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

描述（由申请方提供）：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生物体的免疫反应引起的炎症变化。先天免疫应答在介导B的控制中起关键作用。感染和引发炎症反应。Toll样受体（TLR）是先天模式识别受体的主要家族。TLR 2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。它是对B的炎症反应的关键驱动因素之一。burgdorferi由于其脂蛋白的识别。尽管TLR 2长期以来被认为识别质膜表面的配体，但我们的实验室和其他实验室已经表明TLR 2也可以从内体隔室内发出信号。我们最近发现了一个新的共受体TLR 2，整合素？三个？1，其介导B的内吞作用。burgdorferi和合成的TLR 2配体棕榈酰基-3-Cys-Ser-（Lys）4（Pam 3CSK 4）。响应于B的TLR 2信号传导。burgdorferi或Pam 3CSK 4虽然在许多方面相似，但在诱导的下游细胞因子方面也不同。我们的初步数据表明，B。burgdorferi和Pam 3CSK 4在内吞过程中被运输到不同的内体区室中。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。 在本提案中，我们将确定B. Burgdorferi和Pam 3CSK 4被TLR 2和整合素识别。在目标1中，我们将使用RNAi文库来鉴定参与B摄取和运输的分子。burgdorferi和Pam 3CSK 4.然后，我们将使用共聚焦荧光显微镜对固定和活细胞，以确认加工的B的机制。burgdorferi和Pam 3CSK 4，并确定每种类型的刺激信号室。在目标2中，我们将使用二维差异凝胶电泳（DIGE）来鉴定B之间信号通路激活的差异。burgdorferi和Pam 3CSK 4，并了解整合素？介导的内吞作用影响每种刺激物的信号传导和运输。 亚细胞定位影响TLR信号传导的机制才刚刚开始探索。这些研究的结果将是重要的，为了解从活细菌的合成配体的加工的差异，并确定亚细胞定位的受体和配体到特定的车厢炎症信号和细胞因子释放的作用。

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.