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与成人慢性牙周炎的发生和发展有关的牙周病原体。这种微生物产生主要类别的生物活性鞘脂，称为磷酸化二氢神经酰胺和其他不寻常的脂质。这些脂质是有效的炎症和免疫细胞激活剂。本提案的主要目标是评估这些脂质如何促进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先天免疫系统的参与，特别是通过Toll受体2（TLR2）的参与来介导其对骨骼的影响。了解这些脂质如何促进TLR2依赖性骨丢失与实验动物中牙龈卟啉单胞菌对牙周骨丢失的影响特别相关，并且可能对促进牙周病相关的类风湿性关节炎中的骨丢失很重要。牙龈卟啉单胞菌脂质以这样的方式污染患病的牙周组织，即直接细菌入侵不能解释所观察到的回收的细菌脂质。本申请提出使用碰撞质谱法定量患病组织样品中的细菌脂质水平，以确定用于在骨细胞培养物和实验动物中测试的细菌脂质的适当类型和混合物。将在细胞培养中测试在患病牙周组织中回收的那些脂质抑制骨形成或激活骨吸收的能力，如通过基因表达的特异性变化、骨破坏性细胞因子的分泌增加和介导骨吸收的细胞的直接激活所反映的。此外，将通过向骨表面施用细菌脂质，然后评价骨丢失和骨形成或骨再吸收细胞中的相关改变，在体内评价骨丢失。最后，我们将通过比较从野生型或TLR2敲除动物分离的骨细胞中的细菌脂质效应或通过直接在这些动物中测试脂质来研究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卟啉单胞菌如何促进牙周疾病中的骨丢失至关重要，并可能为与常见全身性疾病（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：据报道，牙周病原体牙龈卟啉单胞菌通过参与先天免疫系统，特别是通过参与Toll受体2，促进牙周骨丢失。该提议与理解牙龈卟啉单胞菌如何介导牙周病中的骨破坏直接相关，因为我们将展示牙龈卟啉单胞菌的组成性脂质阻止骨形成并激活骨破坏，以及该过程如何依赖于通过TLR2参与先天免疫系统。

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.