MNEI/SerpinB1: A modulator of innate pulmonary defense

描述（由申请方提供）：由脱粒或坏死中性粒细胞在炎症部位释放的中性粒细胞丝氨酸蛋白酶（NSP）（弹性蛋白酶、组织蛋白酶G和蛋白酶-3）是囊性纤维化和慢性阻塞性肺病（COPD）病理学的主要贡献者。SerpinB 1/MNEI（单核细胞中性粒细胞弹性蛋白酶抑制剂）天然存在于肺和血细胞中，是三种NSP的高效抑制剂。我们已经产生了mnei缺陷的小鼠，其复制了炎性肺病的蛋白酶过量，并为研究目前被低估的mnei/SerpinB 1对肺保护的贡献提供了模型。mnei缺陷小鼠不能清除铜绿假单胞菌，并且具有增加的促炎细胞因子和表面活性剂蛋白-D（SP-D）（NSP的已知靶标）的消耗。SP-D是细菌摄取和肺泡巨噬细胞清除凋亡中性粒细胞所必需的，在缺乏mnei/serpinb 1的假单胞菌感染小鼠的肺中被发现为非活性片段。坏死的中性粒细胞也会聚集。目的1将检验mnei在两种遗传背景（C57 BL/6和129 S6）中提供针对革兰氏阴性（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）生物的广泛的肺宿主防御保护的假设。将评价Mnei-/-和野生型小鼠的存活率、细菌学以及中性粒细胞的募集和存活率;将分析灌洗液样品的细胞因子、蛋白酶、SP-A和SP-D。目标2将解决在关键时间段（6-24小时）期间导致抗假单胞菌应答缺陷的细胞缺陷，在此期间抗菌防御恶化。将检查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裂解。将评估全身反应（血液细胞因子）。将离体分析中性粒细胞的存活和细菌杀伤活性，并分析肺泡巨噬细胞吞噬坏死细胞的能力。由于mnei在骨髓细胞中以高水平表达，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将测试是否可以通过鼻内递送重组MNEI来重建mnei缺陷小鼠的有缺陷的抗假单胞菌防御和增加的炎症。为了测试SP-D耗竭的作用，我们将确定是否可以通过SP-D的肺特异性过表达来（部分）纠正有缺陷的反应。这些在小鼠中进行的研究描述了侵袭性宿主反应导致肺损伤的机制，如囊性纤维化和COPD（慢性支气管炎、肺气肿）患者中发生的机制，将提供可能导致治疗改善的理解。事实上，该项目正在研究的中心分子mnei是治疗炎症性肺病的候选药物。

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.