Regulation of Inflammation by the Fibrinolytic System

摘要 本课题主要研究纤溶系统及其在调节先天免疫中的作用。我们有 组织型纤溶酶原激活物(TPA)作为促炎反应的拮抗剂发挥作用 由巨噬细胞和小鼠体内的Toll样受体(TLRs)触发。从机制上讲，tPA的活性 是由包括N-甲基-D-天冬氨酸受体(NMDA-R)和低密度脂蛋白受体- 相关蛋白-1(LRP1)。尽管具有酶活性的tPA在先天免疫中的作用受 Serpin、PAI1和通过产生纤溶酶，酶失活tPA(EI-tPA)对这些调节具有抵抗力 小路。Ei-tPA不能抑制模式识别受体(PRRs)介导的促炎反应 而不是TLR；然而，在多个PRR协同作用的小鼠疾病模型中，包括 Ei-tPA对DSS结肠炎模型和K/BxN血清转移性关节炎模型均有效 作为一名候选治疗师。在这项继续支持的申请中，我们提议进行研究以阐明该活性 TPA及其受体在调节炎症中的作用，并确定产生新的抗炎作用的可能性。 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，结构性的 调节先天免疫所需的tPA中的成分将由基因工程确定。小说 将开发和测试Ei-tPA的重组衍生物，目标是优化tPA用作 体内候选抗炎治疗。具体目标2专注于了解抗炎- NMDA-R下游由tPA激活的交配细胞信号通路新的初步结果表明 一种新的系统，涉及通过Src家族激酶反式激活Trk受体，这在以前没有描述过 巨噬细胞和其他炎性细胞。具体目标2中拟议的研究不仅将有助于 我们对tPA信号的总体理解，但也可能识别新的细胞内抗炎靶点- 基于我们对tPA激活的细胞信号的分析的模拟药物开发。在具体目标3中，我们将繁殖 本实验室可用小鼠产生动物，其中巨噬细胞和其他细胞中的LysM 启动子活性不表达NMDA-R。这将使我们能够明确地检验这样的假设： Ei-tPA在体内的炎症活性，例如在中和脂多糖毒性方面，需要NMDA-R和 巨噬细胞和/或中性粒细胞是Ei-tPA的靶细胞。在具体目标4中，我们将研究tPA和 其抗炎受体NMDA-R和LRP1在DSS结肠炎模型中的作用。单细胞转录组 建议进行图谱研究，以无偏见的方式确定Ei-tPA在体内靶向的结肠细胞和 确定EI-tPA在该模型系统中引起良好反应的新途径。总体而言， 这里提出的研究应该进一步加深我们对止血和免疫之间的接口的理解。 进一步挖掘纤溶系统，开发新型抗炎药物。

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.