P. gingivalis: Role of GSK3 in Host Inflammation

描述（由申请人提供）：宿主对牙周相关病原体的免疫应答已被证明是牙周病的关键决定因素，通过调节促炎和抗炎细胞因子的产生来调节这种疾病的严重程度。不能调节宿主炎症反应的性质或持续时间通常可以介导有害的宿主效应，如在慢性炎症性疾病中观察到的。我们的实验室已经确定了激酶糖原合成酶激酶3（GSK 3）作为炎症过程的中心调节因子，其能够在TLR激活后差异调节促炎细胞因子和抗炎细胞因子的水平。我们的具体假设是牙龈卟啉单胞菌与免疫细胞上表达的TLR 2和TLR 4相互作用的能力以及随后由这些TLRs介导的GSK 3活性的差异调节是通过调节宿主炎症反应的性质和程度而导致该生物体发病的主要机制。这一假设基于：1）用牙龈卟啉单胞菌刺激来自TLR 2-或TLR 4-缺陷小鼠的免疫细胞导致对GSK 3的抑制的显著差异; 2）用牙龈卟啉单胞菌刺激的巨噬细胞或树突细胞中的GSK 3的抑制导致促炎细胞因子（即IL-12）水平的严重降低，同时增加抗炎细胞因子IL-10的水平; 3）直接比较用牙龈卟啉单胞菌刺激的TLR 2-和TLR 4-缺陷细胞证明促炎细胞因子如IL-12的水平主要由TLR 4介导，而抗炎细胞因子IL-10的水平主要由TLR 2介导; 4）先天免疫细胞中GSK 3的两种主要同种型的分析揭示树突细胞仅含有可检测水平的GSK-β，而巨噬细胞含有GSK 3的两种同种型，GSK 3-α和GSK 3-β; 5）体内施用GSK 3抑制剂导致IL-1b、IL-6、IL-12、和IFN-γ的水平，而抗炎细胞因子IL-10的水平响应于牙龈卟啉单胞菌而有效地增加;和6）在小鼠中抑制GSK 3导致对牙龈卟啉单胞菌的主要Th 2相关免疫应答。这些观察结果提供了关于所报道的TLR 2和TLR 4介导促炎性和抗炎性细胞因子应答的差异的强有力的理论基础和焦点，并阐明了GSK 3细胞信号传导途径在调节宿主对牙龈卟啉单胞菌的炎症应答中的关键作用。具体目标旨在表征宿主-微生物相互作用如何参与宿主对牙龈卟啉单胞菌的炎症反应的调节。阐明和表征GSK 3参与调节这些免疫应答，并评估可在体内减弱疾病过程的治疗靶标（GSK 3）的功效。

DESCRIPTION (provided by applicant): The host immune response to periodontal-associated pathogens has been shown to be a key determinant involved in periodontal disease by regulating the production of pro- and anti-inflammatory cytokines that regulate the severity of this disease. An inability to regulate the nature or duration of the host's inflammatory response can often mediate detrimental host effects as observed in chronic inflammatory diseases. Our laboratory has identified the kinase glycogen synthase kinase 3 (GSK3) as a central regulator of the inflammatory process by its ability to differentially regulate the levels of pro- and anti-inflammatory cytokines upon TLR activation. Our specific hypothesis is that the ability of P. gingivalis to interact with TLR2 and TLR4 expressed on immune cells and the subsequent differential regulation of GSK3 activity mediated by these TLRs are major mechanisms responsible for the pathogenesis of this organism by regulating the nature and magnitude of the host's inflammatory response. This hypothesis is based on: 1) stimulation of immune cells from TLR2- or TLR4-deficient mice with P. gingivalis resulted in pronounced differences in the inhibition of GSK3; 2) inhibition of GSK3 in macrophages or dendritic cells stimulated with P. gingivalis resulted in the severe reduction in the levels of pro-inflammatory cytokines, i.e. IL-12, while concurrently augmenting the levels of the anti-inflammatory cytokine IL-10; 3) a direct comparison of TLR2- and TLR4- deficient cells stimulated with P. gingivalis demonstrated that the levels of pro-inflammatory cytokines such as IL-12 were largely mediated by TLR4 whereas the levels of the anti-inflammatory cytokine IL-10 were predominantly mediated by TLR2; 4) analysis of the two major isoforms of GSK3 in innate immune cells revealed that dendritic cells contain only detectable levels of GSKS-p, whereas macrophages contain both isoforms of GSK3, GSK3-a and GSK3-p; 5) in vivo administration of a GSK3 inhibitor resulted in a potent reduction in the levels of IL-1b, IL-6, IL-12, and IFN-y whereas the levels of the anti-inflammatory cytokine IL-10 were potently augmented in response to P. gingivalis; and 6) inhibition of GSK3 in mice resulted in a predominant Th2-assoicated immune response to P. gingivalis. These observations provide a strong rationale and focus concerning the reported differences of TLR2 and TLR4 to mediate pro- and anti- inflammatory cytokine responses and elucidate a critical role for the GSK3 cell-signaling pathway in modulating the host's inflammatory response to P. gingivalis. The specific aims are designed to characterize how host-microbial interactions are involved in the regulation of the host inflammatory response to P. gingivalis. to elucidate and characterize the involvement of GSK3 in regulating these immune responses, and to evaluate the efficacy of a therapeutic target (GSK3) that could attenuate the disease process in vivo.