Platelet and megakaryocyte biology in the normal and injured lung

项目总结/摘要 输血的一个主要非感染性并发症是输血相关急性 肺损伤（TRALI）。尽管我们在通过供体移植降低TRALI发生率方面取得了进展， 在美国，它仍然是输血相关死亡的头号原因， 住院期间发病的主要原因。在本申请中，我们将研究激活 血小板促进TRALI中的肺损伤，我们还将研究肺如何反过来成为主要器官。 参与血小板的生物合成。在目标1中，我们将使用以下方法确定肺损伤发展的机制： 抗体介导的TRALI的两事件小鼠模型。线粒体DNA作为一种新的引发剂的作用 将在小鼠模型和来自发生TRALI的人类受试者的血浆样品中测试所述药剂。 然后，我们将使用新的小鼠工具确定同源抗原表达的关键位点， 有条件地删除感兴趣的细胞中的I类MHC。接下来，对TRALI的下游免疫应答将是 通过关注血小板诱导的中性粒细胞胞外陷阱（NET）形成和新型调节剂进行研究 这条路。通过绘制免疫引发的关键决定因素，细胞特异性同源抗原表达， 和NET介导的肺屏障破坏，我们将提高我们对TRALI机制的理解。在目标2中， 我们将把注意力转向肺对血小板生物合成的贡献， 损伤（TRALI）。血小板对肺损伤中的先天免疫反应至关重要，但我们也假设， 肺是血小板生物发生的主要部位。使用荧光报告小鼠，活体显微镜和流式细胞术， 通过流式细胞术，我们将确定巨核细胞在肺中的解剖位置和活性。血小板 在体内平衡和损伤期间测量肺中血管内巨核细胞的产生。 肺血管外巨核细胞的功能将通过肺移植和活体内移植进行检测。 显微镜技术。这些实验将确立肺作为血小板产生的主要部位。在 目的3，我们将确定肺作为巨核细胞和造血前体细胞的小生境的作用。 使用先进的流式细胞分选技术，我们将检测肺中造血前体细胞的存在。 与骨髓相比。这些造血前体的功能将通过使用 涉及肺移植和过继转移来自肺的流式分选细胞的重建策略。 最后，将测试影响肺造血前体的归巢或生态位促进因素。的 该建议的结果将阐明血小板介导的肺损伤的机制， 确定新的治疗或预防途径。这些研究还将确定 正常和受损肺在血小板生物合成和造血细胞维持方面的作用， 公共卫生影响。

Project Summary/Abstract A major non-infectious complication from blood transfusions is the development of transfusion-related acute lung injury (TRALI). Although we have made progress in decreasing the incidence of TRALI through donor management, it remains the number one cause of transfusion-related mortality in the United States and a major cause of in-hospital morbidity. In this application, we will study the mechanisms by which activated platelets promote lung damage in TRALI, and we will also study how the lung in turn is the major organ involved in platelet biogenesis. In Aim 1, we will determine the mechanisms of lung injury development using a two-event mouse model of antibody-mediated TRALI. The role of mitochondrial DNA as a novel priming agent will be tested in the mouse model and in plasma samples from human subjects who developed TRALI. We will then determine the critical site(s) of cognate antigen expression using a new mouse tool to conditionally delete MHC Class I in cells of interest. Next, the downstream immune response to TRALI will be investigated by focusing on platelet-induced neutrophil extracellular trap (NET) formation and novel regulators of this pathway. By mapping critical determinants of immune priming, cell-specific cognate antigen expression, and NET-mediated lung barrier disruption, we will improve our understanding of TRALI mechanisms. In Aim 2, we will turn our attention to the contribution of the lung to platelet biogenesis during homeostasis and lung injury (TRALI). Platelets are critical to the innate immune response in lung injury, but we also hypothesize that the lung is a major site of platelet biogenesis. Using fluorescent-reporter mice, intravital microscopy, and flow cytometry, we will determine the anatomic location and activity of megakaryocytes in the lung. Platelet production from intravascular megakaryocytes in the lung will be measured during homeostasis and injury. The function of extravascular megakaryocytes in the lung will be tested using lung transplantation and intravital microscopy techniques. These experiments will establish the lung as a principle sit of platelet production. In Aim 3, we will determine the role of the lung as a niche for megakaryocytes and hematopoietic precursors. Using advanced flow-based sorting, we will test for the presence of hematopoietic precursors in the lung compared to the bone marrow. The function of these hematopoietic precursors will then be tested using reconstitution strategies involving lung transplantation and adoptive transfer of flow-sorted cells from the lung. Finally, the homing or niche-promoting factors influencing lung hematopoietic precursors will be tested. The results from this proposal will elucidate mechanisms responsible for platelet-mediated lung injury and will identify novel treatment or preventive pathways. These studies will also establish the functional role of the normal and injured lung in platelet biogenesis and hematopoietic cell maintenance, which has far-reaching public health implications.