Epigenetics, dysbiosis and inflammation in epithelial cells

描述（由申请人提供）：牙周炎是一种常见的复杂慢性炎症性疾病，由微生物生物膜引发，是人群的重大健康负担。这种竞争性更新研究了人类牙龈上皮细胞炎症反应的变化，这可能阐明人类对牙周病和其他慢性炎症的易感性，并为治疗这些复杂疾病提供诊断和治疗工具。TLR缺陷和改变的下游信号传导现在在许多疾病病理学中受到重视。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。理解变异的一个主要进展来自我们最近的观察，即在具有功能失调的炎症反应的细胞中存在高甲基化的TLR 2启动子区域，其表现出钝化的细胞因子反应，这可能阻碍抗炎信号传导。因此，炎症反应延长，并有助于炎症负荷。我们的中心问题是：“表观遗传失调会导致生态失调吗？”“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上皮细胞，以测试逆转表观遗传修饰的体内模型和解开疾病易感性中的表观遗传修饰的临床研究。利用DNA甲基转移酶抑制剂抑制DNA的从头甲基化已被认为是一种新的肿瘤治疗方法。然而，这些药物的作用机制和特异性尚不清楚。所提出的体内模型将测试DNA甲基转移酶在恢复表观遗传学沉默的TLR相关基因中的特异性和效用。这项创新的建议涉及在体外，在体内动物和人类的临床样本，以阐明疾病的易感性，并介绍了DNA甲基转移酶抑制剂作为一种潜在的治疗剂牙周病。具体而言，目标1将确定和工程改造牙龈卟啉单胞菌感染后功能失调的上皮细胞中TLR 2启动子DNA的甲基化模式。这将利用DNA甲基转移酶抑制剂5-氮杂胞苷（地西他滨）逆转TLR 2基因启动子的甲基化，以恢复对牙龈卟啉单胞菌的正常TLR 2炎症反应。该目标将测试TLR 2超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的TLR 2功能。目的2：利用小鼠灌胃模型研究牙龈卟啉单胞菌诱导表观遗传学变化的能力。将研究慢性炎症诱导的牙槽骨丢失，并研究细菌诱导的甲基化的后果。该目的还将测试地西他滨作为针对牙周炎的治疗药物的效用，并且在目的3中，提出了临床研究以研究牙周炎个体的上皮细胞中的表观遗传变化。这将解决横截面是否TLR 2启动子超甲基化是相关的牙周病的自然史，如果它影响受试者的疾病的易感性，以及是否实验性牙龈炎诱导牙龈表观遗传改变。

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.