Regulation of NETosis in antibacterial lung defense

NETosis 在抗菌肺防御中的调节

基本信息

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
项目类别：
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

项目摘要

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）：由于无法根除细菌（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（rhinovirus，Rhinovirus，rhinza病毒）的损害负责医院的死亡负责的肺炎。使用小鼠模型，我们确定蛋白酶抑制剂SERPINB1是一种古老的核质蛋白，（i）保护铜绿假单胞菌和流感病毒感染期间的抗微生物剂能力，并且（ii）起到了关键的Netosis负面调节剂。 Netosis（中性粒细胞外陷阱产生）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了覆盖有效抗菌酶的线性DNA：组蛋白，髓过氧化物酶和中性粒细胞粒细胞蛋白蛋白酶（NSPS）。尽管Netosis可以通过防止感染传播来保护，但这种死亡途径经常是病理性的，因为过量的网络会对宿主组织造成严重破坏并破坏肺炎以及其他炎症性和自身免疫性疾病的抗微生物防御。为了检查SERPINB1抑制Netosis的机制，我们将识别并表征与SerpinB1共同功能的所需蛋白酶。我们将在AIM 1中确定网络是否是由弹性酶，组织蛋白酶G或蛋白酶3（颗粒丝氨酸蛋白酶蛋白酶抑制的serpinb1抑制和集体称为NSP的颗粒蛋白酶蛋白酶）和野生型（WT）中性粒细胞的探索和iffifififififer vivifififife forififer ferififer ferefififer ferefififer ferefififer ferefififer ferefififer enderefors everefor enderefors，它们是否是单独，双重，双重和三重删除的中性粒细胞（固定蛋白酶G或蛋白酶3（颗粒丝氨酸蛋白酶））的生产。我们将通过使用不可渗透的丝氨酸蛋白酶抑制剂来确定需求NSP是否位于细胞表面上。第二个目的是基于我们最近的发现，即在Netosis的早期，SERPINB1抑制了PAD4的活性（或激活）（肽基金宁脱氨酸酶-4），这是脱离（citrullinate）组蛋白尾巴残基的基本酶，导致染色素脱孔，并脱离了成分，并导致了纯化的片剂，并导致了纯化的成分。为了剖析这种限制的机制，我们将在AIM 2中确定（I）PAD4蛋白是否在WT和Serpinb1 - / - 中性粒细胞之间（量和分子形式）是否有所不同，以及（II）SERPINB1是否抑制Serpinb1-/ - / - 中性粒细胞破裂的细胞核制剂中PAD4的激活。最终目的将根据发现这些蛋白酶被serpinb1抑制并定位于许多细胞中的细胞核的发现，确定核半胱氨酸结蛋白是否可以在调节Netosis中共同起作用。我们将在AIM 3中确定（i）半胱氨酸结蛋蛋白是在组成型中性粒细胞的核中表达的，无论是组成型还是在Netosis（通过使用基于类别的机制探针）和（ii）calter蛋白抑制剂阻止网络产生的生产。拟议的研究将定义净生产机制的关键要素，并有可能识别治疗的靶标，以减轻过量的肠病，从而减少炎症性损伤并保护肺炎和其他炎症性疾病中的抗微生物防御。