Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases

Abstract

Papillon-Lefèvre syndrome (PLS) is characterized by nonfunctional neutrophil serine proteases (NSPs) and fulminant periodontal inflammation of unknown cause. Here we investigated neutrophil extracellular trap (NET)-associated aggregation and cytokine/chemokine-release/degradation by normal and NSP-deficient human and mouse granulocytes. Stimulated with solid or soluble NET inducers, normal neutrophils formed aggregates and both released and degraded cytokines/chemokines. With increasing cell density, proteolytic degradation outweighed release. Maximum output of cytokines/chemokines occurred mostly at densities between 2 × 10⁷ and 4 × 10⁷ neutrophils/cm³. Assessment of neutrophil density in vivo showed that these concentrations are surpassed during inflammation. Association with aggregated NETs conferred protection of neutrophil elastase against α1-antitrypsin. In contrast, eosinophils did not influence cytokine/chemokine concentrations. The proteolytic degradation of inflammatory mediators seen in NETs was abrogated in Papillon–Lefèvre syndrome (PLS) neutrophils. In summary, neutrophil-driven proteolysis of inflammatory mediators works as a built-in safeguard for inflammation. The absence of this negative feedback mechanism might be responsible for the nonresolving periodontitis seen in PLS.—Hahn, J., Schauer, C., Czegley, C., Kling, L., Petru, L., Schmid, B., Weidner, D., Reinwald, C., Biermann, M. H. C., Blunder, S., Ernst, J., Lesner, A., Bäuerle, T., Palmisano, R., Christiansen, S., Herrmann, M., Bozec, A., Gruber, R., Schett, G., Hoffmann, M. H. Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.

Neutrophil extracellular trap (NET) formation was described to depend on the production of reactive oxygen species (ROS) by NADPH oxidase 2 (NOX2) during the oxidative burst and on histone deamination mediated by peptidyl arginine deiminase 4 (1, 2). ROS-dependent NET formation in neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) does not induce histone deamination (3), and calcium ionophore-induced NET formation does not depend on NADPH oxidase (4), suggesting 2 independent pathways (5). Nonetheless, we and others have shown that infection- and inflammation-related physiologic stimuli are often accompanied by NOX2 activation and also induce histone deamination (6–9). For example, NET formation in response to pristane or monosodium urate (MSU) crystals was reduced in either the absence of functional NOX2 or of functional peptidyl arginine deiminase 4 in vivo (6, 7, 10). Thus, the 2 distinct biologic processes of NET formation induced by defined chemicals in vitro are not mutually exclusive in vivo (11).

Proteolytic degradation of inflammatory mediators by MSU crystal–induced so-called aggregated NETs (aggNETs) results in the resolution of gouty arthritis in humans and mice (10). This process requires the presence of neutrophil serine proteases (NSPs) in high-density neutrophils. Recent evidence from a mouse model of lupus where NET formation is triggered by the alkane oil pristane suggests that similar mechanisms may be involved when NETs are formed in response to other stimuli (6). Thus, the antiinflammatory effect of serine proteases entangled in NETs has a potential impact on different kinds of inflammatory diseases associated with the accumulation of neutrophils.

The pathologic outcome of a complete functional absence of NSP can be seen in individuals with Papillon–Lefèvre syndrome (PLS). In PLS, a rare autosomal-recessive genetic disorder, mutations in cathepsin C prevent posttranslational trimming and therefore functional activation of NSPs (12, 13). PLS patients experience exaggerated and nonresolving periodontal inflammation of unknown cause.

The aim of this study was to investigate if the proresolving effects of aggNETs induced by MSU crystals are also found in high-density NETs induced by other stimuli. Additionally, we intended to shed light on details of NET aggregation, to determine which densities of NETs are required for proteolytic degradation of inflammatory mediators, to descry how binding to aggNETs influences the function of antiproteases, and to shed further light on the importance of these mechanisms for preventing chronic inflammation in vivo. Because eosinophils have also been implicated in the resolution of inflammation (14, 15), we also investigated the potential of eosinophils to degrade inflammatory mediators and modulate MSU crystal–induced inflammation.

We report that release of inflammatory mediators by activated neutrophils is highest at intermediate cell densities (20–40 × 10⁶ cells/cm³). Above such densities, mediator release by normal neutrophils is outweighed by proteolytic degradation in NETs. Thus, proteolysis of inflammatory mediators primarily depends on neutrophil density, but not on the size or the composition of NET aggregates. Additionally, binding to aggNETs confers protection of the serine protease neutrophil elastase (NE) against inactivation by α1-antitrypsin (A1AT). In contrast, neutrophils from individuals with PLS do not form canonical NETs and are unable to degrade cytokines and chemokines even in high cell densities, which might importantly contribute to the exuberant inflammation seen in this condition. We also show that eosinophils do not share the capacities of neutrophils for degradation of inflammatory mediators and thus do not promote the proteolysis-dependent resolution of MSU crystal–induced inflammation.

MATERIALS AND METHODS

Characteristics of 2 patients with PLS and normal healthy donors

In both patients, diagnosis of PLS was made both clinically and by molecular genetic analysis of the CTSC gene encoding cathepsin C. Patient 1, a 28-yr-old man, had the homozygous mutation p.Gly139Ter, while 16-yr-old male patient 2 showed compound-heterozygous mutations p.Lys108Ter and p.Tyr168Ter. We used blood from 6 normal healthy donors (NHDs) during this study (4 male and 2 female donors; mean age, 38.5 yr).

The study was approved by the Institutional Review Boards of the Medical University of Innsbruck and the University Hospital Erlangen, and complied with the principles of the Declaration of Helsinki. All subjects provided written informed consent before study enrollment.

Isolation of neutrophils from human peripheral blood

Neutrophils were freshly isolated from human whole blood obtained by venipuncture from healthy volunteer donors who had provided written informed consent. The blood was anticoagulated with heparin (20 U/ml) and placed on Ficoll (Bio-Rad, Hercules, CA, USA) for density separation. Cells were centrifuged at 1400 rpm for 30 min at room temperature with low acceleration and no break. Suspension above the buffy coat was removed, and the white layer containing the polymorphonuclear leukocytes (PMNs) on the top of the red blood cells was collected. To remove contaminating erythrocytes, PMNs were subjected to short cycles of hypotonic lysis with deionized water. After restitution of normal osmolality with HBSS, viable cells were counted by trypan blue exclusion in a Neubauer chamber. All analyses of human blood samples were performed in accordance to the institutional guidelines and with the approval of the Ethical Committee of the University Hospital Erlangen (Permit 193 13B).

Mice

Ncf1** mice, characterized by a point mutation in the Ncf1 gene (16, 17), and specific eosinophil-deficient ΔdblGATA mice (18) were on the BALB/c background. All mice were housed in a temperature- and humidity-controlled facility at the University of Erlangen with free access to food and water. The animal studies were approved by the local ethical committee (Regierung von Unterfranken, Würzburg, Germany) and conducted according to the guidelines of the Federation of European Laboratory Animal Science Associations. All experiments were performed using age- and sex-matched littermate controls. Mice were allocated randomly into groups by a computer-based random number generator (http://www.randomizer.org) so that each cage contained animals of every group to compensate for possible cage effects. Power analysis was performed on the basis of effect size estimates from previous experiments.

Preparation of MSU crystals

A solution of 10 mM uric acid and 154 mM NaCl (both from Merck, Darmstadt, Germany) was adjusted to pH 7.2 and agitated for 3 d. The resulting crystals were washed with ethanol, dried under sterile conditions, sterilized at 180°C for 2 h, and stored in PBS (pH 7.0).

Air pouch model and MSU crystal–induced paw swelling in mice

Mice were anesthetized with isofluorane, and 3 ml of sterile air was injected subcutaneously into the back to form an air pouch. Two days after the first injection, an additional 2 ml of sterile air was injected into the existing pouch. One day later, neutrophils were depleted by intraperitoneal injection of 500 µg of a Ly6G-specific depleting antibody (clone 1A8; Bio X Cell, West Lebanon, NH, USA). The next day, 5 mg MSU crystals in PBS was injected into the air pouches. The pouch fluid was collected after 24 h, and cytokine and chemokine levels were determined by multiplex bead technology (BioLegend, San Diego, CA, USA) and quantified by cytofluorometry using a Gallios cytofluorometer (Beckman Coulter, Brea, CA, USA). The sizes of newly formed MSU crystal aggregates in the air pouches were analyzed after surgical removal of the air pouch.

Scanning electron microscopy

Isolated human PMNs were cultured for 4 h with or without stimulation in plastic flat-bottomed 96-well plates at different densities, as indicated in the figures. After fixation with 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.2) containing 0.01 M CaCl₂, 0.01 M MgCl₂, 0.09 M saccharose, and 2% glutaraldehyde as a fixative, samples were dehydrated in an ascending ethanol series (2 × 10 min 30% EtOH, 1 × 10 min 50% EtOH, 1 × 10 min 70% EtOH, 3 × 10 min 100% EtOH), followed by 2 × 5 min incubation with 99.9% hexamethyldisilazane ReagentPlusR (MilliporeSigma, Burlington, MA, USA) and evaporation/air drying overnight as described (19). Subsequently, the base of the well was carefully punched out including the cell layer, attached on stubs for electron microscopy, and sputter-coated with Au (Emitech K575X; Quorum Technologies, Laughton, United Kingdom), 2 × 15 mA, 60 s. The samples were then examined using a Tescan Lyra3 field-emitting scanning electron microscope at 5 kV accelerating voltage.

Dual-photon imaging of aggNETs

For investigation of aggNETs via dual-photon and polarization microscopy, human blood neutrophils [1 × 10⁸ cells/ml on Nunc glass-bottomed dishes (Thermo Fisher Scientific, Waltham, MA, USA)] were stained with the green fluorescent membrane dye PKH67 (MilliporeSigma) and the far-red DNA dye SiR-DNA (Spirochrome, Stein am Rhein, Switzerland) and incubated for 4 h with 20 pg per cell MSU crystals or left unstimulated. After fixation with 2% glutaraldehyde, imaging was performed on a Zeiss LSM 880 NLO (Carl Zeiss GmbH, Jena, Germany) equipped with a 680 to 1300 nm tunable and fixed 1040 nm 2-photon laser from Newport SpectraPhysics (Santa Clara, CA, USA) using a ×20 W-Plan Apochromat objective lens. The fluorophores were excited at 760 nm (PKH67) and 1200 nm (SiR-DNA), and specific emission was detected at 503 to 548 nm and 645 to 689 nm, respectively. MSU crystals were imaged by polarization microscopy by inserting 2 polarization foils in the light path, between the transmission light source and the sample, and in front of the detection camera (AxioCam MRm; Zeiss) of the LSM 880 NLO. Fluorescence and transmission stacks were aligned in ImageJ (Image Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/) and Fiji (https://fiji.sc/) by manually selecting corresponding landmark points and rigid registration. Three-dimensional reconstructions were rendered using a self-developed Fiji plugin.

Neutrophil culture and measurement of inflammatory mediators

Isolated human PMNs from healthy donors were cultured at densities ranging from 1 to 100 × 10⁶ cells/ml (200,000 to 20 × 10⁶ cells per well in flat-bottomed 96-well plates) with 20 pg/cell MSU crystals, 100 ng/ml PMA (MilliporeSigma), 5 µg/ml ionomycin (Iono; MilliporeSigma), 1 µM pyocyanin (MilliporeSigma), 15 µM nigericin (NIG; InvivoGen, San Diego, CA, USA), 2.5 µg/ml LPS from Klebsiella pneumoniae (MilliporeSigma), or without stimulus in HBSS medium containing 24 mM bicarbonate and 1 mM calcium at pH 7.4. For the timeline experiments (Supplemental Fig. S2), 60 × 10⁶ cells per well were incubated in flat-bottomed 48-well plates in the presence or absence of PMSF (MilliporeSigma). Because of its short half-life, PMSF was added at a concentration of 300 µM before the start of incubation and again after a 90 min incubation time. Plates were agitated gently before and every 30 min during incubation. After 3 h incubation at 37°C, 5% CO₂ cells were spun down, and supernatants were taken and stored at −20°C for further analysis. Concentrations of cytokines and chemokines in supernatants were measured by multiplex bead technology (BioLegend) and quantified by cytofluorometry using a Gallios cytofluorometer and Kaluza software (Beckman Coulter). Minimum detectable concentrations were as follows: 1.1 pg/ml (IL-1β), 1.8 pg/ml (IL-6), 2.3 pg/ml (CXCL8/IL-8), 1.7 pg/ml (MCP-1), 3.7 pg/ml (monokine induced by IFN-γ; MIG), and 3.0 pg/ml (TNF-α).

Analysis of NE and proteinase 3 activity

For assessment of NE activity in mice, bone marrow neutrophils (1 × 10⁸ cells/ml) isolated from WT and Ncf1** mice were incubated with 200 nM N-formyl-l-methionyl-l-leucyl-phenylalanine for 60 min to induce degranulation. NE activity in supernatants was quantified with the fluorogenic NE substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (Santa Cruz Biotechnology, Dallas, TX, USA). The fluorogenic NE-converted hydrolysis products were then detected at 465 nm on a Tecan Sunrise microplate reader (Tecan, Männedorf, Switzerland).

For investigating protection of NE function in aggNETs, 1 × 10⁸ cells/ml PMNs from human healthy donors were stimulated with PMA, Iono, or MSU crystals for 2 h on flat-bottomed 96-well plates. One hundred microliters of supernatant or the remaining cell pellets were then incubated for 30 min with either 10% autologous plasma, 400 nM of the NE inhibitor sivelestat (MilliporeSigma), or 1 mg/ml of recombinant A1AT (MilliporeSigma) in HBSS medium containing 24 mM bicarbonate at pH 7.4. Remaining NE activity was then determined by measuring hydrolysis of N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (Santa Cruz Biotechnology) on a Tecan Sunrise microplate reader.

To assess proteinase 3 (PR3) activity in human neutrophils, isolated human neutrophils were adjusted to a concentration of 1 × 10⁶ cells/ml, resuspended in 50 µl cold assay buffer from a commercial Cathepsin-G Activity Assay Kit (ab126780; Abcam, Cambridge, United Kingdom), and incubated for 10 min on ice for cell lysis. Lysed neutrophils were then spun down and supernatant collected. PR3 activity was detected in lysates diluted 1:5 using 12.5 µM PR3 substrate [ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO₂)-NH2 (20)] on a Tecan Sunrise Microplate Reader (360/485 nm).

Assessment of degradation of inflammatory mediators in air pouches by Western blot analysis

For analysis of in vivo degradation of cytokines/chemokines, 5 mg MSU crystals were injected into preformed air pouches to induce accumulation of neutrophils and formation of aggNETs. Control mice were injected with PBS. Twenty-four hours after MSU crystal/PBS injection, recombinant IL-6 and MCP-1 biotinylated using the Biotin-XX Protein Labeling Kit (Thermo Fisher Scientific) were injected into the air pouch. After 5 h, air pouches were rinsed with ice-cold PBS containing proteinase inhibitors (Halt proteinase inhibitor cocktail; Thermo Fisher Scientific). Denaturing gradient SDS/PAGE (8–16% bis-acrylamide) and subsequent Western blot analysis with air pouch lavages of biotinylated IL-6 or MCP-1 were performed. Horseradish peroxidase (HRP)-conjugated streptavidin (21130, diluted 1:1000; Thermo Fisher Scientific) was used to detect the respective protein. Membranes were incubated with ECL substrate (34095; Thermo Fisher Scientific) for 90 s, and chemiluminescence signals were measured using the BioStep Celvin S Chemiluminescence Imaging System.

Immunohistochemical analysis of chromatin decondensation and NET formation

Isolated human neutrophils were adjusted to a concentration of 6 × 10⁶ cells/ml in HBSS containing calcium and magnesium. A total of 25 μl of cell suspension was added to each well of an 8-well cell chamber slide (Thermo Fisher Scientific). A total of 150 μl of HBSS containing either 10 ng/ml PMA, 2 µg/ml Iono, 40 pg/ml MSU crystals, or vehicle control were added to the cells. The chamber slide was incubated at 37°C and 5% CO₂ for 3 h. Subsequently, 2% (v/v) paraformaldehyde (Merck) was added to each well and the preparations incubated for 1 h at room temperature, followed by 5 min permeabilization with 0.1% Triton X-100. Samples were subsequently blocked with 10% (v/v) fetal calf serum (Biochrom, Cambridge, United Kingdom); 2% (w/v) bovine serum albumin in PBS for 1 h at room temperature. Primary antibody for NE (ab21595; 1:200; Abcam) was added in 10% fetal calf serum/2% bovine serum albumin in PBS for 18 h at 4°C. Slides were washed 3 times with PBS and secondary Cy5-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added for 1.5 h at room temperature in the dark. Slides were washed with PBS. Staining solution containing 2.5 µM Sytox Green in PBS was added for 15 min at room temperature. Slides were washed with PBS, and samples were embedded in mounting medium (Biozol, Eching, Germany). Slides were analyzed on a BZ-X710 microscope (Keyence, Osaka, Japan). Percentage of cells with decondensated chromatin was defined as Sytox Green–positive events with a 5-fold greater mean nuclear size on 3 random slide sections.

Real-time quantitative PCR

Total RNA was extracted from MSU crystal–injected paws after removal of skin and tendons using peqGold TriFast (Peqlab, Erlangen, Germany). One microgram of total RNA was reverse transcribed and SYBR Green–based real-time quantitative PCR was performed on a Bio-Rad CFX96 Touch Real-Time PCR Detection System. Normalized gene expression values were calculated as the ratio of expression of mRNA of interest to the expression of mRNA for Actb (encoding β-actin). The primer sequences are summarized in Supplemental Table S1.

Immunohistochemistry for approximation of neutrophil density in tissue

MSU crystal–injected mouse paws were fixed with 4% formaldehyde, embedded in paraffin, and sectioned. After dewaxing, antigen retrieval was performed at 80°C in Epitope Retrieval Solution, pH 9 (EB-DEPP-9; Eubio, Atlanta, GA, USA) for 20 min. After equilibration to room tempertature, endogenic oxidase was blocked with 3% hydrogen peroxide for 5 min. After blocking with 0.2% bovine serum albumin in PBS (blocking buffer) for 1 h at room temperature, samples were incubated with anti-mouse Ly6G primary antibody (127601, clone 1A8, 1/1000 in blocking buffer; BioLegend) at 4°C overnight; then, after 3 washing steps, samples were incubated with HRP-conjugated goat–anti-rat secondary antibody (3030-05, 1/500 in blocking buffer; Southern Biotechnology, Birmingham, AL, USA) for 1.5 h at room temperature. The HRP substrate 3,3′-diaminobezidine (DAB Kit SK4100; Vector Laboratories, Burlingame, CA, USA) was used as substrate for HRP. For evaluation of neutrophil density, representative regions were selected, and Ly6G-positive cells were counted in several 50 µm squares. The following formula was used for estimating the cell density: cells/ml = [(√cell count)³ × (8 × 10⁶)].

Statistical analysis

Two-group comparisons were performed using a 2-tailed Student’s t test or, for nonnormally distributed data, a 2-tailed Mann-Whitney U test. Welch’s correction was used in the case of unequal variances. Within each set of experiments shown in a single figure, multiple comparisons of groups were adjusted using Bonferroni’s or Dunnett’s post hoc test. Outliers within data sets were excluded on the basis of a Grubb’s test/extreme Studentized deviate test for variation from a normal distribution. Adjusted values of P < 0.05 were considered statistically significant. All computations were performed and charts produced by Prism 7 software (GraphPad Software, La Jolla, CA, USA).

RESULTS

AggNET formation in PMA, Iono, and MSU crystal–challenged neutrophils

Incubation of human and murine neutrophils with MSU crystals triggers NET formation and, under conditions of high cell density, the generation of large aggregates with proteolytic activity (aggNETs) (7, 10). To investigate if other triggers of NET formation also induce aggNETs, we incubated freshly isolated human PMNs with either PMA, the calcium ionophore Iono, or with MSU crystals (as control). While all NET inducers triggered aggregation, the macroscopic appearance of these aggregates differed from each other: incubation with MSU crystals led to the formation of large agglomerations filling the whole circumference of the well, whereas the aggregates formed in neutrophils stimulated with the soluble agonists PMA and Iono were considerably smaller and often dispersed over the well (Fig. 1A). Unstimulated neutrophils did not form macroscopically visible aggregates. As previously described (7), aggNETs already formed after a couple of minutes, while PMA- and Iono-induced aggregates required 1 h of incubation. The size of MSU crystal–induced aggNETs but also of PMA- and Iono-triggered aggregates depended on the number of cells in the well (Supplemental Fig. S1A).

Figure 1.

Aggregation of neutrophils stimulated with triggers of NET formation. A) Representative photographs from high-density (1 × 10⁸ cells/ml) cultures of human peripheral blood neutrophils incubated with MSU crystals, PMA, Iono, or without stimulants for 3 h. Scale bar, 3 mm. B) Scanning electron microscope micrographs from high-density (1 × 10⁸ cells/ml) neutrophil cultures stimulated as indicated. Scale bars, 20 µm. C) Representative multiphoton images of neutrophils stained with DNA dye SiR-DNA and membrane marker PKH67 and cultured in densities of 1 × 10⁸ cells/ml with MSU crystals or without stimulant. Scale bars, 50 µm. D) Assessment of MSU crystal–induced aggNETs with combination of multiphoton imaging and polarization microscopy. Scale bar, 50 µm. All experiments were performed independently 3 times.

Structural differences of aggNETs induced by PMA, Iono, and MSU crystals

Detailed analysis of NET-associated neutrophil aggregates by scanning electron microscopy confirmed the dependency of the aggregate size on trigger and neutrophil density (Fig. 1B and Supplemental Fig. S1B). Furthermore, MSU crystal–induced aggregates were surrounded by intact neutrophils, suggesting that physical contact of neutrophils with crystals is necessary for NET formation and that neutrophils in the vicinity are continuously recruited to the aggregate after the formation of its crystalline core, thereby increasing its size. In contrast, threadlike structures suggestive of NETs were seen on the majority of cells inside and in the outside layers of the aggregates formed by the soluble agonists PMA and Iono (Fig. 1B). Although a certain amount of aggregation was also to be seen in unstimulated high-density neutrophil cultures, the average size of these aggregates was much smaller (Supplemental Fig. S1C).

For a closer look into the inner structure of aggNETs, we performed multiphoton imaging of high-density neutrophils stained with the fluorescent DNA dye SiR-DNA and the membrane dye PKH67 after incubation with MSU crystals. Heterogeneous dispersion of MSU crystal–stimulated high-density neutrophil cultures into aggregates connected by threadlike NETs was confirmed, whereas unstimulated neutrophils formed homogeneous cell layers (Fig. 1C and Supplemental Videos S1 and S2). Combination with polarization microscopy revealed a distribution into regions with very high MSU crystal densities, attenuating the fluorescence signal of deeper areas, and into regions where only isolated and scattered individual MSU crystals were present (Fig. 1D and Supplemental Video S3).

AggNETs formed after incubation of normal neutrophils with stimulators of NET formation dissipate inflammatory mediators

Serine proteases bound to MSU crystal–induced aggNETs mediate degradation of inflammatory mediators and thus convey resolution of MSU crystal–triggered inflammation (7, 10). We investigated if aggNETs formed after stimulation with different stimulators of NET formation harbor similar proteolytic properties as MSU crystal–induced aggNETs. We therefore incubated cell pellets from high-density neutrophil cultures that had either been stimulated with MSU crystals, PMA, Iono, the bacterial toxins pyocyanin and NIG, or LPS from K. pneumoniae, or were left unstimulated, with exogenous recombinant cytokines and chemokines and measured their concentrations in the supernatants after 18 h. Apart from IL-8 (CXCL8), which has been previously shown to be released in high amounts by MSU crystal–induced aggNETs (7, 10), the concentrations of cytokines and chemokines in the media containing high-density NETs were decreased compared to media that contained unstimulated neutrophils (Fig. 2A). We thus conclude that there is no functional difference in the capacities to degrade inflammatory mediators between high-density NETs induced by different triggers of NET formulation.

Figure 2.

Degradation of inflammatory mediators by neutrophils undergoing NET formation. A) Degradation of exogenously added cytokines/chemokines by cell pellets from high-density cultures (1 × 10⁸ cells/ml) of human neutrophils stimulated with 5 different triggers of NET formation: MSU crystals (MSU), Iono, PMA, pyocyanin (PYO), NIG, and LPS, or by pellets of unstimulated neutrophils (US). Control, values after 18 h of incubation without cell pellets. Dashed line indicates 100%. Bars indicate means ± sem from 4 to 5 individual blood donors. *P < 0.05, ***P < 0.001 (Student’s t test with Dunnett’s post hoc test). B) Fluorescence from creation of fluorogenic hydrolysis products by NE (corresponding to NE activity) in supernatants and cell pellets of high-density neutrophils incubated with PMA, MSU crystals, or Iono, or without stimulant (US). Bars show means ± sem from 4 to 6 individual blood donors. C) Remaining NE activity in supernatants and cell pellets (aggNETs) from PMA-, Iono, or MSU crystal–stimulated neutrophils after incubation with NE inhibitors A1AT or sivelestat, normalized to NE activity in neutrophils not treated with NE inhibitors. Bars show means ± sem from 4 to 5 individual blood donors. **P < 0.01, ***P < 0.001 (2-way ANOVA using Bonferroni’s post hoc test). D) Change in remaining NE activity (remaining activity in supernatants subtracted from remaining activity in aggNETs) after stimulation with PMA, MSU crystals, or Iono and incubation with A1AT or sivelestat. *P < 0.05, **P < 0.01, ***P < 0.001 (2-way ANOVA using Bonferroni’s post hoc tests). E) Relative cytokine and chemokine concentrations in culture supernatants of human neutrophils cultured for 3 h at different densities in presence of MSU crystals, Iono, or PMA, or without stimulant. Graphs show means and sem of 3 individual blood donors. Solid lines indicate concentration normalized to 10⁶ cells; dashed lines, total concentrations; ∇, value below detection limit; dashed red circles, maxima for concentrations/10⁶ cells; solid blue circles, maxima for absolute concentrations.

NE bound to aggNETs is protected from inhibition by antiproteases

The serine protease NE is proteolytically active on NETs and in supernatants of neutrophils that have undergone NET formation (7). NE has a wide spectrum of potential substrates, including extracellular matrix proteins, plasma proteins, cell-surface receptors, other proteases, and cytokines/chemokines. An elaborate system of antiproteases exists to limit the destructive capacity of proteases on tissue. NE is mainly inhibited by A1AT. To assess how binding to aggNETs influences the activity of NE and its inhibition by A1AT, we measured NE activity in cell pellets and supernatants of aggNETs induced with MSU crystals, PMA, and Iono (Fig. 2B, C). NE activity was increased over background levels found in unstimulated neutrophils in both cell pellets and supernatants of aggNETs induced by all stimulators used (Fig. 2B). Incubation with recombinant A1AT decreased proteolytic activities of both aggNET-bound and free NE, but it had a significantly stronger effect on free NE than on aggNET-bound NE (Fig. 2C). These results suggest that binding of NE in aggNETs confers a certain kind of protection against degradation by antiproteases such as A1AT. In contrast, aggNETs did not confer significant protection from inhibition of enzymatic activity by the synthetic NE inhibitor sivelestat. Direct comparison revealed a significantly stronger protection of all kinds of aggNETs against the 54 kDa protein A1AT than against the small molecule sivelestat (Fig. 2D), suggesting that steric inhibition could restrict access of naturally occurring proteinous antiproteases to aggNET-bound proteases.

Density-dependent cytokine/chemokine release and degradation during aggNET formation

To explore in more detail the degradation of inflammatory mediators by neutrophils, we performed titration experiments with unstimulated and stimulated neutrophils in densities ranging from 5 × 10⁶/ml to 1 × 10⁸/ml and analyzed the concentrations of cytokines and chemokines released into the supernatants after 3 h (Fig. 2E). Unstimulated neutrophils released detectable amounts of the chemokines IL-8 and MCP-1, suggesting a certain level of constitutive production and release. IL-8 and MCP-1 were found in concentrations of up to 55 and 20 pg/10⁶ unstimulated neutrophils, respectively. The absolute concentrations of both IL-8 and MCP-1 in supernatants were increased at higher densities of unstimulated neutrophils. Nevertheless, concentration normalized to the cell number was lower in high-density cultures, indicating a degradation process possibly mediated by spontaneous NET formation. The concentrations of all other mediators aside from IL-8 and MCP-1 in the supernatants were below detection limits when neutrophils were left unstimulated.

Upon stimulation with instigators of NET formation, MIG, TNF-α, IL-1β, and IL-6 were also released. Maximum detected amounts of inflammatory mediators in neutrophils stimulated with MSU crystals, PMA, or Iono were as follows: 487 pg/10⁶ cells IL-8, 274 pg/10⁶ cells MCP-1 (CCL2), 167 pg/10⁶ cells MIG (CXCL9), 129 pg/10⁶ cells IL-1β, and 80 pg/10⁶ cells IL-6. Of note, the inflammatory mediators IFN-γ, IL-10, IP-10, and macrophage inflammatory proteins 1α and 1β were below detection limits in the supernatants of all neutrophil cultures (data not shown). Peak production and maximum supernatant concentrations for the cytokines IL-1β and IL-6 and the chemokine MIG (CXCL9) occurred at cell densities of 20–40 × 10⁶/ml. Upon stimulation, most titration curves displayed local maxima indicating the cell concentrations with maximum cytokine/chemokine release. For most cytokines/chemokines and stimulants, these local maxima were to be observed at cell densities of 20–40 × 10⁶ neutrophils/ml. In higher densities, relative and absolute concentrations of cytokines/chemokines were decreased. This suggests that at sites of high neutrophil accumulation, neutrophilic inflammation is a self-limiting process as a result of trapping and cleavage of cytokines and chemokines.

To investigate the temporal profiles of cytokine/chemokine release and degradation, we measured their concentrations in the supernatants of high-density cultures (1 × 10⁸/ml) stimulated with PMA (Supplemental Fig. S2). Interestingly, in cells with functional NSP, mediator concentrations remained low during the whole incubation period. In contrast, supernatants from neutrophil cultures that were additionally treated with the NSP inhibitor PMSF contained significantly higher concentrations of inflammatory mediators, thus further confirming the importance of NSPs for the temporal and spatial shaping of the inflammatory response.

Approximation of neutrophil densities in vivo

To estimate neutrophil densities that occur in vivo in inflamed tissues, we performed immunohistochemical staining of Ly6G in MSU crystal–injected mouse paws during acute inflammation (Fig. 3). Approximation of cell counts at several representative regions showed that densities <1 × 10⁸ neutrophils/ml were reached and exceeded in inflamed tissue of MSU crystal–injected paws (Fig. 3A–C and Table 1), while PBS-injected paws were devoid of neutrophils (data not shown). Thus, the neutrophil-mediated proteolytic degradation processes described above has the potential to be fully functional in vivo.

Figure 3.

Degradation of inflammatory mediators in vivo. A–C) Approximation of neutrophil densities in MSU crystal–induced inflammation in vivo. A) Representative immunohistochemistry of Ly6G-positive neutrophils (stained brown) on serial section of MSU crystal–injected mouse paw. Scale bar, 500 µm. B) Picture of serial section incubated with secondary HRP-conjugated but without primary anti-Ly6G antibody as staining control. Scale bar, 500 µm. C) Calculation of tissue density of Ly6G-positive neutrophils during MSU crystal–induced inflammation. Numbers indicate cell counts/√ (area = 2500 µm²). Scale bars, 50 µm. D) In vivo degradation of biotinylated IL-6 or MCP-1 in PBS (control) or MSU crystal–injected air pouches from WT and NET-deficient (Ncf1**) mice. Each lane indicates 1 mouse. Shown is 1 out of 2 independent experiments.

TABLE 1.

Calculation of neutrophil densities in MSU crystal–induced arthritis

Cell count (2500 µm2)	Conversion to third dimension	Conc/ml
30	164	13.12 × 108
29	156	12.49 × 108
27	140	11.22 × 108
18	76	6.11 × 108
29	156	12.49 × 108
26	133	10.61 × 108
28	148	11.85 × 108
19	82	6.63 × 108

Conc, concentration.

AggNETs are endowed with proteolytic capacities on cytokines and chemokines in vivo

Final concentrations of inflammatory mediators in neutrophil supernatants result from the balance of release and extracellular proteolytic degradation (7, 10). To specifically investigate in vivo degradation, we labeled recombinant cytokines and chemokines with biotin and determined their degradation in MSU crystal–injected air pouches by Western blot analysis (Fig. 3D). Biotinylated IL-6, and to a lesser degree MCP-1, were degraded in air pouches of wild-type (WT) but hardly degraded in ROS-deficient Ncf1** mice, which had previously been shown to be unable to form MSU crystal–induced aggNETs (7, 10). Of note, the activity of the serine protease NE, shown to account for a large part of cytokine degradation in aggNETs (7), did not differ between neutrophils of WT and Ncf1** mice (Supplemental Fig. S3). Thus, the inability to degrade IL-6 and MCP-1 in Ncf1** mice is caused by inhibited apposition of the enzyme in NETs rather than impaired enzymatic activity.

Formation of NETs and defective degradation of inflammatory mediators by neutrophils from individuals with PLS

In order to assess the importance of our findings in a human situation without NSPs, we tested NET formation and proteolytic activity of NSPs in 2 patients with PLS. Individuals with PLS have been previously reported to have impaired canonical NET formation triggered by PMA and bacteria, while other neutrophil functions such as bacterial killing and production of ROS during the oxidative burst are normal (13, 20). When we stimulated neutrophils from 2 PLS patients and a NHD with various NET instigators, we did not observe unambiguous differences in extrusion of threadlike DNA (Fig. 4A) or in chromatin decondensation (Fig. 4B).

Figure 4.

Neutrophils from individuals with PLS exhibit aberrant NET formation and defective degradation of inflammatory mediators. A, B) Morphologic analysis and quantification of NETs in patients with PLS and healthy donors by fluorescence microscopy. A) Representative immunofluorescence images of SYTOX Green- and NE-stained neutrophils isolated from patient with PLS and NHD, and incubated with instigators of NET formation. Right panels show staining controls incubated without primary antibody to NE. B) Quantitative analysis of chromatin decondensation. Bars show means and sd of percentage of cells with decondensated DNA (defined as cells with >2-fold mean nuclear size and low DNA fluorescence intensity in 3 randomly selected fields of view) from 2 PLS patients and 1 NHD. C) PR3 activity in neutrophil lysates from 2 patients with PLS and 1 NHD. Plots show means ± sd of fluorescence units in technical replicates. Baseline mean fluorescence unit values of substrate without cell lysates are subtracted. D) Degradation of exogenously added cytokines/chemokines by cell pellets from high-density cultures (1 × 10⁸ cells/ml) of neutrophils derived from 2 patients with PLS and 1 NHD, and stimulated with triggers of NET formation. Experiments with NHD cells and PLS cells were performed in parallel. Scatter plots indicate means from technical triplicates of individual blood donors. Results from further NHD controls are shown in Fig. 2A.

PLS phenotypes range from specific loss of function of NSPs to complete absence of the enzymes. In the 2 patients enrolled onto our study, the expression of NE in PLS neutrophils was clearly detectable but markedly reduced on extruded DNA after incubation with PMA, Iono, or MSU crystals compared to NHD (Fig. 4A). We tested NSP activity with a newly developed specific substrate for neutrophil PR3 (21) and found completely abrogated enzymatic activity in individuals with PLS (Fig. 4C). Importantly, pellets from PLS-derived neutrophil cultures that had been incubated in high densities (1 × 10⁸ cells/ml) with MSU crystals, PMA, or Iono did not degrade exogenously added IL-1β, IL-6, and MCP-1, leading to higher concentrations of these inflammatory mediators in the supernatants (Fig. 4D). This is in line with the increased concentrations of inflammatory mediators that have been reported in PLS-derived neutrophil cultures on incubation with NET-inducing bacteria (20). An exception again was the chemokine IL-8, which seemed reduced rather than increased in supernatants after incubation with neutrophil pellets from individuals with PLS (Fig. 4D).

Role of eosinophils in degradation of cytokines/chemokines and MSU crystal–induced arthritis

Recently, eosinophils have been shown to dampen chronic arthritis. This proresolving effect is presumably caused by an IL-13–dependent shift of macrophages toward an antiinflammatory phenotype characterized by a high expression of arginase 1 and/or by the production of antiinflammatory lipids such as protectin D1 (15). Although not to the same extent as neutrophils, eosinophils also possess considerable protease activity that might exert immunoregulatory effects similar to neutrophils (22, 23). However, eosinophil granules often stay intact during extracellular trap formation (22), which may possibly affect cytokine degradation.

To determine the role of eosinophils in the degradation of inflammatory mediators, we performed an air pouch model in eosinophil-deficient ΔdblGATA mice that were additionally treated with neutrophil-depleting antibodies (clone 1A8). The absence of eosinophils neither impaired formation of MSU crystal aggregates nor influenced the concentrations of inflammatory mediators in MSU crystal–injected air pouches (Supplemental Fig. S4A). In line with these results, the severity and course of MSU crystal–induced arthritis was not affected in eosinophil-deficient ΔdblGATA compared to WT mice (Supplemental Fig. S4B). Furthermore, in acute MSU crystal–induced arthritis, arginase 1 (Arg1) expression did not differ between ΔdblGATA and WT mice. Likewise, the expression levels of the inflammatory cytokines IL-6 and TNF-α, and the neutrophil-derived chemokines Cxcl1 and Cxcl2 in the MSU crystal–injected joints were not changed in eosinophil-deficient ΔdblGATA mice, nor were the osteoclast markers Nfatc1, Acp5, Tnfrsf11a (receptor activator of nuclear factor κB ligand) or Ctsk (cathepsin K), suggesting that eosinophils do not affect acute MSU crystal–induced arthritis (Supplemental Fig. S4C).

DISCUSSION

Although neutrophils are usually considered proinflammatory cells fueling inflammatory responses, evidence is accumulating that they can also have antiinflammatory and immunoregulatory functions (24). In cancer, immunoregulatory neutrophils are often referred to as granulocytic myeloid-derived suppressor cells. These cells occur as mature and immature (banded) cells, are able to form NETs (25), and suppress T-cell responses by releasing arginase-1, a ureohydrolase degrading arginine essential for T-cell activation and proliferation (26).

Additionally, neutrophils can regulate inflammation by releasing proteases from their intracellular granular storages. Neutrophil granules contain several protease classes, including cysteine proteases (e.g., cathepsin C), metalloproteases (e.g., collagenases and gelatinases), and serine proteases. NSPs comprise NE, PR3, cathepsin G, and neutrophil serine protease 4 and are among the most abundant neutrophil proteins. Release of proteases from their intracellular storages in neutrophils may have pro- as well as antiinflammatory effects. While in the acute situation release of neutrophil enzymes may have a proinflammatory function and their activity can also mediate tissue damage during chronic inflammation (27), these enzymes also have regulatory functions in the resolution of inflammation and in the stimulation of tissue repair. NSPs target a broad array of proteins for modification and degradation: aside from extracellular matrix proteins, they also process cytokines/chemokines and their receptors, and thus modulate their bioactivity. For instance, cleavage of TNF-α, IL-2, IL-6, the cytokine receptor chain IL-2R-α, or the IL-6R 80 kDa chain impairs their functionality. In contrast, cleavage of IL-8 by PR3 reportedly yielded a more active form of this chemokine (28), and NSP-mediated processing increases the biologic activity of IL-1 family members (29, 30).

Degradation of proinflammatory mediators by NSPs was recently shown to dampen sterile inflammation in gout (7, 10) and in an animal model of lupus (6). In gout, agglomerates composed of NETs and MSU crystals (aggNETs) that are formed at high neutrophil densities during acute inflammation, but not at the comparably low neutrophil densities occurring in the peripheral blood, degrade inflammatory mediators via inherent NSPs, thus halting and resolving acute inflammatory attacks (31). It was, however, unclear to date whether instigators of NET formation other than MSU crystals also mediate such cleaving activities if neutrophils are present at high densities, and which neutrophil densities are required to initiate the proteolytic capacities of NETs. As triggers of NET formation, we investigated the canonical but somewhat artificial soluble NET instigators PMA and Iono, and, aside from MSU crystals, 3 further stimuli that are known to induce NET formation under physiologic conditions in vivo: 1) pyocyanin, a toxin derived from the airway pathogen Pseudomonas aeruginosa that induces formation of ROS and NOX2-dependent NET formation (32, 33); 2) NIG, a potassium ionophore toxin derived from Streptomyces hygroscopicus that induces NET formation independently of ROS and myeloperoxidase (34); and 3) LPS. We demonstrate that aggNETs induced by all the used stimuli of NET formation show comparable abilities to degrade inflammatory mediators. These results are in line with a recently published study that showed degradation of histone and casein by NETs independent of the stimulus (34), and that suggests that the mechanism seen in gout could be a general strategy of the body to interrupt the self-amplifying positive feedback loop with neutrophil recruitment, activation and NET formation, inflammation, and further attraction, thus fostering resolution of inflammation and acting as a safeguard for containing neutrophilic inflammation (Fig. 5).

Figure 5.

Model of how density of NETting neutrophils influences inflammation via proteolytic degradation and protection from antiproteases. In areas of low neutrophil densities (e.g., in blood or in affected tissues during early stages of inflammation), individual NETs act in proinflammatory manner, releasing proinflammatory mediators that act as immune modulators and chemotactic agents for further neutrophil influx. At full-blown inflammation, higher numbers of secreting cells in conjunction with functional antiproteases that maintain low proteolytic degradation results in high local cytokine/chemokine concentrations. However, in areas of even higher cell densities (over 20–40 × 10⁶ neutrophils/cm³), NETting neutrophils clump and form aggNETs. In these dense aggregates, function of naturally occurring antiproteases (such as A1AT) is partially inhibited because of restricted access to active sites of proteases. Size and consistency of aggNETs varies depending on physical and chemical nature of trigger. Inducers of NET and aggNET formation may be soluble (e.g., PMA, Iono), oily (e.g., pristane), or solid (e.g., bacteria, crystals). Regardless of their consistency, over certain cell density, proteolytic degradation of inflammatory mediators exceeds their release. Thus, inflammation is halted and resolved, as cytokines and chemokines are degraded faster than they are produced.

Cytokine/chemokine-cleaving effects of NETs essentially depend on neutrophil density. We investigated cytokine/chemokine concentrations in supernatants of neutrophil cultures with densities ranging from those observed in the peripheral blood (5 × 10⁶ cells/cm³) up to densities observed in synovial fluid of patients with gout and septic arthritis (2 × 10⁷ to 1 × 10⁸ cells/cm³) (35). Maximal cytokine/chemokine concentrations were measured at intermediate neutrophil densities (2 × 10⁷ to 4 × 10⁷ cells/cm³), while in higher densities inflammatory mediator concentrations were decreased, most likely owing to proteolytic degradation by NET-associated proteases. An estimation of neutrophil densities in MSU crystal–induced arthritis in vivo showed values that were markedly above 4 × 10⁷ cells/cm³. Thus, the protease-mediated termination of inflammation has the potential to be fully functional in vivo.

Insufficient regulation of NE activity by A1AT importantly contributes to endothelial damage during acute lung injury (36) and nephritis (37) and also promotes tumor progression (38). Although A1AT is able to inhibit both NE released to the pericellular space and membrane-bound NE (39), restricted accessibility of plugged or condensed areas might lower the capacity of inhibition (40) and limit the efficacy of NE inhibition as a therapy for conditions such as cystic fibrosis (41). Our results indicate that different kinds of aggNETs confer protection of proteases from naturally occurring high MW antiproteases, such as A1AT. In comparison, protection from the NE inhibitor sivelestat, a small molecule with a function that is less affected by steric hindrance, was much less pronounced. The conservation of protease function in aggNETs may contribute to the tissue-damaging properties in chronic lung or kidney disease, but it also might enhance its resolving capacities by ensuring sustained degradation of inflammatory mediators.

Although neutrophils produce comparably low amounts of cytokines per cell, their overall contribution to the cytokine and chemokine milieu is of high importance because of their abundance at sites of inflammation compared to other leukocytes (42). The mechanism of degradation described in Fig. 5 is not limited to inflammatory mediators released by the neutrophil itself, but also applies to cytokines/chemokines released by other cell types. In accordance, neutrophil-depleted or -deficient mice develop MSU crystal–induced inflammation with a slower onset but with a chronic course (7, 10), highlighting the effective cytokine cleavage and resolution of inflammation by aggNETs formed by neutrophils. Thus, the neutrophil-mediated degradation of cytokines/chemokines is well designed, not interfering with early neutrophil influx and defense but rather contributing to resolution during advanced stages of an inflammatory response.

The pathologic outcome of a complete functional absence of NSPs is severe early-onset and chronic periodontitis, as observed in PLS, which is surprisingly not caused by an increased susceptibility toward bacterial infections (13, 43). Thus, rather than their antimicrobial action, other functions of NSPs must promote regulation and resolution of inflammation in the oral cavity (44). It is tempting to speculate that defective degradation by PLS neutrophils of inflammatory mediators, as shown in this study, is responsible for inflammation spinning out of control. Interestingly, neutrophil chromatin decondensation and extrusion of DNA into the extracellular space after incubation with the NET instigators MSU crystals, Iono, and PMA did not appear to be reduced in 2 individuals with PLS compared to 1 blood donor with functional NSP who was tested in parallel. This is somewhat surprising because NE and proteinase have been implicated in histone degradation and chromatin decondensation during PMA-induced NETosis in human neutrophils and in vivo in mice (45), and other studies involving PLS patients have reported defective, albeit not completely abrogated, NET formation (13, 21). Although of less importance for the rationale of our current study, further experiments with more PLS patients are needed to unambiguously define the ability of human neutrophils to decondensate chromatin and actively release DNA without functional NSPs.

Another remaining question is the connection among neutrophil aggregation, NET formation and release, and proteolytic degradation of cytokines/chemokines. During the last few years, the technological progress in 2-photon intravital microscopy has enabled the discovery of neutrophil swarming, a phenomenon characterized by highly coordinated series of neutrophil movement, followed by cell accumulation mediated by chemoattractant signals and adhesion molecules (46). Swarming is observed during infection and sterile inflammation in both mouse and human neutrophils (47). Interestingly, cell death, both in the inflamed surrounding tissue and within the neutrophil cluster itself, seems to amplify swarming. It is tempting to hypothesize that neutrophil swarming and the formation of aggNETs might be mutually reinforcing processes, but as for now, the connection between these cellular functions remains elusive. In a recent study, Reátegui et al. (48) used a newly developed microscale array technique to measure the amount of mediators released at different phases of swarming in human neutrophils. The results of this work confirmed a dominant role of leukotriene B4, which had already been suggested following studies in mice (46), and elucidated the contribution of several other protein mediators (among them IL-8) to swarming. Interestingly, release of lipoxin A4 and resolvin E3, 2 lipid mediators of resolution of inflammation (49), was up-regulated from matured swarms, thus acting as an inhibitory signal on swarm growth. If release of lipid mediators from aggNETs also contributes to the proresolving effects of these structures remains to be investigated.

For NET formation in response to MSU crystals, direct physical contact between cells and crystals is necessary. Intact morphology of neutrophils on the exterior side of the MSU crystal–induced aggregates suggests that the aggregates grow by attraction of further cells from the surrounding, possibly in a swarminglike manner. These dynamics might favor the formation of few, but very large, aggregates. In the case of the soluble NET-inducing stimuli PMA and Iono, all cells had undergone NET formation independent of their location, suggesting that the aggregates grow from several condensation nuclei, resulting in a higher number of smaller aggregates. Because degradation of inflammatory mediators did not differ between aggNETs induced by PMA/Iono and MSU crystals, the size of the aggregates is apparently not decisive for its proteolytic activity.

Remarkably, the regulatory effect on cytokines/chemokines was not observed in eosinophils. Our results from the air pouch model and MSU crystal–induced arthritis showed that eosinophils are apparently not involved in aggregate formation and subsequent degradation of inflammatory mediators, suggesting it to be a specific feature of neutrophils. In summary, these data support the notion that neutrophils have phase-specific function during inflammation. They represent the first line of defense during the acute phase of inflammation and trigger cytokine, chemokine, and protease release. If reaching a critical density, however, neutrophils orchestrate the switch to resolution of inflammation by aggNET formation and the cleavage of inflammatory cytokines and chemokines.

Supplementary Material

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

Glossary

A1AT

α1-antitrypsin

aggNET

aggregated NET

HRP

horseradish peroxidase

Iono

ionomycin

MCP-1

monocyte chemotactic protein 1

MIG

monokine induced by IFN-γ

MSU

monosodium urate

NE

neutrophil elastase

NET

neutrophil extracellular trap

NHD

normal healthy donor

NIG

nigericin

NOX2

NADPH oxidase 2

NSP

neutrophil serine protease

PLS

Papillon–Lefèvre syndrome

PMA

phorbol 12-myristate 13-acetate

PMN

polymorphonuclear leukocyte

PR3

proteinase 3

ROS

reactive oxygen species

WT

wild type

AUTHOR CONTRIBUTIONS

J. Hahn and C. Schauer planned and performed most of the animal and in vitro experiments, conducted data analysis, helped write the manuscript, and prepared the figures; C. Czegley ran the experiments with eosinophil-deficient mice and WT control mice, performed Western blot analysis, and conducted data analysis; L. Kling and S. Christiansen performed electron microscopy; B. Schmid and R. Palmisano performed dual-photon imaging; L. Petru carried out the experiments pertaining to protection of NE in aggNETs; D. Weidner and C. Reinwald conducted immunohistochemical stainings; M. H. C. Biermann conducted Western blot analysis; S. Blunder and R. Gruber contributed to experiments using neutrophils from PLS patients and interpretation of the results; J. Ernst set up the combination of dual-photon and polarization microscopy; A. Lesner developed and provided reagents for measurement of PR3 activity; T. Bäuerle was responsible for in vivo imaging; M. Herrmann, A. Bozec, R. Gruber, and G. Schett provided scientific input, strategically planned the experiments, and helped with the manuscript; M. H. Hoffmann supervised the project, planned experiments, conducted data analysis, and wrote the manuscript; and all authors reviewed the final manuscript.