根尖周炎患者外周血单核细胞中TLR-2基因甲基化：一项横断面研究。

TLR-2 gene methylation in peripheral blood monocytes from apical periodontitis individuals: A cross-sectional study.

作者信息

Bordagaray María José, Fernández Alejandra, Pellegrini Elizabeth, Garrido Mauricio, Hernández Patricia, González-Quintanilla David, Navia Gabriel, Rehbein Daniela, Baeza Mauricio, Gebicke-Haerter Peter, Hernández Marcela

机构信息

Laboratory of Periodontal Biology, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.

Department of Conservative Dentistry, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.

出版信息

Int Endod J. 2025 Aug;58(8):1172-1183. doi: 10.1111/iej.14255. Epub 2025 May 23.

DOI:10.1111/iej.14255

PMID:40407270

Abstract

AIM

Apical periodontitis (AP) is a chronic inflammatory disease arising from the contamination of the root canal system. Epigenetic regulation plays a pivotal role in controlling monocyte/macrophage-mediated local and systemic responses to bacterial challenges via toll-like receptors (TLRs). We aimed to explore the relationship between the methylation and expression patterns of TLR-2 in peripheral blood monocytes of individuals with AP and controls.

METHODOLOGY

Cross-sectional study. Otherwise healthy individuals with AP (n = 25) and controls (n = 29) were recruited. Peripheral blood monocytes were isolated from blood samples by Ficoll gradient and negative immunoselection. DNA and RNA were extracted from monocytes. DNA was bisulfite-treated, amplified and sequenced to evaluate the global and cytosine-phosphate-guanine (CpG) single-site methylation pattern of the TLR-2 gene promoter. mRNA relative levels of TLR-2 were assessed by qPCR. The potential associations between AP, TLR-2 DNA methylation and TLR-2 gene expression were explored using generalized structural equation models (GSEM).

RESULTS

TLR-2 expression was significantly upregulated in peripheral blood monocytes from individuals with AP compared to controls (p = 0.005). Though no differences were found in the global methylation pattern, CpG single sites from the TLR-2 gene were differentially methylated at positions -40 and +24 (p < 0.05). The methylated positions at -40 and -20 in TLR-2 were associated with TLR-2 transcriptional upregulation (p < 0.05). Further evaluation with GSEM analysis showed that AP promoted the methylation of the -40 CpG single site on the TLR-2 gene, which, in turn, upregulated TLR-2. Conversely, the methylation of the -20 CpG single site did not act as a mediator of TLR-2 transcription in AP.

CONCLUSIONS

AP diagnosis activates peripheral blood monocytes via -40 CpG single-site methylation, independently promoting TLR-2 expression.

摘要

目的

根尖周炎（AP）是一种由根管系统污染引起的慢性炎症性疾病。表观遗传调控在通过Toll样受体（TLR）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。我们旨在探讨AP患者和对照组外周血单核细胞中TLR-2的甲基化与表达模式之间的关系。

方法

横断面研究。招募了其他方面健康的AP患者（n = 25）和对照组（n = 29）。通过Ficoll梯度和阴性免疫筛选从血样中分离外周血单核细胞。从单核细胞中提取DNA和RNA。对DNA进行亚硫酸氢盐处理、扩增和测序，以评估TLR-2基因启动子的整体和胞嘧啶-磷酸-鸟嘌呤（CpG）单位点甲基化模式。通过qPCR评估TLR-2的mRNA相对水平。使用广义结构方程模型（GSEM）探索AP、TLR-2 DNA甲基化和TLR-2基因表达之间的潜在关联。

结果

与对照组相比，AP患者外周血单核细胞中TLR-2表达显著上调（p = 0.005）。虽然在整体甲基化模式上未发现差异，但TLR-2基因的CpG单位点在-40和+24位置存在差异甲基化（p < 0.05）。TLR-2中-40和-20位置的甲基化与TLR-2转录上调相关（p < 0.05）。用GSEM分析进一步评估表明，AP促进了TLR-2基因上-40 CpG单位点的甲基化，进而上调了TLR-2。相反，-20 CpG单位点的甲基化在AP中不是TLR-2转录的介导因子。