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外陷阱(Net)的能力，Net是一种由突出的染色质细丝网络组成的网络，这些染色质细丝装饰着组蛋白，以及来自细胞质或细胞内颗粒的蛋白质。十年前，蚊帐首次被描述为诱捕(由此得名)各种微生物的结构，因此可以参与杀死它们。自那以后，在各种炎症环境中检测到了蚊帐，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，NETS必须代表着对抗感染的主要防御机制。 尽管如此，网络形成的分子机制以及上游信号通路仍然知之甚少。在过去的十年里，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的凋亡。最近，我们开始探索离散的信号分子如何也控制NETsis(即网络生成)，并在这样做的过程中，观察到这个过程中可能涉及到比以前认识到的更多的步骤。 目标。我们研究计划的总体目标是阐明控制网织红细胞增多症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。这将包括通过活细胞成像观察整个网织红细胞分裂现象，并识别仅影响挤出或细丝形成的过程。我们还将确定细丝是否是移动细胞的物理牵引的结果；细丝的形成是否需要大分子导向器或细胞外支架；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，我们将研究不同的信号成分如何参与控制NETsis(以及在哪些步骤中)，以及它们之间的相互关系；以及在缺乏被认为对这种反应至关重要的活性氧产生的情况下，NETsis是否会发生。 可行性。我们在开展拟议工作所需的所有实验方法方面都有专门知识。 意义重大。拟议的研究计划将通过确定其离散阶段，大大加深我们对NETsis的理解，这是一种看似独特的生物现象。这些研究还将通过确定一些潜在的分子机制，阐明NETsis是如何被调控的各个方面。

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.