PAK1's role in eotaxin-mediated murine eosinophil migration

描述（由申请人提供）：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嗜酸性粒细胞炎症的发展中所起的作用。然而，最近的报道表明，其他t细胞亚群、气道微环境和先天免疫系统也有作用。虽然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以减弱其炎症功能。rho蛋白家族，Cdc42和Rac参与过敏原诱导的气道炎症和高反应性，激活它们的主要效应物丝氨酸/苏氨酸p21活化激酶（PAKs）。通过肌动蛋白结合效应，PAK1调节肥大细胞脱颗粒过程中的肌动蛋白细胞骨架，此外，我们已经证明这种亚型在嗜酸性粒细胞中表达。由过敏致敏和激发过程诱导的Eotaxin通过CCR3信号向肺部强力招募嗜酸性粒细胞。我们假设PAK1调节eotaxin介导的嗜酸性粒细胞迁移和浸润，并使用PAK1敲除（PAK1 -/-）小鼠系统检测其作用。我们之前发表的研究和使用该模型的初步数据表明，在过敏原致敏和Pak1-/-小鼠中，差异血细胞计数和T辅助亚群功能得以保留。此外，Pak1基因缺失会损害致敏小鼠和致敏小鼠体内嗜酸性粒细胞的浸润以及eotaxin介导的嗜酸性粒细胞在体内的浸润和体外的趋化性。在小鼠嗜酸性粒细胞中，PAK1被eotaxin: CCR3信号激活。目前提出的两个目标将集中于描述PAK1调节eotaxin介导的嗜酸性粒细胞迁移的生化机制。在第一个目标中，我们将靶向和恢复PAK1，以彻底探究它在eotaxin介导的迁移和F-actin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。我们预计本项目的结果将为抗pak治疗在哮喘小鼠模型中预防嗜酸性粒细胞炎症发展的试验提供信息。

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.