MNEI/SerpinB1: A modulator of innate pulmonary defense

描述（由申请人提供）：中性粒细胞丝氨酸蛋白酶（NSPs）（弹性酶、组织蛋白酶G和蛋白酶3）在炎症部位由脱颗粒或坏死的中性粒细胞释放，是囊性纤维化和慢性阻塞性肺疾病（COPD）病理的主要因素。SerpinB1/MNEI（单核中性粒细胞弹性酶抑制剂）天然存在于肺和血细胞中，是三种NSPs的高效抑制剂。我们已经产生了mnei缺陷小鼠，它复制了炎性肺病的蛋白酶过剩，并为研究目前被低估的mnei/SerpinB1对肺保护的贡献提供了一个模型。mnei缺陷小鼠无法清除铜绿假单胞菌，促炎细胞因子增加，表面活性剂蛋白- d（一种已知的NSPs靶点）耗竭。SP-D是肺泡巨噬细胞对凋亡中性粒细胞的细菌摄取和清除所必需的，在缺乏mnei/serpinb1的假单胞菌感染小鼠的肺中被发现为无活性片段。坏死的中性粒细胞也会积累。目的1将检验mnei在两种遗传背景（C57BL/6和129s6）中对革兰氏阴性（铜绿假单胞菌、流感嗜血杆菌）和革兰氏阳性（金黄色葡萄球菌）生物提供广泛的肺宿主防御保护的假设。将评估Mnei-/-型和野生型小鼠的存活、细菌学、中性粒细胞的募集和存活；将对灌洗液样本进行细胞因子，蛋白酶，SP-A和SP-D分析。Aim 2将解决在抗菌防御恶化的关键时间段（6-24小时）导致抗假单胞菌反应缺陷的细胞缺陷。将检测感染小鼠的实质细胞、中性粒细胞和肺泡巨噬细胞的活化、细胞损伤和表面受体切割。评估全身反应（血液细胞因子）。将分析中性粒细胞的体外存活和细菌杀灭活性，以及肺泡巨噬细胞吞噬坏死细胞的能力。由于mnei在髓细胞中高水平表达，但也在肺实质中表达，因此将产生嵌合小鼠以确定非造血细胞是否有助于表型。目的3将测试mnei缺陷小鼠抗假单胞菌防御功能缺陷和炎症反应增加是否可以通过鼻腔给药重组mnei来重建。为了测试SP-D耗竭的作用，我们将确定是否可以通过肺特异性过表达SP-D来（部分）纠正缺陷反应。这些小鼠研究旨在描述侵袭性宿主反应导致囊性纤维化和COPD（慢性支气管炎、肺气肿）患者肺损伤的机制，将提供可能导致改进治疗的理解。事实上，该项目研究的中心分子mnei是炎症性肺病的候选治疗药物。

DESCRIPTION (provided by applicant): The neutrophil serine proteases (NSPs) (elastase, cathepsin G and proteinase-3) released at inflammatory sites by degranulating or necrotic neutrophils are major contributors to the pathology of cystic fibrosis and chronic obstructive pulmonary disease (COPD). SerpinB1/MNEI (Monocyte neutrophil elastase inhibitor) is naturally occurring in lungs and blood cells and is a highly efficient inhibitor of the three NSPs. We have produced a mouse deficient for mnei, which replicates the protease excess of inflammatory lung disease and provides a model for studying the currently underestimated contribution of mnei/SerpinB1 to pulmonary protection. The mnei deficient mice fail to clear Pseudomonas aeruginosa and have increased proinflammatory cytokines and depletion of surfactant protein-D (SP-D), a known target of NSPs. SP-D, which is required for bacterial uptake and clearance of apoptotic neutrophils by alveolar macrophages, is found as inactive fragments in lungs of Pseudomonas-infected mice lacking mnei/serpinb1. Necrotic neutrophils also accumulate. Aim 1 will test the hypothesis that mnei provides broad pulmonary host defense protection against both Gram-negative (P.aeruginosa, Haemophilus influenzae) and Gram-positive (Staphylococcus aureus) organisms and in two genetic backgrounds (C57BL/6 and129S6). Mnei-/- and wild type mice will be evaluated for survival, bacteriology, and recruitment and survival of neutrophils; lavage samples will be analyzed for cytokines, proteases, SP-A and SP-D. Aim 2 will address the cellular defects that contribute to the defective anti-Pseudomonas response during the critical time block (6-24 hr) during which antimicrobial defense deteriorates. Parenchymal cells, neutrophils and alveolar macrophages of infected mice will be examined for activation, cell injury, and surface receptor cleavage. Systemic response (blood cytokines) will be assessed. Neutrophils will be analyzed ex vivo for survival and bacterial killing activity, and alveolar macrophages for ability to engulf necrotic cells. Since mnei is expressed at high level in myeloid cells, but is also expressed in lung parenchyma, chimeric mice will be generated to determine whether non-hematopoietic cells contribute to the phenotype. Aim 3 will test whether the defective anti-Pseudomonas defense and increased inflammation of mnei deficient mice can be reconstituted by intranasal delivery of recombinant MNEI. To test the role of SP-D depletion, we will determine whether the defective response can be (partially) corrected by lung-specific overexpression of SP-D. These studies in mice to delineate mechanisms by which an aggressive host response leads to lung injury as occurs in patients with cystic fibrosis and COPD (chronic bronchitis, emphysema) will provide understanding that may lead to improved treatment. Indeed, mnei, the central molecule under study in the project, is a candidate therapeutic for inflammatory lung disease.