ISSN Print 2500–1094
ISSN Online 2542–1204
Bulletin of RSMU
BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)
ORIGINAL RESEARCH
Analysis of TLR gene expression and DEFB1 polymorphisms association in children with bronchial asthma
About authors
1 Department of Immunology, Biomedical Faculty,
Pirogov Russian National Research Medical University, Moscow, Russia
2 Laboratory of Molecular Immunology,Mechnikov Research Institute of Vaccines and Sera, Moscow, Russia
3 Scientific Center of Children's Health, Moscow, Russia
Correspondence should be addressed: Margarita Zaitseva
ul. Svyazistov, d. 10, kv. 68, Krasnoznamensk, Moscow oblast, Russia, 143090; ur.tsil@ecitsa
Received: 2016-06-14
Accepted: 2016-06-23
Published online: 2017-01-05
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Bronchial asthma (BA) is a chronic inflammatory disease of the upper respiratory tract accompanied by bronchial obstruction and hyperresponsiveness. It manifests itself through shortness of breath, wheezing, coughing and choking episodes. It’s prevalence is increasing fast in high- and middle-income countries. According to the Russian Respiratory Society, asthma affects as many as 10 million people in Russia; over 20 % of them are children [1].
It was observed that respiratory infections have a more severe course in patients with BA than in healthy individuals [2, 3]. Acute infections of the upper respiratory tract frequently trigger asthma exacerbations: about 85 % of exacerbations in children and 50 % in adults are caused by respiratory viruses [2]. Pathogens damage ciliated epithelium of the respiratory tract mucosa making it more vulnerable for allergens and toxins and maintaining bronchial hyperresponsiveness. Acute exacerbations can be life-threatening regardless of the BA grade of severity [3].
A lot of contemporary research studies focus on the indepth analysis of BA pathogenesis, including the role of innate immunity components. Of particular interest is a new class of effector molecules (antimicrobial peptides), such as β-defensins. Antimicrobial properties of the latter are due to the electrostatic interactions between negatively charged surface components of the bacterial membrane, such as lipopolysaccharides of gram-negative bacteria and teichoic or lipoteichoic acids of gram-positive bacteria, and a positively charged β-defensin molecule. Critical concentrations of β-defensin on the surface of the target cell trigger pore formation in its membrane followed by cell lysis. Besides, β-defensins exhibit immunoregulatory activity, participating in chemotaxis and adaptive immunity activation, inducing dendritic cell maturation, etc. [4].
The key role in protecting respiratory tract mucosa is played by human β-defensin-1 (HBD-1) synthesized by epithelial cells [5]. β-defensin-1 is encoded by the DEFB1 gene located on the short arm of chromosome 8 (8p23.1) in a highly polymorphic cluster. Due to gene mutations, its expression can be decreased; in turn, insufficient secretion of β-defensins facilitates bacterial adhesion to and invasion of the mucosa and triggers inflammation [6, 7].
Toll-like receptors (TLRs) of the epithelial cells of the respiratory tract mucosa are another important element of the innate immunity. They recognize pathogen-associated molecular patterns (PAMP) of microorganisms and their metabolic byproducts, transmit the signal into the cell and boost leukocyte functional activity, increase pro-inflammatory cytokine and interferon gene expression. The majority of bacterial and viral pathogens are recognized by TLR2, TLR4, and TLR9 that can activate the local mucosal immunity in the respiratory tract.
The aim of this work was to give a comprehensive assessment of the innate immunity markers, namely, the level of expression of the TLR2, TLR4, TLR9 and DEFB1 genes, and to study the association of some single nucleotide polymorphisms (SNPs) in the 5’-untranslated region of the DEFB1 gene with bronchial asthma in children. Three SNPs were studied: rs1799946, rs1800972 and rs11362. They are associated with HIV infection and infections caused by Candida albicans, Pseudomonas aeruginosa and other microorganisms and sepsis development [8, 9], but there are no reports on their association with allergies.
METHODS
The study was carried out in patients of the Rehabilitation. Care Unit for Children with Allergies and Respiratory Tract Diseases of the Scientific Center of Children’s Health (Moscow). The study included 48 asthmatic children aged 3–7 years. The control group included 70 children without respiratory conditions, inflammatory and infectious diseases and allergies. Nasal scrapes were collected at the time of BA exacerbations that were accompanied by an acute respiratory infection.
For DNA extraction, the AmpliPRIME Ribo-sorb kit (InterLabService, Russia) was used. The real time PCR assay was conducted using SYBR Green I PCR Kit by Syntol, Russia. Data were statistically processed in MO Excel 2007 with Statistica 10.0 software (StatSoft, USA). Pearson’s chi squared and Odd Ratio were computed (OR >1 indicated genotype association with BA, OR <1 indicated a genotype protective against BA) [10].
Expression of the DEFB1, TLR2, TLR4 and TLR9 genes was compared to β-actin gene expression. For RNA extraction, the AmpliPRIME Ribo-sorb kit was used. Reverse transcription was performed with the OT-1 kit by Syntol, real time PCR was carried out using the SYBR Green I PCR Kit. For statistical processing, Mann-Whitney test was applied (p <0.05).
The study was approved by the Ethics Committee of Pirogov Russian National Research Medical University. Participants’ parents gave their informed consent.
RESULTS
Genotype frequency distribution of rs1799946, rs1800972 and rs11362 polymorphisms of the DEFB1 gene showed that the following genotypes are associated with the risk of asthma in children: AA of rs11362 and СС of rs1799946, while genotypes GG and AA of rs1799946 and rs1200972 are protective against BA (fig. 1). Distribution of DEFB1 alleles was alike in both groups.
Expression of the DEFB1 gene was 3.5 times lower in children with bronchial asthma, compared to healthy children (fig. 2). A single nucleotide polymorphism in the promoter region can affect the level of gene expression and the amount of the produced protein. We divided patients of the experimental group into 3 subgroups based on the level of β-defensin-1 expression: low expression (> 10,000 times higher than β-actin expression), moderate (10,000–30,000 times higher than β-actin expression ) and high (>30, 000 times higher than β-actin expression). It was found that AG genotype of rs11362 polymorphism is associated with the increased level of β-defensin-1 expression in epithelial cells. For example, the frequency of AG genotype in subgroups with high and low expression of DEFB1 was 0.67 and 0.30, respectively. Genotype AA is associated with reduced expression of the β-defensin gene. Other genotypes of the studied polymorphisms are not associated with changes in the β-defensin gene expression. Patients with bronchial asthma showed a 19.5 times increased expression of the TLR2 gene compared to the controls; TLR9 expression was 9.5 times higher, TLR4 expression was 8.3 times higher. Results are presented in the table below.
DISCUSSION
The obtained data can indicate that chronic inflammation of the bronchial mucosa in asthmatic children is partially associated with mutations in the 5’-untranslated region of DEFB1. Having assessed the expression of DEFB1, TLR2, TLR4 and TLR9, we made a supposition that β-defensin-1 participates in BA pathogenesis. Antimicrobial peptides produced by epithelial cells of the respiratory tract mucosa prevent the invasion of pathogens into the mucosa. However, if antimicrobial peptide production is decreased and bacterial load is high, pathogens are recognized by TLRs of epithelial cells, which triggers a cascade of pro-inflammatory reactions, including synthesis of IL-1β, IL-6 and IL-12, INF-α, INF-β and chemokynes. Besides, through the activation of epithelial TLRs, production of thymic stromal lymphopoietin and IL-33 is induced. The latter interact with dendritic cells, boost activity of CD40 and CD80 costimulatory molecules, regulate Th0 and Th2 differentiation, and come into contact with mast cells inducing their degranulation [11, 12]. It facilitates the development of chronic inflammation (fig. 3).
CONCLUSIONS
Genotype AA of rs11362 and genotype CC of rs1799946 polymorphisms located in the 5’-untranslated region of the DEFB1 gene are reliably associated with bronchial asthma in children. Genotype GG of rs1799946 and genotype AA of rs120097 polymorphisms are protective against asthma. Genotype АА of rs11362 polymorphism is also associated with the reduced expression of the β-defensin-1 gene DEFB1. Thus, some mutations in DEFB1 cause imbalances in the nasal mucosal innate immunity resulting in frequent exacerbations of BA in the setting of respiratory infections.