Outside-in Mechanotransduced Inflammatory Targets

摘要 中性粒细胞在先天性免疫应答中起着早期和关键的防御作用， 病原体，但也与慢性炎症性疾病， 动脉粥样硬化、自身免疫性疾病和癌症。将白细胞募集至 急性炎症是由膜表达启动的精细协调的过程 以及白细胞和内皮细胞粘附分子（CAM）的功能活化 包括选择素、整联蛋白和Ig超家族配体。一系列统一的主题 在R 01的任期内出现了一些新的发现，这些发现提供了分子尺度的见解， PMNs将力整合为从滚动到稳固过渡的空间机械线索 阻滞和跨内皮迁移：1）选择素被赋予机械和 生物化学性质，使他们的解离寿命增加的速度， 拉伸负荷; 2）剪切应力和跨膜钙释放激活的Ca 2 + CRAC通道调节钙通量，其功能是同步整合素介导的钙通道。 剪切下的阻滞和形状取向; 3）两者的亲和力的变构上调 LFA-1和L-选择素分别与ICAM-1和E-选择素结合， 强制集结以触发关键的由外向内信号，这些信号在PMN期间是协作的 招聘在这次竞争性更新中，我们应用了创新的血管模拟物 微流控通道结合实时免疫荧光成像， 具体目标如下：1）建立配体结合和机械转导 人嗜中性粒细胞被激活以通过E-selectin/ICAM-1停滞的事件 通过L-选择素进行信号传导。2)定义机械传导机制， LFA-1通过Kindlin-3和其他途径与ICAM-1结合， 未识别的适配器。3)开发和测试变构小分子拮抗剂 使用血管模拟物阻断中性粒细胞活化和募集的结构域 微流控筛选技术我们的策略需要使用新鲜分离的人类 中性粒细胞的首要目标是确定调节途径和分子 粒细胞炎症性疾病预后和治疗的靶点。 关键词 中性粒细胞，张力结合力，整合素，选择素，炎症，粘附，信号传导

Abstract Neutrophils mount an early and critical defense in the innate immune response to pathogens, but are also associated with chronic inflammatory diseases such as atherosclerosis, autoimmune disease, and cancer. Recruitment of leukocytes to sites of acute inflammation is a finely orchestrated process initiated by membrane expression and functional activation of leukocyte and endothelial cell adhesion molecules (CAMs) including selectins, integrins, and Ig-super family ligands. A set of unifying themes have emerged over the tenure of this R01 which provide molecular scale insight into how PMNs integrate force as a spatio-mechanical cue in the transition from rolling to firm arrest and transendothelial migration: 1) Selectins are endowed with mechanical and biochemical properties that allow their dissociation lifetime to increase with the rate of tensile loading; 2) Shear stress and transmembrane calcium release-activated Ca2+ (CRAC) channels regulate calcium flux which functions to synchronize integrin mediated arrest and shape orientation under shear; 3) allosteric upregulation in affinity of both LFA-1 and L-selectin in binding ICAM-1 and E-selectin, respectively, facilitates tensile force buildup to trigger key outside-in signals that are cooperative during PMN recruitment. In this competitive renewal we apply innovative vascular mimetic microfluidic channels combined with real-time immunofluorescence imaging to pursue the following specific aims: 1) Establish the ligand binding and mechanotransduction events by which human neutrophils are activated to arrest on E-selectin/ICAM-1 via signaling through L-selectin. 2) Define the mechanotransduction mechanism by which LFA-1 bonds to ICAM-1 elicits outside-in signaling via Kindlin-3 and other as yet unidentified adaptors. 3) Develop and test small molecule antagonists to allosteric domains that interrupt neutrophil activation and recruitment using vascular mimetic microfluidic screening technology. Our strategy entails the use of freshly isolated human neutrophils with the overarching goal of identifying regulatory pathways and molecular targets for prognosis and treatment of granulocytic inflammatory diseases. Keywords Neutrophils, Tensile bond force, Integrins, Selectins, Inflammation, Adhesion, Signaling