1. Introduction

Brucella is a gram-negative aerobe and static bacterium without capsule and spore causing brucellosis in human and abortion in the cattle. Some species of these bacteria infecting human include Brucella melitensis, Brucella suis and Brucella canis. Generally, those strains of Brucella with smaller lipopolysaccharide in their external membrane are less pathogenic ^[1]. Brucella is mostly transferred from domesticated animals to human, but human-to-human transfer of these bacteria was also reported ^[2]. Dairy products from infected milk like cheese and yoghurt may contain a high amount of this bacterium, and their consumption could be the main cause of brucellosis ^[3]. Taking raw traditional dishes like liver ^[4], skin abrasion ^[5] and inhaling animal fertilizer particles suspending in the air ^[6] are among other causes of brucellosis. This infectious disease is also considered to be an occupational disease experienced more frequently in shepherds, slaughterhouses, dairy industries workers and microbiology laboratory personnel ^[7]. The endemic regions of this infectious disease are Southern America, Middle East and Mediterranean countries.

Brucella is an intercellular organism mostly living inside macrophages, monocytes in human body organs like lymph glands, spleen, bone marrow and reticoloendothelial system ^[8]. Eradication of the Brucella from these cells requires macrophages activation as well as T-helper cells activation and proliferation ^{[9,10,11,12,13,14]}. Interleukin-2 (IL-2), secreted by T-cells, is one of the major factors contributing to the control of Brucella infection by the induction of T-cells proliferation and activation of cellular immunity. IL-2 is also considered as the activating factor of NK cells. Activated NK cells secrete IFN-ɣ which in turn activates macrophages and increases their antimicrobial activity ^[15]. Interestingly, this cytokine not only plays a significant role in activating different immune system cells but also a key role in controlling the immune responses. It is shown that long term or permanent stimulation of T-cells by IL-2 leads to the cell death or apoptosis ^[7]. Indeed, extensive functions of IL-2 have made this cytokine a critical factor for activation or suppression of cell mediated immunity.

It was revealed that the level of cytokine secretion by the cells of immune system and also the functionality of the secreted cytokines in infected individuals, are affected by different factors such as the nature of the organism, the severity of infection as well as the host genetic background ^[11]. In this regard, previous studies showed that the production of IL-2 is influenced by their gene polymorphisms ^{[12,13,14,15,16,17,18,19]}. Given the key role of IL-2 in the immunity against Brucella and that cytokine genes are in control of their production ^[16,17,18], it seems that IL-2 gene variants can be among factors of susceptibility to this bacterium. So, in the present study, we aimed to investigate the association between the two most famous gene polymorphisms of IL-2 which are at positions +114 (G/T), located in the first intron, and −384 (G/T), located in promoter region (Figure 1) and susceptibility to brucellosis in Iranian population.

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2. Materials and Methods

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2.1. Study groups

A total of 173 brucellosis patients registered and approved by Fars Province health centers included in this study. Brucellosis was diagnosed based on clinical manifestations including fever, night sweat, weight loss, splenomegaly, positive bacterium culture and positive serology tests. Serology tests included Standard Agglutination Test (SAT) and 2-Mercaptoethanol Test (2ME). Positive titer was taken to be > 1.160.

Seventy-five healthy stockbreeders living in endemic areas with infected cattle, having close contact with infected cattle, and using their dairy products yet not infected by brucellosis, were selected as control group. Cattle infection was approved by Fars Province Veterinary Office. Control group members were monitored for six continuous months and their health was confirmed. Blood sampling with EDTA anticoagulation substance was done in both control and patient groups. Then, samples were sent to the molecular laboratory for DNA extraction. This study was carried out under the supervision of ethics committee of Shiraz University of Medical Sciences (Approval number: 1389-712), and informed written consents were obtained from all patients and controls.

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2.2. DNA extraction and genotyping

DNA extraction was carried out by salting out method ^[19]. To determine IL-2 gene polymorphism at position +114, the amount of 250 ng of extracted DNA was added to each microtube. Then, 9 μL of PCR mixture containing 1× buffer, 1.5 μL of MgCl₂ (CinnaGen, Iran), 500 pM direct and reverse primers (Forward: 5ʹ-ATGTACAGGTGCAACTCCT-3ʹ and reverse: 5ʹ-TGGTGAGTTTGGGATTCTTC-3ʹ), 200 μM dNTP and one unit of Taq DNA Polymerase were added to each microtube, and placed in thermocycler. The temperature condition was set as follows: 2 min at 94 ℃ and then 35 cycles at 94 ℃ for 20 s, 52 ℃ for 40 s, 72 ℃ for 20 s, and finally 72 ℃ for 10 min. Upon DNA sequence amplification, the Mwo-Ⅰ (Fermentas, Lithuania) restriction enzyme was added to the tubes and kept at 37 ℃ for 24 hrs. Products were separated on %2.5 of NA agarose gel (GE Halthcare, USA). After being dyed by ethidium bromide, the bands were examined by transilluminator. In the case of the existence of G allele in position of +114, the amplicon digested into two pieces with 111 and 151 bp after being exposed to restriction enzyme. But, If T allele existed, PCR product remained undigested and a 262 bp band appeared. To determine IL-2 gene polymorphism in position −384 of IL-2 gene, PCR was carried out using 5ʹ-ATGTACAGGTGCAACTCCT-3ʹ and 5ʹ-TGGTGAGTTTGGGATTCTTC-3ʹ primers based on the above thermal conditions. For this position Bfa-Ⅰ (Fermentas, Lithuania) restriction enzyme was used. In the case of the present of G allele in position of −384 of Il-2, the amplicon digested into two pieces with 110 and 21 bp after being exposed to restriction enzyme. But, If T allele existed, PCR product remained undigested and a 131 bp band appeared (Table 1).

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2.3. Statistical analysis

The frequencies of alleles and genotypes were calculated in patient and control groups by direct gene counting, and compared using EPI2000 and SPSS16. Hardy-Weinberg equilibrium as well as haplotypes and haplogenotypes were determined using Arlequin3.1. Also, LD2SNPing program V2.0 (http://www.bio.kuas.edu.tw/LD2SNPing) was used to estimate linkage disequilibrium (LD) between the studied polymorphic sites. The study power was calculated via Power SSC software. P value less than 0.05 was considered significant.

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3. Results

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3.1. Allele and genotype distributions

The frequencies of the alleles and genotypes were determined in the control and patient groups. Genotypes of IL-2 in the control group met Hardy-Weinberg equilibrium. As shown in Table 2, at position +144, the frequency of TT genotype was significantly higher in control group, compared to patients (P = 0.016, OR = 0.16, 95%CI = 0.02-0.98). But, there was no significant difference between the groups regarding TG and GG genotypes as well as T and G alleles. Furthermore, at position −384, the frequencies of G allele and GG genotype were higher in the controls compared with patients, however, they were not significantly different (P = 0.44 and 0.68, respectively).

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3.2. Haplotype and haplogenotype distributions

The frequencies of haplotypes and haplogenotypes were determined in the control and patient groups. As shown in Table 3, there was no significant difference between the groups in terms of four existing haplotypes (GG, TG, TT, and GT) in two loci of IL-2 gene (+114 T/G and −384 T/G). Furthermore, among 14 existing haplogenotypes, the frequency of TT/TT haplogenotype was significantly higher in controls, compared to patients (P = 0.049, OR = 0.14, 95%CI = 0.01-1.54). But, regarding other 13 haplogenotypes, there was no significant difference between groups.

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4. Discussion and Conclusion

Cellular immunity and its related cytokines play the main role in immunity against intracellular bacteria such as Brucella ^[20]. IL-2 modulates the activation and proliferation of T-cells and NK cells. It was revealed that IL-2 secretion level increases in the sera of patients with brucellosis ^[9]. Furthermore, some studies showed that the level of cytokine secretion is related to single nucleotide polymorphisms (SNPs) in cytokine genes ^[11]. Based on these studies, we aimed to investigate the potential associations between IL-2 gene polymorphisms and susceptibility to brucellosis in Iranian population. The results of our study indicated that the inheritance of TT genotype in locus +114 of IL-2 significantly affects susceptibility to brucellosis and may be a protective factor against Brucella infection. It is likely that this genotype can increase IL-2 expression ^[21] and secretion by T-cells which in turn can result in Brucella resistance in individuals inheriting this genotype. Results also demonstrated higher frequency of TT/TT haplogenotype (+114 T/G and −384 T/G) in control group, as compared to brucellosis group. Hence, it can be suggested that inheriting TT/TT haplogenotype can affect individual's resistance against Brucella infection. However, our results showed that inheriting different genotypes (TT, TG, and GG) in loci −384 of IL-2 cannot affect resistance or susceptibility to brucellosis. To the best of our knowledge, there is no published article on the relationship between IL-2 gene polymorphisms and susceptibility to Brucella infection in Iranian population.

Some studies have shown the relationship between different cytokine polymorphisms and susceptibility to brucellosis. In this regard, a study on the IL-10 gene polymorphisms demonstrated that the distributions of C allele CC genotypes in loci −819 and −592 were significantly higher in brucellosis patients, compared to healthy stockbreeders ^[17]. Another study on IL-12 and TNF-β gene polymorphisms also showed that the frequencies of AA genotype and A allele related to locus +1188 of IL-12 were higher in the controls than that in the brucellosis patients, but the frequencies of AA genotype and A allele in locus +252 of TNF-β were significantly higher in control group, compared to brucellosis patients ^[18]. Results of a study on the polymorphisms of IFN-ɣ (interferon-ɣ) and IL-4 genes indicated that individuals with AA genotype in locus +874 of IFN-ɣ are further susceptible to brucellosis, while individuals with CC genotype in locus −590 of IL-4 are further brucellosis resistant ^[16].

Furthermore, some studies have shown the relationship between IL-2 gene polymorphisms and susceptibility to various diseases. Accordingly, Matsans et al. studied the IL-2 gene polymorphisms in multiple sclerosis (MS) patients, and showed an association between two TT and GT genotypes in locus −384 of IL-2 and susceptibility to progressive MS ^[19]. In another study, Woo et al. examined the relationship between IL-2 polymorphism and prostate cancer susceptibility. They showed that TT homozygote genotype has a higher frequency in patient, compared to control group ^[20].

Conclusively, the present study results showed significantly higher frequencies of TT genotype in locus +114 as well as TT/TT haplogenotype in loci +114 and −384 in control group as compared to brucellosis group. Hence, it can be suggested that, considering the key role of IL-2 in controlling Brucella infection, inheriting the above genotype and haplogenotype can probably lead to the higher production of IL-2 resulting in higher resistance to brucellosis.

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Acknowledgements

This study was financially supported by the grant No. 89290 awarded by Fasa University of Medical Sciences. Also, our thanks are due to Dr. Hassan Khajehei for linguistic copy editing of the manuscript.

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