Modulation of inflammation and sepsis by bacterial PAL

描述（由申请人提供）： 早期脓毒症反应被微生物产物激活，微生物产物通过Toll样受体（TLR）引发炎症。细菌肽聚糖相关脂蛋白（PAL）是一种普遍存在的革兰氏阴性细菌外膜蛋白。PAL是一种有效的TLR2激动剂，在脓毒症中释放到血液中。中心假设是PAL导致炎症和凝血病，以及败血症中的心血管和肺功能障碍。研究将探索PAL单独的作用，并将测试PAL与通过各种TLR激活的其他促炎细菌产物之间的协同作用。将使用TLR2敲除小鼠评价TLR2在对PAL的脓毒症应答中的作用。具体目标1：探索PAL在细胞和全身炎症和凝血中的作用。将通过测量粘附分子和凝血因子水平来研究内皮细胞活化。将通过流式细胞术分析CD11b表达和趋化性试验评估神经元活化。将使用粘附试验研究中性粒细胞-内皮细胞相互作用。细胞因子水平将用于评价巨噬细胞活化。在注射PAL后，将在小鼠中测量细胞因子和凝血因子的循环水平、血小板和中性粒细胞计数以及中性粒细胞活化。具体目标2：表征PAL和TLR2在心血管和肺反应中的作用。将使用有创和无创方法评估对血管张力和心肌功能的影响，包括超声心动图、左心室传导导管和全身动脉血压监测仪。将通过测量动脉血气、肺湿干重和分析支气管肺泡灌洗液来评估肺反应。具体目标3：评价PAL在脓毒症中促进细菌毒力的机制。将使用缺乏或具有异常PAL的细菌突变体。这些研究将测量细胞和系统的反应，热灭活PAL缺失突变体和纯化的未酰化PAL。此外，将进行研究以比较野生型和PAL突变体E.在腹膜炎脓毒症模型中野生型和TLR2敲除小鼠上的大肠杆菌细菌。这些拟议的研究将使用PAL作为一种工具，以深入了解细菌脂蛋白和TLR2在脓毒症诱导过程中的作用，包括内皮细胞功能障碍，全身性炎症以及心血管和肺功能障碍。

DESCRIPTION (provided by applicant): Early septic responses are activated by microbial products that initiate inflammation through Toll-like receptors (TLRs). Bacterial peptidoglycan-associated lipoprotein (PAL) is a ubiquitous Gram-negative bacterial outer membrane protein. PAL is a potent TLR2 agonist that is released into the blood in sepsis. The central hypothesis is that PAL contributes to inflammation and coagulopathy, and to cardiovascular and pulmonary dysfunction in sepsis. Studies will explore effects of PAL alone and will test for synergy between PAL and additional pro-inflammatory bacterial products that activate through various TLRs. The role of TLR2 in septic responses to PAL will be evaluated using TLR2 knockout mice. Specific Aim 1: Explore the role of PAL in cellular and systemic inflammation and coagulation. Endothelial cell activation will be studied by measuring adhesion molecule and coagulation factor levels. Neutrophil activation will be assessed by FACS analysis for expression of CD11b and chemotaxis assays. Neutrophil-endothelial cell interactions will be studied using adherence assays. Cytokine levels will be utilized to evaluate macrophage activation. Circulating levels of cytokines and coagulation factors, platelet and neutrophil counts, and neutrophil activation will be measured in mice following injection with PAL. Specific Aim 2: Characterize the roles of PAL and TLR2 on cardiovascular and pulmonary responses. Effects on vascular tone and on myocardial function will be assessed using invasive and non-invasive means, including echocardiography, left ventricular conductance catheters, and systemic arterial blood pressure monitors. Pulmonary responses will be assessed by measuring arterial blood gases, lung wet-to-dry weights, and by analysis of bronchoalveolar lavage fluid. Specific Aim 3: Evaluate mechanisms by which PAL contributes to bacterial virulence in sepsis. Bacterial mutants that lack or have abnormal PAL will be utilized. These studies will measure cellular and systemic responses to heatkilled PAL-deletion mutants and to purified unacylated PAL. In addition, studies will be performed to compare effects of wild-type and PAL-mutant E. coli bacteria on wild-type and TLR2 knockout mice in a peritonitis sepsis model. These proposed studies will use PAL as a tool to gain insight into the role of bacterial lipoproteins and of TLR2 in sepsis-induced processes, including endothelial cell dysfunction, generalized inflammation, and cardiovascular and pulmonary dysfunction.