Porphyromonas gingivalis lipids mediate bone loss through TLR2

描述(由申请人提供)：牙龈卟啉单胞菌是一种牙周病原体，与成人慢性牙周炎的发生和发展有关。这种生物产生主要种类的生物活性鞘脂，称为磷酸化二氢神经酰胺和其他不寻常的脂类。这些脂质是强效的炎症和免疫细胞激活剂。这项建议的主要目标是评估这些脂类如何促进与牙周炎相关的骨丢失。另一个主要目标是了解这些脂质如何通过参与先天性免疫系统，特别是通过Toll受体2(TLR2)参与调节其对骨骼的影响。了解这些脂质是如何促进TLR2依赖性骨丢失的，这与已报道的牙龈假单胞菌对实验动物牙周骨丢失的影响有关，并且可能对促进与牙周病相关的类风湿性关节炎的骨丢失具有重要意义。牙龈假单胞菌的脂类污染病变牙周组织的方式，直接的细菌入侵不能解释观察到的细菌脂的恢复。这项应用建议使用碰撞质谱仪来量化病变组织样本中的细菌脂质水平，以便确定用于在骨细胞培养和实验动物中进行测试的适当的细菌脂类的类型和混合物。这些从患病牙周组织中回收的脂质将在细胞培养中进行测试，以确定它们抑制骨形成或激活骨吸收的能力，这反映在基因表达的特定变化、骨破坏性细胞因子的分泌增加以及介导骨吸收的细胞的直接激活上。此外，将在体内通过将细菌脂类注射到骨表面来评估骨丢失，然后评估骨丢失以及骨形成细胞或骨吸收细胞的相关变化。最后，我们将通过比较从野生型或TLR2基因敲除动物分离的骨细胞中的细菌脂质效应，或通过直接测试这些动物的血脂，来研究TLR2在这些过程中的作用。本提案中总结的实验对于解释牙龈假单胞菌如何促进牙周疾病中的骨丢失至关重要，并可能为与常见系统性疾病(如类风湿性关节炎)相关的骨丢失提供另一种机制解释。 公共卫生相关性：牙周病原体牙龈卟啉单胞菌被报道通过参与天然免疫系统，特别是通过参与Toll受体2，促进牙周骨丢失。这一建议与理解牙龈假单胞菌如何在牙周疾病中介导骨破坏直接相关，因为我们将展示牙龈卟啉单胞菌的哪些构成脂防止骨形成并激活骨破坏，以及这一过程如何依赖于通过TLR2参与的天然免疫系统。

DESCRIPTION (provided by applicant): Porphyromonas gingivalis is a periodontal pathogen implicated in the initiation and progression of chronic periodontitis in adults. This organism produces major classes of biologically active sphingolipids, termed phosphorylated dihydroceramides and other unusual lipids. These lipids are potent inflammatory and immune cell activators. The primary goal of this proposal is to evaluate how these lipids promote bone loss associated with periodontitis. Another major goal is to understand how these lipids mediate their effects on bone through engagement of the innate immune system, specifically through Toll Receptor 2 (TLR2) engagement. Understanding how these lipids promote TLR2-dependent bone loss is relevant specifically to the reported effects of P. gingivalis on periodontal bone loss in experimental animals, and may be important in promotion of bone loss in rheumatoid arthritis associated with periodontal disease. P. gingivalis lipids contaminate diseased periodontal tissues in such a way that direct bacterial invasion does not account for the observed bacterial lipids recovered. This application proposes to quantify bacterial lipid levels in diseased tissue samples, using collisional mass spectrometry, in order to determine the appropriate types and mixtures of bacterial lipids for testing in bone cell cultures and experimental animals. Those lipids recovered in diseased periodontal tissues will be tested in cell culture for their capacity to either inhibit bone formation or activate bone resorption as reflected by specific changes in gene expression, increased secretion of bone destructive cytokines and direct activation of cells that mediate bone resorption. Furthermore, bone loss will be evaluated in vivo by administering bacterial lipids to bone surfaces, followed by evaluation of bone loss and the associated alterations in either bone forming or bone resorbing cells. Finally we will examine the role of TLR2 in these processes by comparing bacterial lipid effects in bone cells isolated from either wild type or TLR2 knockout animals or by testing lipids directly in these animals. The experiments summarized in this proposal are critical to explaining how P. gingivalis promotes bone loss in periodontal diseases and may provide another mechanistic explanation for bone loss associated with common systemic diseases such as rheumatoid arthritis. PUBLIC HEALTH RELEVANCE: The periodontal pathogen Porphyromonas gingivalis is reported to promote periodontal bone loss through engagement of the innate immune system, specifically though engagement of Toll Receptor 2. This proposal is directly relevant to understanding how P. gingivalis mediates bone destruction in periodontal disease because we will show which constitutive lipids of P. gingivalis prevent bone formation and activate bone destruction and how this process is dependent on engagement of the innate immune system through TLR2.