Impaired formation of neutrophil extracellular traps in patients with MDS

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.

Graphical abstract

Figure 1.

Diagram of ROS production, enzyme inhibitors, and fluorochromes used to measure ROS.

Figure 2.

HPF signal is subtracted from APF signal to determine hypochloride production by MPO.

Figure 3.

Visual assessment of NET formation. NET formation was assessed microscopically in unstimulated neutrophils and after 30 minutes and 180 minutes of stimulation with PMA, in MDS patients and age-adjusted normal controls (representative examples).

Figure 4.

Automated microscopic assessment of NET formation. MFIs were determined for DAPI (detection of nuclear swelling), PE (detection of histones), and fluorescein isothiocyanate (FITC) (detection of neutrophil elastase). Comparison of MFI pre and poststimulation with PMA for 180 minutes between age-adjusted controls (black columns) and MDS samples (gray columns). *Statistically significant difference.

Figure 5.

Proportion of NETs pre and poststimulation with PMA in age-adjusted controls (black columns) and MDS samples (gray columns). *P = .02.

Figure 6.

Flow cytometric assessment of APF and HPF fluorescence intensity for calculating HOCl production in neutrophils. Granulocytes were gated according to their forward and side scatter (dot blots). The gated cells were then presented according to side scatter and APF/HPF fluorescence intensity (fluorescein isothiocyanate [FITC]). (A) APF stimulation with H₂O₂ plus inhibition of MPO using ABAH/AP. (B) HPF stimulation with H₂O₂ plus inhibition of MPO using ABAH/AP. (C) Histograms representing the findings in (A). (D) Histograms representing the findings in (B). (A-D) A representative example of age-related controls and MDS, respectively. (E) Upon stimulation with H₂O₂, ΔAPF-ΔHPF is a measure of HOCl production. The difference in HOCl production between age-adjusted controls (black columns) and MDS samples (gray columns) only reached statistical significance when MPO activity was diminished by inhibitors. *P = .01.