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

摘要 不受控制的炎症细胞积聚在空气中与血管损伤配对，导致致命的急性 肺损伤（ALI）。免疫细胞持续接触的微血管内皮， 在基础和感染后维持肺内环境稳定中起关键作用。1-磷酸鞘氨醇受体-1 在内皮细胞（EC）表面上表达的G蛋白偶联受体（GPCR）（S1 PR 1）是公知的免疫调节剂。 介导“屏障保护”和“炎症消退”。在这里我们发现S1 PR 1也可以 触发适得其反的信号，从而改变肺血管生态位。项目1的重点是确定 作为一个重要的分子开关，后修饰的S1 PR 1表观遗传修饰EC， 抗炎表型转化为免疫活性表型，导致不可逆的炎性肺损伤。 我们的支持性数据显示：1）炎性细胞因子（例如，炎症损伤时产生的TNFα 磷酸化酪氨酸143上的S1 PR 1（Y143-S1 PR 1）; 2）Y143-磷酸化的S1 PR 1进而信号传导受体 转运和定位到内质网（ER），3）伴侣蛋白BiP（结合 免疫球蛋白蛋白）对于Y143-S1 PR 1移位和结合到ER膜是必需的，4） ER相关的Y143-S1 PR 1随后与细胞内异聚GTP结合蛋白Gi结合，和 当被细胞内S1 P以Gi依赖性方式激活时， 和5）使小鼠EC中的S1 PR 1组成性磷酸化（通过将Y143编辑为D143），增加肺 炎性损伤肺EC还获得了诱导血管发育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活化，表明损伤- EC表观基因组的特异性调节。基于这些令人激动的数据，项目1将检验以下假设： ER驻留的Y143-S1 PR 1是关键信号事件的关键汇合点，具有重新连接细胞的潜力。 EC表观基因组，将EC转变为导致顽固性肺损伤的炎性表型。我们的具体目标 目的#1：阐明S1 PR 1的Y143磷酸化在S1 PR 1的ER定位中的作用， 目的2：研究肺内皮细胞的重编程，从而导致炎性肺损伤; 伴侣蛋白BiP与Y143磷酸化S1 PR 1相互作用在稳定ER膜S1 PR 1中的作用 并介导肺EC向炎性表型的转变。我们将使用转基因小鼠 使用Crisper-Cas9基因编辑以及EC特异性S1 PR 1无效小鼠和Cores中可用的工具， 表观遗传学分析（核心B）、多通道细胞成像（核心C）和肺EC的双光子成像， 活小鼠中的免疫细胞（核心C），以确定EC中ER驻留的Y143-S1 PR 1如何集中在 这些信号事件来控制肺部免疫细胞的命运。我们相信研究会发现新的 免疫活性内皮细胞的标记物和通过以下途径预防炎性肺损伤的靶点 ER-S1 PR 1和BiP信号传导的操纵。

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.