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Die Dynamik der Neutrophil Extracellular Traps (NETs) Bildung

Fachliche Zuordnung Biophysik
Förderung Förderung von 2017 bis 2023
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 346014890
 
Erstellungsjahr 2024

Zusammenfassung der Projektergebnisse

NETosis is a unique form of cell death that involves the release of DNA into the extracellular space by neutrophils, which are the most abundant type of white blood cells. It is a vital part of the immune response against pathogens, particularly bacteria and fungi but also involved in many inflammatory conditions. When neutrophils encounter such threats, they undergo a process called NETosis, wherein they expel their chromatin, a complex of DNA and proteins, to form web-like structures called neutrophil extracellular traps (NETs). These NETs ensnare and neutralize pathogens, preventing their spread. NETosis is initiated by the activation of certain receptors on neutrophils, triggering a cascade of biochemical events that lead to chromatin decondensation and release. Additionally, NETosis can contribute to the pathogenesis of various diseases, including autoimmune disorders and inflammatory conditions, due to excessive or dysregulated NET formation. Understanding the mechanisms underlying NETosis has significant implications for developing therapies targeting immune responses and combating infectious diseases. Overall, NETosis represents a fascinating aspect of the immune system's arsenal highlighting the complexity and versatility of neutrophil-mediated defense mechanisms. However, it is not well understood how chemical and physical are integrated by the cells and what the driving forces are for the dramatic changes in the cells. This project aimed to answer these questions and came to the following insights: 1. We investigated NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process. 2. We investigated the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. Furthermore, we provided experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research. 3. We studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Young’s modulus E) within the physiologically relevant range from 1 to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion. 4. We studied how neutrophils take up nanomaterials and programmed them to release this cargo after a certain time period via NETosis. We hijack this process and demonstrate how neutrophils transport and release functional nanomaterials. 5. Finally, we demonstrated expansion super resolution microscopy of neutrophils to gain a better resolution and study processes related to nuclear structure.

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