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（NETs）的能力-一种用组蛋白以及源自细胞质或细胞内颗粒的蛋白质装饰的挤出染色质细丝的网状结构。NET在十年前首次被描述为捕获（因此得名）各种微生物的结构，因此可以参与杀死它们。NET已经在各种炎症环境中被检测到，并被证明有助于防止微生物在动物模型中的传播。因此，人们普遍认为，NET必须是抵御感染的主要防御机制。 尽管如此，NET形成的分子机制以及上游信号通路仍然知之甚少。在过去的十年中，我们发表了几项研究，记录了中性粒细胞的信号机制，以及它如何控制功能反应，如细胞因子的产生和延迟凋亡。最近，我们开始探索离散的信号分子如何控制NETosis（即NET生成），并且在这样做的过程中，我们观察到这个过程中可能涉及的步骤比以前认识到的更多。 目标.我们研究计划的总体目标是阐明控制NETosis的细胞过程和分子机制。在第一个项目（目标1）中，我们将专注于影响染色质挤出和长DNA纤维形成的细胞过程。这将涉及通过活细胞成像观察整个NETosis现象，并确定仅影响挤出或细丝形成的过程。我们还将确定细丝是否是由移动细胞的物理牵引引起的;细丝的形成是否需要大分子指南或细胞外支架;以及染色质是否可能以某种方式投射到附近的细胞。在第二个项目（目标2）中，我们将研究离散信号成分如何参与控制NETosis（以及在哪些步骤），以及它们的相互关系;以及NETosis是否可以在没有活性氧产生的情况下发生，这被认为是这种反应所必需的。 可行性。我们拥有开展拟议工作所需的所有实验方法的专业知识。 意义拟议的研究计划将大大进一步我们的理解NETosis，一个看似独特的生物现象，通过确定其离散阶段。这些研究还将通过确定一些潜在的分子机制，揭示NETosis如何调控的各个方面。

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 that are decorated with histones, as well as with proteins originating from the cytoplasm or intracellular granules. NETs were first described a decade ago, as structures that entrap (whence the name) various microorganisms and can thus participate in their killing. NETs have since been detected in a variety of inflammatory contexts, and were shown to help in preventing the dissemination of microorganisms in animals models. As a result, it has become a widely held view that NETs must represent a major defense mechanism against infection. Despite this, the molecular mechanisms underlying NET formation, as well as the upstream signaling pathways, remain poorly understood. In the last decade, we published several studies documenting the signaling machinery of neutrophils and how it controls functional responses such as cytokine generation and delayed apoptosis. More recently, we began exploring how discrete signaling molecules also govern NETosis (i.e. NET generation) and in doing so, have observed that there are probably more steps involved in this process than previously appreciated. Objectives. The general goal of our research program is to shed light on the cellular processes and molecular mechanisms that control NETosis. In the first project (Aim 1), we will focus on cellular processes that affect chromatin extrusion and the formation of long DNA fibers. This will involve observing the entire NETosis phenomenon as it unfolds by live cell imaging, and identifying processes that only affect extrusion or filament formation. We will also determine whether filaments result from physical traction by moving cells; whether filament formation requires macromolecular guides or extracellular scaffolds; and whether chromatin might somehow be projected towards nearby cells. In the second project (Aim 2), we will investigate how discrete signaling components participate in controlling NETosis (and at which steps), as well as their inter-relationships; and whether NETosis can occur in the absence of reactive oxygen species production, which are deemed essential for this response. Feasibility. We have expertise in all of the experimental approaches that are required to carry out the proposed work. Significance. The proposed research program will substantially further our understanding of NETosis, a seemingly unique biological phenomenon, by identifying its discrete phases. These investigations will also shed light on various aspects of how NETosis is regulated, by identifying some of the underlying molecular mechanisms.