Regulation of Inflammation by the Fibrinolytic System

纤溶系统对炎症的调节

• 批准号: 10358335
• 负责人: STEVEN L. GONIAS
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-10 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10358335
• 关键词: Activator Appliances; Active Sites; Agonist; Alteplase; Animals; Anti-Inflammatory Agents; Arthritis; Binding; Biological Models; Bone Marrow; Cell physiology; Cells; Chemistry; Chronic; Clinical; Colon; Complex; Data; Disease; Disease Progression; Disease model; Dose; Elements; Engineering; Family; Fibrinolysis; Funding; G-Protein-Coupled Receptors; Gene Deletion; Generations; Genes; Genetic Engineering; Goals; Grant; Hemostatic function; Immunity; In Vitro; Inflammation; Inflammatory; Inflammatory Response; K/BxN model; LDL-Receptor Related Protein 1; Laboratories; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Mediating; Modeling; Mus; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Natural Immunity; Pathogenesis; Pathology; Pathway interactions; Pattern recognition receptor; Peptide Hydrolases; Peritoneal Macrophages; Pharmaceutical Preparations; Phase; Plasmin; Plasminogen; Plasminogen Activator; Plasminogen Activator Inhibitor 1; Process; Progress Reports; Proteins; Publishing; Recombinants; Regulation; Regulatory Pathway; Research Project Grants; Resistance; Serine; Serine Protease; Serpins; Serum; Signal Pathway; Signal Transduction; Structure; Symptoms; System; TLR2 gene; TLR4 gene; TLR7 gene; Testing; Therapeutic; Tissues; Toll-like receptors; Toxic effect; Transactivation; Wild Type Mouse; Work; antagonist; base; cell type; cytokine; dextran sulfate sodium induced colitis; drug development; drug discovery; experimental study; gut inflammation; improved; in vivo; macrophage; mimetics; monocyte; mouse model; neutrophil; novel; promoter; receptor; receptor function; response; src-Family Kinases; therapeutic candidate; tissue injury; transcriptome

Abstract This research project focuses on the fibrinolysis system and its activity in regulating innate immunity. We have shown that tissue-type plasminogen activator (tPA) functions as an antagonist of pro-inflammatory responses triggered by Toll-like Receptors (TLRs) in macrophages and in vivo in mice. Mechanistically, the activity of tPA is mediated by a receptor system that includes the N-methyl-D-aspartate receptor (NMDA-R) and LDL Receptor- related Protein-1 (LRP1). Although the function of enzymatically-active tPA in innate immunity is regulated by the Serpin, PAI1, and by plasmin generation, enzymatically-inactive tPA (EI-tPA) is resistant to these regulatory pathways. EI-tPA fails to inhibit pro-inflammatory responses mediated by Pattern Recognition Receptors (PRRs) other than TLRs; however, in mouse models of disease in which multiple PRRs function in concert, including the Dextran Sulfate Sodium (DSS) colitis model and the K/BxN serum-transfer arthritis model, EI-tPA is efficacious as a candidate therapeutic. In this application for continued support, we propose studies to elucidate the activity of tPA and its receptors in regulating inflammation and determine the potential to generate novel anti-inflam- matory drugs based on the structure of tPA. Four specific aims are proposed. In Specific Aim 1, the structural elements in tPA required for regulation of innate immunity will be determined by genetic engineering. Novel recombinant derivatives of EI-tPA will be developed and tested with the goal of optimizing tPA for use as a candidate anti-inflammatory therapeutic in vivo. Specific Aim 2 is focused on understanding the anti-inflam- matory cell-signaling pathway activated by tPA downstream of the NMDA-R. New preliminary results implicate a novel system, involving Trk receptor transactivation by Src family kinases, which is previously undescribed in macrophages and other inflammatory cells. The proposed studies in Specific Aim 2 will not only contribute to our understanding of tPA signaling in general but also may identify novel intracellular targets for anti-inflam- matory drug development based on our analysis of tPA-activated cell-signaling. In Specific Aim 3, we will breed mice available in our laboratory to generate animals in which macrophages and other cells in which the LysM promoter is active do not express the NMDA-R. This will allow us to definitively test the hypothesis that the anti- inflammatory activity of EI-tPA in vivo, for example in neutralizing LPS toxicity, requires the NMDA-R and that macrophages and/or neutrophils are EI-tPA target cells. In Specific Aim 4, we will study the activity of tPA and its anti-inflammatory receptors, the NMDA-R and LRP1, in the DSS colitis model. Single-cell transcriptome profiling studies are proposed to identify, in an unbiased manner, colon cells targeted by EI-tPA in vivo and identify novel pathways by which EI-tPA elicits a favorable response in this model system. Collectively, the studies proposed herein should further our understanding of the interface between hemostasis and immunity and further efforts to mine the fibrinolysis system for novel anti-inflammatory drug development.

摘要 本研究项目的重点是纤溶系统及其在调节天然免疫中的活性。我们有 显示组织型纤溶酶原激活物（tPA）作为促炎反应的拮抗剂起作用 在巨噬细胞和小鼠体内由Toll样受体（TLR）触发。从机制上讲，tPA的活性 由包括N-甲基-D-天冬氨酸受体（NMDA-R）和LDL受体的受体系统介导。 相关蛋白1（LRP 1）。虽然酶活性tPA在先天免疫中的功能是由 丝氨酸蛋白酶抑制剂、PAI 1和通过纤溶酶产生无酶活性tPA（EI-tPA）对这些调节具有抗性 途径。EI-tPA未能抑制模式识别受体（PRR）介导的促炎反应 然而，在多种PRR共同发挥功能的疾病小鼠模型中， 在右旋糖酐硫酸钠（DSS）结肠炎模型和K/BxN血清转移性关节炎模型中，EI-tPA是有效的 作为候选治疗药物。在本申请中，为了继续获得支持，我们建议进行研究，以阐明 tPA及其受体在调节炎症中的作用，并确定产生新型抗炎症的潜力。 基于tPA结构的炎性药物。提出了四个具体目标。具体目标1： 通过基因工程确定调节先天免疫所需的tPA中的元件。小说 EI-tPA的重组衍生物将被开发和测试，目的是优化tPA用作 候选的体内抗炎治疗剂。具体目标2的重点是了解反通胀- 由NMDA-R下游的tPA激活的炎性细胞信号传导途径。新的初步结果表明， 一种新型系统，涉及Src家族激酶对Trk受体的反式激活，这在以前的文献中未描述 巨噬细胞和其他炎性细胞。具体目标2中拟议的研究不仅有助于 我们对tPA信号传导的一般理解，也可以确定抗炎症的新的细胞内靶点， 基于我们对tPA激活的细胞信号传导的分析的炎性药物开发。在《特定目标3》中， 小鼠在我们的实验室产生的动物，其中巨噬细胞和其他细胞，其中LysM 启动子有活性的不表达NMDA-R。这将使我们能够明确地测试假设，即反- EI-tPA的体内炎性活性，例如中和LPS毒性，需要NMDA-R， 巨噬细胞和/或嗜中性粒细胞是EI-tPA靶细胞。在具体目标4中，我们将研究tPA的活性， 其抗炎受体NMDA-R和LRP 1在DSS结肠炎模型中的作用。单细胞转录组 提出了谱分析研究以无偏倚的方式鉴定EI-tPA体内靶向的结肠细胞， 鉴定EI-tPA在该模型系统中引起有利反应的新途径。统称 本文提出的研究应进一步了解止血和免疫之间的接口 并进一步努力挖掘纤溶系统用于新型抗炎药物的开发。