Role of JAK2 in Innate Immunity

描述（申请人提供）：牙龈卟啉单胞菌被认为是牙周病的主要病因之一。评估宿主对牙龈假单胞菌炎症反应的研究表明，先天免疫受体TLR2和TLR4是主要的tlr，参与识别从牙龈假单胞菌中分离的LPS和各种病原体相关分子模式。然而，体外和体内研究表明，TLR2是主要的TLR，参与调节对整个牙龈假单胞菌的先天免疫反应。研究表明，TLR2缺失可消除或抑制宿主炎症，增强细菌清除，减轻骨质流失。因此，由牙龈假单胞菌诱导的tlr2介导的炎症反应似乎对其加剧疾病进程的能力至关重要。由于TLR2在介导全细胞牙龈假单胞菌的炎症反应中起主要作用，我们关注了TLR2激活的细胞内信号通路，并随后发现Janus激酶2 （JAK2）在控制先天炎症反应中起重要作用。我们的具体假设是JAK2是一种控制TLR2介导的炎症的中枢激酶，因此在疾病过程中起重要作用。这一假设基于：1)用牙龈假单胞菌刺激先天免疫细胞[人牙龈上皮细胞（EC）、树突状细胞（DC）和巨噬细胞]导致JAK2的tlr2依赖性激活，抑制JAK2可减轻炎症反应；(2)牙龈卟啉卟啉菌刺激先天免疫细胞导致活化的JAK2与TLR2胞质结构域相关联；(3) P. gingivalis刺激细胞中TLR2的免疫沉淀表明JAK成员JAK2、JAK3和TYK2与TLR2相关；(4)抑制JAK2导致P. gingivalis介导的TLR2酪氨酸磷酸化缺失，以及TLR2中JAK2、JAK3和TYK2募集缺失；(5)抑制JAK2导致NF-:B p65信号通路的激活消失；(6) JAK2在牙龈假单胞菌攻毒小鼠中被激活；(7)体内抑制JAK2可消除牙龈假单胞菌感染小鼠的宿主炎症反应。这些初步发现首次表征了JAK2在调节TLR2介导的先天免疫反应中的功能细胞信号通路，包括jak是如何被激活和招募到TLR2的，并确定了TLR2激活/招募jak到TLR2的能力是参与牙龈假链球菌炎症反应的一个基本过程。Specific Aims将明确JAK2通路在调节宿主对牙龈卟啉单胞菌炎症反应中的功能作用，并通过确定JAK2抑制如何影响牙龈卟啉单胞菌诱导宿主炎症和骨质流失的能力来评估体内靶向JAK2的重要性。

DESCRIPTION (provided by applicant): Porphyromonas gingivalis is considered to be one of the major etiologic agents of periodontal diseases. Studies assessing the host inflammatory response to P. gingivalis have demonstrated that the innate immune receptors, TLR2 and TLR4, are the major TLRs involved in the recognition of both the LPS and various pathogen associated molecular patterns isolated from P. gingivalis. However, as demonstrated from both in vitro and in vivo studies, TLR2 has been documented to be the predominant TLR involved in regulating the innate immune response to the whole bacterium of P. gingivalis. Studies have shown that the absence of TLR2 results in abrogated or suppressed host inflammation, enhanced bacterial clearance, and attenuated bone loss. Thus, the TLR2-mediated inflammatory response induced by P. gingivalis appears to be critical for its ability to exacerbate the disease process. Due to the primary importance of TLR2 in mediating the inflammatory response to whole-cell P. gingivalis, we focused on the intracellular signaling pathways activated by TLR2 and have subsequently identified that the Janus Kinase 2 (JAK2) plays an essential role in controlling the innate inflammatory response. Our specific hypothesis is that JAK2 is a central kinase controlling TLR2- mediated inflammation by P. gingivalis-stimulated innate immune cells and thus plays an essential role in the disease process. This hypothesis is based on: 1) stimulation of innate immune cells [human gingival epithelial cells (EC), dendritic cells (DC), and macrophages] with P. gingivalis resulted in the TLR2-dependent activation of JAK2, and inhibition of JAK2 attenuated the inflammatory response; (2) stimulation of innate immune cells with P. gingivalis resulted in the association of activated JAK2 with the cytosolic domain of TLR2; (3) immunoprecipitation of TLR2 from P. gingivalis-stimulated cells demonstrated the association of JAK members JAK2, JAK3, and TYK2 with TLR2; (4) inhibition of JAK2 resulted in the loss of P. gingivalis-mediated tyrosine phosphorylation of TLR2 and loss of JAK2, JAK3, and TYK2 recruitment to TLR2; (5) inhibition of JAK2 resulted in the abrogated activation of the NF-:B p65 signaling pathway; (6) JAK2 was activated in mice challenged with P. gingivalis; and (7) in vivo inhibition of JAK2 abrogated the host inflammatory response in mice challenged with P. gingivalis. These preliminary findings are the first to characterize a functional cell- signaling pathway for JAK2 in regulating TLR2-mediated innate immune responses, including how JAKs are activated and recruited to TLR2, and have identified that the ability of TLR2 to activate/recruit JAKs to TLR2 is a fundamental process involved in the inflammatory response to P. gingivalis. The Specific Aims will define the functional role the JAK2 pathway plays in regulating the host inflammatory response to P. gingivalis, and assess the importance of targeting JAK2 in vivo by determining how JAK2 inhibition affects the ability of P. gingivalis to induce host inflammation and bone loss in a mouse model. PUBLIC HEALTH RELEVANCE: Periodontitis is a chronic inflammatory disease. The host's immune response has been shown to play a fundamental role in the disease process by controlling the inflammatory response. Thus, defining and characterizing the how the host controls the inflammatory response is critical for identifying potential therapeutic interventions.