Controlling the upstream migration of neutrophils by manipulating the function of Mac-1 and LFA-1

摘要/摘要 白细胞从血流到炎症部位的运输以寻找感染源 是先天免疫反应的标志。中性粒细胞，髓系白细胞的先天免疫反应， 是所谓的“第一响应者”，并将迅速流量到煽动性侮辱的网站。贩卖人口是 由白细胞粘附级联启动的一种特征良好的逐步事件序列，其中 血液传播的免疫细胞系住、滚动、牢固地阻止并迁移到内皮上，然后进入组织以 发挥免疫细胞功能。这些事件都发生在血管内，通常是毛细血管后小静脉， 细胞在与内皮细胞相互作用和迁移时遇到高剪切率。 最近，一个有趣的现象是某些造血来源的细胞--例如 淋巴细胞(T细胞和B细胞)以及造血干细胞和祖细胞(HSPC)将爬行 上游，与流动方向相反，在出现配体细胞间黏附的表面上停滞后 分子-1(ICAM-1)。上游迁移是由β2整合素αLβ2介导的，也被称为淋巴细胞 功能相关抗原-1(LFA-1)，与ICAM-1结合。中性粒细胞表达LFA-1，以及 ICAM-1的额外受体，巨噬细胞-1抗原(Mac-1)，当中性粒细胞 激活了。我们的实验室推测，中性粒细胞也可以被诱导在ICAM-1上上游爬行 如果MAC-1被禁用或阻止，则允许LFA-1/ICAM-1交互。我们的初步结果 通过阻断MAC-1(用抗体)或通过遗传操作Mac-1(使用CRIPSR-1)来证明这一点 Cas9缺失)，中性粒细胞可以在ICAM-1表面上游迁移，为这一应用奠定了前提。 在这里，我们建议确定控制中性粒细胞上游迁移的关键信号， 中性粒细胞如何在上游迁移时产生力量，并确定 中性粒细胞向上游迁移。在目标1中，我们将确定上游涉及的关键分子信号 中性粒细胞的迁移，首先分析ICAM-1与VCAM-1上发生的差异信号 然后使用CRISPR识别并删除在LFA-1和MAC-1下游运行的信号 正在编辑。具体地说，我们将重点删除Mac-1/ICAM-1结合的下游信号以确定 抑制中性粒细胞上游迁移的信号。在目标2中，我们将测量和量化 由中性粒细胞产生，利用牵引力显微镜进行上游迁移。使用 在目标1中生成的敲除，我们将测量空间排列和大小的差异 上游和下游爬行的中性粒细胞之间的作用力。最后，在目标3中，我们将确定 通过确定中性粒细胞上游迁移的时间变化来确定其生理学相关性 内皮细胞体外迁移所需的改变和失能的中性粒细胞的差异转运 在急性炎症的小鼠模型中进入腹膜。

Summary/Abstract The trafficking of leukocytes from the blood stream to the sites of inflammation to find infectious agents is a hallmark of the innate immune response. Neutrophils, myeloid leukocytes of the innate immune response, are the so-called “first responders” and will rapidly traffic to the sites of inflammatory insult. Trafficking is initiated by the leukocyte adhesion cascade, a well-characterized, stepwise sequence of events in which blood borne immune cells tether, roll, firmly arrest, and migrate on the endothelium and then enter tissues to perform immune cell functions. These events all occur within blood vessels, normally post-capillary venules, where cells encounter high shear rates while interacting with and transmigrating through the endothelium. Recently, an interesting phenomenon wherein certain cells of hematopoietic origin – such as lymphocytes (T-cells and B-cells) as well as hematopoietic stem and progenitor cells (HSPCs) – will crawl upstream, against the direction of flow, after arrest on surfaces that present the ligand intercellular adhesion molecule-1 (ICAM-1). Upstream migration is mediated by the β2 integrin, αLβ2, also known as Lymphocyte Function-associated Antigen-1 (LFA-1), which binds to ICAM-1. Neutrophils express LFA-1, as well as an additional receptor for ICAM-1, Macrophage-1 Antigen (Mac-1), which is upregulated when neutrophils are activated. Our laboratory hypothesized that neutrophils could also be induced to crawl upstream on ICAM-1 if Mac-1 was disabled or blocked, allowing LFA-1/ICAM-1 interactions to dominate. Our preliminary results show that by either blocking MAC-1 (with an antibody) or by genetically manipulating Mac-1 (using CRIPSR- Cas9 deletion), neutrophils can migrate upstream on ICAM-1 surfaces, setting the premise for this application. Here, we propose to determine the critical signals which govern the upstream migration of neutrophils, how neutrophils generate force when migrating upstream, and identify the physiological implications of upstream neutrophil migration. In Aim 1 we will determine the key molecular signals involved in upstream migration in neutrophils, by first analyzing the differential signaling that occurs on ICAM-1 vs. VCAM-1 surfaces and then identify and delete the signals that operate downstream of LFA-1 and Mac-1 using CRISPR editing. Specifically, we will focus on deleting the signals downstream of Mac-1/ICAM-1 binding to determine the signals that inhibit neutrophil upstream migration. In Aim 2, we will measure and quantify the forces generated by neutrophils engineered to migrate upstream using traction force microscopy. Using the knockouts generated in aim 1, we will measure the differences in the spatial arrangement and magnitude of force generation between upstream and downstream crawling neutrophils. Finally, in Aim 3 we will determine the physiological relevance of upstream migration in neutrophils by determining the change in time needed for altered to transmigrate in vitro on endothelium and the differential trafficking of disabled neutrophils into the peritoneal cavity in a murine model of acute inflammation.