Epigenetics, dysbiosis and inflammation in epithelial cells

描述(申请人提供)：牙周炎是一种常见的复杂的慢性炎症性疾病，由微生物生物膜引起，是人群的重大健康负担。这一竞争性更新研究了人类牙龈上皮细胞炎症反应的变化，可能从总体上阐明人类对牙周病和其他慢性炎症的易感性，并提供诊断和治疗工具来治疗这些复杂的疾病。TLR缺陷和下游信号改变现在在许多疾病病理中被认识到。我们的长期目标是解开人类炎症功能障碍，使患者容易患牙周病。我们的目标是确定遗传因素改变炎症反应的机制。在理解变异方面的一个重大进展来自我们最近的观察，即在具有功能障碍的炎症反应的细胞中存在一个高甲基化的TLR2启动子区域，该区域表现出钝化的细胞因子反应，这可能阻碍抗炎信号。因此，炎症反应被延长，并导致炎症负担。我们继续讨论的中心问题是“表观遗传失调是否会造成生物失调？”我们的初步数据表明，上皮细胞中启动子区域的超甲基化通过沉默关键的先天免疫基因而导致宿主反应减弱。本提案旨在通过挑战牙龈卟啉单胞菌来表征体外培养的上皮细胞，测试逆转表观遗传修饰的体内模型，以及揭示表观遗传修饰在疾病易感性中的临床研究。用DNA甲基转移酶抑制剂抑制DNA甲基化已被认为是一种新的肿瘤治疗方法。然而，它们的作用机制和这些药物的特异性还不是很清楚。所提出的体内模型将测试DNA甲基转移酶在修复表观遗传沉默的TLR相关基因方面的特异性和实用性。这一创新方案涉及体外、活体动物和人类临床样本，以阐明疾病的易感性，并引入DNA甲基转移酶抑制剂作为潜在的牙周病治疗剂。具体地说，Aim 1将确定并设计牙龈假单胞菌感染后功能障碍的上皮细胞中TLR2启动子DNA的甲基化模式的改变。这将利用DNA甲基转移酶抑制剂5-氮胞苷(地西他滨)逆转TLR2基因启动子的甲基化，以恢复对牙龈假单胞菌的正常TLR2炎症反应。这一目标将测试TLR2高甲基化是否影响先天免疫失调，以及主动去甲基化是否恢复失调细胞中TLR2的功能。在目标2中，我们将使用经口灌胃的小鼠模型来研究牙龈假单胞菌诱导表观遗传学改变的能力。将研究慢性炎症导致的牙槽骨丢失，以及细菌诱导的甲基化的后果。这一目标还将测试地西他滨作为牙周炎治疗药物的有效性，在目标3中，建议进行临床研究，以调查牙周炎患者上皮细胞的表观遗传学变化。这将横断面研究TLR2启动子超甲基化是否与牙周病的自然历史相关，是否影响受试者对疾病的易感性，以及实验性牙龈炎是否导致牙龈的表观遗传学改变。

DESCRIPTION (provided by applicant): Periodontitis is a common complex chronic inflammatory disease initiated by a microbial biofilm and is a significant health burden for the population. This competing renewal investigates variation in human gingival epithelial cells' inflammatory responses that may elucidate human susceptibility to periodontal disease and other chronic inflammatory conditions in general and provide diagnostic and therapeutic tools to treat these complex diseases. TLR deficiencies and altered downstream signaling is now being appreciated in many disease pathologies. Our long-term goal is to unravel human inflammatory dysfunctions that render patients susceptible to periodontal disease. We aim to define the mechanism whereby genetic factors modify the inflammatory response. A major advance in understanding variation comes from our recent observation that there is a hypermethylated TLR2 promoter region in cells with dysfunctional inflammatory responses that exhibited a blunted cytokine response which could hampers anti-inflammatory signaling. Thus inflammatory responses are prolonged and contribute to the inflammatory burden. Our central question in this continuation is "Does epigenetic dysregulation creates dysbiosis?" Our preliminary data suggest that hyper-methylation of promoter regions in epithelial cells causes a diminished host response by silencing pivotal innate immune genes. The present proposal aims to characterize epithelial cells in vitro by challenging Porphyromonas gingivalis, to test the in vivo model to reverse epigenetic modifications and a clinical study to unravel epigenetic modifications in disease susceptibility. Inhibition of de novo DNA methylation using DNA methyltransferase inhibitors has been regarded as a new therapeutic approach in oncology. Nonetheless, their mechanism of action and specificity of these drugs are not clearly understood. The proposed in vivo model will test the specificity and utility of DNA methyltransferase in restoring the epigenetically silenced TLR related genes. This innovative proposal involves in vitro, in vivo animal and human clinical samples to elucidate disease susceptibility, and introduces DNA methyltransferase inhibitor as a potential therapeutic agent for periodontal disease. Specifically, Aim 1 will determine and engineer the altered methylation pattern of the TLR2 promoter DNA in dysfunctional epithelial cells following P. gingivalis infection. This will utilize DNA methyltransferase inhibitor 5-azacytidine (decitabine) to reverse the methylation of the TLR2 gene promoter to restore the normal TLR2 inflammatory response to P. gingivalis. This aim will test whether TLR2 hypermethylation influences innate immune dysregulation and whether active demethylation restores TLR2 functionality in dysregulated cells. In Aim 2, we will use the oral gavage mouse model to study the ability of P. gingivalis to induce epigenetic changes. The chronic inflammation induced alveolar bone loss will be studied and the consequence of bacterial induced methylation will be investigated. This aim will also test the utility of Decitabine as a therapeutic drug against periodontitis, and in Aim 3, Clinical studies are proposed to investigate epigenetic changes in epithelial cells of individuals with periodontitis. This will address cross-sectionally whether TLR2 promoter hypermethylation is relevant in the natural history of periodontal disease and if it influences a subjects' susceptibility to disease and whether an experimental gingivitis induce epigenetic alteration in the gingiva.