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Release of Cystic Fibrosis Airway Inflammatory Markers from Pseudomonas aeruginosa−Stimulated Human Involves NADPH -Dependent This information is current as Extracellular DNA Trap Formation of September 26, 2021. Dae-goon Yoo, Matthew Winn, Lan Pang, Samuel M. Moskowitz, Harry L. Malech, Thomas L. Leto and Balázs Rada J Immunol 2014; 192:4728-4738; Prepublished online 16 Downloaded from April 2014; doi: 10.4049/jimmunol.1301589 http://www.jimmunol.org/content/192/10/4728 http://www.jimmunol.org/

References This article cites 53 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/192/10/4728.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Release of Cystic Fibrosis Airway Inflammatory Markers from Pseudomonas aeruginosa–Stimulated Human Neutrophils Involves NADPH Oxidase-Dependent Extracellular DNA Trap Formation

Dae-goon Yoo,* Matthew Winn,* Lan Pang,* Samuel M. Moskowitz,†,‡ Harry L. Malech,x Thomas L. Leto,x and Bala´zs Rada*

Cystic fibrosis (CF) airways are characterized by bacterial infections, excess mucus production, and robust recruitment. The main CF airway pathogen is Pseudomonas aeruginosa. Neutrophils are not capable of clearing the infection. Neutrophil

primary components, (MPO) and human neutrophil elastase (HNE), are inflammatory markers in CF Downloaded from airways, and their increased levels are associated with poor lung function. Identifying the mechanism of MPO and HNE release from neutrophils is of high clinical relevance for CF. In this article, we show that human neutrophils release large amounts of neutrophil extracellular traps (NETs) in the presence of P. aeruginosa. are entangled in NETs and colocalize with extracellular DNA. MPO, HNE, and citrullinated histone H4 are all associated with DNA in Pseudomonas-triggered NETs. Both laboratory standard strains and CF isolates of P. aeruginosa induce DNA, MPO, and HNE release from human neutrophils. The

increase in peroxidase activity of neutrophil supernatants after Pseudomonas exposure indicates that enzymatically active MPO is http://www.jimmunol.org/ released. P. aeruginosa induces a robust in neutrophils that is required for extracellular DNA release. Inhibition of the cytoskeleton prevents Pseudomonas-initiated production and DNA release. NADPH oxidase inhibition sup- presses Pseudomonas-induced release of active MPO and HNE. Blocking MEK/ERK signaling results in only minimal inhibition of DNA release induced by Pseudomonas. Our data describe in vitro details of DNA, MPO, and HNE release from neutrophils activated by P. aeruginosa. We propose that Pseudomonas-induced NET formation is an important mechanism contributing to inflammatory conditions characteristic of CF airways. The Journal of Immunology, 2014, 192: 4728–4738.

ystic fibrosis (CF) is an inherited disorder resulting from Pseudomonas aeruginosa represents the predominant pathogen by guest on September 26, 2021 mutations in the CF transmembrane conductance regu- infecting most CF patients (4–6). C lator (CFTR) anion channel (1). Several organs are af- The main pharmacological approaches used in the clinical fected in CF, but lung complications remain the main cause of management of CF airway disease focus on treating bacterial disease morbidity and mortality (2). Lack of apical plasma infections with antibiotics, promoting airway clearance by lysing membrane expression of a fully functional CFTR in respiratory DNA within mucous plugs, and rehydrating the airway surface (7– epithelial cells leads to altered fluid absorption, ion secretion, and 10). Survival and quality of life have improved significantly for mucous retention within the airways. As a result, CF airways people with CF over the last two decades; however, for most become obstructed and infected with bacteria, leading to robust patients, the disease still cannot be adequately managed, let alone neutrophil infiltration and chronic inflammation (3). A wide va- cured, and more effective treatments are urgently needed (8). riety of bacterial species have been detected in CF airways, but Recently, promising new drug candidates have emerged that ad- dress the core problem in CF: the dysfunctional CFTR membrane (11–13). Although these CFTR “protein-assist” therapies are very promising, currently there is only one Food and Drug *Department of Infectious Diseases, College of Veterinary Medicine, University of Administration–approved drug that potentiates CFTR function, in Georgia, Athens, GA 30602; †Department of Pediatrics, Massachusetts General Hos- the case of defective channel gating, and no drugs have been pital, Boston, MA 02114; ‡Department of Pediatrics, Harvard Medical School, Bos- x approved to date to correct the defective cellular trafficking of the ton, MA 02115; and Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852 common CFTR F508del mutation (13). Received for publication June 17, 2013. Accepted for publication March 18, 2014. Airway inflammation in CF is the product of a complex set of This work was supported by startup funds from the Office of the Vice President for innate immune interactions. The neutrophil is a pivotal cellular Research, University of Georgia (to B.R.) and by the Intramural Research Program player influencing the outcome of these interactions. Neutrophil of the National Institutes of Health, National Institute of Allergy and Infectious density in CF sputum has been shown to correlate with CF disease Diseases. severity (measured as forced expiratory volume in 1 s) (14, 15). Address correspondence and reprint requests to Dr. Bala´zs Rada, Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, 501 Sputum and blood concentrations of human neutrophil elastase DW Brooks Drive, Athens, GA 30602. E-mail address: [email protected] (HNE) and myeloperoxidase (MPO) in CF patients are associated Abbreviations used in this article: CF, cystic fibrosis; CFTR, CF transmembrane with declines in lung function (14–18). IL-8, a major neutrophil conductance regulator; CGD, chronic granulomatous disease; citH4, citrullinated chemoattractant that both airway epithelial cells and neutrophils histone H4; DPI, diphenylene iodonium; HNE, human neutrophil elastase; MOI, multiplicity of infection; MPO, myeloperoxidase; NET, neutrophil extracellular trap; produce, has also been associated with CF lung function decline RT, room temperature; UGA, University of Georgia. (15, 16). IL-1b, a proinflammatory cytokine mainly produced by www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301589 The Journal of Immunology 4729 macrophages but also secreted by activated neutrophils, has also activity of 2.34 nmol/106 cells/h (1.04%), and superoxide production in 6 been linked to CF lung damage (14). These data obtained from healthy neutrophils was 226 nmol/10 cells/h (100%) (28, 33). clinical samples of CF patients clearly show that neutrophil re- Human neutrophil isolation cruitment and “activation” are major contributors to lung damage. It is therefore important to understand why recruited neutrophils Neutrophils were isolated as described previously (32). In brief, whole blood was drawn at the Health Center of UGA (Athens, GA) or at the release their granule contents in CF. Formation of neutrophil ex- Department of Transfusion Medicine of the National Institutes of Health tracellular traps (NETs) offers a possible mechanism (19). In NETs, (Bethesda, MD), and coagulation was prevented with . RBCs were extracellular DNA is associated with neutrophil granule compo- removed by dextran sedimentation (GE Healthcare, Amersham, U.K.). nents (HNE, MPO) and histones (19, 20). Neutrophils release NETs Leukocytes were layered on top of a 5-step Percoll gradient (65, 70, 75, 80, in response to bacterial and inflammatory stimuli (19, 21). Negative and 85%; Sigma, St. Louis, MO), and the 70-75-80% Percoll layers con- taining neutrophils were harvested. Cells were stored in a mix of autolo- correlations were found between CF sputum extracellular DNA gous serum and RPMI 1640 medium (Life Technologies, Grand Island, concentrations and lung function measures (16, 22). Neutrophils NY) until use at room temperature (RT). Trypan blue dye extrusion was undergoing NET formation were detected in CF sputum samples used to determine cell viability (.99%). Purity of the neutrophil prepa- (23–25). Histone citrullination, a histone modification characteristic rations was confirmed by cytospin. Serum was prepared from coagulated blood by centrifugation and sterile filtration. Calcium- and magnesium- for NET formation, was also detected in CF sputum samples (23). containing HBSS (Mediatech, Manassas, VA) containing 1% autologous P. aeruginosa is likely to activate neutrophils in CF airways. serum, 5 mM glucose, and 10 mM HEPES was used as assay buffer. This organism is associated with diminished pulmonary function in CF and, similar to many other pathogens, triggers robust NET P. aeruginosa strains release in vivo (15, 26) and in vitro (27, 28). , a major The following P. aeruginosa strains were used in this study: PA14 (kind gift Downloaded from toxin of P. aeruginosa, which can be relatively abundant in the CF from Dr. Frederick Ausubel, Massachusetts General Hospital, Boston, MA), airway, enhances NET formation and has been linked to lung PAO1 (American Type Culture Collection), PA2192 and GFP-expressing function decline in this disorder (26, 28–30). Pseudomonas and PAO1 (kindly provided by Dr. Joanna Goldberg, Emory University, Atlanta, GA), and PA10145 (ATCC 10145). CF isolates of P. aeruginosa were ob- neutrophils are found in close proximity, and neutrophils have been tained as described previously (28). Bacteria were cultured in Luria–Bertani shown to phagocytose bacteria in airway samples of CF patients broth overnight. Late exponential phase cultures were washed twice and 9

(31). These data suggest that Pseudomonas–neutrophil interactions suspended in HBSS. Bacterial density was set to 10 /ml using OD = 0.6 at http://www.jimmunol.org/ are highly relevant to CF airway pathophysiology. The major ob- 600 nm. jective of this study was to provide insight into the mechanism of Quantification of NETs Pseudomonas-induced NET formation, a process highly relevant to neutrophil dysfunction in the CF airway. Our hypothesis was that NETs were quantified essentially as described with some modifications (32). Neutrophils were attached to poly D-lysine–coated 96-well black P. aeruginosa–stimulated NET formation contributes to release of transparent-bottom plates (Thermo Scientific, Rochester, NY) in assay me- MPO, HNE, and extracellular DNA. Our approach to test this dium containing 10 mM Sytox Orange (Life Technologies, Grand Island, hypothesis was to quantify NET formation, MPO, and HNE release NY). For inhibitor tests, cells were pretreated with specific inhibitors for and activities after exposure of human neutrophils in vitro to lab- 15 min before bacterial infection. Neutrophils were infected with 1–50

multiplicity of infection (MOI) P. aeruginosa PA14 for dose-dependent by guest on September 26, 2021 oratory standard strains and CF isolates of P. aeruginosa. extracellular DNA release assay or 10 MOI for inhibitor experiments. Fluorescence (excitation: 530 nm, emission: 590 nm) was measured in Materials and Methods a fluorescence plate reader (Varioskan Ascent, Thermo Scientific) for up to 5 h at 37˚C. Fluorescence of samples containing 500 mg/ml saponin (Sigma) Human subjects with neutrophils was used as the maximal signal for DNA release (100%). The Institutional Review Board of the University of Georgia (UGA) ap- Rise in fluorescence was referred to as “extracellular DNA release” and proved the human subject study to collect peripheral blood from healthy expressed as relative fluorescence units or normalized on saponin-treated as volunteers (UGA #2012-10769-06). Enrolled healthy volunteers were .18 y maximal signal (referred to as “% of max”). The following inhibitor con- of age, were not pregnant, were .110 pounds, had no infectious disease centrations were used: diphenylene iodonium [DPI], NADPH oxidase inhib- complications, and provided written informed consent (32). itor, 10 mM; Sigma), U0126 (MEK1/2 inhibitor, 25 mM; Sigma), MEK162 Human subjects also included CF patients enrolled in an observational (MEK1/2 inhibitor, 50 mM; Sigma), PD98059 (MEK1 inhibitor, 20 mM; study of CF lung disease severity, “Genetics of CF Lung Disease” (Seattle Sigma), and cytochalasin D (cytoskeleton inhibitor, 10 mM; Sigma). Children’s Hospital Institutional Review Board approved protocol 10855 and Partners Healthcare Systems/Massachusetts General Hospital Institu- Immunostaining and fluorescent microscopy tional Review Board approved protocol 2011P000544). The protocols and Neutrophils were seeded on 12-mm glass coverslips (VWR International, informed consent procedures were approved by the Institutional Review Radnor, PA) in 24-well plates (Thermo Scientific, Rochester, NY). PA14 Boards of the Seattle Children’s Hospital and Partners Healthcare Systems/ (opsonized with 10% autologous serum, 37˚C, 30 min) was added to neu- Massachusetts General Hospital, and the studies were performed in ac- trophils and incubated for 3 h at 37˚C. After fixation with 4% paraformal- cordance with the ethical guidelines of the Declaration of Helsinki. All 10 dehyde (Affymetrix, Cleveland, OH), samples were blocked with 5% normal CF patients in this study were homozygous for the F508del allele of the donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA) and CFTR gene and were categorized as having “severe” lung disease: they 0.1% saponin (Sigma) in PBS for 30 min at RT. The following Abs were were in the lowest quintile for age of airway obstruction, as assessed by used: monoclonal mouse anti-human MPO/FITC Ab (1:500, clone MPO-7; their median forced expiratory volume during the initial second of exha- Dako), polyclonal rabbit anti-histone H4 (citrulline 3; 1:1000; Millipore, lation (for more details, see Ref. 28). Billerica, MA), and rabbit anti-HNE (1:1000) overnight at 4˚C. For HNE and Patients with X-linked (gp91phox-deficient) and autosomal (p47phox- citrullinated histone H4 (citH4) staining, Alexa Fluor 594–labeled donkey deficient) chronic granulomatous disease (CGD) and healthy volunteers anti-rabbit secondary Ab was used for 1 h (1:2000; Molecular Probes, Grand were recruited under the National Institute of Allergy and Infectious Island, NY). Samples were stained with DAPI (2 min, RT, 1:20,000; Mo- Diseases Institutional Review Board–approved protocol (National Insti- lecular Probes) and washed in PBS twice. Specimens mounted with ProLong tutes of Health #05-I-0213, “Evaluation of Patients with Immune Function Antifade Kit (Molecular Probes) were analyzed with Zeiss AxioCam HRM Abnormalities”). Human subjects provided written informed consent for fluorescence microscope (Axioplan2 imaging software). participation. Ten milliliters blood was drawn from CGD patients and was processed in parallel with healthy volunteers’ blood. Two X-CGD patients Measurement of superoxide production (Patients 2 and 3) and one p47phox-deficient CGD patient participated in the study; all subjects were characterized in previous studies with regard to Superoxide release was assessed by Diogenes chemiluminescence their genetic defects and residual NADPH oxidase activities: Patient 2 superoxide-detection kit (National Diagnostics, Atlanta, GA). Adherent (1.7 nmol/106 cells/h, gp-146, 0.75%) and patient 3 (2.38 nmol/106 cells/h neutrophils were stimulated by P. aeruginosa PA14 (50 MOI), PMA [1.05%]) (33). The p47phox2/2 patient (p47-16) had NADPH oxidase (100 nM), or were left unstimulated. Chemiluminescence was measured by 4730 NET FORMATION INDUCED BY PSEUDOMONAS AERUGINOSA

Varioskan Flash luminescence microplate reader (Thermo Scientific, determined using the correlation coefficient and the sample size (20) using Logan, UT) for 90 min. Data are shown as kinetics of representative curves online statistical software (Statistics Calculators, version 3.0 Beta). Sta- (relative luminescence units) or integral superoxide production values tistically significant differences were considered as follows: *p , 0.05, (integrated relative luminescence units) obtained by multiplying accumu- **p , 0.01, ***p , 0.001. lated luminescence for the entire duration of the measurement with the ratio of the measurement cycle length and integration time. Results Supernatant collection for ELISA and activity Human neutrophils release extracellular DNA in response to measurements P. aeruginosa Supernatants of neutrophils were used to measure secretion and activities of We show that human neutrophils release extracellular DNA in MPO and HNE. A total of 100,000 neutrophils/well were seeded into 96- response to GFP-expressing P. aeruginosa PAO1 (Fig. 1A). Bac- well poly-D-lysine–coated transparent plates (Thermo Scientific, Rochester, NY), stimulated with 100 nM PMA or P. aeruginosa PA14 (50 MOI), teria are entrapped in NETs (Fig. 1A and insets). Neutrophils also respectively. After 3 h of incubation at 37˚C in HBSS including 1% serum, release DNA in response to another P. aeruginosa strain, PA14 cell supernatants were collected, centrifuged for 10 min at 10,000 3 g to (Fig. 1B, 1C). PA14-induced DNA release starts at the same time remove debris or bacteria, and stored at 280˚C for further analysis or used when induced by the positive control, PMA (Fig. 1B). Bacteria immediately. Each supernatant was diluted 1:30 for HNE ELISA, 1:100 for (PA14) alone do not release any DNA (Fig. 1B). DNA release MPO ELISA, or left undiluted for MPO activity test. from adherent human neutrophils is induced in a dose-dependent MPO and HNE ELISA manner by P. aeruginosa strain PA14 (Fig. 1C). Quantitation m Concentrations of human MPO in neutrophil supernatants were measured reveals an average release of 3.01 ng/ l DNA from 100,000 by commercial ELISA kit (R&D Systems, Minneapolis, MN). Serial neutrophils on PA14 exposure (Fig. 1D). PAO1 and two other Downloaded from dilutions prepared from MPO standard provided in the kit (stock: 125 P. aeruginosa strains (PA10145, PA2192) also stimulate DNA re- ng/ml) were used to quantify MPO concentrations of unknown samples. lease in a dose-dependent manner (Fig. 1E). We next tried to es- HNE release was assessed by ELISA. Supernatant samples diluted with timate the proportion of live bacteria attached to NETs after a 3-h coating buffer (25 mM carbonate, 25 mM bicarbonate, pH 9.6) were in- cubated overnight at 4˚C in 96-well high binding microlon ELISA plates incubation time. The supernatant of Pseudomonas–neutrophil (Greiner bio-one, Fricken-hausen, Germany). After blocking with 1% BSA suspensions was very carefully removed after incubation, did not for 1 h, anti-HNE rabbit polyclonal Ab (1:500 in PBS [Calbiochem], contain NETs, and was therefore termed “NET-free” (Fig. 1F). http://www.jimmunol.org/ 481001 [EMD Millipore]) was added for 2 h at RT. After repeated Absence of DNA in the NET-free fraction was confirmed by washes, samples were incubated with horseradish peroxidase–linked donkey anti-rabbit Ab (1:2000 in PBS, NA934V; GE Healthcare) for 1 h. running the samples on DNA agarose gels (data not shown). An Blue coloration developed in the presence of 3,39,5,59-tetramethylbenzi- equal volume of new assay medium containing DNAse I was dine (Thermo Scientific, Rockford, IL) peroxidase substrate solution. addedbackandwasreferredtoas“NET-linked.”Asshownin Reaction was stopped by adding 1N hydrochloric acid (Sigma), and ab- Fig. 1F, we found that 41.3 6 3.4% of live bacteria (mean 6 SEM, sorption was read at 450-nm wavelength with Eon microplate photometer n = 3) were associated with NETs when a MOI of 10:1 was used (BioTek, Winooski, VT). Purified HNE standard (stock: 1 mg/ml; Cell Sci- 6 6 ences, Canton, MA) was used to determine HNE concentrations in unknown and 29.7 13.6% (mean SD, n = 3) with a MOI of 50:1. These samples. measurements reveal that in vitro NETs are capable of trapping large amounts of P. ae ru gi nos a. by guest on September 26, 2021 Measurement of peroxidase activity Peroxidase activity was measured by hydrogen peroxide–dependent oxi- Pseudomonas-induced NETs contain citH4 dation of Amplex Red. Fifty microliters undiluted neutrophil supernatants The signaling steps leading from stimulation to NET formation in was added to 96-well nontransparent black microplates (Costar, Corning, human neutrophils have not been well defined, but citrullination of NY) and mixed with assay solution containing 100 mM Amplex Red (Molecular Probes, Eugene, OR) and 100 mM hydrogen peroxide (Sigma). histones (H3 and H4) has been implicated and confirmed by several Production of the fluorescent product was measured with fluorescence groups (35–37). Citrullinated histone H3 was detected in CF sputum plate reader (Varioskan Ascent, Thermo Scientific) after 30 min at 560-nm samples by immunofluorescence (23). In this article, we show that excitation and 590-nm emission wavelengths. Standards with known MPO histone H4 is citrullinated on amino acid residue 3 and colocalizes concentrations (stock: 125 ng/ml MPO; R&D Systems) were used to determine peroxidase activities of unknown samples. Results are expressed as “equivalent with extracellular DNA after Pseudomonas exposure (Fig. 2A). ng/ml MPO activity.” This way of data presentation was chosen (instead of showing unit enzymatic activities per time and volume) to enable comparison MPO is associated with Pseudomonas-triggered extracellular between MPO concentration and activity in neutrophil supernatants. DNA traps Measurement of “NET-free” and “NET-linked” bacterial MPO is stored in azurophilic granules in neutrophils, becomes numbers associated with extracellular DNA in NETs, and is also implicated in the progress of NET formation (19, 20). Our indirect immu- PA14 was added to human neutrophils (100,000/well) attached to poly-D- nofluorescence analysis detects colocalization of MPO with ex- lysine–coated transparent 96-well plates (Thermo Scientific, Rochester, NY) in HBSS. Bacteria were added at two different doses: PA14/PMN tracellular DNA and citH4 in Pseudomonas-stimulated NETs MOI = 10:1 or 50:1. After 3 h, supernatants were carefully collected and (Fig. 2A). These data prove that MPO released from neutrophils in referred to as “NET-free” fraction. Absence of DNA in this fraction was the presence of P. aeruginosa is associated with NETs. confirmed by DNA gel electrophoresis. Equal volume of assay medium containing 30 mg/ml DNAse I (Roche Applied Science, Indianapolis, IN) Neutrophil elastase is bound to NETs after Pseudomonas was added back to neutrophils and incubated for 15 min; this volume was challenge referred to as “NET-linked” fraction. After centrifugation, concentration of living PA14 bacteria in each sample was determined by a microplate-based HNE is a major inflammatory marker of CF airways (14–16, 38). bacterial growth assay as described previously (34). HNE is associated with extracellular DNA in NETs stimulated Statistical analysis in vitro by PMA and in CF sputum samples (19, 24). Although HNE is stored in primary granules in resting neutrophils, it Results were analyzed by Student t test or one-way ANOVA with Tukey becomes associated with extracellular DNA after stimulation with posttest for multiple comparisons. Each experiment was independently performed at least three times with neutrophils isolated from different P. aeruginosa PA14 (Fig. 2B, 2C). Thus, the main CF airway donors. Correlation was determined by computing the Pearson correlation pathogen P. aeruginosa activates human neutrophils to release coefficient (r). The significance of correlation was (two-tailed probability) NETs that contain extracellular DNA, HNE, MPO, and cit3H4. The Journal of Immunology 4731 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. P. aeruginosa induces extracellular DNA release in human neutrophils. (A) Adherent neutrophils were incubated with GFP-expressing P. aeruginosa PAO1 (3 h). Sytox Orange extracellular DNA-binding dye was added, and unfixed samples were imaged with a fluorescence microscope (one representative experiment, n = 4). Original magnification 31000 (insets, 34000). 10 MOI. (B) Kinetics of P. aeruginosa PA14-stimulated extracellular DNA release by neutrophils (Sytox Orange fluorescence) was compared with PA14 alone (50 MOI), neutrophils alone, or the positive control PMA (100 nM). One representative result (n = 3). (C) PA14 triggers dose-dependent extracellular DNA release in neutrophils (Sytox Orange fluorescence, 3 h, normalized to unstimulated control as 100%). Mean 6 SEM, n =5.(D) DNA release from neutrophils on PA14 exposure (3 h) was quantitated using Sytox Orange and calf thymus DNA serial dilutions. n =3.(E) Dose-dependent DNA release in human neutrophils exposed to P. aeruginosa strains PAO1, PA10145, and PA2192 (0–30 MOI, 3 h, Sytox Orange fluorescence, mean 6 SEM, n = 3). (F) Numbers of NET-associated (NET-linked) and NET-in- dependent (NET-free) living PA14 bacteria were assessed after 3 h of incubation with neutrophils at two different doses (10 and 50 MOI) according to details found in Materials and Methods. Mean 6 SEM, n =3. 4732 NET FORMATION INDUCED BY PSEUDOMONAS AERUGINOSA

FIGURE 2. NETs stimulated by P. aeruginosa contain citH4, MPO, and HNE. (A) MPO (green) and H4cit3 (red) colocalize with extra- cellular DNA in PA14-induced NETs. Adherent human neutrophils were stimulated by PA14 (50:1 MOI), 100 nM PMA, or were left unstimulated (3 h). Neutrophils were fixed and stained with anti-MPO-FITC Ab or anti-H4cit3 primary and donkey anti- rabbit 594 secondary Abs. DNA was counterstained by DAPI. One repre- sentative experiment, n =5.Original magnification 3400. (B) Localization of HNE in PA14- or PMA-stimulated human neutrophils was detected by immunofluorescence (rabbit anti-HNE Downloaded from primary Ab followed by donkey anti- rabbit 594-linked secondary Ab). Both intracellular and extracellular DNA were stained with DAPI. One rep- resentative measurement, n =4. Original magnification 3400. (C)

Inset of the HNE immunostaining http://www.jimmunol.org/ (B) reveals fine details of HNE lo- calization in NETs after PA14 expo- sure (gray arrowheads). As expected, HNE did not overlap with intact nuclear DNA within resting neu- trophils (white arrowheads). Rep- resentative images, n = 5. Original magnification 32000. by guest on September 26, 2021

MPO and HNE are released after neutrophil exposure to detected 176.0 6 14.5 ng/ml MPO in neutrophil supernatants P. aeruginosa after Pseudomonas (PA-14) stimulation and only 29.2 6 1.7 ng/ml 6 To confirm our immunofluorescence data, we measured MPO and with basal release (mean SEM, n = 8; Fig. 3A). We also ob- HNE release in supernatants of Pseudomonas-stimulated human served an increased peroxidase activity in supernatants of Pseu- neutrophils by ELISA. Throughout the entire article we mea- domonas-activated neutrophils (basal: 28.9 6 6.0, bacterial sured total MPO or HNE concentrations and MPO activity in stimulus: 77.7 6 11.9 ng/ml MPO activity, mean 6 SEM, n =8; our samples. We did not distinguish between NET-associated Fig. 3B). A similarly large increase was observed in HNE release and NET-free . Quantitation of MPO release by ELISA on exposure to PA14 (Fig. 3C). Background release of HNE

FIGURE 3. MPO and HNE are detected in neutrophil supernatants after P. aeruginosa challenge. Adherent human neutrophils were exposed to P. aeruginosa PA14 (50 MOI) for 2.5–3 h. Cell supernatants were assayed for MPO release, HNE release, and peroxidase activity according to Materials and Methods.(A) PA14-induced MPO release in neutrophils measured by ELISA. Mean 6 SEM, n =8.(B) Pseudomonas increases extracellular peroxidase activity in neutrophils. Peroxidase activity was determined by hydrogen peroxide–dependent Amplex Red oxidation in neutrophil supernatants. Mean 6 SEM, n =8.(C) HNE concentrations were measured by ELISA in Pseudomonas-exposed neutrophil supernatants. Mean 6 SEM, n = 8. Significance was evaluated by Student two-tailed t test (**p , 0.01, ***p , 0.001). The Journal of Immunology 4733

(165.1 6 19.8 ng/ml) was enhanced 2.7-fold by bacterial chal- similarly to the laboratory strains and that PAO1 and PA14 are lenge (447.2 6 40.4 ng/ml; mean 6 SEM, n = 8). We did not good models to study Pseudomonas-initiated neutrophil activa- detect any significant peroxidase activity with bacteria alone (data tion. DNA, MPO, and HNE release data show similar patterns not shown). among isolates. To determine the extent of correlation among the parameters we measured, we plotted extracellular DNA, MPO, and Human neutrophils release DNA, MPO, and HNE in the HNE release of clinical isolates against each other (Fig. 4D–F). We presence of CF isolates of P. aeruginosa calculated the Pearson correlation coefficients (r) to measure the To confirm that our previous findings are not limited to Pseudo- strength of a linear association and the significance between any monas laboratory strains, we exposed human neutrophils to CF of these two variables: MPO versus HNE: r = 0.89157226975503, clinical isolates of P. aeruginosa and measured DNA, MPO, and p = 0.00000013; MPO versus DNA: r = 0.80978489595945, p = HNE releases. All clinical isolates induced release of extracellular 0.00001517; and HNE versus DNA: r = 0.62058387267863, p = DNA (Fig. 4A), MPO (Fig. 4B), and HNE (Fig. 4C) at comparable 0.00350493. Correlations in each combination were determined to levels with PAO1. This confirms that clinical isolates behave be significant. Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. CF isolates of P. aeruginosa stimulate release of DNA, MPO, and HNE from human neutrophils. Attached neutrophils were incubated with 20 P. aeruginosa CF isolates (10 MOI, 3 h). Supernatants were assayed for release of extracellular DNA (ecDNA) (A), MPO (B), and HNE (C). DNA release was measured by Sytox Orange fluorescence. MPO and HNE releases were quantitated by ELISA. Mean 6 SEM, n =4.(D) Plotting MPO data against extracellular DNA data obtained in (A)–(C). (E) Plotting HNE data against extracellular DNA data obtained in (A)–(C). (F) Plotting MPO data against HNE data obtained in (A)–(C). (D–F) Each dot represents a CF isolate of P. aeruginosa. Pearson correlation coefficient (r). 4734 NET FORMATION INDUCED BY PSEUDOMONAS AERUGINOSA Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. The NADPH oxidase is required for Pseudomonas-stimulated NET formation, MPO release, and HNE release. Respiratory burst activity of neutrophils was measured by Diogenes luminescence superoxide assay. (A) P. aeruginosa PA14 induces superoxide production in adherent human neu- trophils in a dose-dependent manner (1–50 MOI). Kinetics are shown; chemiluminescence was measured for 90 min. One representative measurement, n =4. (B) PA14-triggered NADPH oxidase activity is blocked by DPI (10 mM). Inhibitor was added 15 min before stimulation. PA14/neutrophil MOI = 10. Integrated chemiluminescence for 90 min is shown. Mean 6 SEM, n =4.(C) X-CGD neutrophils do not respond with superoxide production to P. aeruginosa PA14 or PMA (100 nM). Experimental data obtained on both patients are shown. Mean 6 SD. (D) Extracellular DNA release (increase in Sytox Orange fluorescence, 3 h) from human neutrophils induced by P. aeruginosa PA14 (10 MOI) is inhibited by DPI (10 mM). Mean 6 SEM, n =5.(E) CGD neutrophils (X-CGD, left; p47-deficient, right) do not release extracellular DNA (ecDNA) in response to P. aeruginosa (PA14 or PAO1). One experiment/CGD donor; assays were performed in triplicates. Mean 6 SD. P. aeruginosa PA14-induced MPO release (F), peroxidase activity (G), and HNE secretion (H) were inhibited by 10 mM DPI. Mean 6 SEM, n = 8. Significance was evaluated by Student two-tailed t test (*p , 0.05, ***p , 0.001). The Journal of Immunology 4735

The neutrophil respiratory burst is activated by P. aeruginosa Pseudomonas-stimulated respiratory burst and DNA release NADPH oxidase activity is required for NET formation triggered requires an intact cytoskeleton by bacteria and PMA (39, 40). The requirement of neutrophil Human neutrophils phagocytose bacteria; however, the extent to respiratory burst activity for Pseudomonas-induced NETs has not which this process is required for Pseudomonas-activated NET been studied. First, we wanted to see whether adherent human formation is unknown. We stimulated human neutrophils with neutrophils respond to P. aeruginosa with superoxide production. P. aeruginosa PA14 in the presence of the cytoskeleton inhibitor Neutrophils stimulated by PA14 released large amounts of su- cytochalasin D and measured superoxide production and NET peroxide that was dependent on the dose of bacterial challenge formation. Cytochalasin D treatment blocked both the Pseudo- (Fig. 5A). As expected, the flavoenzyme inhibitor DPI diminished monas-triggered respiratory burst (Fig. 6A) and DNA release the Pseudomonas-initiated respiratory burst (Fig. 5B). We also (Fig. 6B). exposed neutrophils of X-linked CGD patients to PA14 and Role of MEK/ERK signaling in Pseudomonas-triggered NET measured the chemiluminescence signal. CGD neutrophils were formation entirely unresponsive to the bacterium, whereas oxidase-competent control neutrophils responded with large respiratory burst activity Activation of the MEK/ERK signaling pathway has been impli- (Fig. 5C). Thus, human neutrophils respond to P. aeruginosa with cated in PMA-stimulated NET formation (41, 42). We tested the oxidative activity. effect of U0126, a compound with known inhibitory effect on MEK1/2, on Pseudomonas-triggered neutrophil activation. U0126 Reactive species are required for neutrophil activation reduced PA14-induced extracellular DNA release and caused by P. aeruginosa a 67.0% decline in PA14-stimulated superoxide production Downloaded from We then examined whether treating neutrophils with the NADPH (Fig. 7A). U0126 had an inhibitory action of 42.6% on Pseudo- oxidase inhibitor DPI before Pseudomonas challenge would affect monas-triggered MPO release, but entirely blocked peroxidase extracellular DNA release and observed a significant reduction by activity in the supernatant (Fig. 7A). The fact that U0126 blocked prior DPI treatment (Fig. 5D). Neutrophils from both a gp91phox- MPO activity but had only moderate-to-medium inhibitory action and a p47phox-deficient patient remained unresponsive to PAO1 or on the other parameters raised concerns that U0126 can interfere

PA14, further confirming the requirement for a functional NADPH with MPO activity (Fig. 7A). We therefore tested U0126 and http://www.jimmunol.org/ oxidase in Pseudomonas-induced NET formation (Fig. 5E). DPI two other, well-characterized MEK inhibitors (MEK162 and pretreatment also greatly reduced Pseudomonas-induced total PD98059) in a cell-free system measuring MPO activity. Sur- MPO release (Fig. 5F) and extracellular peroxidase activity prisingly, we found that U0126 strongly inhibited in vitro MPO (Fig. 5G). PA14-triggered MPO release (146.8 6 14.8 ng/ml) was function, whereas MEK162 and PD98059 were without effect reduced by up to 77.5% by DPI (33.2 6 7.0 ng/ml; mean 6 SEM, (Fig. 7B). n = 8; Fig. 5F). PA14-stimulated extracellular peroxidase activity We next studied the effects of MEK162 and PD98059 on (51.6 6 7.3) was inhibited by up to 63.6% by NADPH oxidase neutrophils. Both inhibitors had minor but significant inhibition inhibition (18.8 6 4.6; relative fluorescence units, mean 6 SEM, of Pseudomonas-triggered extracellular DNA release: MEK162 n = 8; Fig. 5G). PA14-triggered secretion of total HNE (282.1 6 inhibited 33% of DNA release (n = 4) and PD98059 inhibited 38% by guest on September 26, 2021 29.4 ng/ml) was also reduced by 61.5% after DPI pretreatment of DNA release (n = 4; Fig. 7C, first panel). MEK162 and (108.7 6 23.2 ng/ml; mean 6 SEM, n = 8; Fig. 5H). Our results PD98059 did not influence release or activity of MPO (Fig. 7C). demonstrate that NADPH oxidase mediates Pseudomonas-in- MEK162 (but not PD98059) mildly but significantly reduced HNE duced NET formation, and this process is responsible for the release (Fig. 7C). These data confirm that whereas MEK/ERK majority of MPO and HNE release from neutrophils on Pseudo- signaling has minor contribution to extracellular DNA secretion, monas challenge. it does not generally influence release of MPO and HNE.

FIGURE 6. Cytochalasin D inhibits Pseudomonas- stimulated respiratory burst and DNA release. (A) Human neutrophils were stimulated with PA14 (10 MOI, 3 h) in the presence or absence of 10 mM cytochalasin D. Superoxide production was measured with Diogenes superoxide-specific chemiluminescence kit (integrated relative luminescence units, int. RLU). Mean 6 SEM, n = 3. ANOVA, Tukey’s post hoc analysis. (B) Extracellular DNA release (Sytox Orange fluorescence) was measured in neutrophils exposed to PA14 (10 MOI, 3 h) in the presence of 10 mMcy- tochalasin D. PA14-stimulated values are shown after subtraction of background activity. Values are expressed as relative fluorescence units (RFU). Mean 6 SEM, n =3.*p , 0.05, **p , 0.01, Student t test. 4736 NET FORMATION INDUCED BY PSEUDOMONAS AERUGINOSA

FIGURE 7. Minimal contribution of MEK/ERK signaling to Pseudomonas-induced DNA release. (A) Human neutrophils were exposed to PA14 (50 MOI) for 3 h in the presence or absence of U0126 (25 mM), and the following parameters were measured according to the detailed descriptions in Materials and Methods: extracellular DNA (ecDNA) release (Sytox Orange, n = 5), superoxide production (Diogenes, n = 4), MPO re- lease (ELISA, n = 8), peroxidase activity (Amplex Red, Downloaded from n = 8), and HNE release (ELISA, n = 8). Mean 6 SEM. Student t test. (B) Enzymatic activity of 500 mM MPO (Amplex Red oxidation) was measured in a cell-free system in the presence of indicated concentrations of U0126, MEK-162, and PD98059. Results are normal- ized on the inhibitor-free sample (mean 6 SEM, n = 2). (C) Neutrophils were exposed to PA14 (50 MOI) for 3 h http://www.jimmunol.org/ in the presence or absence of MEK-162 (50 mM) or PD98059 (20 mM), and the following parameters were measured: extracellular DNA release (Sytox Orange, n = 4), MPO release (ELISA, n = 4), peroxidase activity (Amplex Red, n = 4), and HNE release (ELISA, n =4). Mean 6 SEM. ANOVA, Tukey’s post hoc analysis. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 26, 2021

Discussion strongest NET inducers, whereas PAO1 and PA2192 release NETs Neutrophil airway recruitment and activation in CF is poorly to a lesser extent (Fig. 1). Similarly, CF isolates show a wide understood and has not been targeted therapeutically. In this study, variation in NET induction: most strains stimulate DNA release we explored details of neutrophil responses to the main CF airway at levels comparable with that of PAO1, whereas a few (#28, pathogen, P. aeruginosa. This mechanism is of high importance #64, #300) provoke marked responses from human neutrophils to understand CF lung inflammation because both presence of (Fig. 4A). Although our data used human neutrophils isolated from P. aeruginosa and neutrophil activation have been strongly linked peripheral blood of healthy volunteers, these data confirm that to diminished lung function in CF (14–18, 26, 38, 43–45). P. aeruginosa is a strong inducer of DNA release from neutrophils We found that the main effector response of neutrophils in and suggest that P. aeruginosa is likely to stimulate NET forma- response to PA14 in vitro is NET formation (Figs. 1–3). The tion in CF airways. backbone of NETs is extracellular DNA (19). DNA has been Neutrophil components MPO and HNE are detected in CF airways, and its presence correlates with acceler- found in large amounts in CF airways and contribute to tissue ated decline of lung function in CF patients (16, 22). Moreover, destruction through and proteolytic damage (14, DNAse treatment is routinely used to ease CF respiratory symp- 16, 18). Recently, HNE was found in the airways of CF infants, toms (16, 22, 46, 47). Our data show that neutrophils stimulated where its presence was associated with persistent bronchiectasis by different P. aeruginosa strains (PA14, PAO1, PA10145, and (38). Colocalization of neutrophil granule proteins (HNE, MPO) PA2192) release large amounts of DNA (Fig. 1). This is consistent and histones with DNA is required to define the mechanism of with data obtained by several other groups (25, 27, 28, 48–50). DNA release as NET formation (19, 40). Citrullination of histones Our data allow comparison of different bacterial strains used in (H3, H4) by peptidyl arginine deiminase 4 is required for in vitro Pseudomonas research and found that PA14 and PA10145 are the and in vivo NET release (35–37, 51). Citrullinated histone H3 has The Journal of Immunology 4737 previously been detected in CF sputum samples (23). Pseudo- tivation as well. U0126 has been previously used to implicate the monas-stimulated NETs contain citH4, MPO, and HNE that MEK-ERK signaling pathway in PMA- or Helicobacter pylori– colocalize with extracellular DNA, as detected by immunofluo- stimulated NET formation (41). However, this supports that MPO rescence (Fig. 2). Our data show that when neutrophils are in the activity is required for NET formation stimulated by PMA but presence of Pseudomonas in vitro, DNA release occurs mainly also by P. aeruginosa (55, 57). The requirement of MPO for through NET formation. Physical association of DNA, MPO, and Pseudomonas-induced NETs is, however, controversial because HNE was found previously in CF sputum samples (23, 24). Ab- other studies found that MPO-deficient human neutrophils or mu- solute quantitation of total MPO release detected by ELISA versus rine neutrophils do not have impaired NET release (48, 49). The enzymatic activity data suggests that ∼45–50% of released MPO mechanism of inhibitory action of U0126 on MPO function is not is enzymatically active after Pseudomonas challenge (Figs. 3, 5F, known. Its chemical structure (four primary amines and two 5G, 7A, and 7C). MPO has been shown to remain active in its disulfide bonds) suggests that U0126 could be an excellent scav- NET-bound state (52). enger of hypochlorous ions (55, 58). MEK162- and PD98059- Our survey of CF isolates of P. aeruginosa shows for the first mediated blockade of the MEK-ERK pathway decreased Pseu- time, to our knowledge, that these clinical strains stimulate release domonas-stimulated DNA release but had no significant effect on of extracellular DNA, MPO, and HNE from human neutrophils MPO measures or HNE release (except for MEK162 on HNE (Fig. 4). Previously, only NET-mediated killing of CF isolates of ELISA; Fig. 7). This suggests that the MEK/ERK pathway con- P. aeruginosa was measured (27). Linear correlation between tributes to Pseudomonas-induced NET release to a similar extent extracellular DNA, MPO, and HNE indicate that their release is as in PMA induction (41). However, this minor effect on NET not independent but coordinated, through mechanisms such as inhibition does not appear to contribute significantly to MPO or Downloaded from NET formation (Fig. 4D–F). All clinical isolates tested trigger HNE release, as NET formation is not their only release mecha- DNA/MPO/HNE release, but to a different extent. Future studies nism. are required to determine whether the extent of NET induction by P. aeruginosa acquires resistance to NET-mediated killing in the clinical isolates correlates with clinical measures of the CF CF airways (27). Clinical data clearly indicate that neutrophils are patients from whom they were isolated. unable to remove Pseudomonas effectively from the CF lung, as

Oxidative stress has been implicated in the pathogenesis of CF both NET-mediated and classical intracellular killing mechanisms http://www.jimmunol.org/ (53). In the inflammatory CF lung, neutrophils are the main source fail. Neutrophils instead release their dangerous granule contents of released . P. aeruginosa stimulates a into the airway lumen. Although the CF airway environment is robust neutrophil respiratory burst that is required for DNA release very complex and multiple mechanisms can be responsible for and contributes to MPO and HNE release (Fig. 5). Our data neutrophil dysfunction, our detailed in vitro characterization of confirm previous findings that DPI inhibits extracellular DNA Pseudomonas-induced NET formation (DNA associated with ac- release from neutrophils stimulated by a clinical isolate of tive MPO and HNE) presents a likely mechanism for excess in- P. aeruginosa of unknown origin (49). Beyond producing tissue- flammatory mediator release from neutrophils in CF. Our study damaging reactive oxygen species, neutrophil NADPH oxidase helps to understand neutrophil dysfunction, a clinically relevant is required for maximal release of DNA, MPO, and HNE after mechanism of CF airway disease. by guest on September 26, 2021 Pseudomonas challenge. This suggests that NADPH oxidase– dependent NET formation stimulated by Pseudomonas occurs in Acknowledgments CF airways. The need for NADPH oxidase to mediate NET We thank the staff of the UGA Health Center: Angela Standridge and formation could depend on the nature of the stimuli (54). The Vickie Cromer for phlebotomy; and Adam Davis, Houston Taylor, and observation that DPI blocks DNA/NET release in Pseudomonas- Dr. Ronald L. Forehand for their continuous support. We thank Sandra treated neutrophils suggests that DPI treatment can distinguish Anaya-O’Brien for CGD patient recruitment and Dr. Douglas Kuhns between NET-dependent and -independent release mechanisms for archived patient data (National Institutes of Health, National Institute (Fig. 5). Incubation with DPI inhibited a major part of both MPO of Allergy and Infectious Diseases). We are grateful to Drs. Brian Condie and HNE release (Fig. 5F–H), suggesting that NETs are the main and Kristina Buac (Department of Genetics, UGA) for providing access mechanism of neutrophil inflammatory marker release. to and help with the AxioZeiss fluorescence microscope. We also thank Our previous data indicate that NET formation triggered by Dr. Joanna B. Goldberg (Emory University) for providing the GFP- expressing PAO1 and the PA2192 strains. calcium pyrophosphate dihydrate crystals requires an intact cy- toskeleton (32). Similarly, bacteria represent microscopic particles that neutrophils routinely phagocytose. The observation that cy- Disclosures tochalasin D inhibits Pseudomonas-stimulated superoxide pro- The authors have no financial conflicts of interest. duction and NET formation further suggests that NET release requires an intact cytoskeleton (Fig. 6). Cytochalasin B treatment References has a similar effect on Staphylococcus aureus–triggered NET 1. Kim Chiaw, P., P. D. Eckford, and C. E. Bear. 2011. Insights into the mechanisms formation (55). It is not clear whether of microbes underlying CFTR channel activity, the molecular basis for cystic fibrosis and is required or not for microbe-stimulated NET formation. One strategies for therapy. Essays Biochem. 50: 233–248. 2. Ciofu, O., C. R. Hansen, and N. Høiby. 2013. Respiratory bacterial infections in school of thoughts argues that neutrophils kill microbes either by cystic fibrosis. Curr. Opin. Pulm. 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