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。先天免疫反应在介导控制伯氏疏螺旋体感染和启动炎症反应中起关键作用。Toll样受体（Toll like receptor, TLRs）是一类主要的先天模式识别受体。TLR2识别细菌、真菌和病毒产物的成分，包括脂蛋白、非典型脂多糖、？？葡聚糖，血凝素和糖磷脂酰肌醇。由于其脂蛋白的识别，它是对伯氏疏螺旋体炎症反应的关键驱动因素之一。虽然TLR2长期以来一直被认为识别等离子体表面膜上的配体，但我们的实验室和其他人已经证明TLR2也可以从内体腔室内发出信号。我们最近发现了TLR2的一种新的共受体，整合素3？1，介导伯氏螺旋体和合成TLR2配体palmityl -3- cys - Ser-(Lys)4 （Pam3CSK4）的内噬作用。B. burgdorferi或Pam3CSK4对TLR2信号的响应虽然在许多方面相似，但在下游细胞因子诱导下也存在差异。我们的初步数据表明，伯氏疏螺旋体和Pam3CSK4在内吞过程中被转运到不同的内体区室。其他受体的研究表明，亚细胞定位可能影响信号通路的激活。

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.