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）在美国每年影响超过190，000名患者， 仍然有30-40%的死亡率。我们的研究小组最近报告说， 血红蛋白（CFH）在来自ARDS患者远端气隙的肺水肿液中升高， 增加的肺泡-毛细血管通透性与增加的肺泡-毛细血管通透性有关，这表明CFH 可能参与了ARDS的发病机制，并可能成为ARDS治疗的新靶点。本提案的目标 是为了提高我们对CFH在空域中的细胞和分子后果的认识，并测试 CFH靶向治疗在空域。我们的初步数据建立了一个独特的模型系统，我们将 来定义肺部急性炎症的近端调节因子。注入纯化的内毒素- 游离CFH进入小鼠的空气空间足以诱导强烈的肺泡炎症， 肺泡巨噬细胞是CFH的关键靶点。此外，TLR 4缺陷的小鼠具有减弱的 炎症反应CFH，确定其机制中的一个关键途径。我们还确定， CFH仅被空气中的巨噬细胞亚群吞噬，并且这些含CFH的细胞 巨噬细胞比缺乏CFH的细胞具有更少的促炎细胞因子产生。根据这些数据，我们 提出肺泡内CFH可能调节急性肺损伤时的促炎和抗炎通路， 这些途径之间的平衡可以在空域中操纵，作为一种新的治疗方法 关于ARDS在这项提案中，我们将测试CFH通过TLR 4引起空域炎症的假设， 肺泡巨噬细胞NF-κB依赖性活化及CFH摄取增加 与触珠蛋白联合使用将通过减弱巨噬细胞依赖性炎症来改善急性肺损伤。 在目标1中，我们将使用巨噬细胞特异性TLR 4缺失或NF-κB抑制的转基因小鼠来确定 巨噬细胞TLR 4和NF-κB活化在CFH介导的气道炎症中的作用。在目标2中，我们 使用CD 163缺失小鼠和磁性细胞分选的新应用来确定CFH的摄取是否进入 肺泡巨噬细胞改变巨噬细胞向抗炎状态的极化。在目标3中，我们将测试 肺泡内结合珠蛋白作为一种新的治疗ARDS的方法， 确定急性呼吸窘迫综合征（ARDS）期间空域中触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介质的细胞特异性和分子机制， 测试了一种新的局部化ARDS疗法，该疗法具有快速转化为人体临床试验的高潜力。在 此外，通过这些研究，一个有前途的年轻医生科学家将获得新的技能， 在一个高度成功的专家指导委员会的指导下， ARDS、炎症和巨噬细胞生物学。这些新的指导技能将构成这一基础 初级研究员，以实现一个长期的目标，是一个独立资助的学术医生科学家。

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.