Mechanisms of airspace inflammation caused by cell-free hemoglobin during ARDS

项目摘要 急性呼吸窘迫综合征(ARDS)在美国和美国每年影响19万多名患者 仍然有30%-40%的死亡率。我们的研究小组最近报告说，无细胞水平 ARDS和ARDS患者肺水肿液中的血红蛋白(CFH)升高 空气中CFH的增加与肺泡-毛细血管通透性的增加有关，提示CFH 可能参与ARDS的发病机制，并可能成为ARDS治疗的新靶点。这项提案的目标是 是促进我们对空气中CFH的细胞和分子后果的了解，并测试 空域中的CFH靶向治疗。我们的初步数据建立了一个独特的模型系统，我们将 确定急性肺部炎症的近端调节因子。注射纯化的内毒素- 进入小鼠体内的游离CFH足以引起强烈的肺泡炎，并已发现 肺泡巨噬细胞作为CFH的关键靶点。此外，缺乏TLR4的小鼠已经变弱 炎症与CFH的反应，确定其机制中的一个关键途径。我们还确定， CFH只被空气中的一小部分巨噬细胞吞噬，这些含有CFH的 与缺乏CFH的细胞相比，巨噬细胞产生的促炎细胞因子较少。根据这些数据，我们 提示肺泡内CFH对急性肺损伤的促炎和抗炎通路均有调节作用 作为一种新的治疗方法，这些通路之间的平衡可以在空中进行操纵 为ARDS做准备。在这个提案中，我们将测试CFH通过TLR4导致空域炎症的假设- 肺泡巨噬细胞依赖的核因子-κB活化及巨噬细胞摄取CFH的增加 结合珠蛋白可通过减轻巨噬细胞依赖的炎症来减轻急性肺损伤。 在目标1中，我们将使用巨噬细胞特异性TLR4缺失或NF-κB抑制的转基因小鼠来确定 巨噬细胞TLR4和NF-κB活化在CFH介导的肺泡炎症中的作用在目标2中，我们将 使用CD163缺失小鼠和一种新的磁性细胞分选应用来确定CFH在体内的摄取 肺泡巨噬细胞改变巨噬细胞的极化状态，使其处于抗炎状态。在目标3中，我们将测试 肺泡内注射结合珠蛋白作为治疗ARDS的新方法，采用细菌性肺炎的小鼠模型，并将 确定ARDS期间空隙中结合珠蛋白的临床重要性。通过完成这些 研究，我们将确定一种新的ARDS和ARDS介导物的细胞特异性和分子机制 测试了一种新的本地化ARDS疗法，该疗法具有快速转化为人类临床试验的高潜力。在……里面 此外，通过这些研究，一位有前途的年轻内科科学家将获得基本和 在卓有成效的专家指导委员会的指导下进行的ARDS的翻译研究 ARDS、炎症和巨噬细胞生物学。这些新的指导技能将构成这一点的基础 初级研究员要实现长期目标，成为一名独立资助的学术内科科学家。

Project Summary The acute respiratory distress syndrome (ARDS) affects more than 190,000 patients each year in the US and continues to have a 30-40% mortality rate. Our research group recently reported that levels of cell-free hemoglobin (CFH) are elevated in pulmonary edema fluid from the distal airspaces of patients with ARDS and that increased airspace CFH is associated with increased alveolar-capillary permeability, suggesting that CFH may contribute to ARDS pathogenesis and may be a new target for ARDS therapy. The goals of this proposal are to advance our knowledge of the cellular and molecular consequences of CFH in the airspace and test a CFH-targeted therapy in the airspace. Our preliminary data establish a unique model system that we will harness to define the proximal regulators of acute inflammation in the lung. Instillation of purified endotoxin- free CFH into the airspace of mice is sufficient to induce robust alveolar inflammation and has identified alveolar macrophages as a key target of CFH. Furthermore, mice deficient in TLR4 have attenuated inflammation in response to CFH, identifying one key pathway in its mechanism. We also have determined that CFH is engulfed by only a subpopulation of macrophages in the airspace and that these CFH-containing macrophages have less pro-inflammatory cytokine production than cells lacking CFH. Based on these data, we propose that intra-alveolar CFH may modulate both pro- and anti-inflammatory pathways during acute lung injury and that the balance between these pathways could be manipulated in the airspace as a novel therapy for ARDS. In this proposal, we will test the hypothesis that CFH causes airspace inflammation through TLR4- dependent NF-κB activation in alveolar macrophages and that augmentation of CFH uptake into macrophages with haptoglobin will ameliorate acute lung injury through attenuation of macrophage-dependent inflammation. In Aim 1, we will use transgenic mice with macrophage-specific TLR4 deletion or NF-κB inhibition to determine the role of macrophage TLR4 and NF-κB activation in CFH-mediated airspace inflammation. In Aim 2, we will use CD163 null mice and a novel application of magnetic cell sorting to determine whether uptake of CFH into alveolar macrophages alters macrophage polarization towards an anti-inflammatory state. In Aim 3, we will test intra-alveolar haptoglobin as a new therapy for ARDS using a mouse model of bacterial pneumonia and will determine the clinical importance of haptoglobin in the airspace during ARDS. By the completion of these studies, we will have identified the cell-specific and molecular mechanisms of a novel mediator of ARDS and tested a new localized ARDS therapy that has high potential for rapid translation into human clinical trials. In addition, through these studies, a promising young physician scientist will gain new skills in basic and translational studies of ARDS under the guidance of a highly accomplished mentorship committee of experts in ARDS, inflammation, and macrophage biology. These new mentored skills will form the foundation for this junior investigator to achieve a long-term goal to be an independently funded academic physician scientist.