Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation

Abstract

Cystic fibrosis (CF) airways are characterized by bacterial infections, excess mucus production, and robust neutrophil recruitment. The main CF airway pathogen is Pseudomonas aeruginosa. Neutrophils are not capable of clearing the infection. Neutrophil primary granule components, myeloperoxidase (MPO) and human neutrophil elastase (HNE), are inflammatory markers in CF 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. Bacteria 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 released. P. aeruginosa induces a robust respiratory burst in neutrophils that is required for extracellular DNA release. Inhibition of the cytoskeleton prevents Pseudomonas-initiated superoxide production and DNA release. NADPH oxidase inhibition suppresses 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.

FIGURE 1. Pseudomonas aeruginosa induces extracellular DNA release in human neutrophils

(A) Adherent neutrophils were incubated with GFP-expressing Pseudomonas aeruginosa PAO1 (3hrs). Sytox Orange extracellular DNA-binding dye was added and unfixed samples were imaged with a fluorescence microscope (one representative experiment, n=4). Original magnification ×1000 (insets, ×4000) 10 MOI. (B) Kinetics of Pseudomonas 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, 3hrs, normalized to unstimulated control as 100%). Mean±SEM, n=5. D) DNA release from neutrophils upon PA14 exposure (3 hrs) was quantitated using Sytox Orange and calf thymus DNA serial dilutions. n=3. E) Dose-dependent DNA release in human neutrophils exposed to Pseudomonas aeruginosa strains PAO1, PA 10145 and PA2192 (0–30 MOI, 3hrs, Sytox Orange fluorescence, mean+/−S.E.M., n=3). F) Numbers of NET-associated (NET-linked) and NET-independent (NET-free) living PA14 bacteria were assessed after 3 hrs of incubation with neutrophils at two different doses (10 and 50 MOI) according to details found in Methods section. Mean+/−S.E.M., n=3.

Figure 2. NETs stimulated by Pseudomonas aeruginosa contain citrullinated histone H4, myeloperoxidase and human neutrophil elastase

A) MPO (green) and H4cit3 (red) co-localize with extracellular DNA in PA14-induced NETs. Adherent human neutrophils were stimulated by PA14 (50:1 MOI), 100 nM PMA or were left unstimulated (3hrs). Neutrophils were fixed and stained with anti-MPO-FITC antibody or anti-H4cit3 primary and donkey anti-rabbit 594 secondary antibodies. DNA was counterstained by DAPI. One representative experiment, n=5. Original magnification ×400. B) Localization of HNE in PA14- or PMA-stimulated human neutrophils was detected by immunofluorescence (rabbit anti-HNE primary antibody followed by donkey anti-rabbit 594-linked secondary antibody). Both intracellular and extracellular DNA was stained with DAPI. One representative measurement, n=4. Original magnification ×400. C) Inset of the HNE immunostaining (B) reveals fine details of HNE localization in NETs after PA14 exposure (gray arrowheads). As expected, HNE did not overlap with intact nuclear DNA within resting neutrophils (white arrowheads). Representative images, n=5. Original magnification ×2000.

Figure 3. MPO and HNE are detected in neutrophil supernatants after Pseudomonas aeruginosa challenge

Adherent human neutrophils were exposed to Pseudomonas aeruginosa PA14 (50 MOI) for 2.5 to 3 hrs. Cell supernatants were assayed for MPO release, HNE release and peroxidase activity according to the Materials and Methods. (A) PA14-induced MPO release in neutrophils measured by ELISA. Mean+/−S.E.M., 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+/−S.E.M., n=8. (C) HNE concentrations were measured by ELISA in Pseudomonas-exposed neutrophil supernatants. Mean+/−S.E.M., n=8. Mean+/−S.E.M., n=8. Significance was evaluated by student’s two tailed T-test (**P<0.01, ***P<0.001)

Figure 4. Cystic fibrosis isolates of Pseudomonas aeruginosa stimulate release of DNA, MPO and HNE from human neutrophils

Attached neutrophils were incubated with twenty P. aeruginosa CF isolates (10 MOI, 3hrs). Supernatants were assayed for release of extracellular DNA (A), MPO (B) and HNE (C). DNA release was measured by Sytox Orange fluorescence. MPO and HNE releases were quantitated by ELISA. Mean+/−S.E.M., 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. In D–F each dot represents a CF isolate of P. aeruginosa. Pearson correlation coefficient (r).

Figure 5. The NADPH oxidase is required for Pseudomonas-stimulated NET formation, MPO and HNE release

Respiratory burst activity of neutrophils was measured by Diogenes luminescence superoxide assay. (A) Pseudomonas aeruginosa PA14 induces superoxide production in adherent human neutrophils in a dose-dependent manner (1 to 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 (10μM). Inhibitor was added 15 min prior to stimulation. PA14:neutrophil MOI=10. Integrated chemiluminescence for 90 min is shown. Mean+/−S.E.M., n=4. (C) X-CGD neutrophils do not respond with superoxide production to Pseudomonas aeruginosa PA14 or PMA (100 nM). Experimental data obtained on both patients are shown. Mean+/−S.D. D) Extracellular DNA release (increase in Sytox Orange fluorescence, 3 hrs) from human neutrophils induced by Pseudomonas aeruginosa PA14 (10 MOI) is inhibited by DPI (10 μM). Mean+/−S.E.M., n=5 (E) CGD neutrophils (X-CGD, left; p47-deficient, right) do not release DNA in response to Pseudomonas aeruginosa (PA14 or PAO1). One experiment/CGD donor, assays were performed in triplicates. Mean+/−S.D. Pseudomonas aeruginosa PA14-induced MPO release (F), peroxidase activity (G) and HNE secretion (H) were inhibited by 10 μM DPI. Mean +/−S.E.M., n=8. Significance was evaluated by student’s two tailed T-test (*P<0.05, ***P<0.001)

Figure 6. Cytochalasin-D inhibits Pseudomonas-stimulated respiratory burst and DNA release

A) Human neutrophils were stimulated with PA14 (10 MOI, 3 hrs) in the presence or absence of 10 μM cytochalasin-D. Superoxide production was measured with Diogenes superoxide-specific chemiluminescence kit. Mean+/−S.E.M., 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 hrs) in presence of 10 μM cytochalasin-D. PA14-stimulated values are shown after subtraction of background activity. Values are expressed as % of maximal DNA release. Mean+/−S.E.M., n=3. Student’s t-test, *P<0.05, **P<0.01.

Figure 7. Minimal contribution of MEK/ERK signaling to Pseudomonas-induced DNA release

(A) Human neutrophils were exposed to PA14 (50 MOI) for 3hrs in the presence or absence of U0126 (25 μM) and the following parameters were measured according to the detailed descriptions found in the Methods section: extracellular DNA release (Sytox Orange, n=5), superoxide production (Diogenes, n=4), MPO release (ELISA, n=8), peroxidase activity (Amplex Red, n=8) and HNE release (ELISA, n=8). Mean+/−S.E.M. Student’s t-test (B) Enzymatic activity of 500 μM 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 normalized on the inhibitor-free sample (mean+/−S.D., n=2). (C) Neutrophils were exposed to PA14 (50 MOI) for 3hrs in the presence or absence of MEK-162 (50 μM) or PD98059 (20 μM) 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+/−S.E.M. ANOVA, Tukey’s post-hoc analysis. *P<0.05, **P<0.01, ***P<0.001.