P13Kdelta Modulation of Neutrophil Trafficking

说明(申请人提供)：中性粒细胞在炎症状态中起着重要作用，如果不加以控制，可能会导致组织损伤。因此，选择性地抑制对它们进入组织至关重要的通路将具有治疗价值。在这一点上，I类PI3激酶(PBK)，包括α，P，γ和增量亚型，成为有吸引力的靶点，因为它们在这些细胞的趋化因子定向迁移中发挥着关键作用。许多研究都集中在它们的功能上，但由于缺乏1)无毒以便可以在动物模型中进行研究的抑制剂，以及2)选择性地靶向主要在白细胞中表达的PDK，如Delta亚型，这一研究进展受到了阻碍。利用最近开发的PI3K Delta小分子抑制剂IC87114和p110 Delta缺陷的小鼠，我们提供了证据，表明该信号通路是药物开发的有效靶点。给小鼠口服该化合物可减少趋化因子介导的迁移，有趣的是，中性粒细胞与血管壁的粘连相互作用类似于在p110 Delta缺失动物中观察到的方式。因此，本研究的目的是确定PI3K Delta在炎症部位中性粒细胞定位中的生物学作用，以及它调节细胞因子刺激的内皮细胞致炎状态的能力。我们的研究将针对三个具体目标。在目标1中，我们将通过复制人类疾病状态的小鼠模型来评估PI3K Delta在促进炎症肺和关节中中性粒细胞聚集方面的作用。在目标2中，我们将确定PI3K/伽马和p38MAPK信号通路在体内和体外促进中性粒细胞活化和迁移的重要性，以响应内源性和细菌来源的趋化物质。后者将通过使用活体显微镜直接观察表达GFP的细胞的行为来完成。在目标3中，我们将确定在内皮细胞的功能复合体中是否存在1a和Ib类PBK，并探讨它们参与这类细胞招募中性粒细胞能力的机制(S)。总之，这些研究将确定PI3K Delta在炎症中中性粒细胞招募中的重要性，并为限制它们迁移到组织中的治疗策略提供洞察力。此外，我们的结果将为I类PBK定义一个新的角色，即调节炎症的内皮细胞的粘附性。

DESCRIPTION (provided by applicant): Neutrophils play a prominent role in inflammatory conditions, which if uncontrolled, can result in tissue injury. Thus, selective inhibition of a pathway critical for their movement into tissues would be of therapeutic value. In this regard, class I PI3 kinases (PBKs), consisting of a, P, gamma, and delta isoforms, make attractive targets as they play a pivotal role in chemokine-directional migration of these cells. Much research has concentrated on their functions, but progress has been hampered by the lack of inhibitors that 1) are nontoxic so that they may be studied in animal models, and 2) selectively target PDKs that are primarily expressed in leukocytes such as the delta isoform. Utilizing the recently developed small molecule inhibitor of PI3Kdelta, IC87114, and mice deficient in p110delta we provide evidence that this signaling pathway is a valid target for drug development. Oral administration of this compound to mice reduced chemokine-mediated migration and interestingly, adhesive interactions of neutrophils with the vessel wall in a manner analogous to that observed in p110delta null animals. Thus, the purpose of this application is to define the biological role of PI3Kdelta in neutrophil localization at sites of inflammation and its ability to modulate the proinflammatory state of cytokine-stimulated endothelium. Our studies will be directed at three specific aims. In Aim 1, we will evaluate the role of PI3Kdelta in promoting neutrophil accumulation in inflamed lung and joints using murine models that replicate disease states in man. In Aim 2, we will determine the importance of this isoform relative to PI3K/gamma and the p38MAPK signaling pathways in promoting neutrophil activation and migration in response to endogenous vs. bacteria-derived chemoattractants in vitro and in vivo. The latter will be accomplished by direct visualization of the behavior of GFP-expressing cells using intravital microscopy. In Aim 3, we will determine whether class la and Ib PBKs exist in functional complexes in endothelium and explore the mechanism(s) by which they participate in the ability of this cell type to recruit neutrophils. Together, these studies will determine the importance of PI3Kdelta in neutrophil recruitment in inflammation as well as provide insight into therapeutic strategies designed to limit their migration into tissues. Moreover, our results will define a new role for class I PBKs, regulation of the adhesive properties of inflamed endothelium.