Lack of eosinophil extracellular trap formation due to failure of plasma membrane breakdown in the absence of elastase

Abstract

Activated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell's DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.

Graphical abstract

Figure 1.

Absence of ET formation by eosinophils. Eosinophils and neutrophils were preincubated with 10 μM DPI or 0.1% DMSO as vehicle control where indicated. To induce ET formation, eosinophils and neutrophils were stimulated with (A-C,E) PMA (100 ng/mL), (D) MSU crystals (200 μg/mL), or opsonized C. albicans for 4 hours at 37°C. (A-B,D) DNA release of eosinophils and neutrophils was measured in real time by Sytox Green Nucleic Acid Stain and (C) the area under the curve was calculated (mean + standard deviation , n = 6-7 for PMA vehicle condition, n = 6-7 for MSU crystals condition, n = 4-5 for C. albicans condition, n = 3-4 for DPI conditions). (E) ETs were visualized by staining for NE (green, 488), DNA (Hoechst, blue, 405), and myeloperoxidase (MPO, magenta, 633). Images were acquired using a Leica SP8 confocal microscope, original magnification ×400. Scale bar = 20 μm. Results are representative of 3 independent experiments. DMSO, dimethyl sulfoxide.

Figure 2.

Failure of plasma membrane breakdown and extracellular DNA release by eosinophils. To induce ET formation, eosinophils and neutrophils were stimulated with (A-B, D-G) PMA (100 ng/mL), (C) MSU crystals (200 μg/mL), or opsonized C. albicans for 4 hours (unless otherwise indicated) at 37°C. (A-C) Images were acquired using a Leica SP8 confocal microscope. (A) NETosis dynamics upon PMA stimulation were assessed by live cell imaging using the permeable DNA dye DRAQ5 (cyan) and the impermeable DNA dye Sytox Green (magenta). Asterisks indicate an eosinophil. Time is displayed in hours, scale bar = 20 μm. Refer to supplemental Video 1. Results are representative of 3 independent experiments. (B) Dynamics of eosinophils upon PMA stimulation. Scale bar = 20 μm. Refer to supplemental Video 1. Results are representative of 2 independent experiments. (C) Dynamics of eosinophils and neutrophils upon exposure to MSU crystals (upper panel) or C. albicans (lower panel). Scale bar = 100 μm. Refer to supplemental Videos 2-3. Results are representative of 2 independent experiments. (D-G) Images were acquired using the ZEISS LSM 980 Airyscan 2 confocal microscope, original magnification ×400. (D-E) Dynamics of eosinophils upon PMA stimulation were assessed by live cell imaging using the plasma membrane dye CellMask Orange (orange), permeable DNA dye DRAQ5 (magenta), and the impermeable DNA dye Sytox Green (green). Time is displayed in hours, scale bar = 20 μm. Refer to supplemental Video 4. (E) Stills of supplemental Video 5, with a 360º zoom around an eosinophil illustrating that the plasma membrane of the eosinophils does not breakdown and DNA remains inside of the cell. Scale bar = 5 μm. (F-G) NETosis dynamics upon PMA stimulation. Scale bar = 20 μm. Refer to supplemental Video 6. (D) Stills of supplemental Video 7, with a 360º zoom around a neutrophil illustrating that the plasma membrane of the neutrophil breaks down, resulting in extracellular release of DNA and thus NETosis. Scale bar = 5 μm.

Figure 3.

Impaired NET formation by neutrophils derived from a patient with a mutation in ELANE. (A) Protease release and proteolytic activity of neutrophils and eosinophils upon stimulation with CytoB/fMLF (5 μg/mL; 1 μM) and CytoB/PMA (5 μg/mL; 100 ng/mL) were determined by DQ-green BSA. A Triton X-100 lysate was used as 100% (mean + standard deviation, n = 3). (B) Representative cytospins of isolated granulocyte fractions from controls and patient (original magnification ×400; May-Grünwald/Giemsa stain). These cell suspensions were used in panel F. ∗eosinophil, ^¥neutrophil, ^xlymphocyte. (C) Protease release and proteolytic activity by control (n = 5) and ELANE patient (n = 3) granulocytes (mean + standard deviation). (D-F) To induce ET formation, cells were stimulated with PMA (100 ng/mL) for 4 hours, and (D) DNA release was measured in real-time by Sytox Green Nucleic Acid Stain (mean + standard deviation, n = 4 for controls, n = 3 for ELANE patient) and (E) ETs were visualized by staining for neutrophil elastase (NE, green, 488), DNA (Hoechst, blue, 405), and myeloperoxidase (MPO, magenta, 633). Images were acquired using a Leica SP8 confocal microscope, original magnification ×400. Scale bar = 50 μm. Results are representative of 3 independent experiments. (F) NETosis dynamics upon PMA stimulation were assessed by live cell imaging using the permeable DNA dye DRAQ5 (cyan) and the impermeable DNA dye Sytox Green (magenta). Asterisk indicates an eosinophil. Time is displayed in hours, scale bar = 20 μm. Refer to supplemental Video 8. Results are representative of 2 independent experiments. (A) Statistics were performed by unpaired t test; ∗∗P < .01; ∗∗∗P < .001.