Shear Stress and NETosis
by Lukas Frederik Mrowietz
Date of Examination:2024-12-17
Date of issue:2024-11-29
Advisor:Prof. Dr. Michael P. Schön
Referee:Prof. Dr. Michael P. Schön
Referee:Prof. Dr. Sarah Köster
Persistent Address:
http://dx.doi.org/10.53846/goediss-10893
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Abstract
English
Neutrophil granulocytes, the most abundant type of circulating immune cells, engage in a process called NETosis when encountering certain pathogens and danger signals or experimentally induced by phorbol-12-myristate-13-acetate (PMA). During NETosis the chromatin decondenses and through a passive swelling process this leads to the rupture of the nuclear and subsequently of the plasma membrane. A meshwork of desoxyribonucleic acid (DNA) decorated with nuclear and cytosolic proteins is released into the extracellular space forming Neutrophil Extracellular Traps (NETs). It has been shown that this process can be divided into two distinct phases. The first phase, being energy-dependent, starts at stimulation and ends at chromatin decondensation. The second phase consists of a passive swelling process ending at the rupture of the plasma membrane and the release of the NET. There is experimental evidence that fluidic shear stress generated by the bloodstream to adherent neutrophils attached to the vessel’s wall is promoting NETosis. The underlying mechanism, however, remains unknown. The aim of this study was to establish an experimental setup and to investigate the effect of fluidic shear stress on adherent neutrophils undergoing NETosis in greater detail focusing on changes of the different phases of NETosis. All experiments were performed with human neutrophils and analyzed by live cell imaging. For this purpose, a novel cell culture medium was developed to avoid unspecific cellular activation and provide optimal cell adherence and survival. Fluid chambers and cell dishes, two different pump systems for flow application, a DNA live stain to visualize chromatin, and PMA as a stimulus for NETosis were used. With a circular pump continuous shear stress was generated and with a syringe-pump neutrophils were pre-treated with shear stress before stimulation. Videos obtained by live cell imaging were analyzed either by computational analysis or by the naked eye. The setup proved to be suitable for investigating NETosis under shear stress. All variables such as staining, stimuli, and shear stress quality and quantity could be modified. Analysis of live cell imaging allowed to clearly characterize distinct elements of NETosis. The results of the investigation showed that neutrophils exposed to either continuous shear stress or shear stress pre-treatment underwent an altered process of NETosis in some donors in comparison to cells that were not exposed to shear stress. The most common observation was that neutrophils treated with shear stress during or before undergoing NETosis showed a prolonged first phase until the swelling of chromatin. Under continuous shear stress a shortened second phase until the rupture of the plasma membrane was observed. These findings indicate that shear stress did influence NETosis, altering the duration of its phases. Previous studies suggested a link between shear stress and cytoskeletal changes, calcium-homeostasis, or mechanotransduction. The development of the methodology employed in this work by creating different experimental setups offers the possibility to further investigate the molecular mechanisms behind NETosis under the influence of shear stress adapted to experimental needs.
Keywords: neutrophil granulocytes; neutrophil extracellular traps; NETosis; innate immunity; shear stress; flow chamber; chromatin swelling; programmed cell death; flow
