NETs and their modulating enzymes in age-related inflammatory diseases

描述(由申请人提供)：随着人口寿命的普遍增加，慢性炎症性和血栓性疾病的患病率正在上升。我们的应用解决了中性粒细胞胞外陷阱(Net)在此类年龄相关疾病中的作用，Net是由激活的中性粒细胞释放的染色质。我们假设，衰老促进了网络的形成，而网络反过来又加剧了慢性炎症和血栓性疾病。我们认为调节NETase的基因，如PAD4，影响衰老。我们的初步结果表明，老年小鼠的中性粒细胞产生更多的NETs，而PAD4，它协调NETase，促进老年小鼠的心脏纤维化。我们观察到线粒体脱乙酰基酶sirtuin3(SIRT3)，其对活性氧物种(ROS)的调节与衰老有关，它由中性粒细胞和血小板表达，是中性粒细胞ROS产生和NET代谢的有力下调因子。我们将通过野生型(WT)和基因工程突变小鼠的体外研究和疾病模型来解决我们的假设。我们提出了三个目标：目的1.研究WT小鼠NET代谢与衰老的关系，热量限制(CR)对NET代谢的影响以及由此引起的炎症、血栓和纤维化反应。我们将研究小鼠年龄对心肺纤维化易感性的影响。我们将评估帮助清除Net的DNase1是否会减少实验性肺纤维化。我们将研究CR，已知可以延缓衰老，对网络形成和血栓形成和器官纤维化的易感性的影响。目的2.研究PAD4基因突变是否会影响衰老以及与年龄相关的炎症性和血栓性疾病。我们将研究PAD4缺乏对年龄相关性心脏纤维化和实验性肺纤维化易感性的影响。我们将跟踪PAD4-/-和WT窝产仔的老化、健康和寿命。我们将对中性粒细胞过度产生PAD4的转基因小鼠进行基因改造。我们预计，它们将更容易产生Net，并有更多的倾向于血栓形成和间质肺纤维化以及延迟伤口愈合。目的3.评价SIRT3在NET病和涉及NETs的疾病模型中的作用。血小板SIRT3在血小板活化及其启动中的作用研究 中性粒细胞向网织红细胞增多。我们热衷于评估SIRT3在中性粒细胞、血小板中的作用以及在血小板和中性粒细胞之间的相互作用中可能增强NETsis的作用。SIRT3-/-和已经产生的SIRT3过表达的小鼠将接受炎症和血栓形成的评估。骨髓移植嵌合动物将准备区分血细胞和组织SIRT3的作用。我们希望发现新的药理靶点，以减少NET在慢性炎症和血栓形成过程中的作用，并希望这最终可能改善我们老龄化人口的健康。

DESCRIPTION (provided by applicant): The prevalence of chronic inflammatory and thrombotic diseases is rising with the general increase in population longevity. Our application addresses the role of neutrophil extracellular traps (NETs), chromatin released by activated neutrophils, in such age-related diseases. We hypothesize that aging promotes NET formation and NETs in turn escalate chronic inflammatory and thrombotic diseases. We propose that genes regulating NETosis, such as peptidylarginine deiminase 4 (PAD4), impact aging. Our preliminary results indicate that neutrophils from old mice make more NETs and PAD4, which orchestrates NETosis, promotes cardiac fibrosis in older mice. We observed that the mitochondrial deacetylase sirtuin 3 (SIRT3), whose regulation of reactive oxygen species (ROS) is implicated in aging, is expressed by neutrophils and platelets and is a potent down-regulator of neutrophil ROS production and NETosis. We will address our hypothesis with in vitro studies and disease models in wild type (WT) and genetically engineered mutant mice. We propose three Aims: Aim 1. To study the relationship of NETosis and aging in WT mice, the effect of calorie restriction (CR) on NETosis and resulting inflammatory, thrombotic, and fibrotic responses. We will examine NETosis and the susceptibility to cardiac and lung fibrosis as a function of mouse age. We will evaluate whether DNase1, that helps clear NETs, will reduce experimental lung fibrosis. We will study the effect of CR, known to slow down aging, on NET formation and susceptibility to thrombosis and organ fibrosis. Aim 2. To investigate whether mutations in PAD4, an enzyme required for NET formation, affect aging and age-related inflammatory and thrombotic diseases. We will study the effect of PAD4- deficiency on age-related heart fibrosis and susceptibility to experimental lung fibrosis. We will follow aging healt and longevity of PAD4-/- and WT littermates. We will genetically engineer transgenic mice overproducing PAD4 in neutrophils. We anticipate that they will produce NETs more readily and have an increased propensity towards thrombosis and interstitial lung fibrosis as well as delayed wound healing. Aim 3. To evaluate the role of SIRT3 in NETosis and in disease models involving NETs. Study the function of platelet SIRT3 in platelet activation and in their priming of neutrophils towards NETosis. We are keen to evaluate the role of SIRT3 in neutrophils, platelets and in the interaction between platelets and neutrophils that may enhance NETosis. SIRT3-/- and already generated SIRT3- overexpressing mice will be evaluated in inflammation and thrombosis. Bone marrow transplant chimeric animals will be prepared to distinguish the role of blood cell versus tissue SIRT3. We hope to uncover new pharmacological targets to reduce NETs' contribution to chronic inflammatory and thrombotic processes and that this may eventually improve the health of our aging population.