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

通过操纵Mac-1和LFA-1的功能来控制中性粒细胞的上游迁移

• 批准号: 10446740
• 负责人: Daniel A Hammer
• 金额: $ 31.09万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-02 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10446740
• 关键词: Actins; Acute; Adhesions; Antibodies; B-Lymphocytes; Binding; Biological Assay; Blood; Blood Vessels; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complication; Disabled Persons; Disease; Endothelial Cells; Endothelium; Engineering; Event; Extravasation; Generations; Goals; Greater sac of peritoneum; HL-60 Cells; Hematopoietic; Hematopoietic stem cells; Image; Immune; In Vitro; Infectious Agent; Inflammation; Inflammatory; Innate Immune Response; Integrin alpha4beta1; Integrins; Intercellular adhesion molecule 1; Knock-out; Laboratories; Lead; Leukocyte Trafficking; Leukocytes; Ligands; Lymphocyte; Lymphocyte Function-Associated Antigen-1; Lymphoid; Macrophage-1 Antigen; Measurement; Measures; Mediating; Molecular; Monoclonal Antibodies; Myelogenous; Neutrophil Infiltration; Paper; Pathway interactions; Patient-Focused Outcomes; Physiological; Population; Receptor Signaling; Role; Secure; Signal Pathway; Signal Transduction; Site; Stimulus; Stream; Stress; Sum; Surface; T-Lymphocyte; Testing; Therapeutic; Time; Tissues; Traction; Traction Force Microscopy; Vascular Cell Adhesion Molecule-1; Work; experimental study; fascinate; first responder; genetic manipulation; improved; in vivo; mechanotransduction; migration; monolayer; mouse model; mutant; neutrophil; polymerization; postcapillary venule; preference; receptor; recruit; trafficking

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.

摘要/摘要 白细胞从血流中运输到炎症部位以寻找传染源 是先天免疫反应的标志中性粒细胞，骨髓白细胞的先天免疫反应， 是所谓的“第一反应者”，并将迅速交通到炎症性损伤的地点。贩运是 由白细胞粘附级联反应启动，这是一种特征良好的逐步事件序列， 血液携带的免疫细胞在内皮上系留、滚动、牢固地阻滞和迁移，然后进入组织， 执行免疫细胞功能。这些事件都发生在血管内，通常是毛细血管后小静脉， 其中细胞在与内皮相互作用并穿过内皮时遇到高剪切速率。 最近，一个有趣的现象，其中某些造血来源的细胞-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，在存在配体细胞间粘附的表面上停滞后 分子-1（ICAM-1）。上游迁移由β2整合素αLβ2介导，也称为淋巴细胞 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的另一个受体，巨噬细胞-1抗原（Mac-1），当中性粒细胞被激活时， 激活我们的实验室假设中性粒细胞也可以被诱导在ICAM-1上向上爬 如果Mac-1被禁用或阻断，则允许LFA-1/ICAM-1相互作用占主导地位。我们的初步结果 表明通过阻断MAC-1（用抗体）或通过遗传操纵Mac-1（使用CRIPSR- Cas9缺失），嗜中性粒细胞可以在ICAM-1表面上向上游迁移，这为该应用奠定了前提。 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， 中性粒细胞在向上游迁移时如何产生力，并确定 上游中性粒细胞迁移。在目标1中，我们将确定上游参与的关键分子信号， 通过首先分析ICAM-1与VCAM-1上发生的差异信号传导， 表面，然后使用CRISPR识别和删除在LFA-1和Mac-1下游运行的信号 编辑.具体来说，我们将重点删除Mac-1/ICAM-1结合的下游信号，以确定 抑制中性粒细胞向上游迁移的信号。在目标2中，我们将测量和量化 产生的中性粒细胞工程迁移上游使用牵引力显微镜。使用 在aim 1中产生的敲除，我们将测量空间排列和大小的差异， 在上游和下游爬行的中性粒细胞之间产生力。最后，在目标3中，我们将确定 通过确定时间变化，确定中性粒细胞上游迁移的生理相关性 需要改变内皮细胞的体外迁移和失能中性粒细胞的差异运输 在急性炎症的小鼠模型中注入腹膜腔。