Outside-in Mechanotransduced Inflammatory Targets

摘要 中性粒细胞在先天免疫反应中发挥早期和关键的防御作用 病原体，但也与慢性炎症性疾病有关，如 动脉粥样硬化、自身免疫性疾病和癌症。白细胞向外周血中的聚集 急性炎症是由膜表达启动的精心策划的过程。 和功能激活的白细胞和内皮细胞黏附分子(Cam) 包括选择素、整合素和免疫球蛋白超家族配体。一系列统一的主题具有 在R01的任期内出现，它提供了分子级的洞察 PMN集成力作为从滚动到坚固转变的空间力学线索 阻止和跨内皮细胞迁移：1)选择素被赋予机械和 生物化学特性使它们的解离寿命以 拉伸负荷；2)剪应力与跨膜钙释放激活的钙离子 (CRAC)通道调节钙离子流量，使整合素介导的钙离子流量同步 剪切下的停滞和形状取向；3)变构上调两者的亲和力 LFA-1和L-选择素分别与ICAM-1和E-选择素结合促进张力 强制增强以触发PMN期间协作的关键自外向内信号 招聘。在这场竞争的更新中，我们应用了创新的血管模拟 微流控通道结合实时免疫荧光成像追踪 具体目标如下：1)建立配体结合和机械转导 人中性粒细胞被激活以阻止E-选择素/ICAM-1通过 通过L-选择素传递信号。2)定义机械转导机制， LFA-1与ICAM-1的结合通过Kindlin-3等途径诱导内外信号传导 适配器身份不明。3)开发和测试变构小分子拮抗剂 利用血管模拟物阻断中性粒细胞激活和募集的结构域 微流控筛选技术。我们的策略需要使用新隔离的人类 中性粒细胞，首要目标是确定调节途径和分子 粒细胞性炎症性疾病的预后和治疗指标。 关键词 中性粒细胞，拉伸粘合力，整合素，选择素，炎症，黏附，信号

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