Regulation of Inflammation by the Fibrinolytic System

纤溶系统对炎症的调节

• 批准号: 10557130
• 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/10557130
• 关键词: Activator Appliances; Active Sites; Agonist; Alteplase; Animals; Anti-Inflammatory Agents; Arthritis; Binding; Biological Models; Bone Marrow; Breeding; 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; Genetic Engineering; Goals; Grant; Hemostatic function; Immunity; In Vitro; Inflammation; Inflammatory; Inflammatory Response; K/BxN model; LDL-Receptor Related Protein 1; Laboratories; Lipopolysaccharides; Macrophage; 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; aspartate receptor; cell type; cytokine; dextran sulfate sodium induced colitis; drug development; drug discovery; experimental study; gut inflammation; improved; in vivo; mimetics; monocyte; mouse model; neutrophil; novel; promoter; receptor; receptor function; response; src-Family Kinases; therapeutic candidate; tissue injury; transcriptomic profiling

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.

抽象的 该研究项目的重点是纤维蛋白溶解系统及其调节先天免疫的活性。我们有 研究表明组织型纤溶酶原激活剂 (tPA) 可作为促炎症反应的拮抗剂 由巨噬细胞和小鼠体内的 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 受体在内的受体系统介导 相关蛋白-1 (LRP1)。尽管酶活性 tPA 在先天免疫中的功能受 Serpin、PAI1 以及通过纤溶酶生成，无酶活性的 tPA (EI-tPA) 对这些调节具有抵抗力 途径。 EI-tPA 无法抑制模式识别受体 (PRR) 介导的促炎症反应 TLR 除外；然而，在多个 PRR 协同作用的小鼠疾病模型中，包括 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，EI-tPA有效 作为候选治疗剂。在此持续支持申请中，我们提出研究来阐明该活动 tPA 及其受体在调节炎症中的作用，并确定产生新型抗炎药物的潜力 基于tPA结构的合成药物。提出了四个具体目标。在具体目标 1 中，结构 tPA 中调节先天免疫所需的元素将通过基因工程确定。小说 将开发和测试 EI-tPA 的重组衍生物，目标是优化 tPA，使其用作 候选体内抗炎治疗。具体目标 2 侧重于了解抗炎作用 NMDA-R 下游的 tPA 激活成熟细胞信号传导通路。新的初步结果暗示 一个新的系统，涉及 Src 家族激酶的 Trk 受体反式激活，这在之前的文献中未曾描述过 巨噬细胞和其他炎症细胞。具体目标 2 中提出的研究不仅有助于 我们对 tPA 信号传导的总体理解，但也可能确定新的细胞内抗炎靶点 基于我们对 tPA 激活细胞信号转导的分析的成熟药物开发。在具体目标 3 中，我们将培育 我们实验室中可用小鼠来产生巨噬细胞和其他细胞中含有 LysM 的动物 启动子有活性，不表达 NMDA-R。这将使我们能够明确地检验以下假设：反 EI-tPA 体内的炎症活性，例如中和 LPS 毒性，需要 NMDA-R，并且 巨噬细胞和/或中性粒细胞是EI-tPA靶细胞。在具体目标 4 中，我们将研究 tPA 的活性和 DSS 结肠炎模型中的抗炎受体 NMDA-R 和 LRP1。单细胞转录组 建议进行分析研究，以公正的方式识别体内 EI-tPA 靶向的结肠细胞， 确定 EI-tPA 在该模型系统中引起良好反应的新途径。总的来说， 本文提出的研究应进一步加深我们对止血与免疫之间相互作用的理解 并进一步挖掘纤溶系统用于新型抗炎药物的开发。