Dynamics of neutrophil extracellular trap (NET) formation

中性粒细胞是人类血液系统中最丰富的免疫细胞类型，并且是先天免疫的中心。最近，人们发现它们能够通过排出由其自身DNA、瓜氨酸化组蛋白和抗菌肽（中性粒细胞胞外陷阱，NET）制成的原纤维网络来捕获和杀死病原体，如细菌和真菌。在（自杀性）NETosis期间，细胞内的物质发生剧烈的重排。在几个小时的时间范围内，细胞核的DNA内容物膨胀并最终从细胞中释放出来，最终使中性粒细胞死亡。到目前为止，人们对控制这一复杂过程的机制知之甚少。已知NETosis由细菌或真菌诱导，但也由诸如脂多糖（LPS）、趋化因子或佛波醇肉豆蔻酸酯乙酸酯（PMA）的物质诱导。然而，问题仍然是如何协调DNA内容的解压缩和挤出。该项目的目的是了解NETosis期间细胞的染色质和细胞骨架如何重排以及DNA如何离开细胞（主动或被动运输）。我们将通过生命细胞成像以及经典的生物化学方法如蛋白质印迹来研究NETosis过程中染色质和细胞骨架的非平衡重塑。此外，我们将评估细胞骨架抑制剂如何影响NET的形成。细胞内部的重排表明细胞的机械性质在NETosis期间发生变化。因此，我们将使用时间分辨原子力显微镜（AFM）的中性粒细胞，并找出是否细胞改变其机械性能和释放的DNA内容（被动）在一个突发/内爆或如果一个主动（生物）运输机制参与。我们还将施加外力来测试电池的机械稳定性在NETosis期间丢失并且最终（外部）力触发对于释放NET是必要的假设。 接下来，我们将测试粘附对NETosis的影响。有人提出，整合素如Mac-1有助于NETosis所必需的信号传导。因此，我们将量化化学定义明确的表面上的NETosis，这些表面完全防止粘附或以明确定义的密度呈现整合素配体并支持粘附。这种方法将使我们能够理解粘附和NETosis之间的相互作用。总之，我们的目标是从生物物理学的角度来理解这种类型的免疫防御机制。此外，NETosis可以作为一个模型来理解控制细胞结构（如染色质）重组的一般原理。通过这项工作确定的分子参与者和过程可能会对NETosis以外的其他生物过程产生影响。

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.