Molecular Regulation of Neutrophil Transcellular Migration'

 描述（由申请人提供）：中性粒细胞是细胞防御的第一道防线，可快速移动至组织中的感染部位。这种迁移受到严格的调控;事实上，中性粒细胞的异常聚集会导致组织损伤。尽管临床上的重要性，中性粒细胞迁移到组织的机制仍然知之甚少。我们的长期目标是确定限制中性粒细胞组织浸润和炎症的信号通路。一 该过程中的关键步骤是中性粒细胞穿过内皮的迁移，称为“血渗出”。“细胞渗出不仅仅是一个迁移步骤。它对中性粒细胞活化和随后通过嗜中性粒细胞-内皮细胞相互作用的功能以及内皮屏障完整性也很重要。因此，它直接参与炎症。有趣的是，明确的证据表明，渗出可以发生在内皮细胞之间或通过内皮细胞（分别为细胞旁或跨细胞）。但是，分别控制这两种迁移模式的机制却知之甚少。不同的迁移路径如何影响炎症结果尚不清楚。了解这些机制对于我们理解中性粒细胞生物学和炎症非常重要。更广泛地说，它将揭示可能适用于其他细胞的原理，包括癌细胞。这在临床上是基本的，因为仅针对一种迁移模式可以提供新的方法来抑制过度炎症，同时保留一些宿主防御机制。我们发现Ras邻近1（Rap 1）亚型Rap 1b限制中性粒细胞渗出和炎症。Rap 1b-/-中性粒细胞表现出增加渗出，由于选择性增加跨细胞迁移。这是由增强的PIP 3-Akt活性介导的侵入性突起引起的。Rap 1b的丢失增强了炎症，这是通过体内Akt抑制来防止的。因此，通过信号输出的中性粒细胞活化控制渗出途径， 炎症结果。我们假设，由Rap 1b，磷酸酶，PIP 3和金属蛋白酶（MMPs）调节的信号传导的时空组织，通过炎症介导的嗜中性粒细胞-内皮细胞相互作用指定了渗出途径，并因此调节炎症反应。我们将追求以下目标。目的1将研究Rap 1b，磷酸酶和PIP 3在渗出中的作用。目的2将检验特定的中性粒细胞侵入伪足组分，包括PIP 3、蛋白酶和MMP的底物，控制中性粒细胞/内皮细胞相互作用以进行跨细胞迁移的假设。目的3利用活体成像技术研究中性粒细胞渗出的机制。它将检查路径选择和体内炎症之间的关系。临床影响应该是显著的，因为拟议的目标集中在与炎症，免疫疾病和癌症相关的分子（即AKT，CD 11b，磷酸酶[SHP-1，PTEN，SHIP-1/2]）。我们预期这项研究将揭示中性粒细胞渗出的全新机制。它将为设计新的炎症治疗机会铺平道路，更广泛地说，它将为转移性细胞外渗到组织中铺平道路，因为Rap 1b失活和后续靶点与一些转移性癌症相关。

 DESCRIPTION (provided by applicant): Neutrophils are the first line of cellular defense by moving rapidly to sites of infection in tissue. This migration is tightly regulated; indeed, aberrat accumulation of neutrophils causes tissue injury. Despite the clinical importance, the mechanisms of neutrophil migration into tissues remain poorly understood. Our long-term goal is to identify the signaling pathways that limit neutrophil tissue infiltration and inflammation. A critical step in that process is neutrophil migration across the endothelium, known as "diapedesis." Diapedesis is not solely a migration step. It is also important for neutrophil activation and subsequent functions via neutrophil-endothelial cell interactions, and for endothelial barrier integrity. Hence, it directly participates in inflammation. Interestingly, unequivocal evidence indicates that diapedesis can occur between or through endothelial cells (paracellular or transcellular, respectively). But, the mechanisms that separately control these two modes of migration are poorly understood. How distinct migration paths impact outcome of inflammation is not known. Understanding these mechanisms is important for our understanding of neutrophil biology and inflammation. More broadly, it will reveal principle that may apply to other cells, including cancer cells. It is fundamental clinically because targeting only one mode of migration could offer new ways to dampen hyperinflammation while preserving some host defense mechanism. We found that Ras proximity 1 (Rap1) isoform Rap1b limits neutrophil diapedesis and inflammation. Rap1b-/- neutrophils exhibit increased diapedesis, due to a selective increase in transcellular migration. This is caused by enhanced PIP3-Akt activity-mediated invasive protrusions. Rap1b loss enhanced inflammation, which is prevented by Akt inhibition in vivo. Hence, neutrophil activation via signaling outputs control diapedesis route and inflammation outcome. We hypothesize that the spatiotemporal organization of signaling, regulated by Rap1b, phosphatase, PIP3 and metallo-proteinases (MMPs), specifies diapedesis route via protrusions-mediated neutrophil-endothelial cell interactions - and, as a result modulates inflammatory reactions. We will pursue the following aims. Aim1 will study the role of Rap1b, phosphatases and PIP3 in diapedesis. Aim 2 will test the hypothesis that specific neutrophil invadopodia components, including substrates of PIP3, proteases and MMP, control neutrophil/endothelial cell interactions for transcellular migration. Aim 3 will use intravital imaging to investigate mechanism of neutrophil diapedesis in vivo. It will examine the relationship between route choice and inflammation in vivo. The clinical impact should be significant, since the proposed aims focus on molecules (ie, AKT, CD11b, phosphatase [SHP-1, PTEN, SHIP-1/2]) that are associated with inflammatory, immune disorders, and cancer. We anticipate the study will uncover completely novel mechanisms of neutrophil diapedesis. It will pave the way to designing novel therapeutic opportunities to inflammation, and more broadly to metastatic cell extravasation into tissues, as Rap1b inactivation and subsequent targets are associated with some metastatic cancers.