S1PR1 Mislocalization in Lung Endothelium Regulates Innate Immune Function and Mediates Inflammatory Lung Injury

S1PR1 在肺内皮细胞中的错误定位调节先天免疫功能并介导炎症性肺损伤

基本信息

批准号：
10706510
负责人：
DOLLY MEHTA
金额：
$ 42.85万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-20 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10706510
关键词：
Acute Lung Injury Address Anti-Inflammatory Agents Area Bacterial Infections Binding Blood Vessels Calcium Cell Differentiation process Cell surface Cells Chromatin Data Development Endoplasmic Reticulum Endothelial Cells Endothelium Epigenetic Process Event G-Protein-Coupled Receptors GTP-Binding Proteins Gases Gene Modified Genes Homeostasis IGBP1 gene Immune Immunoglobulin binding proteins Immunoglobulins Immunologic Markers Immunologics Individual Infection Inflammation Inflammatory Injury Innate Immune System Knock-in Knockout Mice Leukocytes Ligation Lung Lung infections Mediating Mediator Membrane Modeling Modification Molecular Molecular Chaperones Mus Myeloid Cells Phenotype Phosphorylation Play Positioning Attribute Post-Translational Protein Processing Protein Tyrosine Kinase Proteins Pseudomonas Pseudomonas aeruginosa Receptor Activation Receptor Signaling Resolution Role Signal Transduction Signaling Protein Site Sphingosine-1-Phosphate Receptor Surface TNF gene Testing Tissues Tyrosine Vascular Endothelium Virus Diseases cellular imaging cytokine epigenome immune activation innate immune function lung imaging lung injury mouse model mutant mouse model novel novel marker prevent programs receptor recruit tool transcriptome two-photon ultra high resolution vascular injury

项目摘要

ABSTRACT Uncontrolled accumulation of inflammatory cells in the airspace paired with vascular injury causes lethal acute lung injury (ALI). The microvascular endothelium, with which immune cells are continuously in contact with, plays a critical role in maintaining lung homeostasis basally and after infection. Sphingosine-1-phosphate receptor-1 (S1PR1), a G-protein coupled receptor (GPCR) expressed on the endothelial cell (EC) surface, is a well-known mediator of “barrier protection” and “resolution of inflammation”. Here we have discovered that S1PR1 can also trigger counterproductive signaling, thus changing the lung vascular niche. The focus of Project 1 is to identify post-translationally modified S1PR1 as a significant molecular switch that epigenetically modifies EC from an anti-inflammatory phenotype to an immune-active phenotype, leading to irreversible inflammatory lung injury. Our Supporting Data show that: 1) inflammatory cytokines (e.g., TNFα) produced during inflammatory injury phosphorylates S1PR1 on tyrosine143 (Y143-S1PR1); 2) Y143-phosphorylated S1PR1 in turn signaled receptor translocation and localization to the endoplasmic reticulum (ER), 3) the chaperone protein BiP (Binding immunoglobulin Protein) was essential for the translocation and binding of Y143-S1PR1 to the ER membrane, 4) the ER-associated Y143-S1PR1 subsequently bound to the intracellular heteromeric GTP binding protein Gi, and when activated by intracellular S1P augmented store-operated calcium entry (SOCE) in a Gi-dependent manner, and 5) rendering S1PR1 constitutively phosphorylated (by editing Y143 to D143) in EC of mice increased lung inflammatory injury. Lung EC also gained epigenetic modifications of genes inducing vascular development and myeloid cells differentiation and activation following injury by Pseudomonas aeruginosa, suggesting injury- specific modulation of EC epigenome. Based on these provocative data, Project 1 will test the hypothesis that ER-resident Y143-S1PR1 is a crucial point of confluence for key signaling events with the potential to rewire the EC epigenome, shifting EC into an inflammatory phenotype leading to intractable lung injury. Our Specific Aims are: Aim #1: to address the role of Y143 phosphorylation of S1PR1 in the ER positioning of S1PR1 and reprogramming of lung endothelium, and thereby leading to inflammatory lung injury; and Aim #2: to study the role of chaperone protein BiP interaction with Y143 phosphorylated-S1PR1 in stabilizing S1PR1 at ER membrane and mediating the shift to inflammatory phenotype in lung EC. We will use genetically modified mice generated using Crisper-Cas9 gene editing as well as EC-specific S1PR1 null mice and tools available in Cores such as epigenetic analysis (Core B), multichannel cellular imaging (Core C), and 2-photon imaging of lung EC and immune cells in living mouse (Core C) to define mechanistically how ER-resident Y143-S1PR1 in EC centralizes these signaling events to control the fate of immune cells in the lungs. We believe studies will identify novel markers of the immunologically active endothelium and targets for preventing inflammatory lung injury through manipulation of ER-S1PR1 and BiP signaling.

抽象的炎症细胞在空腔中不受控制的积聚与血管损伤相结合，导致致命的急性炎症肺损伤（ALI）中，免疫细胞持续接触的微血管内皮发挥着作用。 1-磷酸鞘氨醇受体 1 在维持肺部稳态方面发挥着关键作用。 (S1PR1) 是一种在内皮细胞 (EC) 表面表达的 G 蛋白偶联受体 (GPCR)，是一种众所周知的在这里我们发现S1PR1也可以作为“屏障保护”和“炎症消退”的调节剂。触发适得其反的信号传导，从而改变肺血管生态位项目 1 的重点是确定。翻译后修饰的 S1PR1 作为一个重要的分子开关，通过表观遗传修饰 EC 抗炎表型转变为免疫活性表型，导致不可逆的炎症性肺损伤。我们的支持数据表明：1）炎症损伤过程中产生的炎症细胞因子（例如 TNFα） 2) Y143-磷酸化的S1PR1依次信号受体易位并定位至内质网 (ER)，3) 伴侣蛋白 BiP（结合免疫球蛋白）对于 Y143-S1PR1 易位和结合到 ER 膜至关重要，4) ER相关的Y143-S1PR1随后与细胞内异聚GTP结合蛋白Gi结合，并且当被细胞内 S1P 增强的钙库操作钙进入 (SOCE) 以 Gi 依赖性方式激活时， 5) 在小鼠 EC 中使 S1PR1 组成型磷酸化（通过将 Y143 编辑为 D143）增加肺肺内皮细胞还获得了诱导血管发育和炎症损伤的基因的表观遗传修饰。铜绿假单胞菌损伤后骨髓细胞分化和激活，表明损伤- 基于这些令人兴奋的数据，项目 1 将检验以下假设： ER 驻留 Y143-S1PR1 是关键信号事件汇合的关键点，有可能重新连接 EC 表观基因组，将 EC 转变为炎症表型，导致顽固性肺损伤。目标#1：解决 S1PR1 的 Y143 磷酸化在 S1PR1 的 ER 定位中的作用，以及肺内皮细胞重新编程，从而导致炎症性肺损伤；目标#2：研究伴侣蛋白 BiP 与 Y143 磷酸化-S1PR1 相互作用在稳定 ER 膜上的 S1PR1 中的作用并介导肺内皮细胞向炎症表型的转变我们将使用产生的转基因小鼠。使用 Crisper-Cas9 基因编辑以及 EC 特异性 S1PR1 缺失小鼠和 Core 中提供的工具，例如表观遗传分析（核心 B）、多通道细胞成像（核心 C）和肺 EC 和 2 光子成像活体小鼠免疫细胞（核心 C）从机制上定义 EC 中 ER 驻留 Y143-S1PR1 如何集中我们相信研究将发现新的信号事件来控制肺部免疫细胞的命运。免疫活性内皮的标志物和通过预防炎性肺损伤的靶标 ER-S1PR1 和 BiP 信号传导的操纵。