Platelet and megakaryocyte biology in the normal and injured lung

项目摘要/摘要 输血的一个主要非传染性并发症是与输血相关的急性 肺损伤(TRALI)。虽然我们在通过供者减少TRALI发生率方面取得了进展 管理方面，它仍然是美国输血相关死亡的头号原因，而且 住院发病率的主要原因。在这个应用中，我们将研究激活的机制 血小板促进TRALI中的肺损伤，我们还将研究肺是如何反过来成为主要器官的 参与了血小板的生物生成。在目标1中，我们将确定肺损伤发生的机制 抗体介导TRALI的二事件小鼠模型。线粒体DNA作为一种新的启动因子的作用 该试剂将在小鼠模型和患有TRALI的人类受试者的血浆样本中进行测试。 然后我们将使用一种新的小鼠工具来确定同源抗原表达的关键部位(S) 有条件地删除感兴趣单元格中的MHC I类。下一步，对TRALI的下游免疫反应将是 通过集中研究血小板诱导的中性粒细胞胞外陷阱(NET)的形成和新的调节剂 在这条道路上。通过绘制免疫启动的关键决定因素，细胞特异性同源抗原表达， 以及网络介导的肺屏障破坏，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在动态平衡和肺中对血小板生物生成的贡献 伤害(TRALI)。在肺损伤中，血小板是先天免疫反应的关键，但我们也假设 肺是血小板生物生成的主要部位。使用荧光报告小鼠、活体显微镜和Flow 通过细胞学检查，我们将确定巨核细胞在肺内的解剖位置和活性。血小板 肺内血管内巨核细胞的产量将在动态平衡和损伤期间进行测量。 肺内血管外巨核细胞的功能将通过肺移植和活体内试验进行。 显微镜技术。这些实验将使肺成为产生血小板的主要场所。在……里面 目的3，我们将确定肺作为巨核细胞和造血祖细胞的小生境的作用。 使用先进的基于血流的分类，我们将测试在肺中是否存在造血祖细胞 与骨髓相比。这些造血祖细胞的功能随后将用 重建策略包括肺移植和过继转移来自肺的流动分选细胞。 最后，将测试影响肺造血前体的归巢或生态位促进因素。这个 这一提议的结果将阐明血小板介导的肺损伤的机制，并将 确定新的治疗或预防途径。这些研究还将确立 正常肺和损伤肺在血小板的生物生成和造血细胞的维持方面具有深远的意义 对公共健康的影响。

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.