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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: J Allergy Clin Immunol. 2015 Jun 9;137(1):258–267. doi: 10.1016/j.jaci.2015.04.041

Eosinophil ETosis-derived DNA traps: their presence in secretions and their functional attributes

Shigeharu Ueki a,b, Yasunori Konno a, Masahide Takeda a, Yuki Moritoki a, Makoto Hirokawa a, Yoshinori Matsuwaki c, Kohei Honda d, Nobuo Ohta e, Shiori Yamamoto f, Yuri Takagi f, Atsushi Wada f, Peter F Weller b
PMCID: PMC4674385  NIHMSID: NIHMS690041  PMID: 26070883

Abstract

Background

Activated human eosinophils, as well as neutrophils, can release extracellular chromatin to form DNA traps through cytolytic extracellular trap cell death (ETosis). Although formations of neutrophil DNA traps are recognized in various inflammatory conditions, neither the presence of ETosis-derived eosinophil DNA traps in human allergic diseases nor the characteristics of these DNA traps have been studied.

Objective

We investigated the presence of ETosis-derived DNA traps in eosinophil-rich sinus and ear secretions and the functional attributes of ETosis DNA traps.

Methods

Eosinophil-rich secretions obtained from patients with eosinophilic chronic rhinosinusitis (ECRS) and eosinophilic otitis media (EOM) were studied microscopically. In vitro studies of ETosis and DNA trap formation used blood-derived eosinophils and neutrophils, and binding capacities of DNA traps used labeled bacteria and fluorescent microbeads. Stabilities of DNA traps were evaluated by fluorescence microscopy.

Results

Abundant nuclear histone H1-bearing DNA traps had formed in vivo in the eosinophilic secretions and contributed to their increased viscosity. In vitro, following brief shear flow, eosinophil ETosis-elicited DNA traps assembled to form stable aggregates. Eosinophil DNA traps entrapped bacteria and fungi and by hydrophobic interactions microbeads. In comparison with neutrophil-derived DNA traps, eosinophil DNA traps ultrastructurally exhibited thicker fibers with globular structures and were less susceptible to leukocyte-derived proteolytic degradation, likely due to the lesser protease activities of eosinophils.

Conclusions

In human allergic diseases, the local cytolysis of eosinophils not only releases free eosinophil granules but also generates nuclear-derived DNA traps that are major extracellular structural components within eosinophil-rich secretions.

Keywords: eosinophilic chronic rhinosinusitis, eosinophils, ETosis, extracellular DNA traps, neutrophils

Introduction

A novel form of rapid cell death, namely extracellular trap cell death (also called ETosis1), was first recognized in neutrophils.2 In contrast to other known cell death types, including apoptosis and necrosis, ETosis is characterized by the striking final morphology: release of web-like chromatin structures (DNA traps) through breakdown of nuclear and plasma membranes. We recently reported that eosinophil ETosis (EETosis) mediates eosinophil cytolysis that is well-recognized in tissues in association with diverse eosinophil-associated diseases.3

Similar to neutrophil ETosis (NETosis), EETosis is a rapid cell death and actively mediated through production of reactive oxygen species (ROS) by NADPH-oxidase. In eosinophils stimuli elicited loss of the bi-lobular form of the eosinophil nucleus followed by nuclear envelope disintegration with nuclear chromatin spilling into the cytoplasm. Finally, the eosinophil plasma membrane ruptures allowing extrusion of the nuclear DNA traps as well as the release of intact eosinophil granules. EETosis has several differences relative to NETosis. In the process of NETosis, neutrophil granule membranes are lost within the cell and granule proteins are mixed intracellularly with nuclear chromatin, thereby binding granule proteins to nuclear DNA when it is released as DNA traps.4,5 In contrast, eosinophil granules are released as intact structures and eosinophil granule proteins rather remain within granules that themselves could bind to DNA traps.3 Scanning electron microscopy revealed that neutrophil DNA traps consist of 5–10 nm smooth stretches (composed of stacked cylindrical nucleosome) and 25–50 nm globular domains,2,6 although eosinophil DNA traps consisted mostly of fibers with diameters of 25–35 nm.3

Neutrophil DNA traps, containing antibacterial proteins such as histones and granule enzymes (e.g., elastase and myeloperoxidase), are considered to play a role in innate immunity by trapping various microorganisms.2, 7, 8 On the other hand, neutrophil DNA traps could be pathogenic in themselves when they are produced in excess.9,10 For instance, neutrophil DNA traps provide a scaffold for cell localization including red blood cells and platelet aggregation and serve to promote coagulation and thrombosis.11 With their sticky nature, neutrophil DNA traps contribute to sputum viscosity and pulmonary dysfunction in cystic fibrosis patients; therefore, inhalation of DNase is currently used to degrade DNA aggregates.10,12

To date, neutrophil DNA traps have been shown to be present in various infectious diseases and pathological conditions. However, it is still unknown whether EETosis-mediated DNA traps are found in vivo in human allergic diseases. Here, we investigated the presence of eosinophilic DNA traps in two human diseases that specifically are noted by predominantly eosinophil rich exudative secretions. To clarify the characteristics of EETosis-derived DNA traps that are important for understanding their functions following eosinophil cytolysis, the trapping capacities and differences relative to neutrophils were also investigated in this study.

Materials and Methods

Sample collection and cell preparations

Written informed consent was obtained from donors under Institutional Review Board (IRB)-approved protocols. Sinus specimens were obtained from three surgical patients with eosinophilic chronic rhinosinusitis (ECRS) according to the diagnostic criteria.13 We studied this form of rhinosinusitis, prevalent in Japan, because its exudative mucin contains mostly eosinophils (>96%) with negligible neutrophils amongst nucleated cells. In addition, we studied ear secretions from patients with eosinophilic otitis media (EOM).14 For in vitro studies eosinophils were purified from normal donor blood by negative selection as previously described.15 Briefly, venous blood was collected into a 6% dextran saline solution, and red blood cells (RBCs) were allowed to sediment. Buffy coat was centrifuged over Ficoll-Paque (GE Healthcare, Pittsburgh, PA) to separate granulocyte pellets. Eosinophils were isolated by incubation with a depletion antibody cocktail (StemSep™, StemCell Technologies, Vancouver, BC, Canada) followed by passage over magnetized columns (Miltenyi Biotec, Auburn, CA). Purity of isolated eosinophils was >98% of nucleated cells and viability >99%. Neutrophils (>90% neutrophils, viability >98%) were obtained from buffy coats followed by hypotonic lysis of RBCs. In some experiments, eosinophils were isolated by a MACS™ system (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described.16,17

Microscopic study for eosinophilic mucin

Eosinophilic mucin was fixed in 10% formalin and embedded in paraffin. For histone H1 staining, the deparaffinized sample slides were incubated with 3% bovine serum albumin (BSA) and 1% saponin containing PBS for 30 min. The slides were then incubated with primary mouse anti-human nuclear histone H1 mAb (AE-4, 2 μg/ml, Abcam, 3 h, at RT). Anti-human neutrophil elastase (G-2, 1:100, Santa Cruz, 1 h, at RT) was used to study the presence of neutrophils. Alexa-488 conjugated Ab (goat anti-mouse IgG; Invitrogen, Carlsbad, CA, 1:300, 30 min, at RT) was used for secondary incubation. Control Abs and Hoechst 33342 (Invitrogen) were used for each experiment. To study the 3D structure of DNA traps, fixed mucin was stained with SYTOX green (Invitrogen). Images were obtained with a Carl Zeiss LSM510 confocal microscope. Z-stack images were obtained using Zeiss LSM software.

Visualization of DNA trap aggregates in culture condition

Cells (1 × 105 cells in 100 μl) were stimulated with A23187 (Sigma-Aldrich, St. Louis, MO), phorbol 12-myristate 13-acetate (PMA; Sigma) or immobilized human IgG (Sigma) in 96-well flat-bottom tissue culture plates, in 0.1% BSA containing phenol red-free HEPES-buffered RPMI 1640. To induce necrotic cell death, eosinophils were heated at 60°C for 5 min and then cultured for 24 h at 37°C. Apoptosis was induced by culturing for 48 h in medium containing anti-Fas activating mAb (Millipore, Temecula, CA).3 SYTOX green (1:10000) was added to the medium. Without fixation, culture plates were shaken with an MS1 plate shaker (IKA Works, Wilmington, NC) for up to 120 sec at 1000 rpm. The plates were viewed by inverted microscopy (Eclipse TE300, Nikon, Tokyo, Japan) equipped with a Cooled Color Digital camera (Spot 1.3.0, Diagnostic Instruments, Sterling Heights, MI) in conjunction with image analysis software IP Lab (Scanalytics, Fairfax, VA). Hoescht 33342 (1:10000) and acridine orange (3 μM, Sigma) were used to stain DNA and lysosomal eosinophil granules, respectively. Stained aggregates were seeded on poly-L-lysine-coated glass slides, and images were obtained with a BX62 Olympus microscope. To study the stability of DNA traps, neutrophils and eosinophils were stimulated with 2 μM A23187 for 24 h in the presence of SYTOX to induce ETosis in the culture plate and shaken to visualize aggregates. 5% BSA-PBS, heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY), DNase I (20 U/ml, New England Biolabs), proteinase K (20 mg/ml), trypsin (0.25%, Life Technologies), elastase from human leukocytes (0.1 U/ml, Sigma), neutrophil lysate (1:5), and proteinase inhibitor cocktail (Complete Mini, Roche) were gently added to the medium and further incubated for indicated time points at 37°C. For preparation of neutrophil lysates, neutrophils were resuspended in PBS at 1 × 107 cells and subjected to three freeze-thaw and sonication cycles; and supernatants were obtained by centrifugation at 10000 × G for 10 min.

Microorganism trapping assay

For scanning electron microscopy (SEM), purified eosinophils (2 × 105 cells in 0.1% BSA RPMI) were stimulated with 2 μM A23187 for 3 h on glass slides. After EETosis induction, culture medium was removed, and 1 McFarland saline suspensions of Escherichia Coli (E. coli: ATCC 25922), Staphylococcus aureus (S. aureus: ATCC 29213), or Candida albicans (in-house) were gently added. Plates were shaken with an MS1 plate shaker for 5 min at 600 rpm. The samples were then prepared for SEM observation as described previously,18 with slight modifications. Briefly, samples were fixed with 1.25% glutaraldehyde −1% paraformaldehyde, dehydrated in a graded series of ethanols, and then reduced to t-butyl alcohol. They were then freeze-dried, decorated with osmium using an osmium coater (NEOC, Meiwafosis, Tokyo, Japan), and observed on a field emission scanning electron microscope, SU-8020 (Hitachi High-Technologies, Tokyo, Japan). For fluorescence microscopic study, purified live eosinophils were loaded with Hoechst 33342 (1:10000) for 10 min and washed twice. Hoechst-loaded cells (1 × 105 cells in 100 μl) were stimulated with indicated stimuli in 96-well flat-bottom tissue culture plates. After EETosis induction, Alexa 594-labeled E. coli and S. aureus (Invitrogen, 10 μg in 20 μl PBS/well) were gently added. The plate was shaken with an MS1 plate shaker for 30 sec at 1000 rpm and viewed by inverted microscopy.

Microbead trapping assay

Fluorescent 1 micron microspheres (Molecular Probes, Eugene, OR), both hydrophobic (sulfate microspheres (F8851)) and hydrophilic, positively charged (amine microspheres (F8765)), were used. For quantification of trapped beads, cells (1 × 105 cells in 100 μl) underwent induction of EETosis with A23187 for 24 h in RPMI medium. Twenty μl of DNase I (150 U/ml) or vehicle was added to each well and incubated for 90 min at 37°C. Medium was aspirated carefully and changed to the indicated buffer; HBSS (with calcium and magnesium), HBSS with 1% BSA, 10% FBS, 0.3% tween 20 (Sigma), 5 mM EDTA, or 600 mM NaCl. After adding the 20 μl of microsphere suspension (diluted to 1:10 with PBS), the plate was shaken with an MS1 plate shaker for 3 min at 800 rpm. Immediately after shaking, 10 μl of buffer was collected from each well and resuspended in 100 μl of PBS in a 96-well flat-bottom culture plate, followed by measurement of the fluorescence intensity of the beads using a GloMax-Multi detection system (Promega, Madison, WI). The fluorescence intensity of DNase-treated wells was considered as 100% microbead recovery.

Measurement of protease activity

Protease activity was measured with an Amplite universal protease activity assay kit (AAT Bioquest, Sunnyvale, CA) according to the manufacturer’s instruction. Fluorescence intensities were measured using a FLUOstar Omega (BMG Labtech, Offenburg, Germany).

Transmission electron microscopy (TEM)

Eosinophils and neutrophils were seeded on Aclar film (Nishin EM, Tokyo, Japan), stimulated with 10 ng/ml of PMA for 3 h, and immediately fixed in a mixture of freshly prepared aldehydes (1.25% glutaraldehyde and 1% paraformaldehyde) in 0.1 M cacodylate buffer. After incubation for 2 h in 0.1 M cacodylate buffer containing 1% OsO4, the specimens were dehydrated in an ethanol series, passed through propylene oxide, and embedded in epoxy resin. Ultrathin sections (80 nm) were collected on copper grids and stained for 20 min in 4% uranyl acetate and 0.5% lead citrate. The specimens were viewed with a Hitachi H-7650 transmission electron microscope at 100 KV.

Statistical Analysis

Results were expressed as mean ± SD. Differences between groups were evaluated using two-tailed Student’s t test, with the level of statistical significance taken as p<0.05.

Results

Presence of EETosis-derived extracellular DNA traps in eosinophilic exudative secretions

Neutrophil DNA traps that consist of filamentous chromatin structures are found in a variety of secretions such as abscesses and sputum and contribute to their viscosity. ECRS is characterized by massive infiltration of eosinophils (within negligible potentially confounding neutrophils) in tissues and the sinus cavity, which is filled with highly viscous fluid called “eosinophilic mucin”.13,19,20 We therefore searched for EETosis cells and DNA traps in eosinophilic mucin from patients with ERCS. Present in ECRS eosinophilic mucin, in addition to intact eosinophils, were significant numbers of eosinophils that showed defining nuclear characteristics of EETosis: decondensated nuclei, loss of lobulation, and/or disrupted nuclei. Clusters of free intact eosinophil granules were found near EETosis cells (Fig. 1A). To ascertain the presence of nuclear-derived genomic DNA, we stained the eosinophilic mucin with a DNA dye and an anti-histone H1Ab and an isotype-matched control Ab. We observed co-localizations of DNA and histone H1 showing fibrous structures with morphological DNA trap characteristics (Fig. 1B). Staining of ERCS mucin failed to detect neutrophil elastase (not shown). Confocal Z-stacks and 3D reconstruction for SYTOX DNA staining showed the presence of abundant DNA traps that spread and aggregated (Fig. 1C). Furthermore, DNase I treatment of the sinus secretion significantly inhibited the thread-forming ability (Supplemental video 1, 2). Similarly, DNA traps were observed in ear secretions from patients with eosinophilic otitis media (EOM) 14 (Fig. S1). Control isotype Ab for the anti-histone H1 Ab yielded no staining in ERCS (not shown) and EOM (Fig. S1). These results indicated that EETosis-derived eosinophil DNA traps are major components of eosinophil-rich exudative secretions in human “allergic” diseases and contribute to their viscosity.

Figure 1. Presence of abundant nuclear DNA traps and free granules in eosinophilic chronic rhinosinusitis (ECRS) sinus exudative secretions.

Figure 1

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(A) Infiltrated eosinophils and their free granules are abundantly present in the surgically obtained specimen from the ethmoidal sinus of a patient with eosinophilic chronic rhinosinusitis (ECRS). The mucin contains massive infiltration of eosinophils (>96 % of nucleated cells) with negligible numbers of other cells including neutrophils. Light microscopic images were obtained from hematoxylin and eosin stained sections of eosinophilic mucin (left panel, x20 objective). Arrows show Charcot-Leyden crystals, a characteristic of eosinophil infiltration. Right panel is the boxed area seen at higher magnification (x100 objective). Arrowheads show lytic eosinophils with decondensated nuclei and some are showing a filamentous structure. (B) Section of eosinophilic mucin was stained for linker-histone H1 (green) and DNA (Hoechst 33342; blue) and analyzed using a laser scanning confocal microscope. The scale bar shows 20 μm. (C) Pseudocolor representation of DNA traps evaluated throughout the depths of eosinophilic mucin. Fixed eosinophilic mucin was stained for DNA using SYTOX green. Depth’s level serial Z-stack images were pseudocolored according to the indicated color depth scale and projected to 3D using Zeiss LSM software. The white scale bar indicates 20 μm for XY axis. Experiments were repeated with samples from three ECRS patients with similar results.

Detection of EETosis-mediated DNA traps in culture conditions

To investigate the characteristics of EETosis-mediated DNA traps in vitro, we next aimed to mimic the DNA trap aggregate using purified human eosinophils. EETosis was induced by calcium ionophore, PMA, or immobilized IgG in SYTOX (cell-impermeable DNA-specific dye) containing medium and studied with fluorescence microscopy. Consistent with our previous report,3 EETosis cells demonstrated SYTOX-positive nuclei and loss of nuclear lobular formation. Filamentous DNA traps were released around the originating cells and difficult to visualize in static culture conditions (Fig. S2A). We developed a simple method to visualize extracellular DNA traps by inducing larger DNA aggregates with brief experimental shear flow (Fig. 2A, S2B). In this system, EETosis eosinophils remained adherent to the culture plate and DNA traps released from EETosis cells formed large aggregates (Fig. 2Ai, ii). The aggregates consisted of accumulated DNA traps and cell debris, including acridine orange-stained, approximately 1 μm-size structures and their clusters (Fig. 2B, arrowheads), indicative of intact cell-free eosinophil granules. To ascertain the difference among necrosis, apoptosis, and EETosis, we induced necrosis and apoptosis by brief heating and anti-Fas Abs, respectively. DNA trap aggregates were never observed in necrotic (Fig. 2Aiii) and apoptotic cells (Fig. 2Aiv). Using the extracellular histone staining that can recognize EETosis cells,3, we confirmed the DNA traps retained histone H1 (Fig. S3).

Figure 2. Detection of EETosis-mediated DNA trap aggregation in culture condition.

Figure 2

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(A) Purified eosinophils were stimulated with A23187 (2 μM) for 24 h in SYTOX (cell-impermeable DNA-specific dye)-containing medium and studied with inverted fluorescence microscopy (x4 objective). With brief shear flow stress, DNA traps were clearly visualized and formed aggregates in the middle of the culture plate (i, arrow). Stretched DNA traps emergent from adherent dead cells aggregated to form larger bundles (ii). Neither heat-induced necrotic cells (iii) nor apoptotic cells induced by anti-Fas-activating mAb (iv) produced DNA traps and formed aggregates. Small dots are nuclei of dead cells. Scale bars show 200 μm (i, iii, iv) and 100 μm (ii). Data are representative of >3 experiments from independent donors with similar results. (B) Microscopic image of shear flow-induced DNA trap aggregates (left: phase contrast image, right: fluorescence image, scale bar 10 μm). The aggregates consisted of DNA (blue: Hoechst 33342) and cell debris including acridine orange (AO)-stained intact free granules (red). Arrowheads designate clusters of free eosinophil granules.

Although EETosis is a rapid cell death and develops within 30–120 min of stimulation, we incubated eosinophils for a longer time period to determine the nuclear shape change as a fate of cell death. After 24 h of stimulation, approximately 95% of cells underwent cell death (SYTOX-positive) in our settings. In the presence of an NADPH oxidase inhibitor, diphenyleneiodium chloride (DPI), or an extracellular calcium chelator, EDTA, there was no significant release of extracellular DNA (Fig. S2A) and no formation of the DNA aggregates (Fig. S2B). In contrast, a pan-caspase inhibitor did not inhibit the nuclear decondensation and aggregate formation (Fig. S2A, B). These results confirmed that EETosis is dependent on reactive oxygen production and calcium, but independent of caspase-mediated processes.

EETosis-mediated DNA traps entrap microorganisms by passive contact

Neutrophil DNA traps are known to entrap microorganisms. To show the association between DNA traps and Gram-positive and negative bacteria, or fungi, EETosis-induced eosinophils were incubated with S. aureus, E. coli, Candida albicans or Aspergillus spp. As shown in Fig. 3A, these microorganisms (Aspergillus spp.; data not shown) were captured by filamentous DNA structures. To confirm the interaction between DNA traps and bacteria, we stimulated DNA dye-loaded eosinophils with different stimuli for 24 h to induce EETosis. Thereafter, Alexa Fluor 594-labeled S. aureus or E. coli was added to the culture medium and incubated under shear flow conditions. As shown in Fig. 3B, large DNA-bacteria aggregates were observed after 30 sec of shaking with EETosis cells but not in nonstimulated cells. No significant differences were observed in terms of the EETosis induced with different stimuli or among microbial species. These results indicate that EETosis-mediated DNA traps are able to ensnare microorganisms by passive contact.

Figure 3. Eosinophil extracellular DNA traps are able to capture microorganisms.

Figure 3

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(A) Microorganisms trapped by eosinophil DNA traps were observed by scanning electron microscope. (B) DNA dye (Hoechst 33342: blue)-loaded eosinophils were stimulated with 2 μM A23187, 10 ng/ml PMA, or immobilized IgG (1mg/ml) for 24 h to induce EETosis. Thereafter, inactivated bacteria (Alexa Fluor 594-labeled S. aureus or E. coli) were added to the culture medium, and DNA aggregates were induced with brief shear flow. Control, non-stimulated control cells did not produce DNA traps. Fluorescence-labeled bacteria not bound to DNA were out of focus. Extracellular DNA released from EETosis cells bound abundant bacteria. Data are representative of >3 experiments from independent donors with similar results. Scale bar: 100 μm.

Eosinophil extracellular DNA traps provide adhesive surfaces mediated by non-electrostatic forces

Although a charge-mediated binding has been proposed for the trapping capacity of DNA structures,9 the detailed mechanisms are poorly understood. To study the mechanism of the adhesive property of eosinophil-derived DNA traps, we utilized two different 1 μm-size fluorescent microbeads; hydrophobic sulfate-modified beads and hydrophilic, positively charged amine-modified beads (Fig. 4A). Notably, sulfate beads were preferentially bound to DNA traps to form aggregates with brief shear flow (Fig. 4B). We quantified the trapping capacity by measuring the fluorescence intensity of unbound microbeads (i.e. those not entrapped bound to eosinophil DNA traps) in comparison to DNase-treated EETosis cells (Fig. 4C). Approximately 30% of total sulfate beads were trapped to DNA in our system, although amine beads were not. To study the mechanism of binding, different medium conditions were tested. Sulfate bead trapping was inhibited by the presence of albumin (BSA) or serum (FBS), and by a non-ionic detergent (Tween 20) that inhibits hydrophobic interactions. Cationic ions have been reported to affect nucleosome structure and high salt condition inhibits electrostatic interactions,21,22 but neither EDTA nor high salt-containing medium affected the trapping of beads. These results suggest that non-electrostatic binding, especially by hydrophobic interactions, plays a predominant role in the binding capacity of eosinophil-derived nuclear DNA traps.

Figure 4. Eosinophil extracellular DNA entrap microbeads by non-electrostatic forces.

Figure 4

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(A) Two different-colored 1 μm-size fluorescent microbeads (sulfate-modified beads: red, amine-modified beads: green) were used to study the mechanism of interactions between DNA traps and particles. Eosinophils were stimulated with A23187 (2 μM, 24 h) to induce EETosis, followed by addition of sulfate and amine-modified beads. Aggregation was induced on a plate shaker and viewed by inverted microscopy. (B) Sulfate beads preferentially bind DNA traps to form large aggregates. Upper panel shows bright field image (x4 objective). Fluorescence image of boxed area is shown in lower panel (the scale bar shows 20 μm). (C) Quantification of bead trapping in different conditions. The assays measure unbound beads, so decreases are a measure of beads adherent to DNA traps. Medium conditions were; HBSS (control), 1% BSA, 10% FBS, 0.3% tween 20, 5 mM EDTA, or 600 mM NaCl. Immediately after shaking, medium was collected from each well and the fluorescence intensity of beads was measured. Fluorescence intensities of DNase treated wells were considered as 100% recovery. Data are expressed as mean ± SD (n=3, independent donors). *p<0.05 vs DNase treated well.

Lesser eosinophil protease activities contribute to the stability of eosinophil DNA traps

Human neutrophils undergo ETosis (NETosis) in response to calcium ionophore or PMA.23 To test whether NETosis can form similar aggregates in our system, experiments were conducted using purified neutrophils. NETosis-mediated DNA trap aggregates were elicited by A23187 or PMA (Fig. S4A). Neutrophil DNA traps formed larger aggregates compared to eosinophils. Detachment of scattered cells from culture plates was observed after experimental shear flow (Fig. S4B). Aggregated neutrophil DNA trap structures showed a white-yellow, gel-like appearance (Fig. S4C). These results indicated the different characteristics of eosinophil- and neutrophil-derived DNA traps.

In an attempt to compare the stability of NETosis- and EETosis-mediated DNA traps, the aggregates were incubated in culture conditions for longer periods. Once the aggregates were formed, EETosis-mediated DNA traps were very stable for days, keeping their shape for >7 days (data not shown). In contrast, neutrophil DNA traps showed spontaneous dissociation and were more susceptive to increased concentrations of albumin or FBS, or treatment with DNase, indicating their lesser stability (Fig. 5A).

Figure 5. Eosinophil DNA traps showed higher stability due to lesser leukocyte protease activity.

Figure 5

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(A) Eosinophil DNA traps formed stable aggregates. Neutrophil ETosis and eosinophil ETosis were induced by stimulation with A23187 in SYTOX-containing medium, followed by DNA trap aggregate formation. Then, indicated agents (PBS, 1% BSA, 10% FBS, 20 U/ml DNase I) were added to the culture medium and studied using fluorescence microscopy at indicated time points. Data are representative of >3 experiments from independent donors with similar results. (B) Eosinophil DNA traps were degraded by proteases. Indicated proteases were gently added to the culture medium and studied using fluorescence microscopy at indicated time points. Vehicle, proteinase K, trypsin, elastase, neutrophil lysate (Neu Lys), and proteinase inhibitors (PI) were added to the medium and further incubated for indicated time points at 37°C. Data are representative of >3 experiments from independent donors with similar results. (C) Neutrophil contains abundant proteases. Neutrophils and eosinophils were lysed and protease activity was measured by fluorescence plate reader for indicated time points. Data are expressed as mean ± SD (n=4, independent donors). (D) Analysis of ultrathin sections of DNA traps using TEM indicates thicker and more globular structures of eosinophil DNA traps compared to neutrophil DNA traps. The scale bar shows 200 nm.

Since neutrophil elastase has been reported to enhance DNA trap solubilizaton by cleaving histones,24 we hypothesized that lesser eosinophil protease activities might play a role in the stability of eosinophil DNA traps. Incubation with proteinase K, trypsin, neutrophil elastase, or neutrophil lysates did solubilize eosinophil DNA traps (Fig. 5B). Eosinophil lysates, however, did not induce the solubilization of eosinophil DNA traps (data not shown). The effect of neutrophil lysates was not observed in the presence of proteinase inhibitors (Fig. 5B). Indeed, neutrophils contained significantly greater proteinase activities than did eosinophils (Fig. 5C). TEM showed globular components of eosinophil DNA traps, suggesting the presence of intact chromatin (Fig. 5D). Taken together, eosinophil DNA traps are more stable than neutrophil DNA traps due, at least in part, to lesser exposures to endogenous leukocyte-derived proteinases that can cleave the protein nucleosome components of nuclear-derived DNA traps.

Discussion

Eosinophilic secretions in ECRS and EOM patients were chosen to study the in vivo presence of eosinophil-derived DNA traps in lesional sites. In line with a previous reports,20,14 these tenacious eosinophilic secretions displayed a highly viscous consistency. Within the secretions tightly clustered eosinophils often showed their nuclei to be smudged and elongated, sometimes forming collections of nuclear debris, and exhibiting cytolytic release of cell-free extracellular eosinophil granules. Here, we demonstrated that eosinophil DNA traps represent major extracellular structural components scaffolding the eosinophilic secretions and increasing their viscosity. Activated eosinophils after luminal entry can undergo EETosis to produce nuclear-derived DNA traps that contribute to the pathogenesis of eosinophilic diseases. Regulating excess EETosis could be a novel therapeutic target.

Blood-derived eosinophils were utilized to study the characteristics of EETosis-mediated DNA traps. The process of EETosis is mediated through ROS production and calcium signaling. ETosis releases nuclear contents as filamentous chromatin structures, and neither apoptosis nor necrosis induce DNA traps.3,4 We developed a simple method to visualize extracellular DNA traps by inducing formation of large DNA aggregates with brief experimental shear flow. The aggregates contained cell debris including intact granules, mimicking the in vivo clinical findings. Another advantage of our simple plate-based system is the assessment of the characteristics of ETosis-derived DNA traps without fixation that could possibly affect their nature.

Live eosinophils, in response to specific stimuli, can release mitochondrial DNA in a non-cytolytic catapult-like manner.25 However, generally small amounts of mitochondrial DNA (approximately 1% of the total DNA) are present in cells.26 As evidenced by the histone staining, the bulk of extracellular DNA traps in this study, found both in vivo and in vitro, was derived from the nucleus. In contrast to active projection of mitochondrial DNA, ETosis-mediated DNA release was a result of cell death; therefore, spreading DNA traps in the fluid phase was by a passive process. Released eosinophil nuclear DNA traps rapidly assembled together and ensnared small particles such as intact eosinophil granules and microorganisms. Hydrophobic interactions played a major role in the adhesive capacity and DNA trap folding to make stable aggregates. These characteristics of extracellular DNA traps are important for ensnarling microorganisms to prevent their dissemination, but excessive formation might be counterproductive, preventing proper clearance and increasing the viscosity of secretions, as observed in samples from ECRS and EOM patients.

Our study highlights the different characteristics between eosinophil and neutrophil DNA traps. In NETosis, the granule membranes disappear intracellularly, followed by translocations of neutrophil elastase and myeloperoxidase to the neutrophil nucleus.4,5 This intracellular degranulation process appears to be essential for binding of these proteins to nuclear chromatin before neutrophils rupture. In contrast, eosinophil granules are released as intact structures and eosinophil granule proteins remain largely within released granules.3 In neutrophil DNA traps, nuclear core histones were the most abundant proteins and accounted for 70% of all net-associated proteins.6 A long strand of DNA is wrapped around core histones to form a nucleosome structure like “beads on a string.”27 Neutrophil elastase promotes DNA traps’ chromatin decondensation in cystic fibrosis patient sputum by proteolytic processing of histones.24 Eosinophil DNA traps from clinical and experimental sources were uniformly stained using a monoclonal antibody against nuclear linker-histone H1, which plays a key role to form 30-nm chromatin fibers.28 Notably, the ultrastructural morphology of neutrophil DNA traps showed thinner and less globular fibers in contrast to the ultrastrucuture of eosinophil DNA traps. Taken together, we propose that eosinophil DNA traps are well-conserved with nucleosomes that are less susceptible to the limited proteolytic degradatory capacities of human eosinophils (Fig. 6).

Figure 6. ETosis-mediated eosinophil and neutrophil DNA traps.

Figure 6

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Proposed differential characteristics between eosinophil and neutrophil DNA traps. Both activated eosinophils and neutrophils undergo NADPH-oxidase-dependent ETosis to release DNA traps that can spread and provide adhesive surfaces. In contrast to granule lysis and release of abundant protease in neutrophils, eosinophils contain much less protease and granules are intact after ETosis. Eosinophils release globular chromatin fibers that contribute to the stability and viscosity of secretions.

Eosinophil DNA traps provide adhesive surfaces that can entrap intact eosinophil granules and microorganisms. In concert with our studies that demonstrated that cell-free eosinophil granules can continue to function as independent extracellular secretion-competent structures,3,15,29 our current findings shed light on the roles of residual eosinophil “cell debris,” including entrapped intact eosinophil granules, which have been overlooked both experimentally and clinically. Cytolytic release from eosinophils of nuclear DNA traps and free extracellular granules may exert long-lasting effects even after lysed eosinophils are no longer recognizable.

Supplementary Material

1. Figure S1. Presence of DNA traps in ear secretions from eosinophilic otitis media (EOM) patients.

The highly viscous ear secretions obtained from an EOM patient were placed on a slide glass, followed by fixation and permeabilization. Light microscopic images were obtained from Giemsa stained eosinophilic secretion (left panel, x20 objective). Note the massive eosinophil infiltration and presence of filamentous structures. The slides were then incubated with control isotype-matched Ab (middle panel) or primary mouse anti-human nuclear histone H1 mAb (green) (right panel), as described in Materials & Methods. DNA was stained with Hoechst 33342 (blue) and analyzed using a laser scanning confocal microscope. Images were obtained with a Carl Zeiss LSM710 confocal microscope. Experiments were repeated with samples from four EOM patients with similar results. The scale bars show 20 μm.

2. Figure S2. Nuclear decondensation and flow-induced DNA trap aggregation with various inhibitors and stimuli.

(A) Nuclear shape of dead cells was studied with fluorescence microscopy in static culture conditions. Purified eosinophils were stimulated with 2 μM A23187, 10 ng/ml PMA, or immobilized IgG (1 mg/ml) for 24 h in SYTOX green (cell-impermeable DNA-specific dye)-contained medium. A23187-, PMA-, and IgG-stimulated dead cells showed decondensed nuclei, although release of extracellular DNA was difficult to observe. In the presence of the NADPH oxidase inhibitor diphenyleneiodium chloride (DPI; 30 μM, Sigma) or the extracellular calcium chelator EDTA (5 mM), nuclear decondensation was inhibited. In the presence of DPI, A23187-stimulated dead cells showed loss of nuclear lobulation, although release of extracellular DNA was not significant. A pan-caspase inhibitor (Z-VAD (OMe)-I; 50 μM, EMD Chemicals, Gibbstown, NJ), added 20 min before stimulation, failed to inhibit the nuclear decondensation. Filamentous DNA traps were difficult to observe due to association with culture plate or dead cells. More than 90% of cells underwent cell death, showing SYTOX-positive nuclei in these conditions. The scale bars show 20 μm.

(B) Brief experimental shear flow induced aggregation of released DNA traps. Filamentous DNA emerged from plate-attached ETosis cells and assembled to form large aggregates. Culture plates were shaken with a plate shaker for up to 120 sec at 1000 rpm. Intact cells did not produce DNA traps in our experimental settings. Data are representative of three experiments from independent donors with similar results. The scale bars show 200 μm.

3. Figure S3. Extracellular histone H1 staining detected chromatin from lysed eosinophils.

Eosinophils were stimulated with 2 μM A23187, 10 ng/ml PMA, or immobilized IgG (1 mg/ml) for 24 h, followed by fixation without permeabilization. Intact cells with intact nuclei were single positive for fixed cell permeable PI (red) but not stained with histone H1 (green). Control and non-stimulated cells did not produce extracellular DNA traps. The DNA traps were uniformly stained with linker-histone H1 mAb. Each image is representative of at least two experiments with similar results. The scale bars show 20 μm.

4. Figure S4. Comparison of NETosis- and EETosis-mediated DNA traps.

(A) Purified eosinophils and neutrophils (1 × 105 cells/well in 96-well culture plate) were stimulated with A23187 or PMA for 24 h in SYTOX containing medium. Plates were shaken with a plate shaker for up to 120 sec at 1000 rpm and viewed with an Eclipse TE300 inverted microscope (x4 objective). Each image is representative of duplicates with similar results.

(B) Increased numbers of cells (5 × 106 cells/well in 12-well culture plate) were stimulated for 24 h with 2 μM A23187, and the plate was shaken with a plate shaker for 5 min. Macroscopic images (upper panels) indicate the neutrophils underwent NETosis form the large aggregates, although EETosis did not. Lower panels indicate the bright field images obtained with an Eclipse TE300 inverted microscope (x4 objective). Neutrophil DNA traps were detached from culture plate. Data are representative of >3 experiments from independent donors with similar results.

(C) Neutrophil aggregates were able to pick up with tweezers from the plate and showing sputum-like appearance with high viscosity and spinnability.

5. Supplemental video 1.

Nasal secretion obtained from ECRS patient was incubated overnight at 37°C with equal volume of calcium and magnesium containing HBSS. The secretion showed a high viscosity with significant thread-forming capacity under the effect of traction.

Download video file (735.2KB, mp4)
6. Supplemental video 2.

Nasal secretion obtained from same ECRS patient was incubated with DNase I (100 U/ml) in similar condition as that in supplemental video 1. DNase treatment decreased the thread-forming capacity.

Download video file (493.9KB, mp4)
7

Key messages.

  • Eosinophil ETosis-derived DNA traps are abundantly present in eosinophilic secretions and contribute to increasing their viscosity.

  • Eosinophil DNA traps provide the long-lasting adhesive surfaces for postmortem functions.

Acknowledgments

Funding:

This study was funded in part by Uehara Memorial Foundation and Grant-in-Aid for Scientific Research 13383320 (SU), 25462675 (NO), NIH R37-AI020241 (PFW), and R01-AI051645 (PFW).

We are grateful to Kristen Young, Noriko Tan, and Shinsuke Chida for their outstanding technical assistance. We also thank Mari Kono for critical reading of the manuscript.

Abbreviations

BSA

Bovine serum albumin

ECRS

Eosinophilic chronic rhinosinusitis

EETosis

Eosinophil extracellular trap cell death

ETosis

Extracellular trap cell death

EOM

Eosinophilic otitis media

FBS

Fetal bovine serum

NETosis

Neutrophil extracellular trap cell death

ROS

Reactive oxygen species

Footnotes

The authors declare no relevant conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1. Figure S1. Presence of DNA traps in ear secretions from eosinophilic otitis media (EOM) patients.

The highly viscous ear secretions obtained from an EOM patient were placed on a slide glass, followed by fixation and permeabilization. Light microscopic images were obtained from Giemsa stained eosinophilic secretion (left panel, x20 objective). Note the massive eosinophil infiltration and presence of filamentous structures. The slides were then incubated with control isotype-matched Ab (middle panel) or primary mouse anti-human nuclear histone H1 mAb (green) (right panel), as described in Materials & Methods. DNA was stained with Hoechst 33342 (blue) and analyzed using a laser scanning confocal microscope. Images were obtained with a Carl Zeiss LSM710 confocal microscope. Experiments were repeated with samples from four EOM patients with similar results. The scale bars show 20 μm.

NIHMS690041-supplement-1.tif (432.7KB, tif)
2. Figure S2. Nuclear decondensation and flow-induced DNA trap aggregation with various inhibitors and stimuli.

(A) Nuclear shape of dead cells was studied with fluorescence microscopy in static culture conditions. Purified eosinophils were stimulated with 2 μM A23187, 10 ng/ml PMA, or immobilized IgG (1 mg/ml) for 24 h in SYTOX green (cell-impermeable DNA-specific dye)-contained medium. A23187-, PMA-, and IgG-stimulated dead cells showed decondensed nuclei, although release of extracellular DNA was difficult to observe. In the presence of the NADPH oxidase inhibitor diphenyleneiodium chloride (DPI; 30 μM, Sigma) or the extracellular calcium chelator EDTA (5 mM), nuclear decondensation was inhibited. In the presence of DPI, A23187-stimulated dead cells showed loss of nuclear lobulation, although release of extracellular DNA was not significant. A pan-caspase inhibitor (Z-VAD (OMe)-I; 50 μM, EMD Chemicals, Gibbstown, NJ), added 20 min before stimulation, failed to inhibit the nuclear decondensation. Filamentous DNA traps were difficult to observe due to association with culture plate or dead cells. More than 90% of cells underwent cell death, showing SYTOX-positive nuclei in these conditions. The scale bars show 20 μm.

(B) Brief experimental shear flow induced aggregation of released DNA traps. Filamentous DNA emerged from plate-attached ETosis cells and assembled to form large aggregates. Culture plates were shaken with a plate shaker for up to 120 sec at 1000 rpm. Intact cells did not produce DNA traps in our experimental settings. Data are representative of three experiments from independent donors with similar results. The scale bars show 200 μm.

NIHMS690041-supplement-2.tif (950.1KB, tif)
3. Figure S3. Extracellular histone H1 staining detected chromatin from lysed eosinophils.

Eosinophils were stimulated with 2 μM A23187, 10 ng/ml PMA, or immobilized IgG (1 mg/ml) for 24 h, followed by fixation without permeabilization. Intact cells with intact nuclei were single positive for fixed cell permeable PI (red) but not stained with histone H1 (green). Control and non-stimulated cells did not produce extracellular DNA traps. The DNA traps were uniformly stained with linker-histone H1 mAb. Each image is representative of at least two experiments with similar results. The scale bars show 20 μm.

NIHMS690041-supplement-3.tif (780.8KB, tif)
4. Figure S4. Comparison of NETosis- and EETosis-mediated DNA traps.

(A) Purified eosinophils and neutrophils (1 × 105 cells/well in 96-well culture plate) were stimulated with A23187 or PMA for 24 h in SYTOX containing medium. Plates were shaken with a plate shaker for up to 120 sec at 1000 rpm and viewed with an Eclipse TE300 inverted microscope (x4 objective). Each image is representative of duplicates with similar results.

(B) Increased numbers of cells (5 × 106 cells/well in 12-well culture plate) were stimulated for 24 h with 2 μM A23187, and the plate was shaken with a plate shaker for 5 min. Macroscopic images (upper panels) indicate the neutrophils underwent NETosis form the large aggregates, although EETosis did not. Lower panels indicate the bright field images obtained with an Eclipse TE300 inverted microscope (x4 objective). Neutrophil DNA traps were detached from culture plate. Data are representative of >3 experiments from independent donors with similar results.

(C) Neutrophil aggregates were able to pick up with tweezers from the plate and showing sputum-like appearance with high viscosity and spinnability.

NIHMS690041-supplement-4.tif (987.5KB, tif)
5. Supplemental video 1.

Nasal secretion obtained from ECRS patient was incubated overnight at 37°C with equal volume of calcium and magnesium containing HBSS. The secretion showed a high viscosity with significant thread-forming capacity under the effect of traction.

Download video file (735.2KB, mp4)
6. Supplemental video 2.

Nasal secretion obtained from same ECRS patient was incubated with DNase I (100 U/ml) in similar condition as that in supplemental video 1. DNase treatment decreased the thread-forming capacity.

Download video file (493.9KB, mp4)
7
NIHMS690041-supplement-7.docx (42.1KB, docx)

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