Regulation of NETosis in antibacterial lung defense

NETosis 在抗菌肺防御中的调节

• 批准号: 8510275
• 负责人: EILEEN REMOLD-O'DONNELL
• 金额: $ 30.63万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-15 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8510275
• 关键词: Anti-Bacterial Agents; Arginine; Autoimmune Diseases; Bacteria; Bacterial Infections; Biological Assay; Cathepsin G; Cathepsins; Cell Count; Cell Nucleus; Cell surface; Cells; Cessation of life; Charge; Chromatin; Chronic; Citrulline; Cysteine; Cysteine Protease; Cytoplasm; Cytoplasmic Granules; DNA; Data; Disease; Elastases; Elements; Enzymes; Event; Failure; Goals; Granulopoiesis; Haemophilus influenzae; Histones; Hospitalization; In Vitro; Infection; Inflammation; Inflammation Mediators; Inflammatory; Injury; Knowledge; Left; Lung; Measures; Modeling; Molecular; Morphology; Mus; Neutrophil Activation; Nuclear; Outcome; PRTN3 gene; Pathology; Pathway interactions; Peptide Hydrolases; Peroxidases; Pneumonia; Preparation; Production; Protease Inhibitor; Protein-arginine deiminase; Proteinase 3; Proteins; Pseudomonas aeruginosa; Publishing; Regulation; Rest; Rhinovirus; Serine; Serine Protease; Serine Proteinase Inhibitors; Shapes; Signal Transduction; Specificity; Staging; Staphylococcus aureus; Streptococcus pneumoniae; Structure of parenchyma of lung; Tail; Testing; Tissues; Viral; Virus; Virus Diseases; antimicrobial; base; chemokine; cytokine; extracellular; fighting; in vivo; influenzavirus; inhibitor/antagonist; injured; killings; lung injury; migration; mouse model; neutrophil; pathogen; prevent; programs; public health relevance; response; therapeutic target

DESCRIPTION (provided by applicant): Pneumonias resulting from failure to eradicate bacterial (Streptococcus pneumonia, Haemophilus influenza, Staphylococcus aureus, Pseudomonas aeruginosa) and viral (rhinovirus, influenza virus) infections are responsible for a tremendous burden of hospitalizations and deaths. Using mouse models, we established that the protease inhibitor SerpinB1, an ancient nucleocytoplasmic protein, (i) protects anti-microbial capacity during P. aeruginosa and influenza virus infections and (ii) acts as a critical negative regulator of NETosis. NETosis (Neutrophil Extracellular Trap production) is a programmed death pathway induced by pathogens and inflammatory mediators in which dying neutrophils extrude linearized DNA coated with potent antimicrobial enzymes: histones, myeloperoxidase and neutrophil serine proteases (NSPs). Although NETosis can be protective by preventing dissemination of infection, this death pathway is frequently pathological because excess NETs inflict serious damage to host tissue and destroy anti-microbial defenses in pneumonia and other inflammatory and autoimmune diseases. To examine the mechanisms by which SerpinB1 inhibits NETosis, we will identify and characterize the required proteases that co-function with SerpinB1. We will determine in Aim 1 whether NETs are produced by neutrophils that are singly, doubly and triply deleted for elastase, cathepsin-G or proteinase 3 (granule serine proteases inhibited by SerpinB1 and collectively known as NSPs) and by wild-type (WT) neutrophils treated with inhibitors and will verify the findings in vivo. We will determine whether the require NSP is located on the cell surface by the use of non-permeable serine protease inhibitors. The second aim builds on our recent findings that SerpinB1 migrates into the nucleus early during NETosis where it inhibits the activity (or activation) of PAD4 (peptidylarginine deiminase-4), the essential enzyme that deiminates (citrullinates) histone tail arginine residues, causing chromatin decondensation, expansion of the nucleus, and extrusion of NETs. To dissect the mechanism of this restriction, we will determine in Aim 2 whether (i) PAD4 protein differs (in amount and molecular form) between WT and serpinb1-/- neutrophils and whether (ii) SerpinB1 inhibits PAD4 activation in a broken cell nuclear preparation from serpinb1-/- neutrophils. The final aim will determine whether nuclear cysteinyl cathepsins might co-function in regulating NETosis based on the findings that these proteases are inhibited by SerpinB1 and localize to the nucleus in a number of cells. We will determine in Aim 3 whether (i) cysteinyl cathepsins are expressed in the nucleus of murine neutrophils, either constitutively or during NETosis (by the use of class-specific mechanism-based probes) and whether (ii) cathepsin inhibitors block NETs production. The proposed studies will define key elements of the mechanism of NET production and potentially identify targets for therapy to dampen excess NETosis and thereby decrease inflammatory injury and protect anti-microbial defense in pneumonia and other inflammatory diseases.

描述（由申请人提供）：未能根除细菌（肺炎链球菌、流感嗜血杆菌、金黄色葡萄球菌、铜绿假单胞菌）和病毒（鼻病毒、流感病毒）感染而导致的肺炎造成了巨大的住院和死亡负担。使用小鼠模型，我们确定蛋白酶抑制剂 SerpinB1（一种古老的核细胞质蛋白）（i）在铜绿假单胞菌和流感病毒感染期间保护抗微生物能力，并且（ii）充当 NETosis 的关键负调节因子。 NETosis（中性粒细胞胞外陷阱产生）是一种由病原体和炎症介质诱导的程序性死亡途径，其中垂死的中性粒细胞挤出涂有有效抗菌酶的线性化 DNA：组蛋白、髓过氧化物酶和中性粒细胞丝氨酸蛋白酶 (NSP)。尽管 NETosis 可以通过防止感染传播来发挥保护作用，但这种死亡途径通常是病理性的，因为过量的 NET 会对宿主组织造成严重损害，并破坏肺炎和其他炎症和自身免疫性疾病的抗菌防御。为了研究 SerpinB1 抑制 NETosis 的机制，我们将鉴定并表征与 SerpinB1 共同起作用的所需蛋白酶。我们将在目标 1 中确定 NET 是否由弹性蛋白酶、组织蛋白酶-G 或蛋白酶 3（受 SerpinB1 抑制的颗粒​​丝氨酸蛋白酶，统称为 NSP）单次、两次和三次删除的中性粒细胞以及用抑制剂处理的野生型 (WT) 中性粒细胞产生，并将在体内验证结果。我们将通过使用非渗透性丝氨酸蛋白酶抑制剂来确定所需的 NSP 是否位于细胞表面。第二个目标基于我们最近的发现，即 SerpinB1 在 NETosis 早期迁移到细胞核中，抑制 PAD4（肽基精氨酸脱亚胺酶 4）的活性（或激活），PAD4 是组蛋白尾部精氨酸残基脱亚胺（瓜氨酸化）的必需酶，导致染色质解缩、细胞核扩张和 NET 挤出。为了剖析这种限制的机制，我们将在目标 2 中确定 (i) PAD4 蛋白在 WT 和 serpinb1-/- 中性粒细胞之间是否存在差异（数量和分子形式），以及 (ii) SerpinB1 是否在来自 serpinb1-/- 中性粒细胞的破碎细胞核制剂中抑制 PAD4 活化。最终目标将确定核半胱氨酰组织蛋白酶是否可能在调节 NETosis 中发挥共同作用，基于这些蛋白酶被 SerpinB1 抑制并定位于许多细胞的细胞核的发现。我们将在目标 3 中确定 (i) 半胱氨酰组织蛋白酶是否在鼠中性粒细胞的细胞核中表达，无论是组成型还是在 NETosis 期间（通过使用基于类特异性机制的探针），以及 (ii) 组织蛋白酶抑制剂是否阻止 NET 的产生。拟议的研究将确定 NET 产生机制的关键要素，并可能确定抑制过度 NETosis 的治疗靶标，从而减少炎症损伤并保护肺炎和其他炎症性疾病的抗菌防御。