MNEI/SerpinB1: A modulator of innate pulmonary defense

描述(申请人提供)：中性粒细胞脱颗粒或坏死性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疾病(COPD)的主要病理因素。SerpinB1/MNEI(单核细胞中性粒细胞弹性蛋白酶抑制物)天然存在于肺和血细胞中，是一种高效的三种NSP的抑制剂。我们已经培育了一只mnei缺乏的小鼠，它复制了炎症性肺部疾病中过量的蛋白酶，并为研究mnei/serpinB1目前被低估的肺保护作用提供了一个模型。Mnei缺陷小鼠不能清除铜绿假单胞菌，并增加了促炎细胞因子和表面活性蛋白D(SP-D)的耗竭，SP-D是NSPs的已知靶标。在缺乏mnei/serpinB1的假单胞菌感染小鼠的肺部，发现SP-D是细菌摄取和肺泡巨噬细胞清除中性粒细胞凋亡所必需的非活性片段。坏死性中性粒细胞也会积聚。目的1验证肺炎支原体对革兰氏阴性菌(铜绿假单胞菌、流感嗜血杆菌)和革兰氏阳性菌(金黄色葡萄球菌)在两个遗传背景(C57BL/6和129S6)中提供广泛的肺宿主防御的假设。Mnei-/-和野生型小鼠将接受生存、细菌学以及中性粒细胞的招募和存活的评估；灌洗液样本将进行细胞因子、蛋白酶、SP-A和SP-D的分析。目标2将解决在抗菌素防御恶化的关键时间段(6-24小时)内导致抗假单胞菌反应缺陷的细胞缺陷。将对感染小鼠的实质细胞、中性粒细胞和肺泡巨噬细胞进行激活、细胞损伤和表面受体切割的检查。将评估全身反应(血液细胞因子)。将在体外分析中性粒细胞的存活和杀菌活性，并分析肺泡巨噬细胞吞噬坏死细胞的能力。由于mnei在髓系细胞中高水平表达，但在肺实质中也表达，因此将产生嵌合小鼠来确定非造血细胞是否对表型有贡献。目的研究重组MNEI鼻腔给药能否重建Mnei缺陷小鼠的抗假单胞菌防御功能和炎症反应。为了测试SP-D耗竭的作用，我们将确定肺特异性SP-D的过度表达是否可以(部分)纠正这种缺陷反应。这些在小鼠身上进行的研究描述了攻击性宿主反应导致肺损伤的机制，就像囊性纤维化和慢性阻塞性肺病(慢性支气管炎、肺气肿)患者所发生的那样，这将提供了解，可能导致改进治疗。事实上，该项目正在研究的中心分子mnei是治疗炎症性肺部疾病的候选药物。

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.