Role of sphingosine-1-phosphate receptor 2 in the pathogenesis of periodontitis

 描述（由申请人提供）：牙周炎是一种细菌驱动的炎性骨丢失疾病，影响美国47%的成年人。口腔病原体，如伴放线菌聚集杆菌（Aggregatibacteractinomycetemcomitans，Aa），与局部侵袭性牙周炎相关的病原体，刺激哺乳动物细胞产生和释放鞘氨醇-1-磷酸（S1 P）。S1 P与5种G蛋白偶联受体结合，启动多种细胞信号通路，影响多种生理和病理生理过程。然而，目前尚不清楚S1 P信号如何调节口腔病原体诱导的炎性骨丢失反应。长期目标是了解S1 P信号在调节对口腔病原体的免疫反应中的作用，并开发一种新的牙周炎治疗策略。我们的初步研究表明，S1 P信号在调节对Aa的免疫应答中是至关重要的。在牙周炎性骨丢失动物模型中，小鼠（鞘氨醇激酶1 KO小鼠）中S1 P生成的缺陷减弱了牙周白细胞浸润，减轻了对Aa刺激的牙槽骨丢失。此外，药理学抑制或RNA沉默小鼠骨髓源性巨噬细胞（BMM）中的S1 P受体2（S1 PR 2）可显著减弱Aa诱导的考克斯-2、IL-1 β、IL-6和TNF mRNA表达。此外，药理学抑制S1 PR 2在BM衍生的前破骨细胞抑制破骨细胞诱导的AA刺激的条件培养基。本申请的总体目的是建立S1 PR 2介导的信号传导作为调节对口腔病原体Aa的免疫应答的关键调节剂。我们假设Aa诱导的促炎性骨丢失反应是通过S1 PR 2信号传导介导的。阻断S1 PR 2信号传导将减少促炎性细胞因子的产生，减弱破骨细胞生成，并减轻Aa诱导的牙槽骨丢失。我们将测试这两个特定目的以确定1）体外S1 PR 2缺乏是否会显著降低ERK、PLC、Rho和NF-κ B蛋白激酶活性，减少促炎细胞因子产生，并减弱对Aa暴露的响应的破骨细胞生成。此外，我们将确定阻断S1 PR 2是否会降低破骨细胞生成因子RANK、RANKL和M-CSF。2)体内S1 PR 2缺乏会减少牙周白细胞浸润，减轻促炎细胞因子的产生，并减轻牙槽骨丢失。在第一个目标下，我们将使用BMM或来自S1 pr 2 +/+和S1 pr 2-/-小鼠的前破骨细胞来确定细胞中的S1 PR 2缺陷是否会降低对Aa的免疫应答。此外，我们将确定阻断S1 PR 2是否会降低Aa在BMM或成骨细胞中诱导的RANK、RANKL和M-CSF。在第二个目标下，我们将使用Aa诱导的牙周炎性骨丢失动物模型来确定小鼠中S1 PR 2缺陷是否会减少Aa诱导的炎性骨丢失反应。本研究将阐明S1 PR 2信号通路在牙周炎发病机制中的作用。本研究将为牙周炎的治疗提供新的靶点，为进一步研究以S1 PR 2为靶点的牙周炎治疗策略奠定基础。

 DESCRIPTION (provided by applicant): Periodontitis is a bacteria-driven inflammatory bone loss disease affecting 47% of adults in the United States. Oral pathogens, such as Aggregatibacter actinomycetemcomitans (Aa), the pathogen associated with localized aggressive periodontitis, stimulate mammalian cells to generate and release sphingosine-1-phosphate (S1P). S1P binds to five G protein-coupled receptors, which initiates various cellular signaling pathways and affects many physiological and pathophysiological processes. However, it is unknown how S1P signaling modulates the inflammatory bone loss response induced by oral pathogens. The long-term goal is to understand the role of S1P signaling in regulating the immune response to oral pathogens and to develop a novel therapeutic strategy for periodontitis. Our preliminary study demonstrated that S1P signaling is critical in regulating the immune response to Aa. In a periodontal inflammatory bone loss animal model, deficiency in generation of S1P in mice (sphingosine kinase 1 KO mice) attenuated periodontal leukocyte infiltration and alleviated alveolar bone loss in response to Aa stimulation. Additionally, pharmacological inhibition or RNA silencing of S1P receptor 2 (S1PR2) in murine bone marrow-derived macrophages (BMM) significantly attenuated COX-2, IL-1ß, IL-6, and TNF mRNA expressions induced by Aa. Furthermore, pharmacological inhibition of S1PR2 in BM-derived preosteoclasts suppressed osteoclastogenesis induced by Aa-stimulated conditioned media. The overall objective of this application is to establish S1PR2-mediated signaling as a key modulator in regulating the immune response to the oral pathogen Aa. We hypothesize that the Aa-induced proinflammatory bone loss response is mediated through S1PR2 signaling. Blocking S1PR2 signaling will reduce proinflammatory cytokine production, attenuate osteoclastogenesis, and alleviate alveolar bone loss induced by Aa. We will test these two specific aims to determine if 1) S1PR2 deficiency in vitro will significantly decrease ERK, PLC, Rho, and NF-ĸB protein kinase activities, reduce proinflammatory cytokine production, and attenuate osteoclastogenesis in response to Aa exposure. Additionally, we will determine if blocking S1PR2 will decrease osteoclastogenic factors RANK, RANKL, and M-CSF. 2) S1PR2 deficiency in vivo will decrease periodontal leukocyte infiltration, alleviate proinflammatory cytokine production, and attenuate alveolar bone loss. Under the first aim, we will use BMM or preosteoclasts derived from S1pr2+/+ and S1pr2-/- mice to determine if S1PR2 deficiency in cells will reduce the immune response to Aa. Additionally, we will determine if blocking S1PR2 will decrease RANK, RANKL, and M-CSF induced by Aa in BMM or osteoblasts. Under the second aim, we will use an Aa-induced periodontal inflammatory bone loss animal model to determine if S1PR2 deficiency in mice will reduce the inflammatory bone loss response induced by Aa. This study will elucidate the role of S1PR2 signaling, a novel key mechanism, in the pathogenesis of periodontitis. This study will define a new therapeutic target and lay the foundation to develop a novel therapeutic strategy for periodontitis by targeting S1PR2.