Dynamics of neutrophil extracellular trap (NET) formation

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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.