MNEI/SerpinB1: A modulator of innate pulmonary defense

MNEI/SerpinB1：先天肺防御的调节剂

• 批准号: 7798071
• 负责人: EILEEN REMOLD-O'DONNELL
• 金额: $ 35.94万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-29 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7798071
• 关键词: Address; Alveolar Macrophages; Anti-Bacterial Agents; Apoptosis; Apoptotic; Bacteria; Bacteriology; Biological Assay; Blood; Blood Cells; Cathepsin G; Cells; Chronic Bronchitis; Chronic Obstructive Airway Disease; Complement Receptor; Cystic Fibrosis; Cytokine Degradation; Defect; Elastases; Epithelial Cells; Genetic; Goals; Haemophilus influenzae; Harvest; Hematopoietic; Host Defense; Human; Immune; Immune response; Immunohistochemistry; Infection; Inflammation; Inflammatory; Injury; Irrigation; Lead; Leukocyte L1 Antigen Complex; Life; Lung; Lung diseases; Matrix Metalloproteinases; Modeling; Mus; Myeloid Cells; Nature; Necrosis; Organism; Pathology; Peptide Hydrolases; Peroxidases; Phagocytosis; Phenotype; Predisposition; Process; Property; Protease Inhibitor; Protein C Inhibitor; Proteinase 3; Proteins; Proteolysis; Pseudomonas; Pseudomonas Infections; Pseudomonas aeruginosa; Pulmonary Emphysema; Pulmonary Surfactant-Associated Protein D; Recombinants; Role; Sampling; Serine Protease; Site; Specimen; Staphylococcus aureus; Structure of parenchyma of lung; Study models; Surface; Testing; Therapeutic; Time; Transgenic Mice; Western Blotting; Wild Type Mouse; antimicrobial; cell injury; chemokine; cystic fibrosis patients; cytokine; improved; inhibitor/antagonist; killings; lung injury; monocyte; neutrophil; neutrophil elastase inhibitor; overexpression; pathogen; prevent; receptor; reconstitution; response; surfactant; uptake

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.

描述（由申请人提供）：脱颗粒或坏死的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疾病（COPD）病理学的主要贡献者。 SerpinB1/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。我们培育了一种 mnei 缺陷的小鼠，它复制了炎症性肺病的蛋白酶过量，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，表面活性蛋白 D (SP-D) 消耗，表面活性蛋白 D 是 NSP 的已知靶标。 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/serpinb1 的假单胞菌感染小鼠的肺部中被发现为无活性片段。坏死的中性粒细胞也会积聚。目标 1 将检验以下假设：mnei 针对两种遗传背景（C57BL/6 和 129S6）的革兰氏阴性菌（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）生物体提供广泛的肺部宿主防御保护。将评估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；将分析灌洗样品的细胞因子、蛋白酶、SP-A 和 SP-D。目标 2 将解决在抗菌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。将检查受感染小鼠的实质细胞、中性粒细胞和肺泡巨噬细胞的激活、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灭活性，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对表型有贡献。目标 3 将测试 mnei 缺陷小鼠的抗假单胞菌防御缺陷和炎症增加是否可以通过鼻内递送重组 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 SP-D 过度表达来（部分）纠正缺陷反应。这些在小鼠中进行的研究旨在阐明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）患者发生的情况，这将提供可能导致改善治疗的理解。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。