. Eur Cytokine Netw 2010;21(4):292-296.   [PubMed]
  13. Mahdaviani SA, Rezaei N, Moradi B, Dorkhosh S, Amirzargar AA, Movahedi M. Proinflammatory cytokine gene polymorphisms among Iranian patients with asthma. J Clin Immunol 2009;29(1):57-62.   [PubMed]
  14. Rezaei N, Amirzargar AA, Shakiba Y, Mahmoudi M, Moradi B, Aghamohammadi A. Proinflammatory cytokine gene single nucleotide polymorphisms in common variable immunodeficiency. Clin Exp Immunol 2009;155(1):21-27.   [PubMed]
  15. Amirzargar AA, Bagheri M, Ghavamzadeh A, Alimoghadam K, Khosravi F, Rezaei N, et al. Cytokine gene polymorphism in Iranian patients with chronic myelogenous leukaemia. Int J Immunogenet 2005;32(3):167-171.   [PubMed]
  16. Amirzargar AA, Rezaei N, Jabbari H, Danesh AA, Khosravi F, Hajabdolbaghi M, et al. Cytokine single nucleotide polymorphisms in Iranian patients with pulmonary tuberculosis. Eur Cytokine Netw 2006;17(2):84-89.   [PubMed]
  17. Tahghighi F, Ziaee V, Moradinejad MH, Rezaei A, Harsini S, Soltani S, et al. Tumor necrosis factor-alpha single nucleotide polymorphisms in juvenile systemic lupus erythematosus. Hum Immunol 2015;76(8):533-536.   [PubMed]
  18. Mahmoudi M, Tahghighi F, Ziaee V, Harsini S, Rezaei A, Soltani S, et al. Interleukin-4 single nucleotide polymorphisms in juvenile systemic lupus erythematosus. Int J Immunogenet 2014;41(6):512-517.   [PubMed]
  19. Rezaei A, Ziaee V, Sharabian FT, Harsini S, Mahmoudi M, Soltani S, et al. Lack of association between interleukin-10, transforming growth factor-beta gene polymorphisms and juvenile-onset systemic lupus erythematosus. Clin Rheumatol 2015;34(6):1059-1064.   [PubMed]
  20. Ziaee V, Tahghighi F, Moradinejad MH, Harsini S, Mahmoudi M, Rezaei A, et al. Interleukin-6, interleukin-1 gene cluster and interleukin-1 receptor polymorphisms in Iranian patients with juvenile systemic lupus erythematosus. Eur Cytokine Netw 2014;25(2):35-40.   [PubMed]
  21. Amirzargar AA, Naroueynejad M, Khosravi F, Dianat SS, Rezaei N, Mytilineos J, et al. Cytokine single nucleotide polymorphisms in Iranian populations. Eur Cytokine Netw 2008;19(2):104-112.   [PubMed]
  22. Miller S, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16(3):1215.   [PubMed]
  23. Armstrong PW. Left ventricular dysfunction: causes, natural history, and hopes for reversal. Heart 2000;84 (Suppl 1):i15-17:discussion i50.   [PubMed]
  24. Willerson JT, Ridker PM. Inflammation as a cardiovascular risk factor. Circulation 2004;109(21 suppl 1):II2-10.   [PubMed]
  25. Mishra A, Srivastava A, Mittal T, Garg N, Mittal B. Role of inflammatory gene polymorphisms in left ventricular dysfunction (LVD) susceptibility in coronary artery disease (CAD) patients. Cytokine 2013;61(3):856-861.   [PubMed]
  26. Seaman WE. Natural killer cells and natural killer T cells. Arthritis Rheum 2000;43(6):1204-1217.
  27. Amirzargar AA, Naroueynejad M, Khosravi F, Dianat S, Rezaei N, Mytilineos J, et al. Cytokine single nucleotide polymorphisms in Iranian populations. Eur Cytokine Netw 2008;19(2):104-112.   [PubMed]
  28. Ding R, Gao W, Ostrodci DH, He Z, Song Y, Ma L, et al. Effect of interleukin-2 level and genetic variants on coronary artery disease. Inflammation 2013;36(6):1225-1231.   [PubMed]
  29. Cappuzzello C, Di Vito L, Melchionna R, Melillo G, Silvestri L, Cesareo E, et al. Increase of plasma IL-9 and decrease of plasma IL-5, IL-7, and IFN-g in patients with chronic heart failure. J Transl Med 2011;9:28.   [PubMed]
  30. Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol 1989;135(1):169-175.   [PubMed]
  31. Pravica V, Perrey C, Stevens A, Lee JH, Hutchinson IV. A single nucleotide polymorphism in the first intron of the human IFN-gamma gene: Absolute correlation with a polymorphic CA microsatellite marker of high IFN-gamma production. Hum Immunol 2000;61(9):863-866.   [PubMed]
  32. Licastro F, Chiapelli M, Caldarera CM, Caruso C, Lio D, Corder EH. Acute myocardial infarction and proinflammatory gene variants. Ann N Y Acad Sci 2007;1119:227-242.   [PubMed]
  33. Tiroch K, von Beckerath N, Koch W, Lengdobler J, Joost A, Schömig A, et al. Interferon-gamma and interferon-gamma receptor 1 and 2 gene polymorphisms and restenosis following coronary stenting. Atherosclerosis 2005;182(1):145-151.   [PubMed]
  34. Buono C, Come CE, Stavrakis G, Maguire GF, Connelly PW, Lichtman AH. Influence of interferon-gamma on the extent and phenotype of diet-induced atherosclerosis in the LDLR-deficient mouse. Arterioscler Thromb VascBiol 2003;23(3):454-460.   [PubMed]
  35. Whitman SC, Ravisankar P, Daugherty A. IFN-gamma deficiency exerts gender-specific effects on atherogenesis in apolipoprotein E-/-mice. J Interferon Cytokine Res 2002;22(6):661-670.   [PubMed]
  36. Garg PR, Saraswathy KN, Kalla AK, Sinha E, Ghosh PK. Pro-inflammatory cytokine gene polymorphisms and threat for coronary heart disease in a North Indian Agrawal population. Gene 2013;514(1):69-74.   [PubMed]