Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α
Abstract
:1. Introduction
2. Results
2.1. Establishment of Hypoxia Conditions and HIF-1α-Deficient Neutrophils to Study NET Formation
2.2. NET Formation via Cholesterol Depletion Is Independent of HIF-1α Expression in Murine Neutrophils and Independent of Oxygen
2.3. NET Formation via Cholesterol Depletion Is Oxygen Independent in Human Neutrophils
2.4. Statins Induce NET Formation in Human Neutrophils in an Oxygen Independent Manner
2.5. MβCD Leads to Strongly Reduced Cellular Cholesterol Levels in Murine Bone Marrow-Derived as Well as Human Blood-Derived Neutrophils after 3 h, Independent of Oxygen Level
3. Discussion
4. Materials and Methods
4.1. Transgenic Mice Breeding and Genotyping
4.2. Isolation of Murine Neutrophils from Bone Marrow
4.3. Purity Analysis via Flow Cytometry
4.4. Neutrophil Isolation from Human Blood
4.5. Neutrophil Stimulation and Incubation under Normoxia and Hypoxia
4.6. Immune Fluorescence Staining and Confocal Laser Scanning Microscopy
4.7. Scanning Electron Microscopy
4.8. NET Analysis
4.9. RNA Isolation and RT-qPCR
4.10. Oxygen Measurement during In Vitro Experiments
4.11. Neutrophil Stimulation under Hypoxia and Normoxia for Lipid Isolation and HPLC
4.12. Statistical Analysis
4.13. Ethical Approval
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
References
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Henneck, T.; Mergani, A.; Clever, S.; Seidler, A.E.; Brogden, G.; Runft, S.; Baumgärtner, W.; Branitzki-Heinemann, K.; von Köckritz-Blickwede, M. Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α. Int. J. Mol. Sci. 2022, 23, 3195. https://doi.org/10.3390/ijms23063195
Henneck T, Mergani A, Clever S, Seidler AE, Brogden G, Runft S, Baumgärtner W, Branitzki-Heinemann K, von Köckritz-Blickwede M. Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α. International Journal of Molecular Sciences. 2022; 23(6):3195. https://doi.org/10.3390/ijms23063195
Chicago/Turabian StyleHenneck, Timo, AhmedElmontaser Mergani, Sabrina Clever, Anna E. Seidler, Graham Brogden, Sandra Runft, Wolfgang Baumgärtner, Katja Branitzki-Heinemann, and Maren von Köckritz-Blickwede. 2022. "Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α" International Journal of Molecular Sciences 23, no. 6: 3195. https://doi.org/10.3390/ijms23063195