Department of Oral Diagnosis and Surgery, School of Dentistry at Araraquara, UNESP- São Paulo State University, São Paulo, Brazil; Department of Clinics and Surgery, School of Dentistry, Alfenas Federal University (UNIFAL-MG), Minas Gerais, Brazil.
Department of Oral Diagnosis and Surgery, School of Dentistry at Araraquara, UNESP- São Paulo State University, São Paulo, Brazil.
Arch Oral Biol. 2019 Nov;107:104508. doi: 10.1016/j.archoralbio.2019.104508. Epub 2019 Jul 24.
The aim of this study was to investigate the functionality of ATC/TTC (Hap-1) and ATT/TTC (Hap-2) Interleukin (IL) 8 gene haplotypes in the response of neutrophils to Gram-negative bacteria associated with periodontitis.
Neutrophils were isolated by gradient centrifugation from whole peripheral blood of systemically healthy individuals presenting the two IL8 gene haplotypes. Neutrophils were stimulated with P. gingivalis, A. actinomycetemcomitans and PMA/ionomycin. Cytokine gene expression (RT-qPCR) and migration/chemotaxis (boyden chamber assay) were compared according to the presence of Hap-1 or Hap-2 haplotypes. Protein production was also evaluted in the multiplex assay using the mixed population of leukocytes present in the whole blood from the same individuals. The influence of these two haplotypes on the IL8 promoter activity was assessed in gene-reporter experiments.
Hap-1 haplotype in neutrophils and leukocytes exacerbated the response to stimulation with Gram-negative bacteria, with higher levels of TNF-α (mRNA and protein), IL-1β, IL-2R and IFN-γ (protein) and with increased chemotaxis. Presence of the T allele at the rs4071 polymorphism (alias -251) was associated with increased activity of IL8 proximal promoter.
Neutrophils and leukocytes carrying the Hap-1 haplotype (ATC/TTC) in the IL8 gene present an enhanced response to stimulation with Gram-negative bacteria associated with periodontitis. Presence of the T allele (rs4073) in the IL8 proximal promoter increases transcription activity.
本研究旨在探讨白细胞介素（IL）8 基因 ATC/TTC（Hap-1）和 ATT/TTC（Hap-2）单倍型在中性粒细胞对与牙周炎相关的革兰氏阴性菌反应中的功能。
通过梯度离心从具有两种 IL8 基因单倍型的健康个体的全外周血中分离中性粒细胞。用 P. gingivalis、A. actinomycetemcomitans 和 PMA/离子霉素刺激中性粒细胞。根据是否存在 Hap-1 或 Hap-2 单倍型，比较细胞因子基因表达（RT-qPCR）和迁移/趋化（Boyden 室测定）。还使用来自同一个体全血中存在的混合白细胞群体在多重测定中评估蛋白产生。使用基因报告实验评估这两种单倍型对 IL8 启动子活性的影响。
中性粒细胞和白细胞中的 Hap-1 单倍型加剧了对革兰氏阴性细菌刺激的反应，导致 TNF-α（mRNA 和蛋白）、IL-1β、IL-2R 和 IFN-γ（蛋白）水平升高，趋化作用增强。rs4071 多态性（别名-251）处 T 等位基因的存在与 IL8 近端启动子活性增加相关。
携带白细胞介素 8 基因 Hap-1 单倍型（ATC/TTC）的中性粒细胞和白细胞对与牙周炎相关的革兰氏阴性菌刺激表现出增强的反应。IL8 近端启动子中 T 等位基因（rs4073）的存在增加转录活性。
