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

• 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
• 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

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 空域中炎性细胞与血管损伤配对的不受控制的积累会导致致命急性 肺损伤（ALI）。免疫细胞连续接触的微血管内皮 在基本和感染后基本维持肺稳态方面的关键作用。鞘氨醇1-磷酸受体1 （S1PR1）是在内皮细胞（EC）表面表达的G蛋白偶联受体（GPCR），是一个众所周知的 “屏障保护”和“注射清除”的调解人。在这里，我们发现S1PR1也可以 触发适得其反的信号传导，因此改变了肺血管生态位。项目1的重点是确定 翻译后修饰的S1PR1作为一种显着的分子开关 免疫活性表型的抗炎表型，导致不可逆的炎症肺损伤。 我们的支持数据表明：1）在炎症损伤期间产生的炎性细胞因子（例如TNFα） 在酪氨酸143（Y143-S1PR1）上磷酸化S1PR1; 2）Y143磷酸化的S1PR1依次签名的接收器 易位和定位到内质网（ER），3）伴侣蛋白BIP（结合 免疫球蛋白蛋白）对于Y143-S1PR1与ER膜的易位和结合至关重要，4） 与ER相关的Y143-S1PR1随后结合到细胞内杂体GTP结合蛋白GI和 当通过细胞内S1P增强商店经营钙进入（SOCE）以GI依赖性方式激活时， 5）渲染S1PR1组成型磷酸化（通过编辑Y143至D143）在EC中增加了肺 炎症伤害。肺EC还获得了基因诱导血管发育的表观遗传修饰和 铜绿假单胞菌损伤后的分化和激活，表明损伤 EC表观基因组的特定调节。基于这些挑衅性数据，项目1将检验以下假设。 ER居住的Y143-S1PR1是关键信号事件的关键点，有可能重新连接 EC表观基因组，将EC转移到炎症表型中，导致棘手的肺损伤。我们的具体目标 是：目标＃1：解决y143 s1pr1磷酸化在S1PR1和ER定位中的作用 重新编程肺部内皮，从而导致炎症性肺损伤；和目标2：研究 伴侣蛋白BIP与Y143磷酸化的S1PR1相互作用在ER膜上稳定S1PR1中的作用 并介导肺EC中炎症表型的转变。我们将使用生成的一般修饰的小鼠 使用Crisper-Cas9基因编辑以及EC特异性的S1PR1 NULL小鼠和核心中可用的工具 表观遗传分析（核心B），多通道细胞成像（Core C）和2光子成像的肺EC和 活小鼠中的免疫细胞（核心C）机械定义了EC中的ER居民Y143-S1PR1如何集中 这些信号事件以控制肺中免疫细胞的命运。我们认为研究将确定新颖 免疫活性内皮的标记和靶标，以防止通过 ER-S1PR1和BIP信号传导的操作。