Impaired formation of neutrophil extracellular traps in patients with MDS
Carolin Brings1, Julia Fröbel2, Patrick Cadeddu3
1Department of Vascular and Endovascular Surgery, Heinrich Heine University, Düsseldorf, Germany.
Abstract:
Neutrophil extracellular traps (NETs) are networks of extracellular fibers primarily composed of DNA and histone proteins, which bind pathogens. We investigated NET formation in 12 patients with myelodysplastic syndrome (MDS) and 15 age-adjusted normal controls after stimulation with phorbol-12-myristate-13-acetate (PMA). Histones and neutrophil elastase were visualized by immunostaining. Since NET formation is triggered by reactive oxygen species (ROS), mainly produced by reduced NADP-oxidase and myeloperoxidase (MPO), ROS were analyzed by flow cytometry using hydroethidine, 3'-(p-aminophenyl) fluorescein, and 3'-(hydroxyphenyl) fluorescein. On fluorescence microscopy, PMA-stimulated MDS neutrophils generated fewer NETs than controls (stimulated increase from 17% to 67% vs 17% to 85%) (P = .02) and showed less cellular swelling (P = .04). The decrease in mean fluorescence intensity (MFI) of 4',6-diamidino-2-phenylindole, indicating chromatin decondensation, was significantly less in MDS neutrophils than controls (ΔMFI 3467 vs ΔMFI 4687, P = .03). In addition, the decrease in MFI for fluorescein isothiocyanate, indicating release of neutrophil elastase from cytoplasmic granules, was diminished in patients with MDS (P = .00002). On flow cytometry, less cell swelling after PMA (P = .02) and a smaller decrease in granularity after H2O2 stimulation (P = .002) were confirmed. PMA-stimulated ROS production and oxidative burst activity did not reveal significant differences between MDS and controls. However, inhibition of MPO activity was more easily achieved in patients with MDS (P = .01), corroborating the notion of a partial MPO defect. We conclude that NET formation is significantly impaired in MDS neutrophils. Although we found abnormalities of MPO-dependent generation of hypochloride, impaired ROS production may not be the only cause of deficient NETosis in MDS.
Insights
Neutrophil extracellular traps (NETs) formation is impaired in myelodysplastic syndrome (MDS) patients. MDS neutrophils showed reduced NETs, less cellular swelling, and diminished elastase release compared to controls, indicating a defect in NETosis.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Neutrophil extracellular traps (NETs) are crucial in pathogen binding and innate immunity.
- Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia.
- NET formation involves decondensation of chromatin and release of antimicrobial proteins, a process influenced by reactive oxygen species (ROS).
Purpose of the Study:
- To investigate and compare NET formation in neutrophils from patients with myelodysplastic syndrome (MDS) and healthy controls.
- To analyze the role of reactive oxygen species (ROS) and myeloperoxidase (MPO) activity in NET deficiency in MDS.
- To evaluate cellular changes, including chromatin decondensation and granule release, during NETosis in MDS.
Main Methods:
- Neutrophil extracellular trap (NET) formation was induced using phorbol-12-myristate-13-acetate (PMA) in neutrophils from 12 MDS patients and 15 age-matched controls.
- Immunostaining was used to visualize histones and neutrophil elastase.
- Reactive oxygen species (ROS) production was assessed by flow cytometry using specific fluorescent probes.
- Cellular swelling and degranulation were quantified using flow cytometry and fluorescence microscopy.
Main Results:
- PMA-stimulated neutrophils from MDS patients generated significantly fewer NETs compared to controls (67% vs 85%).
- MDS neutrophils exhibited reduced cellular swelling and diminished release of neutrophil elastase.
- While ROS production and oxidative burst activity showed no significant differences, MPO activity inhibition was easier in MDS patients, suggesting a partial MPO defect.
Conclusions:
- NET formation is significantly impaired in neutrophils from patients with myelodysplastic syndrome.
- Deficient NETosis in MDS is associated with abnormalities in chromatin decondensation and elastase release.
- A partial MPO defect may contribute to impaired NET formation in MDS, but impaired ROS production is not the sole cause.
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