Regulation of neutrophil extracellular trap formation

中性粒细胞是先天免疫系统不可或缺的组成部分。这些细胞的一个关键功能反应是它们释放所谓的中性粒细胞胞外陷阱（NET）的能力-一种由各种抗菌蛋白修饰的挤出染色质细丝的网络。NET捕获（因此得名）微生物并促进其在体外和动物模型中的杀灭。因此，NET已经成为一种主要的抗菌防御机制。 在过去的3年中，我们证明了酶PAD 4在NET生成中的关键作用;相反，我们表明，对于大多数生理NET诱导剂，NADPH氧化酶系统是必需的，以引起这种反应。我们还发现，NET形成的特点是由离散信号通路控制的即时早期和晚期事件-这是几类生理中性粒细胞刺激共有的特征。在驱动NET形成的晚期细胞过程中，我们最近揭示了自分泌/旁分泌反馈回路的存在，内源性因素介导了这一现象。然而，尽管有这些进展，我们对NET形成的分子基础的理解仍然是零碎的。 目标.我们研究计划的总体目标是阐明NET形成的关键细胞过程和分子，以及控制这一现象的上游信号中间体。在第一个项目（目标1）中，我们将研究一些驱动NET形成的晚期细胞过程，重点是（i）确定一些反馈中性粒细胞以触发NET释放的内源性因素，以及控制其释放的信号通路;（ii）影响染色质挤出和长DNA纤维形成的过程。在后一种情况下，我们将确定细丝的形成是否需要大分子向导或细胞外支架;以及染色质是否以某种方式投射到附近的细胞。在第二个项目（目标2）中，我们将研究如何磷酸肌醇3-磷酸激酶（PI 3 K）途径，这是迟发性的，是核心的NET生成，控制这种反应。特别是，我们将探讨哪些PI 3 K亚型参与，以及它们是否保守在几类生理刺激之间的（或不同的）; PI 3 K下游的或与PI 3 K相关的各种信号传导组分中的哪一种有助于NET诱导，以及它们的相互关系;以及NET形成的哪个后期步骤（即内源性介质产生、染色质解凝聚、NET挤出）受上述相关信号分子的影响。 可行性。我们精通开展拟议工作所需的所有实验方法。 意义拟议的研究计划将大大进一步我们的理解NET一代，一个看似独特的生物现象，通过确定一些潜在的分子机制。

Neutrophils are indispensable components of the innate immune system. A key functional response of these cells is their ability to release so-called neutrophil extracellular traps (NETs) - a meshwork of extruded chromatin filaments decorated with various antimicrobial proteins. NETs entrap (whence the name) microorganisms and promote their killing, both ex vivo and in animal models. As a result, NETs have emerged as a major antimicrobial defense mechanism. Over the last 3 years, we demonstrated the pivotal role of the enzyme, PAD4, for NET generation; conversely, we showed that for most physiological NET inducers, the NADPH oxidase system is dispensable to elicit this response. We also found that NET formation features both immediate-early and late events that are controlled by discrete signaling pathways - a characteristic shared across several classes of physiological neutrophil stimuli. Among the late cellular processes driving NET formation, we recently unveiled the existence of an autocrine/paracrine feedback loop whereby endogenous factors mediate the phenomenon. Despite these advances however, our understanding of the molecular underpinnings of NET formation remains fragmentary. Objectives. The overarching goal of our research program is to shed light on key cellular processes and molecules underlying NET formation, and on the upstream signaling intermediates controlling the phenomenon. In the first project (Aim 1), we will investigate some of the late cellular processes driving NET formation, with a focus (i) on identifying some of the endogenous factors that feed back on neutrophils to trigger NET release, and the signaling pathways controlling their release; and (ii) on processes that affect chromatin extrusion and the formation of long DNA fibers. In the latter instance, we will determine whether filament formation requires macromolecular guides or extracellular scaffolds; and whether chromatin is somehow projected towards nearby cells. In the second project (Aim 2), we will investigate how the phosphoinositol 3-phosphate kinase (PI3K) pathway, which acts belatedly and is central to NET generation, controls this response. In particular, we will explore which PI3K isoforms are involved and whether they are conserved (or differ) among several classes of physiological stimuli; which of the various signaling components downstream of, or associated with, PI3K contribute to NET induction, as well as their inter-relationships; and which late step of NET formation (i.e. endogenous mediator generation, chromatin decondensation, NET extrusion) is affected by relevant signaling molecules identified above. Feasibility. We are well versed in all of the experimental approaches required to carry out the proposed work. Significance. The proposed research program will substantially further our understanding of NET generation, a seemingly unique biological phenomenon, by identifying some of the underlying molecular mechanisms.