Regulation of B-Cell Apoptosis by TLR Signaling in Periodontal Disease

描述（由申请人提供）：牙周炎是由宿主对牙周生物膜中的病原微生物的免疫反应引发的炎性疾病。B淋巴细胞在感染部位密集浸润，并且是NF-：B配体（RANKL）的受体活化剂的主要来源，RANKL是在破骨细胞分化和炎性骨吸收中起关键作用的细胞因子。我们的长期目标是确定控制免疫细胞介导的牙周骨吸收的分子机制，以预防和治疗牙周炎。Toll样受体（TLR）识别病原体相关分子模式（PAMP），TLR信号传导途径在调节免疫细胞功能中起重要作用，包括细胞因子产生、吞噬作用和程序性细胞死亡（凋亡）。虽然各种B淋巴细胞亚群表达多种TLR，包括TLR 4和TLR 9，但TLR信号传导对B细胞凋亡的作用完全不清楚。本申请的中心假设是TLR 4/TLR 9信号传导的共激活诱导RANKL产生B细胞的凋亡，从而减少B细胞介导的牙周骨吸收。我们的假设是基于我们的初步结果，即用TLR 4/9激动剂、LPS和CpG ODN共刺激培养的大鼠脾细胞，增加TLR 4和TLR 9的表达，并增加B细胞凋亡。在本申请中，我们将探索TLR 4/TLR 9信号传导如何使用体外和体内模型调节B细胞凋亡。在特定目标1中，我们将使用体外破骨细胞生成模型确定TLR 4和TLR 9的共激活是否诱导表达RANKL的B细胞凋亡，从而抑制B细胞介导的破骨细胞生成。在具体目标2中，我们将确定TLR 4/TLR 9的共激活是否诱导B细胞凋亡并减少体内B细胞介导的牙周骨吸收。我们已经开发了一种体内模型的过继B细胞转移/牙龈抗原注射，可用于进行拟议的研究。拟定研究的基本原理是，一旦了解TLR信号传导如何控制RANKL表达的B细胞凋亡，基于诱导RANKL表达的B细胞凋亡的治疗策略，因此，抑制B细胞介导的破骨细胞生成可能有效预防牙周炎患者的骨吸收和牙齿脱落。我们还希望，这里学到的东西可以适用于其他免疫介导的骨吸收疾病的理解。

DESCRIPTION (provided by applicant): Periodontitis is an inflammatory disease triggered by host immune response to pathogenic microorganisms in the periodontal biofilm. B lymphocytes are densely infiltrated at the site of infection and are a primary source of receptor activator of NF-:B ligand (RANKL), a cytokine that plays a pivotal role in osteoclast differentiation and inflammatory bone resorption. Our long-term goal is to determine the molecular mechanisms which control immune cell-mediated periodontal bone resorption in periodontitis for preventive and therapeutic purposes. Toll-like receptors (TLRs) recognize pathogen associated molecular patterns (PAMPs) and TLR signaling pathways play an important role in regulating immune cell functions, including cytokine production, phagocytosis, and programmed cell death (apoptosis). While various B lymphocyte subsets express multiple TLRs, including TLR4 and TLR9, the role of TLR signaling on B cell apoptosis is entirely unclear. The central hypothesis of this application is that co-activation of TLR4/TLR9 signaling induces apoptosis of RANKL-producing B cells, thus diminishing B cell-mediated periodontal bone resorption. Our hypothesis is based on our preliminary results demonstrating that co-stimulation with TLR4/9 agonists, LPS and CpG ODN, elevated expressions of TLR4 and TLR9 and increased B cell apoptosis in cultured rat splenocytes. In this application we will explore how TLR4/TLR9 signaling regulates B cell apoptosis using both in vitro and in vivo models. In Specific Aim 1, we will determine if co-activation of TLR4 and TLR9 induce RANKL-expressing B cell apoptosis, thereby inhibiting B cell-mediated osteoclastogenesis, using an in vitro osteoclastogenesis model. In Specific Aim 2, we will determine whether co-activation of TLR4/TLR9 induces B cell apoptosis and decreases B cell-mediated periodontal bone resorption in vivo. We have developed an in vivo model of adoptive B cell transfer/gingival antigen injection that can be used to undertake the proposed research. The rationale for the proposed research is that, once it is known how TLR signaling controls RANKL-expressing B cell apoptosis, therapeutic strategies based on induction of RANKL-expressing B cell apoptosis, therefore, inhibition of B cell-mediated osteoclastogenesis may be effective in preventing bone resorption and tooth loss in people with periodontitis. It is also expected that what is learned here could be applicable to the understanding of other immune-mediated bone-resorptive disorders.