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 将白细胞从血流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，先天免疫反应的髓系白细胞， 是所谓的“第一响应者”，并将迅速流量到煽动性侮辱的网站。 由白细胞粘附级联启动，这是一个特征明确的逐步事件序列，其中 血源性免疫细胞在内皮上束缚、滚动、牢固地停留和迁移，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，通常是毛细血管后微静脉， 细胞在与内皮细胞相互作用并迁移时遇到高剪切速率。 最近，一个有趣的现象包括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) – 会爬行 上游，逆流向，在存在配体细胞间粘附的表面上停留后 分子-1 (ICAM-1) 上游迁移由 β2 整合素 αLβ2（也称为淋巴细胞）介导。 功能相关抗原 1 (LFA-1)，与 ICAM-1 结合，表达 LFA-1。 ICAM-1 巨噬细胞 1 抗原 (Mac-1) 的额外受体，当中性粒细胞处于 我们的实验室提出，中性粒细胞也可以被诱导向 ICAM-1 上游爬行。 如果 Mac-1 被禁用或阻止，则 LFA-1/ICAM-1 相互作用将占主导地位。 表明通过阻断 MAC-1（用抗体）或通过基因操作 Mac-1（使用 CRIPSR- Cas9 缺失），中性粒细胞可以在 ICAM-1 表面向上游迁移，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， 中性粒细胞向上游迁移时如何产生力，并确定其生理意义 在目标 1 中，我们将确定上游中性粒细胞迁移的关键分子信号。 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 表面，然后使用 CRISPR 识别并删除 LFA-1 和 Mac-1 下游的信号 具体来说，我们将重点删除 Mac-1/ICAM-1 结合下游的信号以确定。 在目标 2 中，我们将测量和量化抑制中性粒细胞上游迁移的信号。 由使用牵引力显微镜向上游迁移的中性粒细胞产生。 目标 1 中产生的敲除，我们将测量空间排列和大小的差异 最后，在目标 3 中，我们将确定上游和下游爬行中性粒细胞之间的力产生。 通过确定时间变化来确定中性粒细胞上游迁移的生理相关性 改变内皮细胞体外迁移和残疾中性粒细胞的差异运输所需的 进入小鼠急性炎症模型的腹膜腔。

项目成果

Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

让我们开始滚动：精确控制微流体测定条件以重现白细胞粘附级联的选择素介导的滚动相互作用。

• 发表时间: 2024-04
• 作者: Amoabediny, Zeinab;Mittal, Aman;Guin, Subham;Buffone Jr, Alexander
• 通讯作者: Buffone Jr, Alexander

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

并非所有徘徊的（细胞）都会丢失：上游迁移是变形虫细胞运动的普遍模式。

• 发表时间: 2023
• 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