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ENGLISH VERSION: FEATURES OF THE IMMUNE STATUS OF PATIENTS WITH ALLERGIC RHINITIS, DEPENDING ON TLR 2,4 AND 10 «GALECTIN» GENE POLYMORPHISM
Sakevych V. D., Shlykova O.A., Kaidashev I. P.
Research Institute for Genetic and Immunological Bases of Pathology and Pharmacogenetics of Higher State Education Establishment of Ukraine " Ukrainian Medical Stomatological Academy, Poltava
The study presented theoretical generalization and a new resolve of scientific task in Immunology and Allergology concerning the definition of genetic determinism in allergic rhinitis by examining the role of Toll-ike receptors of innate immunity by polymorphisms TLR2 (rs5743708), TLR 4 (rs4986790) and CLC-10 (rs420297). In examined patients in 76% of cases, AR is predominantly hereditary nature of the mother (36°%), mostly begin in childhood and adolescence (88%) and 44%o is accompanied by other allergic diseases. In the study of the immune status of patients with AR in 15°% of patients one observed moderate and high eosinophilia, increase the average level of total immunogiobuin E, which is significantly higher values have been established in patients with moderate and severe forms of motion (p = 0.0008) increase in the relative number of CD4 CD25 MPER Foxp3 cells with reducing the amount of IL-10 and IL-4 increase. Significant differences between the frequencies of genotypes in the control group and patients with AR gene polymorphism by TLR 4 (Asp299Giy) were found. For the first time the population-based prevalence of polymorphism rs420297 gene «Galectin»u-10 among persons living in Poltava region (SS-76%, ST-22%, TT-2%) revealed significant differences between the frequencies of genotypes in the control group and patients with AR (p = 0.04), rs420297 polymorphism of the gene CLC-10 was significantly more common in patients wtth RA. Significant association between the presence of polymorphic alleles of the gene CLC-10 levels and CD4+, CD4+ CD25+, in the group of homozygous carriers of polymorphic alleles T and C alleles of the gene CLC-10 were revealed. The authentic association between the presence of polymorphic alleles of the gene CLC-10 levels and CD4+CD25+Foxp3+, CD4+was found.
Keywords: allergic rhinitis, cellular and humoral immunity polymorphism, Toll-like receptors, «Galectin»-10.
Allergic diseases, including asthma, allergic rhinitis, atopic dermatitis every year become more urgent and serious problem. Allergic rhinitis (AR) is a disease characterized by IgE-mediated immunological inflammation that is caused by penetration of allergens on the nasal mucosa [1].
Currently recognized point of view, according to which AR is a multifactorial disease (MFZ). Now prevalent concept is an imitation of allergic diseases and identified quite a number of genes that mediate their demonstration. However, the results of numerous studies are controversial molecular genetic basis of disease formation yet insufficiently studied. This turns the AR into a serious medical and social problem and necessitates the study of the causes and im-munological mechanisms of this disease to develop adequate therapeutic and preventive measures, taking into account ethnicity, immunological reactivity and genetic characteristics of patients.
The aim of the study was to determine the role of polymorphisms of genes that control the structural and regulatory elements of nonspecific resistance of the organism (Toll-like receptors: 2258G /A gene TLR2 (rs5743708), 896A / G gene TLR4 (rs4986790) and «Galectin»u-10 gene (rs420297 C / T)) in the pathogenesis of RA, to further the understanding of the immunological mechanisms of this disease.
Materials and methods.
To solve the nominated tasks examined 45 patients aged 19 to 65 patients with AR at the stage of clinical remission who were dispensary in outpatient department of the Fourth City Hospital and Allergic department of Poltava Regional Hospital im.Sklifosovskoho. Comparison group consisted of 95 healthy persons from DNA of-bank Research-Research Institute for Genetic and im-munological bases of pathology and pharmacogenetics State Higher School of Ukraine "Ukrainian Medical
To cite this English version: Sakevych V. D., Shlykova O.A., Kaidashev I. P. Features of the immune status of patients with allergic rhinitis, depending on the gene polymorphism TLR 2,4 and 10 halektynu/ /Problemy ekologii ta medytsyny. - 2014. - Vol 18, № 3-4. - P. 39 -43.
Stomatological Academy", which were surveyed and clinically examined to rule out allergies.
The diagnosis is established based on the AR diagnostic criteria ARIA (2008) diagnostic algorithm adopted in Ukraine and approved by the Ministry of Health of Ukraine. The quality of life of patients was determined using generally recognized questionnaires (Adult Rhino-conjunctivitis Quality of Life Questionnaire). Allergic survey conducted by the conventional method by setting skin prick-tests (lLc «Immunologist ', Vinnitsa, Ukraine).
Getting the peripheral blood of patients was done by sampling blood from the vein kubitalnoyi fasting disposable plastic syringe in a volume of 1 ml in sterile dry glass tube with heparin (25 U / ml) to obtain a suspension of peripheral blood mononuclear cells ; in a volume of 4 ml in a vacuum tube with EDTA (8.4 mg K3EDTA) for DNA isolation ; and in the volume of 5 ml of sterile dry glass tube without the addition of anticoagulants for serum.
Getting the suspension of peripheral blood with mononuclear cells was carried out by centrifugation in density gradient fikol-verohrafinu (1,077g / ml). Serum was isolated from peripheral blood collected on an empty stomach in a sterile dry glass tube without the addition of anticoagulants by incubation and centrifugation [2]. Bold genomic DNA was performed by phenol-chloroform extraction. Determination of polymorphisms Toll-like receptors: 2258G / A gene TLR2, 896A / G TLR4 gene carried by polymerase chain reaction (PCR). Amplification was performed using Thermocyclers "Tertsyk" ("DNA Technology ", Moscow) for the corresponding program using specific oligonucleotide primers followed by restriction analysis [3]. Detection of amplification products carried by electrophoresis in 3% agarose gel («Helikon», Moscow) in 1 x TBE (50 mM Tris-H3BO3 and 2 mM EDTA, pH 8.0), followed by visualization of the results in UV light and taking pictures.
To determine gene polymorphism isolation of ge-nomic DNA from peripheral blood of subjects with a set of "DNA Express-blood" (OOO NPF «Lyteh", Russia), amplified by allele-specific PCR in 35 ml of the reaction mixture with the addition of 5 pkmol specific primers was performed. Products gene amplification «Galectin»u 10 identified using fluorystsentnoyi registration accumulation of DNA fluorescence channels.
The phenotype of lymphocytes was analyzed by determining the expression levels of cell surface antigens CD4, CD25 and intracellular Foxp3 protein by flow tsyto-flyuorymetriyi («EPICS XL-MCL» («Beckman Coulter», USA) by the standard method using the appropriate monoclonal antibody (production of " sorbent " Russia; «eBioscience», USA).
In order to determine the status of humoral immunity of patients with AR serum total IgE vyznachaly in serum were determined on the basis dvosaytovoho (sandwich) ELISA using ELISA test kits for the quantitative determination of total IgE (LLC "Ukrmed Don", Ukraine). Assay interleykinu-4 and interleukin-10 in patients with AR was performed using ELISA test kits for the quantitative determination of interleukin-4 and interleukin-10 of "Ukrmed Don" (Ukraine) according to the instructions sets. Optical density of the samples was determined by enzyme-linked immunosorbent analyzer "Stat-Fax 2100" (USA) at a wavelength of 450 nm.
Statistical analysis of the results conducted by using the statistical package STATISTICA 6.0 (StatSoft Inc., USA) according to the recommendations [4]. Comparison
of genotype frequencies between the study groups was performed by analysis of contingency tables using Fisher's exact test. To compare allele frequencies used criterion x2. To assess the reliability of differences between groups using Fisher's exact two-sided test (for small groups). For all types of analysis considered differences statistically significant at p <0.05.
Results and discussion.
The study examined 45 patients with AR aged 19 to 65 years (of which women accounted for 49% and men 51%) showed that, depending on the frequency of allergen exposure, the nature of the course and frequency of symptoms in 27% (12 patients) found year-round (or persistent) AR and 73% (33 patients) seasonal (or intermittent) AR. The severity of disease was assessed on the basis of generally accepted diagnostic criteria ARIA (2008) [5] on the basis of anamnes-tic indicators of severity of clinical manifestations and their impact on the overall status and quality of life (hard work, education, leisure, daily activities, sleep, etc.), the frequency of use of drugs and their effectiveness. Based on this set easy course-in 11 (25%), medium-heavy-in 32 (71%), heavy-in 2 (4%) patients suffering from RA. Complicated aR detected in 32% of patients. Among the most common were: rhinosinusitis polypoznyy-18%, otitis media-9%, dysfunction of Eustachian tubes and laryngitis-6%.
Medical history revealed that the disease is associated with allergen exposure on the body mainly in child-hood-56% (25 patients) and in adolescence and young respectively-32% (14 patients) and 12% (6 patients), due to features of the immune system in children [6]. The presence of allergic diseases in relatives and II degree relatives of the mother was found in 35% of the father-in 30% of both parents-11% of surveyed patients with RA, which is consistent with the scientific evidence regarding preferential connection with allergic diseases of the mother [7].
When examining patients with AR in 89% of patients were found positive skin tests to pollen, fungal, household, epidermal and food allergens, with 7% occurred sensitization to one allergen group, 29%-to two groups, 36%-up to three groups, 13%-up to four groups, and 4%-to all five groups of allergens.
The concept of inheritance AR implemented dysfunction of the immune system, proven by clinical observations and nowadays there is no doubt [7]. In the pathogenesis of AR is usually considered major IgE-mediated immune responses [6]. Numerous studies have prompted scientists to explore new concepts immunopathogenesis of RA. Opening of T regulatory (Treg) cells determines the path to understanding the mechanisms of peripheral tolerance and the induction of Th1-and Th2-mediated immune responses, an imbalance which accompanies the development of RA. We know that it is important to not only the ratio of Th1 > < Th2, as appropriate activation of T regulatory immune response [8]. In addition, it is considered proven only in the early stages of forming AR dominated Th1 effect and later the imbalance of Th1 / Th2 determined in favor of Th2. It should be noted that in most cases, sneezing, itchy nose, rhinorrhea, nasal zak-ladennist were more pronounced in late phase of allergic reaction type 1, where the leading role was played by Th2 [9].
Were studied peripheral blood and indicators of immune status of patients with AR in remission (WBC, the relative number of lymphocytes, the relative number of eosinophils) generally answered the normal range of healthy individuals (tabl..1) [10].
npoSAeMH eKOAoriï Ta MeanuHHH
Table 1
Immunological parameters examined patients with AR
Index Indicators of healthy individuals Performance of patients with AR, n=45
Total IgE, IU / mL 0 - 130 198,2 ± 11,42
CD4+,% 39 ± 5 40,5 ± 1,02
CD4+/CD25+,% 9,4 ± 2,05 16,5 ± 1,9 (0,25)
CD4+CD25+Foxp3+,% 5-10% of the CD4+ 4,66± 0,38
IL-10 pg / ml 0-50 pg / ml 0,35 ± 0,016
IL-4 pg / ml 0-20 pg / ml 50,34 ± 3,58
However, in 15% of patients had eosinophilia with increasing absolute and relative number of eosinophils in determining the level of total IgE in 76% of patients have a significant increase in performance, the level of total IgE was 198,2 ± 11,42 IU / ml, which confirms the leading role of IgE-mediated immune responses in atopy [11].
It should be noted increase in the relative number of CD4 CD25 Foxp3 MPER cells compared to healthy people, which is consistent with the results of current research [12]. These data support the concept that the immune response to allergens in healthy individuals and patients with allergies are the result of the ratio between allergenspecific MPER cells and Th2 cells [12].
The identified violations of cytokine regulation in the investigation of patients with RA, namely increasing the content of IL-4 (84%)-which enhances the survival of eosinophils. The average figure was 50,34 ± 3,58 pg / ml, which is 2.5 times higher than the values of healthy individuals.
Important components of the cellular immune response is MPER cells that regulate the function of T-helper and T-cytotoxic cells, providing direction Th1 / Th2 type immune response. Recent studies indicate the existence of inducible IL-10-producing cells MPER [13 ]. According to our data, the level of suppressor cytokine IL-10 in patients with AR averaged 0,35 ± 0,016 pg / ml.
According to current research, atopy genes and associated states are concentrated mainly in the 10 areas of the human genome [14]. There are data on the association of allergic rhinitis with gene polymorphism of TIM-1, SD14, TLR 2-4, RNAse.
To determine the role of polymorphisms of individual genes that control the structural and regulatory elements of nonspecific resistance of the organism in the development of allergic rhinitis, determined the prevalence of gene polymorphisms TLR2 (rs5743708), TLR 4 gene (rs4986790) and CLC-10 gene (rs420297) in group supervision and group of population control, the analysis of immunological parameters and clinical manifestations in patients with polymorphic variants studied genes. By molecular genetic study among patients with AR were allocated carriers of polymorphic alleles and analyzed the distribution of genotypes for the studied polymorphisms.
In individuals who were part of the control group, the frequency of TLR2 GG genotype was 97.8%; frequency of heterozygous genotype GA-2,2%, polymorphic genotype AA was not detected. In the group of patients with Ar corresponding results were as follows: GG-93,3%, GA-6,6% and AA was also not detected. There was no significant association between the frequency of occurrence polymorphic allele A in the control group and patients with AR (x2 = 0,74; p = 0.34). The results are the lack of association of polymorphism 2258G / A gene TLR2 with AR are consistent with other scientific data [15].
To study the gene polymorphism rs4986790 TLR4 in the control group the frequency of polymorphic genotype
AA was 95.6%, heterozygous genotype AG-4,5%, polymorphic genotype GG was not found. In patients with AR, respectively: AA-92,3%, AG-7,7% and GG-not found. Reliably was significantly higher difference between the frequencies of genotypes in the control group and patients with AR (p = 0.03). The frequency of polymorphic allele G in the group of patients with AR was 7,7% (x2 = 3,58; p = 0.06) compared with the control group, the difference was found at the level of a statistical trend [16].
Functional TLR4 gene polymorphism is the replacement of aspartic acid glycine Asp299Gly1187 (rs4986790). As a result of reduced ability to recognize relevant TLR4 ligands or for the intracellular signals, leading to a less pronounced activation of immune cells after meeting with the pathogen..Funktsionalnyy TLR4 polymorphism violates the regulation of the innate immune response that is essential imbalance T1 / T2-helper. This mechanism plays a crucial role in the formation of chronic inflammation and attracts attention as a potential risk factor for the development of atopic diseases, including AR [17].
Group, the frequency of CC genotype was 75.56% heterozygous genotype CT-22,22%, genotype TT-2,22%. In patients with AR, respectively: CC-57,78%, CT-24,44% and TT-17,78%.
Revealed significant differences between the frequencies of genotypes in the control group and patients with AR (p = 0.04). Reliably was significantly higher difference in frequency polymorphic allele T in a group of patients with AR-30% (x2 = 6,42; p = 0.011) compared with the control group. In studies of foreign scientists study the association of allergic rhinitis with gene polymorphism «Galectin»u-10 (CLC-10) has recently been given special attention [18].
Genetic markers may determine susceptibility to disease or be associated with the relevant pathogenetic significant figures. Therefore, within the proposed research studied the effect of the gene polymorphism of TLR2 (rs5743708), TLR 4 gene (Asp299Gly) and CLC-10 gene (rs420297) on the course and features of the clinical manifestations of RA. When analyzing the differences in the incidence of seasonal and year-round shape flow between groups of patients with AR depending on genotype polymorphism 2258G / A gene TLR2 and TLR 4 gene (Asp299Gly) had no statistical significance.
Note the presence of possible differences between groups based on genotype polymorphism of the gene TLR4 Asp 299 Gly in the presence of concomitant allergic diseases. Significantly, more commonly in those patients with AR showed concomitant asthma (p = 0.0003), concomitant AD (0.0031) and asthma in combination with AD (p = 0.0005). Probable differences between groups based on genotype polymorphism rs420297 C / T gene «Galectin»u-10 were detected in the clinical forms of AR. Significantly, more commonly in patients with AR polymorphic allele «Galectin»u-10 showed year-round shape AR (p = 0.0001).
As noted earlier, the imbalance T1 \ T2-helper cells with a predominance of Th-2 immune response plays a major role in the development of RA. In response to exposure to allergens in patients with AR is the release of cytokines and Th2-induced activation of eosinophils, mast cells and increased synthesis of IgE. There is evidence that these reactions occur indirectly through TLR2,4. We also know that «Galectin»-10 is necessary for the regulatory activity of Treg cells.
In order to detect differences between patients with AR, depending on the gene polymorphisms the study of immunological parameters was carried out by comparison of groups using Mann-Whitney.
Differences in immunological parameters of patients with AR
Significant differences between the groups of patients with AR with the presence of allele A TLR2 gene and homozygous carriers of allele G in terms of CD4 (U (n = 42; n = 3) = 12,00; p = 0,020) were found.
Significant differences between the groups of patients with AR with the presence of polymorphic alleles of the T gene CLC-10 and homozygous carriers of allele C in terms of CD4 (U (n = 26; n = 9) = 139,50; p = 0,014) were revealed. It should be noted that the level of expression of CD4 molecules in patients with AR gene al-lele T «Galectin»u 10 on average tended to increase, and in patients with AR allele C carriers should not go beyond indicators of healthy individuals (Table 2).
Table 2
depending on genotype polymorphism of the gene «Galectin»u 10
Index Patients with genotypes CT AR IT, (n = 19) Patients with AR genotype CC, (n = 26)
CD4+,% 44,14±1,92 37,92±1,37
U, р U(n=26;n= 19) = 139,50; p=0,014
CD4+ CD25+,% 11,09±1,32 21,16±2,96
U, р U(n=26;n= 19) = 136,00; p=0,012
Total IgE, IU / mL 234,86±12,46 171,48±15,74
U, р U(n=26;n= 19) = 138,50; p=0,013
IL-4 pg / ml 61,61±5,30 42,11± 4,22
U, р U(n=26;n= 19) = 123,00; p=0,004
IL-10 pg / ml 0,303±0,11 0,394±0,024
U, р U(n=26;n= 19) = 136,50; p=0,011
U, p-the differences between the groups on the criterion Manna-Whitney
Also the group of patients with AR with T allele of the gene CLC-10 differed significantly at higher levels of the value CD4 CD25 (U (n = 26; n = 9) = 136,00; p = 0,012) from a group of patients with AR homozygous carriers of allele C. So «Galectin»-10 is necessary for the regulatory activity of Treg cells.
Comparing indicators of total IgE in patients based on genotype polymorphism of the gene CLC-10 was significantly higher values were established IgE levels in patients with AR gene allele T CLC-10 (U (n = 26; n = 9) = 138.50; p = 0.013), which was 234,86 ± 12,46 IU / ml.
Identified violations cytokine regulation with a significant increase in indicators of IL-4, which enhances the survival of eosinophils. Between groups of patients based on gene polymorphisms CLC-10 set significantly higher values of IL-4 in patients with AR gene allele T CLC-10 (U (n = 26; n = 9) = 123.00, p = 0.004), the level of which was 61,61 ± 5,30 pg / ml.
Level suppressor cytokine IL-10 in patients with AR averaged 0,35 ± 0,016 pg / ml. This may be associated with different forms of AR as in patients with a chronic
form of the process of the concentration of IL-10 can significantly decrease performance compared to the acute stage of the disease (may be due to an increase in the concentration of serum cytokine profile of Th-1) or to remain elevated in accordance with indicators norm [6]. Between groups of patients based on gene polymorphisms «Galectin»u-10 was detected difference in performance level of IL10; which was significantly higher values were established in patients with AR homozygous carriers of allele C (U (n = 26 ; n = 9) = 136.50, p = 0.038) and amounted to 0,394 ± 0,024 pg / ml.
Significant differences between the groups of patients with AR homozygous carriers of the T allele of the gene CLC-10 and homozygous carriers of allele C in terms of CD4 (U (n = 26; n = 19) = 37,00; p = 0,006 were revealed. Also the group of patients with AR homozygous carriers of the T allele of the gene CLC-10 differed significantly at higher levels of the value of CD4 CD25 Foxp3% (U (n = 26; n = 8) = 52,50; p = 0,037) from a group of patients with homozygous carriers of AR allele of the gene CLC-10 (Table. 3).
Table 3
Differences in immunological parameters of patients with AR depending on genotype polymorphism of the gene «Galectin»u 10
homozygous carriers
Index Patients with genotype TT AR gene «Galectin» 10, Patients with genotype SS AR gene «Galectin» 10,
(n = 8) (n = 26)
CD4+,% 46,96±2,30 37,91 ± 1,37
U, р U(n=8;n= 26) = 37,00; p=0,006
CD4+CD25+Foxp3+,% 6,61±1,54 4,08± 0,35
U, р U(n=8;n= 26) = 52,50; p=0,037
U, p-the differences between the groups on the criterion Manna-Whitney.
These results confirm that, despite a significant intracellular expression of «Galectin»u-10 in CD25 Treg cells, it is not involved in the suppression CD25 Treg cells, but the specific blockade «Galectin»u-10 restores proliferative capacity of CD25 Treg cells and enhances their suppressive function. So «Galectin»-10 is necessary for the regulatory activity of Treg cells.
Thus, detected changes and the relationship of clinical and immunological parameters in patients with AR and polymorphic alleles of genes TLR2, TLR 4, CLC-10 suggest the importance of these polymorphisms in determining the course of RA, presence of comorbidity, polyvalent allergy in patients AR and implementation of immune mechanisms in the pathogenesis of the disease.
npoSAeMH eKOAorii Ta MeanuHHH
Findings
1. In patients examined AR in 76% of cases there is largely hereditary nature of the mother (36%), wich predominantly begins in childhood or adolescence (88%) and 44% is accompanied by other allergic diseases. clinical forms AR-27% year-round and seasonal 73%, severity found: light-32%, 62%-in intermedius, hard-6% of the disease were defined. With Allergic examination in 89% of patients with AR positive skin tests for domestic, epidermal, pollen, food and fungal allergens were revealed. In 82% of patients polyvalent sensitization to two or more groups of allergenswas found.
2. In the study of the immune status of patients with AR in 15% of patients one observed eosinophilia, increase in the average level of total immunoglobulin E, increase the relative number of CD4 CD25 Foxp3 cells MPER of reducing the amount of IL-10 and IL-4 increase.
3. In the group of patients with AR TLR2 (rs5743708):gene polymorphism prevalence was found: GG genotype was 93.3%, genotype GA-6,6%, genotype AA met. Significant differences between the groups of patients with AR with the presence of polymorphic alleles of TLR2 gene and homozygous carriers of allele G in terms of CD4 (U (n = 42; n = 3) = 12,00; p = 0,020) were revealed.
4. In the group of patients with AR TLR 4 (Asp299Gly): gene polymorphism prevalence AA genotype was 92.3%, genotype AG-7,7%, genotype GG was found. Significant differences between the frequencies of genotypes in the control group and patients with AR (p = 0.03) were revealed. In patients with AR allele G carriers for polymorphism 896A / G TLR4 gene atopic lesions concomitant asthma (p = 0.0003), concomitant AD (0.0031) and asthma in combination with AD (p = 0.0005) were revealed..
5. Prevalence gene polymorphism rs420297 «Galectin»-10 among persons living in Poltava Oblast is the SS-76%; ST-22%; TT-2%. Revealed significant differences between the frequencies of genotypes in the control group and patients with AR (p = 0.04) gene polymorphism rs420297 CLC-10 was significantly more common in the group of patients with RA. Revealed significant differences in frequency of T allele CLC-10 in the group of patients with AR-30%, compared with the control group-13% (x2 = 6,42; p = 0.011). The development and course of allergic rhinitis is associated with a gene polymorphism rs420297 CLC-10 (significantly more often in patients with AR mutant allele CLC-10 showed year-round shape AR (p = 0.0001)).
6. The significant association between the presence of polymorphic alleles of the gene CLC-10 levels and CD4 (p = 0,014), CD4 CD25 (p = 0,012), the group of homozygous carriers of polymorphic alleles T and C alleles of the gene CLC-10 were observed; significant association between the presence of polymorphic alleles of the gene CLC-10 levels and CD4 CD25 Foxp3 (p=0,037), CD4 (p = 0,014) were determined. It has bee established that persons who are polymorphic allele of the gene
CLC-10 have significantly higher levels of IgE (p = 0.013)
and IL-4 (p = 0.004) and lower levels of IL10 (p = 0.038).
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