Fernández Alejandra, Bordagaray María José, Garrido Mauricio, Pellegrini Elizabeth, Baeza Mauricio, Chaparro Alejandra, Hernández Patricia, Hernández Marcela
Faculty of Dentistry, Universidad Andres Bello, Santiago, Chile.
Laboratory of Periodontal Biology, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
Int Endod J. 2025 Feb;58(2):284-294. doi: 10.1111/iej.14162. Epub 2024 Nov 6.
Epigenetic regulation of the key inflammatory genes plays a crucial role in controlling monocyte/macrophage-mediated local and systemic responses to bacterial challenges. However, it has not been addressed in apical periodontitis (AP). We aimed to explore the methylation pattern of the TNF-α gene promoter and its association with the inflammatory phenotype of peripheral blood monocytes from individuals with AP and controls.
A cross-sectional study was conducted, including otherwise healthy individuals with AP (n = 25) and controls (n = 29). Monocytes were isolated from the volunteer's blood samples using a Ficoll gradient followed by negative immunoselection. RNA and DNA were extracted. The DNA methylation profiles of the TNF-α gene promoter region were analyzed using bisulfite sequencing PCR. The mRNA expression levels of DNA methyltransferases 3a (DNMT3a) and Ten Eleven Translocation enzymes 1(TET1) were assessed by qPCR. A fraction of primary monocytes was also cultured for 24 h, and the supernatant was collected to measure cytokine levels through a Luminex assay. Generalized structural equation models (GSEM) evaluated the association between AP, DNA methylation, and TNF-α protein expression controlled for potential covariates. Models included the effect of the methylation of TNF-α gene promoter as a mediator of the association between AP and TNF-α protein expression levels.
Monocytes from AP individuals exhibited a heightened secretion of TNF-α and IL-1β and hypomethylation of the TNF gene promoter (p < .05). AP diagnosis was associated with the TNF-α gene promoter´s hypomethylated profile and enhanced pro-inflammatory cytokine levels, while lower methylation of the gene promoter region and -163 CpG single site mediated TNF-α overexpression (p < .05).
DNA hypomethylation at the TNF-α gene mediates a proinflammatory phenotype in monocytes from AP patients, supporting a role in the systemic response.
关键炎症基因的表观遗传调控在控制单核细胞/巨噬细胞介导的对细菌挑战的局部和全身反应中起关键作用。然而，在根尖周炎（AP）中尚未得到研究。我们旨在探讨肿瘤坏死因子-α（TNF-α）基因启动子的甲基化模式及其与AP患者和对照组外周血单核细胞炎症表型的关系。
进行了一项横断面研究，纳入了健康的AP患者（n = 25）和对照组（n = 29）。使用Ficoll梯度离心法从志愿者血液样本中分离单核细胞，随后进行阴性免疫筛选。提取RNA和DNA。使用亚硫酸氢盐测序PCR分析TNF-α基因启动子区域的DNA甲基化谱。通过定量PCR评估DNA甲基转移酶3a（DNMT3a）和十一转位酶1（TET1）的mRNA表达水平。一部分原代单核细胞也培养24小时，收集上清液通过Luminex测定法测量细胞因子水平。广义结构方程模型（GSEM）评估了AP、DNA甲基化和TNF-α蛋白表达之间的关联，并对潜在协变量进行了控制。模型包括TNF-α基因启动子甲基化作为AP与TNF-α蛋白表达水平之间关联的中介作用。
AP患者的单核细胞表现出TNF-α和白细胞介素-1β分泌增加以及TNF基因启动子低甲基化（p < 0.05）。AP诊断与TNF-α基因启动子的低甲基化谱和促炎细胞因子水平升高相关，而基因启动子区域和-163 CpG单一位点的低甲基化介导了TNF-α的过表达（p < 0.05）。
TNF-α基因的DNA低甲基化介导了AP患者单核细胞中的促炎表型，支持其在全身反应中的作用。
