Role of Endosomal Localization in Toll-like receptor 2 Responses to Borrelia

描述(申请人提供)：伯氏疏螺旋体是人类莱姆病的病原体。莱姆病的主要表现是由于宿主对机体的免疫反应而引起的炎症变化。天然免疫应答在控制伯氏杆菌感染和启动炎症反应中起着关键作用。Toll样受体是一类主要的先天模式识别受体。TLR2识别细菌、真菌和病毒产品的成分，包括脂蛋白、非典型脂多糖、葡聚糖、血凝素和糖磷脂肌醇。由于对伯氏杆菌脂蛋白的识别，它是伯氏杆菌炎症反应的关键驱动因素之一。尽管TLR2长期以来一直被认为是识别质膜上的配体，但我们的实验室和其他实验室已经表明，TLR2也可以从内体内部发出信号。我们最近发现了一种新的TLR2的共受体，整合素？3？1，它能介导伯氏杆菌和合成的TLR2配体棕榈酰基-3-半胱氨酸-丝氨酸-(Lys)4(Pam3CSK4)的内吞作用。伯氏杆菌或Pam3CSK4对TLR2信号的响应虽然在许多方面相似，但在诱导下游细胞因子方面也有所不同。我们的初步数据表明，伯氏杆菌和Pam3CSK4在内吞过程中被运输到不同的内吞体内。对其他受体的研究表明，亚细胞定位可能会影响信号通路的激活。 在这项建议中，我们将确定伯氏杆菌和Pam3CSK4被TLR2和整合素？识别的机制。在目标1中，我们将使用RNAi文库来鉴定参与伯氏杆菌和Pam3CSK4摄取和运输的分子。然后，我们将在固定细胞和活细胞上使用共聚焦荧光显微镜来确认Burgdorferi和Pam3CSK4的处理机制，并确定每种类型刺激的信号室。在目标2中，我们将使用双向差异凝胶电泳法(DGE)来鉴定伯氏杆菌和Pam3CSK4在信号通路激活方面的差异，并了解整合素是如何？介导的内吞作用影响每种刺激的信号和运输。 亚细胞定位影响TLR信号转导的机制才刚刚开始探索。这些研究结果将对了解活菌合成配体的加工过程中的差异以及确定受体和配体的亚细胞定位在炎症信号和细胞因子释放中的作用具有重要意义。

DESCRIPTION (provided by applicant): Borrelia burgdorferi is the causative agent of human Lyme disease. The major manifestations of Lyme disease are caused by inflammatory changes due to the host immune response to the organism. The innate immune response plays a key role in mediating control of B. burgdorferi infection and in initiating inflammatory responses. Toll like receptors (TLRs) are a major family of innate pattern recognition receptors. TLR2 recognizes components of bacterial, fungal and viral products including lipoproteins, atypical lipopolysaccarides, ??glucans, hemagglutinin and glycophosphatidylinositol. It is one of the key drivers of inflammatory responses to B. burgdorferi due to recognition of its lipoproteins. Although TLR2 has long been thought to recognize ligands at the plasma surface membrane, our lab and others have shown that TLR2 can also signal from within endosomal compartments. We have recently identified a new co-receptor for TLR2, integrin ?3?1, that mediates endocytosis of both B. burgdorferi and the synthetic TLR2 ligand palmitoyl-3-Cys- Ser-(Lys)4 (Pam3CSK4). TLR2 signaling in response to B. burgdorferi or Pam3CSK4 while similar in many respects, also differs in the downstream cytokines induced. Our preliminary data suggests that B. burgdorferi and Pam3CSK4 are trafficked into different endosomal compartments during the endocytic process. Studies of other receptors have suggested that subcellular localization may affect signaling pathway activation. In this proposal, we will determine the mechanisms by which B. burgdorferi and Pam3CSK4 are recognized by TLR2 and integrin ????. In Aim 1, we will use an RNAi library to identify molecules involved in uptake and trafficking of B. burgdorferi and Pam3CSK4. We will then use confocal fluorescent microscopy on fixed and live cells to confirm the mechanisms of processing of B. burgdorferi and Pam3CSK4, and to identify signaling compartments for each type of stimulus. In Aim 2, we will use 2-dimensional difference gel electrophoresis (DIGE) to identify differences in signaling pathway activation between B. burgdorferi and Pam3CSK4 and to understand how integrin ???? mediated endocytosis affects signaling and trafficking of each stimulus. The mechanisms by which subcellular localization affects TLR signaling are just beginning to be explored. The results of these studies will be important for understanding the differences in processing of synthetic ligands from live bacteria and determining the role of subcellular localization of receptors and ligands into specific compartments on inflammatory signaling and cytokine release.