Dynamics of neutrophil extracellular trap (NET) formation

中性粒细胞胞外陷阱（NET）形成的动力学

• 批准号: 346014890
• 负责人: Professorin Dr. Luise Erpenbeck
• 金额: --
• 依托单位: Lehrstuhl für Physikalische Chemie II: Laserspektroskopie und Biophotonik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/346014890?language=en
• 关键词: Dynamics; neutrophil; extracellular; trap; NET

Neutrophils are the most abundant type of immune cells in the human blood system and central for innate immunity. Recently, it was found that they are able to catch and kill pathogens such as bacteria and fungi by expelling a fibril network made from their own DNA, citrullinated histones and antimicrobial peptides (neutrophil extracellular traps, NETs). During (suicidal) NETosis, a drastic rearrangement of the materials inside the cell takes place. Within the time frame of a few hours, the DNA-content of the nucleus expands and is finally released from the cell, ultimately leaving the neutrophils to die. So far, the mechanisms that govern this complex process are poorly understood. It is known that NETosis is induced by bacteria or fungi but also by substances such as lipopolysaccharides (LPS), chemokines or phorbol myristate acetate (PMA). However, the question remains how decondensation and extrusion of the DNA-content is orchestrated. The aim of this project, is to understand how the chromatin and the cytoskeleton of the cell is rearranged during NETosis and how the DNA leaves the cells (active or passive transport). We will study the non-equilibrium remodeling of the chromatin and the cytoskeleton during NETosis by life-cell imaging as well as classical biochemical approaches such as Western blots. Additionally, we will evaluate how inhibitors of the cytoskeleton affect the formation of NETs. The rearrangement of the cell's interior suggests that mechanical properties of cells change during NETosis. Therefore, we will use time-resolved atomic force microscopy (AFM) of neutrophils and find out whether the cell changes its mechanical properties and releases the DNA content (passively) in a burst/implosion or if an active (biological) transport mechanism is involved. We will also apply external forces to test the hypothesis that the mechanical stability of the cell is lost during NETosis and a final (external) force trigger is necessary for release of NETs. Next, we will test the influence of adhesion on NETosis. It has been suggested that integrins such as Mac-1 contribute to the signaling that is necessary for NETosis. Therefore, we will quantify NETosis on chemically well-defined surfaces that either completely prevent adhesion or that present integrin ligands at a well-defined density and support adhesion. This approach will allow us to understand the interplay between adhesion and NETosis. In summary, our goal is to understand this type of immune defense mechanism from a biophysical perspective. Additionally, NETosis can serve as a model to understand general principles that govern the reorganization of cellular structures such as chromatin. It is likely that the molecular players and processes identified through this work will have implications for other biological processes beyond NETosis.

中性粒细胞是人体血液系统中最丰富的免疫细胞类型，是天然免疫的中枢。最近，人们发现它们能够通过排出由它们自己的DNA、瓜氨酸组蛋白和抗菌肽(中性粒细胞外陷阱，Nets)组成的纤维网络来捕捉和杀死细菌和真菌等病原体。在自杀过程中，细胞内的物质会发生剧烈的重新排列。在几个小时的时间内，细胞核的DNA含量会膨胀，最终从细胞中释放出来，最终让中性粒细胞死亡。到目前为止，人们对管理这一复杂过程的机制知之甚少。众所周知，蚊虫肺炎可由细菌或真菌引起，但也可由脂多糖(LPS)、趋化因子或佛波酯(PMA)等物质引起。然而，问题仍然是DNA内容的解浓缩和挤出是如何安排的。这个项目的目的是了解细胞的染色质和细胞骨架在网络分裂过程中是如何重新排列的，以及DNA是如何离开细胞的(主动或被动运输)。我们将通过生命细胞成像以及经典的生化方法，如Western blotts，来研究NETsis过程中染色质和细胞骨架的非平衡重构。此外，我们还将评估细胞骨架的抑制剂如何影响Net的形成。细胞内部的重排表明，细胞的机械性能在网络分裂过程中发生了变化。因此，我们将使用中性粒细胞的时间分辨原子力显微镜(AFM)，并找出细胞是否在爆炸/内爆中改变其机械性能并(被动)释放DNA内容，或者是否涉及主动(生物)运输机制。我们还将施加外力来测试假设，即在蚊虫感染期间细胞的机械稳定性丢失，最终(外部)力触发是释放蚊帐所必需的。接下来，我们将测试粘连对网织红细胞增多症的影响。已有研究表明，整合素如Mac-1参与了NETsis所必需的信号转导。因此，我们将对化学定义良好的表面上的NETsis进行量化，这些表面要么完全防止粘连，要么以定义明确的密度呈现整合素配体并支持粘连。这种方法将使我们能够理解粘连和网织红细胞增多症之间的相互作用。综上所述，我们的目标是从生物物理学的角度来理解这种免疫防御机制。此外，NETsis可以作为一个模型来理解支配细胞结构重组的一般原理，如染色质。通过这项工作确定的分子参与者和过程很可能会对NETsis以外的其他生物过程产生影响。