DataSheet_1_Mapping of DNA methylation-sensitive cellular processes in gingival and periodontal ligament fibroblasts in the context of periodontal tissue homeostasis.pdf

Interactions between gingival fibroblasts (GFs) and oral pathogens contribute to the chronicity of inflammation in periodontitis. Epigenetic changes in DNA methylation are involved in periodontitis pathogenesis, and recent studies indicate that DNA methyltransferase (DNMT) inhibitors may protect against epithelial barrier disruption and bone resorption. To assess the impact of DNMT inhibition on GFs, cells were cultured with decitabine (5-aza-2’-deoxycytidine, DAC) for 12 days to induce DNA hypomethylation. We observed several potentially detrimental effects of DAC on GF biological functions. First, extended treatment with DAC reduced GF proliferation and induced necrotic cell death. Second, DAC amplified Porphyromonas gingivalis- and cytokine-induced expression and secretion of the chemokine CCL20 and several matrix metalloproteinases (MMPs), including MMP1, MMP9, and MMP13. Similar pro-inflammatory effects of DAC were observed in periodontal ligament fibroblasts. Third, DAC upregulated intercellular adhesion molecule-1 (ICAM-1), which was associated with increased P. gingivalis adherence to GFs and may contribute to bacterial dissemination. Finally, analysis of DAC-induced genes identified by RNA sequencing revealed increased expression of CCL20, CCL5, CCL8, CCL13, TNF, IL1A, IL18, IL33, and CSF3, and showed that the most affected processes were related to immune and inflammatory responses. In contrast, the genes downregulated by DAC were associated with extracellular matrix and collagen fibril organization. Our observations demonstrate that studies of DNMT inhibitors provide important insights into the role of DNA methylation in cells involved in periodontitis pathogenesis. However, the therapeutic potential of hypomethylating agents in periodontal disease may be limited due to their cytotoxic effects on fibroblast populations and stimulation of pro-inflammatory pathways.

牙龈成纤维细胞（GFs）与口腔病原体之间的相互作用，可促进牙周炎的炎症慢性化进程。DNA甲基化的表观遗传改变参与牙周炎的发病机制，近期研究显示，DNA甲基转移酶（DNMT）抑制剂或可抵御上皮屏障破坏与骨吸收。为评估DNMT抑制对GFs的影响，本研究将细胞与地西他滨（5-氮杂-2’-脱氧胞苷，DAC）共培养12天以诱导DNA低甲基化。我们观测到DAC对GFs生物学功能存在多项潜在有害影响：其一，延长DAC处理会降低GFs的增殖活性并诱发坏死性细胞死亡；其二，DAC可放大牙龈卟啉单胞菌及细胞因子介导的趋化因子CCL20与包括MMP1、MMP9、MMP13在内的多种基质金属蛋白酶（MMPs）的表达与分泌。在牙周膜成纤维细胞中，同样可观测到DAC的这类促炎效应；其三，DAC可上调细胞间黏附分子-1（ICAM-1），该现象与牙龈卟啉单胞菌对GFs的黏附增强相关，或可促进细菌播散；最后，对RNA测序鉴定出的DAC诱导基因进行分析后发现，CCL20、CCL5、CCL8、CCL13、TNF、IL1A、IL18、IL33及CSF3的表达均显著上调，且受影响最显著的生物学过程与免疫及炎症应答密切相关。与之相反，DAC下调的基因则与细胞外基质及胶原纤维组织过程相关。本研究结果表明，针对DNMT抑制剂的相关研究可为阐明DNA甲基化在牙周炎发病进程中相关细胞内的作用提供重要见解。然而，低甲基化药物在牙周疾病中的治疗潜力或存在局限，因其会对成纤维细胞群体产生细胞毒性作用并激活促炎症通路。