Start of funding 01.07.2024

The impact of immune cell membrane fluidity on membrane rupture during neutrophil extracellular trap release

Prof. Dr. Christoph Westerhausen
University of Augsburg
Institute of Theoretical Medicine

Prof. Dr. Hawa Racine Thiam
Stanford University
Department of Bioengineering



NETosis is a process by which certain immune cells, particularly neutrophils, release extracellular traps (NETs) composed of DNA, histones, antimicrobial and cytotoxic proteins to capture and neutralize pathogens. This process plays a crucial role in the immune system's defense against infections but can also damage the host and correlate with the worsening of chronic inflammatory diseases. While the pathophysiological relevance and the molecular mechanism of NETosis are accumulating, we have a limited understanding of the biophysical mechanism driving NETosis. For instance, for neutrophils to release NETs to the extracellular environment, the DNA must breach the plasma membrane. The mechanism by which this is accomplished is currently not known.

Our project therefore aims to investigate the biophysical properties of plasma membranes during NETosis formation. We will leverage the Westerhausen lab’s expertise in measuring membrane fluidity and the Thiam lab’s expertise in measuring membrane tension to investigate how membrane fluidity impacts membrane tension and the downstream cellular process of NETosis. Ultimately, our project aims to comprehensively characterize NETosis and its influence on plasma membrane biophysics, shedding light on the underlying mechanisms of immune response.

Final report:
Activities
Our project aims to elucidate the dynamics of plasma membrane biophysical properties in neutrophils, specifically lipid order and membrane tension, and to determine how these properties influence the downstream cellular process of NETosis. By combining the Westerhausen lab’s expertise in measuring membrane fluidity with the Thiam lab’s expertise in neutrophil cell culture and membrane tension measurements, the project seeks to establish a much-needed understanding of how immune cells regulate the physico-chemical properties of their membrane to execute host defense functions while maintaining host homeostasis. To achieve this goal, Paul Täufer, a master’s student in the Westerhausen lab, spent six months (October 2024 – March 2025) as a visiting researcher in the Thiam lab at Stanford University. During this research stay, he characterized changes in membrane tension and lipid order in neutrophils during NETosis and investigated the functional importance of regulating these membrane properties for NETosis efficiency. Upon returning to Augsburg, Paul Täufer transferred the acquired expertise in neutrophil culture and experimental techniques to the Westerhausen lab and is currently continuing lipid order characterization experiments on neutrophils.

Outcomes
We found that lipid order in the plasma membrane decreases consistently, while membrane tension increases, as neutrophils progress toward NETosis. To assess the functional relevance of these dynamics, we subsequently perturbed membrane biophysical properties by modulating cellular cholesterol levels. Increasing cholesterol levels impaired NETosis, whereas cholesterol depletion enhanced NETosis efficiency, indicating that proper regulation of cellular cholesterol levels, and the associated control of plasma membrane lipid order and tension, is critical for efficient NET formation. These findings provide a biophysical framework that may guide future strategies to modulate NETosis, with potential implications for improving human health. The project outcomes were presented by Paul Täufer at the 15th Congress of the European Biophysical Societies’ Association (EBSA) in Rome1. Furthermore, the results form the basis of his master’s thesis, and a manuscript2 summarizing the project findings is currently in preparation.

Outlook
Building on the results of this project, the collaboration between the Westerhausen lab and Thiam lab will be further strengthened through continued characterization of neutrophil plasma membrane properties in Augsburg. Furthermore, membrane lipid order and tension will be modulated using approaches beyond cholesterol perturbation to confirm that changes in NETosis efficiency are directly driven by altered membrane mechanical properties, and not by additional effects of cholesterol modulation on cellular signaling pathways. In the longer term, this work aims to identify effective strategies to tune membrane biophysical properties for therapeutic purposes, either to reduce excessive NETosis in autoimmune and inflammatory diseases or to enhance NET formation for improved host defense against pathogens.

1 S142 European Biophysics Journal (2025) 54 (Suppl 1):S1–S273.

2 P. Täufer, C. Westerhausen. H. R. Thiam, “Tight Regulation of Neutrophils Plasma Membrane Lipid Order and Tension is Required for Efficient Neutrophil Extracellular Trap Release”, in Vorbereitung, 2026.