PAK1's role in eotaxin-mediated murine eosinophil migration

描述(申请人提供)：几十年来，基础科学家认为哮喘炎症是由Th2合成和Th2分泌的细胞因子和生长因子协调的CD4+T辅助II(Th2)细胞反应。因此，许多研究都集中在T辅助细胞亚群的异常信号在哮喘典型的嗜酸性炎症的发展中所起的作用。然而，最近的报告表明，其他T细胞亚群、呼吸道微环境和先天免疫系统也起到了作用。尽管嗜酸性粒细胞是传播慢性哮喘炎症的数量最多的免疫效应细胞类型，但很少有信号分子被成功地靶向减弱其炎症功能。与变应原诱导的呼吸道炎症和高反应性有关的Rho家族蛋白CDC42和Rac激活其主要效应分子丝氨酸/苏氨酸p21激活的蛋白激酶(PAK)。通过肌动蛋白结合效应器，PAK1调节肥大细胞脱颗粒过程中的肌动蛋白细胞骨架，此外，我们还证明了该异构体在嗜酸性粒细胞中表达。变态反应性致敏和激发过程诱导的嗜酸性粒细胞通过CCR3信号将嗜酸性粒细胞强大地招募到肺内。我们假设PAK1调节嗜酸性粒细胞的迁移和渗透，并使用Ak1基因敲除(Ak1-/-)小鼠系统检验其影响。我们之前发表的工作和我们使用该模型的初步数据表明，在过敏原致敏和挑战的pak1-/-小鼠中，差异血细胞计数以及T辅助细胞亚群功能保持不变。此外，在过敏原致敏和激发的小鼠中，嗜酸性粒细胞的浸润以及在体内由嗜酸性粒细胞介导的嗜酸性粒细胞的浸润和体外的趋化作用都会受到pak1基因缺失的影响。在这里，我们展示了PAK1是由嗜酸性粒细胞中的嗜酸性粒细胞趋化因子：CCR3信号激活的。两个拟议的目标现在将重点描述PAK1调节嗜酸性粒细胞迁移的生化机制。在第一个目标中，我们将靶向并修复PAK1，以深入了解它在嗜酸性粒细胞趋化蛋白介导的迁移和F-肌动蛋白聚合和解聚中所起的作用。此外，我们第二个目标的研究将利用PAK1突变体的慢病毒表达来剖析PAK1发挥这种作用的具体机制。我们预测，该项目的结果将有助于测试抗PAK疗法在预防哮喘小鼠模型嗜酸性炎症发展中的作用。

DESCRIPTION (provided by applicant): For many decades, basic scientists considered asthmatic inflammation as a CD4+ T-helper II (Th2) cell response orchestrated by Th2-synthesized and Th2-secreted cytokines and growth factors. Consequently, many studies have centered around the role aberrant signaling of T helper cell subsets plays in the development of the eosinophilic inflammation typical of asthma. Recent reports, however, suggest roles for other T-cell subsets, the airway microenvironment, and the innate immune system. Although the eosinophil is the most numerous immune effector cell type that propagates chronic asthmatic inflammation, few signaling molecules have successfully been targeted to attenuate its inflammatory functions. The Rho-family of proteins, Cdc42 and Rac implicated in allergen-induced airway inflammation and hyper-reactivity, activate their main effectors serine/threonine p21-activated kinases (PAKs). Through actin-binding effectors, PAK1 modulates the actin cytoskeleton in mast cell degranulation, and additionally we have shown that this isoform is expressed in eosinophils. Eotaxin induced by the allergic sensitization and challenge process powerfully recruits eosinophils to the lungs via CCR3 signaling. We hypothesize that PAK1 modulates eotaxin-mediated eosinophil migration and infiltration and examine its effect using a Pak1 knockout (Pak1-/-) murine system. Previous work we have published and our preliminary data using this model suggest that differential blood cell counts as well as T helper subset function is preserved in the allergen-sensitized and challenged Pak1-/- mouse. Furthermore, eosinophil infiltration in the allergen-sensitized and challenged mouse as well as eotaxin-mediated eosinophil infiltration in vivo and chemotaxis in vitro are impaired by Pak1 genetic deletion. Here we show that PAK1 is activated by the eotaxin: CCR3 signaling in murine eosinophils. Two proposed aims will now focus on delineating the biochemical mechanism by which PAK1 modulates eotaxin-mediated eosinophil migration. In the first aim, we will target and restore PAK1 to thoroughly interrogate the role it plays in eotaxin-mediated migration and F-actin polymerization and depolymerization. Moreover, studies in our second aim will utilize lentiviral expression of PAK1 mutants to dissect out the specific mechanism by which PAK1 exerts this effect. We predict that the results in this project will inform the testing of anti-PAK therapy in preventing the development of eosinophilic inflammation in murine models of asthma.