Regulation of NETosis in antibacterial lung defense

描述（由申请方提供）：由于未能根除细菌（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）感染而导致的肺炎是造成住院和死亡的巨大负担的原因。使用小鼠模型，我们确定了蛋白酶抑制剂SerpinB1（一种古老的核质蛋白）（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，（ii）作为NETosis的关键负调节因子。NETosis（Neutrophil Extracellular Trap production）是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被有效的抗菌酶（组蛋白、髓过氧化物酶和中性粒细胞丝氨酸蛋白酶（NSP））包被的线性化DNA。虽然NETosis可以通过防止感染传播来保护，但这种死亡途径通常是病理性的，因为过量的NETs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中的抗微生物防御。为了研究SerpinB1抑制NETosis的机制，我们将鉴定和表征与SerpinB1共同作用的所需蛋白酶。我们将在目标1中确定NETs是否由弹性蛋白酶、组织蛋白酶-G或蛋白酶3（被SerpinB1抑制的颗粒丝氨酸蛋白酶，统称为NSP）单、双和三缺失的中性粒细胞和用抑制剂处理的野生型（WT）中性粒细胞产生，并将在体内验证发现。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。第二个目标建立在我们最近的发现基础上，即SerpinB1在NETosis期间早期迁移到细胞核中，在那里它抑制PAD 4（肽基精氨酸脱亚胺酶-4）的活性（或激活），PAD 4是使组蛋白尾部精氨酸残基脱亚胺（瓜氨酸化）的必需酶，导致染色质解凝聚，细胞核扩张和NET挤出。为了剖析这种限制的机制，我们将在目的2中确定（i）WT和serpinb 1-/-中性粒细胞之间的PAD 4蛋白是否不同（在量和分子形式上），以及（ii）在来自serpinb 1-/-中性粒细胞的破碎细胞核制备物中，serpinB 1是否抑制PAD 4活化。最终的目标将确定核半胱氨酰组织蛋白酶是否可能共同作用于调节NETosis，基于这些蛋白酶被SerpinB1抑制并定位于许多细胞中的细胞核的发现。我们将在目标3中确定（i）半胱氨酰组织蛋白酶是否在鼠嗜中性粒细胞的细胞核中表达，无论是组成性的还是在NETosis期间（通过使用基于类别特异性机制的探针），以及（ii）组织蛋白酶抑制剂是否阻断NETs产生。拟议的研究将定义NET产生机制的关键要素，并可能确定治疗目标，以抑制过量的NETosis，从而减少炎症损伤并保护肺炎和其他炎症性疾病的抗微生物防御。

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.