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: 10616779
• 负责人: Daniel A Hammer
• 金额: $ 31.13万
• 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/10616779
• 关键词: Actins; Acute; Adhesions; Antibodies; B-Lymphocytes; Binding; Biological Assay; Blood; Blood Vessels; Cell Adhesion Molecules; 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; Leukocyte Trafficking; Leukocytes; Ligands; Lymphocyte; Lymphocyte Function-Associated Antigen-1; Lymphoid; Macrophage-1 Antigen; Measurement; Measures; Mediating; Migration Assay; Molecular; Monoclonal Antibodies; Myelogenous; Neutrophil Infiltration; PIK3CG gene; Paper; Pathway interactions; Patient-Focused Outcomes; Physiological; Polymers; Population; Role; Secure; Signal Pathway; Signal Transduction; Site; Stimulus; Stream; Stress; Surface; T-Lymphocyte; Testing; Therapeutic; Time; Tissues; Traction; Traction Force Microscopy; Vascular Cell Adhesion Molecule-1; Work; cell motility; experimental study; fascinate; first responder; genetic manipulation; improved; in vivo; lymphocyte function associated antigen; mechanotransduction; migration; monolayer; mouse model; mutant; neutrophil; polymerization; postcapillary venule; preference; receptor; recruit; stem cells; 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 从血液流到炎症部位的白细胞贩运以发现感染剂 是先天免疫响应的标志。中性粒细胞，先天免疫响应的髓样白细胞， 是所谓的“第一响应者”，将迅速访问炎症性伤害部位。贩运是 由白细胞粘附级联反应发起的 血液传播的免疫细胞束缚，滚动，首先逮捕并在内皮上迁移，然后进入时间到达 执行免疫细胞功能。这些事件全部发生在血管内，通常是毛细血管后静脉， 细胞在与内皮相互作用和迁移时遇到高剪切速率的地方。 最近，一个有趣的现象，其中某些造血存在的细胞 - 例如 淋巴细胞（T细胞和B细胞）以及造血茎和祖细胞（HSPC） - 将爬网 上游，朝着流动方向，在呈现配体间细胞间粘合剂的表面上进行逮捕。 分子1（ICAM-1）。上游迁移是由β2整合素αLβ2（也称为淋巴细胞）介导的 与ICAM-1结合的功能相关的抗原1（LFA-1）。中性粒细胞表达LFA-1，以及 ICAM-1，巨噬细胞-1抗原（MAC-1）的其他受体，当中性粒细胞为 活性。我们的实验室假设中性粒细胞也可能被诱导到ICAM-1上游爬网 如果MAC-1被禁用或阻止，则允许LFA-1/ICAM-1交互占主导地位。我们的初步结果 证明通过阻断MAC-1（使用抗体）或通过遗传操纵MAC-1（使用Cripsr-- CAS9删除），中性粒细胞可以在ICAM-1表面上游迁移，为该应用程序设置前提。 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， 中性粒细胞在上游迁移时如何产生力，并确定 上游中性粒细胞迁移。在AIM 1中，我们将确定上游涉及的关键分子信号 通过首先分析ICAM-1与VCAM-1上发生的差异信号传导，在中性粒细胞中的迁移 表面，然后使用CRISPR识别并删除LFA-1和MAC-1下游操作的信号 编辑。特别是，我们将重点删除Mac-1/ICAM-1绑定下游的信号以确定 抑制上游迁移的信号。在AIM 2中，我们将测量和量化力 由中性粒细胞生成的，设计用于使用牵引力显微镜在上游迁移。使用 在AIM 1中产生的敲除，我们将测量空间排列和幅度的差异 上游和下游爬行中性粒细胞之间产生。最后，在目标3中，我们将确定 通过确定时间变化，上游迁移在中性粒细胞中的身体相关性 需要改变内皮和残疾中性粒细胞的差异运输所需的改变 在急性炎症的鼠模型中进入腹膜腔。

项目成果

Not all (cells) who wander are lost: Upstream migration as a pervasive mode of amoeboid cell motility.

• DOI: 10.3389/fcell.2023.1291201
• 发表时间: 2023
• 期刊: FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY
• 影响因子: 5.5
• 作者: Buffone Jr, Alexander;Hammer, Daniel A.;Kim, Sarah Hyun Ji;Anderson, Nicholas R.;Mochida, Ai;Lee, Dong-Hun;Guin, Subham
• 通讯作者: Guin, Subham