Pososome Regulated Monocyte/Macrophage Tissue Infiltration

描述（由申请人提供）：粒体调节的单核细胞/巨噬细胞组织浸润摘要虽然单核细胞/巨噬细胞（MD）是先天免疫的重要组成部分，但它们在某些病理条件下也会对宿主造成不利，例如慢性炎症性疾病或癌症。已经提出，包括MD在内的一系列白细胞采用足状体、富含F-肌动蛋白的腹侧粘附和膨胀结构，不仅介导基质降解，而且在MD趋化性和组织募集中发挥作用。Wiskott-Aldrich综合征蛋白（WASP）是一种仅在造血细胞中表达的肌动蛋白成核剂Arp 2/3复合物的激活剂，是足体形成所需的，并且对适当的MD趋化性和基质降解至关重要。我们最近还鉴定了WASP和普遍表达的家族成员N-WASP的非冗余作用。该建议将确定WASP和N-WASP在趋化性、跨内皮迁移（或渗出）和侵袭中的确切作用。我们以前的工作已经证明，WASP的酪氨酸磷酸化是一个关键因素，在podosome营业额和趋化性和初步的数据表明，Src家族酪氨酸激酶Hck介导的WASP磷酸化，这是需要MD渗出通过内皮细胞。在特定目标1中，我们将确定Intersectin 2L（一种仅与磷酸化WASP结合的蛋白质）在介导WASP功能中的作用。Intersectin 2L是Cdc 42的鸟嘌呤核苷酸交换因子。我们推测WASP通过在趋化过程中募集Intersectin 2L来促进Cdc 42活性的维持。将使用多种技术来检验该假设，包括siRNA、体外和体内监测Cdc 42活性以及可光活化形式的Intersectin 2的产生。在特异性目标2中将探讨足体在趋化性、侵袭和渗出中的作用。我们将使用各种新的成像方法，包括使用生物传感器来分析本地化的活动，podosome相关的肌动蛋白的光转化，以监测动力学和贡献的富肌动蛋白的车厢在趋化性，和光消融的WASP或其他组件的podosome的趋化性。除了WASP，我们已经证明，N-WASP也需要的podosome介导的基质降解和初步数据表明，在跨内皮细胞迁移过程中的WASP磷酸化的要求，可能通过影响podosome动力学在上下表面的内皮细胞。我们将使用活细胞成像监测渗出过程中足粒的定位和WASP活性。将使用MMP生物传感器以及抑制MMP的药理学和遗传学方法来测定渗出和侵袭过程中足粒的蛋白水解功能。最后，在目标3中，我们将确定Hck、WASP和N-WASP在体内MD募集中的作用。将使用Rag 2-/-小鼠中的原位肿瘤监测MD渗出，在野生型、Hck-或WASP-缺陷小鼠中使用GFP标记的内源性巨噬细胞，或通过尾静脉注射WASP、N-WASP或Intersectin 2中下调或表达突变的GFP标记的巨噬细胞。此外，我们将使用一种新的活体成像技术来获得单核细胞迁移到活体小鼠乳腺肿瘤中的高分辨率图像。表征介导MD趋化性和外渗的分子事件将导致更好地理解这些细胞向肿瘤的募集。此外，这项工作的结果可能适用于慢性炎症性疾病，其中MD似乎发挥致病作用，这表明WASP抑制可能在治疗几种不同的人类疾病中具有治疗益处。 公共卫生相关性：虽然单核细胞/巨噬细胞（MD）是先天免疫的重要组成部分，但它们也可能在某些病理条件下造成不利影响，如慢性炎症性疾病或癌症。因此，巨噬细胞是治疗的主要靶点，但重要的是要阐明它们在组织中被招募和激活的机制。该项目的重点是表征介导MD趋化性和外渗的分子事件，这将有助于更好地了解这些细胞如何被招募到肿瘤中。此外，这项工作的结果可能适用于慢性炎症性疾病，其中MDs似乎发挥致病作用，并有助于确定治疗几种不同人类疾病的潜在治疗靶点。

DESCRIPTION (provided by applicant): Podosome regulated monocyte/macrophage tissue infiltration ABSTRACT While monocyte/macrophages (MDs) are essential components of innate immunity, they can also pose disadvantages to the host in certain pathological conditions such as chronic inflammatory diseases or cancer. It has been proposed that podosomes, F-actin rich ventral adhesion and protrusive structures, are employed by a range of leukocytes including MDs to not only mediate matrix degradation but to also play a role in MD chemotaxis and tissue recruitment. Wiskott-Aldrich syndrome protein (WASP), an activator of the actin nucleator Arp2/3 complex expressed exclusively in hematopoietic cells, is required for podosome formation and is critical for proper MD chemotaxis and matrix degradation. We have also recently identified non- redundant roles for WASP and the ubiquitously expressed family member N-WASP. This proposal will determine the precise role of WASP and N-WASP in chemotaxis, transendothelial migration (or diapedesis) and invasion. Previous work by us has demonstrated that tyrosine phosphorylation of WASP is a key factor in podosome turnover and chemotaxis and preliminary data suggest that the Src family tyrosine kinase Hck mediates WASP phosphorylation, which is required for MD diapedesis across an endothelium. In Specific Aim 1 we will determine the role of Intersectin 2L, a protein that only binds to phosphorylated WASP, in mediating WASP function. Intersectin 2L is a guanine nucleotide exchange factor for Cdc42. We hypothesize that WASP promotes the maintenance of Cdc42 activity by recruitment of Intersectin 2L during chemotaxis. A variety of techniques including siRNA, monitoring Cdc42 activity both in vitro and in vivo, and the creation of a photo- activatable form of Intersectin 2 will be utilized to test this hypothesis. The role of podosomes in chemotaxis, invasion and diapedesis will be explored in Specific Aim 2. We will determine the specific requirement of podosomes for chemotaxis using a variety of novel imaging approaches including the use of biosensors to analyze localized activity, photoconversion of podosome-associated actin to monitor kinetics and contributions of actin-rich compartments during chemotaxis, and photoablation of WASP or other components in podosomes by chromophore-assisted laser inactivation. In addition to WASP, we have demonstrated that N-WASP is also required for podosome-mediated matrix degradation and preliminary data suggest a requirement for WASP phosphorylation during transendothelial migration, potentially by affecting podosome dynamics at both the upper and lower surface of the endothelium. We will monitor the localization of podosomes and WASP activity during diapedesis using live cell imaging. The proteolytic function of podosomes during diapedesis and invasion will be assayed using an MMP biosensor and both pharmacological and genetic methods of inhibiting MMPs. Finally in Aim 3 we will determine the role of Hck, WASP and N-WASP in MD recruitment in vivo. MD diapedesis will be monitored using orthotopic tumors in Rag2-/- mice either with endogenous macrophages labeled with GFP in wild-type, Hck- or WASP-deficient mice or by tail vein injection of GFP labeled macrophages with down-regulation or expressing mutations in WASP, N-WASP or Intersectin2. In addition, we will use a new intravital imaging technique to obtain high resolution images of monocytes migrating into breast tumors in live mice. Characterizing the molecular events mediating MD chemotaxis and extravasation will lead to a better understanding of the recruitment of these cells to tumors. In addition, the results of this work may be applicable to chronic inflammatory diseases where MDs appear to play pathogenic roles, suggesting that WASP inhibition may have therapeutic benefits in the treatment of several different human diseases. PUBLIC HEALTH RELEVANCE: Project Narrative While monocyte/macrophages (MDs) are essential components of innate immunity; they can also pose disadvantages in certain pathological conditions such as chronic inflammatory diseases or cancer. Macrophages are therefore a prime target for therapies, but it is important to elucidate the mechanisms by which they are recruited to and activated in tissues. This project focuses on characterizing the molecular events mediating MD chemotaxis and extravasation that will lead to a better understanding of how these cells are recruited to tumors. In addition, the results of this work may be applicable to chronic inflammatory diseases where MDs appear to play pathogenic roles and aid in the identification of potential therapeutic targets in the treatment of several different human diseases.