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Relationship between CD14-159C/T gene polymorphism and acute brucellosis risk

Objective To investigate the association between the cluster of differentiation 14 (CD14)-159C/T (rs2569190) gene polymorphism and susceptibility to acute brucellosis in an Iranian population. Methods The study included 153 Iranian patients with active brucellosis and 128 healthy individuals as the control group. Genotyping of the CD14 variant was performed using an amplification refractory mutation system-polymerase chain reaction method. Results The prevalence of CD14-159 TT and CT genotypes were associated with increased risk of brucellosis [odds ratio (OR) = 1.993, 95% confidence interval (95% CI) = 1.07–3.71, P = 0.03 for CT; OR = 3.869, 95% CI = 1.91–7.84, P = 0.01 for TT genotype. Additionally, the minor allele (T) was significantly more frequently present in brucellosis patients than in controls (61% vs. 45%, respectively), and was a risk factor for brucellosis (OR = 3.058, 95% CI = 1.507–6.315, P = 0.01). Conclusions The findings provided suggestive evidence of association of the CD14-159C/T gene polymorphism with susceptibility to acute brucellosis in the Iranian population.


Introduction
Brucellosis (or Malta fever, Gibraltar fever, Cyprus fever, Danube fever, undulant fever, Mediterranean fever and Bang's disease) is one of the most frequent zoonotic infections worldwide, transmitted to humans by eating contaminated, unpasteurized dairy products, direct contact with infected animal, and through the inhalation of infected aerosolized particles [1][2][3][4]. The majority of cases arise in the Mediterranean countries of Europe and Africa, Mexico, and Central and South America, India, Central Asia and Middle East including Iran [4] .
Brucella spp. (pleomorphic, gram-negative, non-sporeforming coccobacillus bacteria) causes severe debilitating and disabling disease that is accompanied with manifestations such as fever, weakness, sweating, weight loss, headache, persisted joint pain, neurological complications, endocarditis and testicular or bone abscess formation [1,2,4,5] . Brucella spp. are facultative intracellular bacteria that have the ability to evade from killing mechanism of immune system and multiply within in organs with frequent macrophages such as liver, spleen, lung, bone marrow and synovium (reticuloendothelial system) [4,6] . Among all species of Brucella genus, three species including Brucella melitensis, Brucella abortus, and Brucella suis are the most commonly associated with human disease [7].
Cluster of differentiation 14 (CD14) is a glycosylphosphatidylinositol-anchored molecule that is mainly expressed on cell membrane surfaces of monocyte/macrophage lineage [8,9] , and acts as a pattern recognition receptor in innate immunity for detection of variety of ligands such as bacterial products, microbial cell wall components and even whole bacteria. Particularly, CD14 is the specific receptor for the lipopolysaccharide (LPS) of gram-negative bacteria [10,11] .
LPS is the major component of the outer membrane of gramnegative bacteria and is called 'bacterial endotoxin' [12] . LPS is the main virulence factor of Brucella and has a role in cell entry and immune evasion of the infected cell [6]. LPS also has an ability to induce the production of interleukin (IL)-12 resulting in promotion of T helper (Th) 1 responses and inhibition of Th-2 responses in the host [13]. CD14 as a coreceptor for the toll-like receptor (TLR) and together with TLR4 forms a complex that activates the innate immune system and starts a variety of effector functions including secretion of pro-inflammatory cytokines such as IL-1, IL-6, tumor necrosis factor-a, nitric oxide, oxygen radicals and complement components as well as cell proliferation, subsequent binding to LPS [8,[13][14][15] . Nevertheless, high production of these molecules may cause intense harmful consequences including severe inflammatory responses, septic shock and death [15]. Moreover, a soluble type of CD14 (sCD14) is produced by monocytes, hepatocytes and endothelial cells and is present in the circulation and other body fluids. sCD14 is derived both from secretion of CD14 and from enzymatically cleaved glycosylphosphatidylinositol-anchored membrane type of CD14 [10,16] . Since sCD14 plasma levels increases during inflammation and infection, it is believed that sCD14 is an acute-phase protein, which is increased in several inflammatory processes [8,15,17] .
Thus far, the CD14-159C/T gene polymorphism has been examined in several inflammatory and infectious diseases including tuberculosis [8,9,21] , brucellosis [22] , Chlamydia pneumoniae infection [23,24] , Helicobacter pylori infection [25] , hepatitis B and C [26,27] and severe acute respiratory syndrome [28], but the results have been inconsistent and conflicting. Therefore, the current study aimed to evaluate the possible association between the CD14-159C/T (rs2569190) gene polymorphism and susceptibility to acute brucellosis in an Iranian population.

Study population
The current case-control retrospective study included 153 patients (102 men and 51 women) suffering from active brucellosis, age range 6-76 years old and mean ± SD = 31.24 ± 16.6 and 128 healthy individuals as the control group (93 men and 35 women), age range 19-64 years and mean ± SD = 34.04 ± 13.69. Blood sample from each participant was collected in EDTA-containing tubes for DNA isolation after taking an informed consent from all patients and healthy individuals. All patients were either milk farmers (including diagnosed infected animals) or had a history of consuming raw milk and unpasteurized dairy products. Demographic characteristics of patients and their clinical complications are shown in Table 1. Brucellosis was diagnosed according to the clinical manifestations (including fever, night sweating, weakness, malaise, weight loss, splenomegaly, lymphadenophathy, myalgia and arthralgia) and positive blood cultures as described previously [29][30][31][32]. The control group included healthy blood donors with no evidence of brucellosis and genetic disorders and matched for age, sex, and geographic area. The control subjects had the same background as patients and were at the same risk of exposure for brucellosis.

Culture and identification of organism
Brucella spp. were cultivated on 5% sheep blood-agar plates and incubated at 37 C in the contact to 5%-10% of CO 2 conditions for 48 h. A typical and well-isolated Brucella like colony is tiny, transparent, raised, and convex, with an entire edge and smooth and glistening surface along the streak lines by examining macroscopically by Gram's stain [33] . Serological tests, defined as wright titre 1/160 plus mercaptoethanol test 1/ 80 or coomb's wright 1/320 certified the brucellosis infection. The complete details of the organism identification methods have been described previously in papers by this research team [29][30][31][32].

Genotyping of CD14-159C/T (rs2569190) SNP
Genomic DNA was extracted from the peripheral blood leukocytes by the 'salting-out' method as explained previously [34]. The quality of the isolated DNA was checked by electrophoresis on 1% agarose gel, quantitated spectrophotometrically and stored at −20 C still further use.
The CD14-159C/T SNP, rs2569190, was genotyped using an amplification refractory mutation system-polymerase chain reaction method as described previously by Karhukorpi et al [25,35] . This allele-specific, single-tube polymerase chain reaction method uses four primers including two allele specific primers (cfors for the C allele and trevs for the T allele) and two outer primers (cdp1 and cdp2) for the control band. An additional mismatch was inserted at the penultimate 3 0 nucleotide (underlined) of the allele-specific primers to increase the specificity of the PCR reaction. The primer sequences were as follows: cfors: 5 0 -CTC CAG AAT CCT TCC TGT TAC GAC-3 0 , trevs: 5 0 -TGT AGG ATG TTT CAG GGA GGG GTA-3 0 , cdp1: 5 0 -TTG GTG CCA ACA GAT GAG GTT CAC-3 0 , cdp2: 5 0 -TTC TTT CCT ACA CAG CGG CAC CC-3 0 . The PCR conditions for the CD14-159C/T (rs2569190) polymorphism were as followed: an initial denaturation at 95 C for 5 min; 30 cycles at 95 C for 30 s, 60 C for 30 s and 72 C for 1 min; the final extension step proceeded at 72 C for 5 min [25,35] . The PCR products were separated by standard electrophoresis on 2% agarose gel containing ethidium bromide. The PCR products included a 381 bp band for the T allele, a 227 bp band for the C allele and a 561 bp band for the control.

Statistical analysis
All statistical analyses were performed applying the SPSS software for Windows, version 18.0 (SPSS Inc, Chicago IL, USA). The association among genotypes and brucellosis was calculated by reckoning the odds ratio (OR) and 95% confidence interval (95% CI) from logistic regression analyses. P values below 0.05 were considered statistically significant. The Hardy-Weinberg equilibrium was tested with the c 2 test for the SNP under consideration.

Results
The CD14 polymorphism, -159C/T, was successfully genotyped in 153 brucellosis patients and 128 control subjects. No deviation from Hardy-Weinberg equilibrium was observed in the studied case and control groups (P > 0.05). The genotype and allele frequency of the SNP in the studied groups were shown in Table 2.
The results showed that the CD14 polymorphism, -159C/T, was associated with the risk of brucellosis in our population. At genotypic level, the CD14 CT, and TT genotypes were more frequent in the patients group compared to cases, and were associated with increased risk of brucellosis in the codominant model (OR = 1.993, 95% CI = 1.07-3.71, P = 0.03 for CT; OR = 3.869, 95% CI = 1.91-7.84, P = 0.01 for TT genotype). Additionally, the CT + TT vs. CC genotype in the dominant model was a risk factor for brucellosis (OR = 2.514, 95% CI = 1.34-4.73, P = 0.02). Likewise, in the recessive model, the TT genotype vs. CC + CT was associated with the risk of brucellosis (OR = 2.379, 95% CI = 1.33-4.27, P = 0.02). At allelic level, the minor allele (T) was significantly more frequently present in brucellosis patients than in controls (61% vs. 45%, respectively), and was a risk factor for brucellosis (OR = 3.058, 95% CI = 1.507-6.315, P = 0.01).

Discussion
Our study provided data on the association between one important SNP in the 5 0 flanking region of the CD14 gene and acute brucellosis using genotype analysis. The results showed that the prevalence of −159 homozygote TT genotype as well as T allele were much higher in brucellosis patients than in controls (36.6% vs. 19.5% and 61% vs. 44%, respectively). The carriers of the TT genotype and T allele were at 3.8-fold or 3-fold higher risk of brucellosis, respectively, than those with the CC genotype or C allele.
Several studies have been conducted to show the impact of the CD14-159C/T SNP on the risk of infectious diseases [9,18,[21][22][23]. To the best of our knowledge, there is only one study assessing the association of CD14, -159C/T polymorphism and brucellosis [22]. Haidari et al [22], in their study on brucellosis in a Western Iranian population have shown that the frequency of the TT genotype was higher in the patients than in controls (34.5% vs. 15.5%). They suggested that the homozygousity for T allele of promoter of CD14 gene may confer an increased risk of brucellosis, which supports our findings. Moreover, our results regarding CD14-159C/T polymorphism are in agreement with several infectious studies on pulmonary tuberculosis [9,18,21] and C. pneumoniae infection [23]. In contrast, other studies reported no association between CD14-159C/T SNP and Chlamydia trachomatis infection [36], Campylobacter jejuni infection [37] and severe acute respiratory syndrome [28].
Most Brucella species are highly virulent causing an acute infection in their hosts. The LPS of this bacterium is one of the essential components for the first steps in the establishment of an intracellular replication niche, in which Brucella survives and multiplies [7] . LPS is a potent stimulator of monocytes and macrophages [25] . Activation of macrophages is one of the early events in the innate immunity to intracellular pathogens. CD14 is a high-affinity receptor for bacterial LPS and is expressed mainly by cells of monocyte/macrophage lineage. The binding of LPS to CD14 results in cellular activation and initiates a range of effector functions including Table 2 The genotype and allele frequencies of CD14-159C/T SNP between brucellosis patients and controls.

SNPs
Allele cytokine secretion and cell proliferation [8,19] . CD14 as a coreceptor for the TLR is transferring LPS to the TLR4/MD-2 signaling complex. Engagement of this complex results in the activation of innate immunity mechanisms such as the release of proinflammatory cytokines and subsequently severe inflammatory responses as well as in worse circumstances of septic shock and death [15,19]. Some studies have been performed to explain the impact of the CD14-159C/T SNP on CD14 protein expression [8,38] . The CD14-159C/T polymorphism is located at a GC box close to the transcription start site, which serves as a binding site for Sp1-Sp2/Sp3 transcription factors that are involved in the regulation of CD14 gene transcription [15] . The T variant of this SNP is associated with high expression of CD14 on the surface of monocytes and macrophages, which further increases the inflammatory response [39,40]. Thus, this SNP could be a genetic factor for inter-individual differences in susceptibility to infectious diseases like brucellosis. Our results highlight the role of CD14 in the inflammatory response and the effect of its promoter polymorphism on the host immune defense.
In conclusion, our study provided evidence that the CD14-159, CT and TT genotypes may serve as risk factors for brucellosis infection in our population. Further studies on larger populations with different ethnicities are required to validate our findings. More inclusive understanding of the basic mechanisms underlying infections pathogenesis may lead to the development and application of efficient products for the treatment of brucellosis.

Conflict of interest statement
The authors declare that there is no conflict of interest.