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. 2009;1(6):527-42.
doi: 10.1159/000235860. Epub 2009 Aug 27.

DNase I inhibits a late phase of reactive oxygen species production in neutrophils

Daniela B Munafo  1 , Jennifer L Johnson, Agnieszka A Brzezinska, Beverly A Ellis, Malcolm R Wood, Sergio D Catz
Affiliations

DNase I inhibits a late phase of reactive oxygen species production in neutrophils

"VSports手机版" Daniela B Munafo et al. J Innate Immun. 2009.

Abstract

Neutrophils kill bacteria on extracellular complexes of DNA fibers and bactericidal proteins known as neutrophil extracellular traps (NETs). The NET composition and the bactericidal mechanisms they use are not fully understood. Here, we show that treatment with deoxyribonuclease (DNase I) impairs a late oxidative response elicited by Gram-positive and Gram-negative bacteria and also by phorbol ester. Isoluminol-dependent chemiluminescence elicited by opsonized Listeria monocytogenes-stimulated neutrophils was inhibited by DNase I, and the DNase inhibitory effect was also evident when phagocytosis was blocked, suggesting that DNase inhibits an extracellular mechanism of reactive oxygen species (ROS) generation. The DNase inhibitory effect was independent of actin polymerization VSports手机版. Phagocytosis and cell viability were not impaired by DNase I. Immunofluorescence analysis shows that myeloperoxidase is present on NETs. Furthermore, granular proteins were detected in NETs from Rab27a-deficient neutrophils which have deficient exocytosis, suggesting that exocytosis and granular protein distribution on NETs proceed by independent mechanisms. NADPH oxidase subunits were also detected on NETs, and the detection of extracellular trap-associated NADPH oxidase subunits was abolished by treatment with DNase I and dependent on cell stimulation. In vitro analyses demonstrate that MPO and NADPH oxidase activity are not directly inhibited by DNase I, suggesting that its effect on ROS production depends on NET disassembly. Altogether, our data suggest that inhibition of ROS production by microorganism-derived DNase would contribute to their ability to evade killing. .

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Figures

Fig. 1
Fig. 1
ROS production is attenuated by DNase I. Human neutrophils were stimulated with opsonized E. coli (a) or HKLM (b)in the presence or absence of 100 U/ml DNase I. ROS production was measured as luminol-dependent chemiluminescence as described in Materials and Methods. Kinetic results are the mean ± SEM of 1 representative experiment performed in triplicate (left panels). Unstimulated samples showed no significant luminescence (data not shown). The right panels show the maximum chemiluminescence at the indicated time. Results are the mean ± SEM of 3 independent experiments with samples from different donors. Maximum chemiluminescence in samples treated with DNase I at 75 min but not at 30 min was significantly different from control (* p<0.05). c E. coli and L. monocytogenes stimulate NET formation. Human neutrophils were seeded into 96–well plates and stimulated with non-opsonized heat-killed E. coli or HKLM or with the same bacteria previously opsonized with autologous serum (Ops.). Experiments were carried out in the absence (grey columns) or in the presence (white columns) of 100 U/ml DNase I for 1 h at 37°C. Next, the non-cell-permeant DNA-binding dye Sytox Green was added to a final concentration of 5 μM, and NET formation was evaluated fluorometrically with a setting of 485 nm (excitation) and 527 nm (emission). Cells incubated in the absence of bacteria and DNase I were used as control (C). d Phagocytosis in the presence of DNase I (100 U/ml) or cytochalasin D (Cyto D) was measured using fluorescently labeled E. coli as described in Materials and Methods. e Luminol-dependent chemiluminescence in response to PMA was monitored for 2 h in DNase-I-treated or –untreated human neutrophils. Kinetic results are the mean ± SEM of 3 independent experiments, each of them performed in duplicate or triplicate. The right panel shows the maximum chemiluminescence at the indicated times. Results are the mean ± SEM of 3 independent experiments with samples from different donors. * p<0.05. f The kinetics of NET formation in response to PMA stimulation was followed using Sytox Green as described above. g ROS production by HL–60 cells differentiated to granulocytes was monitored by luminol-dependent chemiluminescence after preincubation in the absence or presence of 100 U/ml DNase I. The cells were preincubated in the absence or presence of LPS (100 ng/ml) and stimulated with 1 μM fMLF. Data are represented as the mean ± SD from duplicate wells. h Isoluminol, a cell-impermeant analog of luminol, was used to analyze the effect of DNase I on opsonized HKLM-induced extracellular chemiluminescence. Circles indicate the basal luminescence level in the absence of HKLM. Results are represented as the mean ± SD of duplicates from 1 experiment representative of 2 experiments with similar results. RFU = Relative fluorescence unit; RLU = relative light unit.
Fig. 2
Fig. 2
The inhibitory effect of DNase I on ROS production is independent of actin depolymerization and phagocytosis. Human neutrophils were stimulated with non-opsonized (non-ops.; a, b) or opsonized (ops.; c, d) HKLM in the presence (▴) or absence (▪) of 100 U/ml DNase I. ROS production was measured as luminol-dependent chemiluminescence as described in figure 1 except that, where indicated, cells where preincubated in the presence of 10 μg/ml cytochalasin D for 10 min before the addition of bacteria. Kinetic results are the mean ± SD from 1 experiment representative of 2 independent experiments. Unstimulated samples showed no significant luminescence (data not shown). RLU = Relative light unit.
Fig. 3
Fig. 3
Localization of MPO on NETs in wild-type and Rab27a-deficient neutrophils. Neutrophils from wild-type (WT) or ashen (Rab27a/) mice were stimulated with PMA (100 ng/ml) for 60 min, and MPO was detected on NETs by immunofluorescence confocal microscopy analysis as described in Materials and Methods. Upper and middle panels: MPO localization on NETs detected using the DNAbinding fluorescent probe DAPI in wild-type and Rab27a-deficient neutrophils. Lower panels: MPO staining in a cell that is not forming NETs is shown. Control antibodies do not recognize structures on NETs when used under the same experimental conditions. Scale bars = 5 μm.
Fig. 4
Fig. 4
Localization of NADPH oxidase subunits on NETs. Neutrophils were stimulated with PMA (100 ng/ml), LPS (100 ng/ml) or HKLM for 90 min, and the NADPH oxidase subunits were detected on NETs by immunofluorescence confocal microscopy analysis as described in Materials and Methods. a Immunofluorescence analysis showing that p22phox is present on extracellular DNA fibers in fields where there is significant NET formation in neutrophils stimulated with phorbol ester (upper panels). The lower panels show a higher magnification of a field where p22phox can be detected in punctate structures on NETs. Arrows indicate p22phox-specific staining on NETs; arrowheads indicate p22phox-specific staining on intact neutrophils. Scale bars = 10 μm. b The upper and middle panels show localization of p67phox and p22phox, respectively, on NETs detected using DAPI. NADPH oxidase subunits were detected in close proximity to the ectosome marker CD11b. Some of these structures are indicated with arrows. The lower panels show the magnification of the area highlighted on the middle panels. Scale bars = 10 μm. c Control antibodies do not recognize structures on NETs when used under the same experimental conditions. Scale bar = 10 μm. d Immunofluorescence analysis shows that the cytosolic subunit p47 phox and the cytochrome b558 component p22phox colocalize on punctate structures distributed on NETs. Some of these structures are indicated with arrows. Neither NETs nor NADPH oxidase subunits were detected extracellularly in the absence of stimuli. Scale bars = 10 μm. e Immunofluorescence analysis of the distribution of NADPH oxidase subunits on NETs in neutrophils stimulated with 100 ng/ml LPS for 90 min in the absence (upper panels) or presence (lower panels) of 100 U/ml DNase I. Scale bars = 10 μm.
Fig. 4
Fig. 4
Localization of NADPH oxidase subunits on NETs. Neutrophils were stimulated with PMA (100 ng/ml), LPS (100 ng/ml) or HKLM for 90 min, and the NADPH oxidase subunits were detected on NETs by immunofluorescence confocal microscopy analysis as described in Materials and Methods. a Immunofluorescence analysis showing that p22phox is present on extracellular DNA fibers in fields where there is significant NET formation in neutrophils stimulated with phorbol ester (upper panels). The lower panels show a higher magnification of a field where p22phox can be detected in punctate structures on NETs. Arrows indicate p22phox-specific staining on NETs; arrowheads indicate p22phox-specific staining on intact neutrophils. Scale bars = 10 μm. b The upper and middle panels show localization of p67phox and p22phox, respectively, on NETs detected using DAPI. NADPH oxidase subunits were detected in close proximity to the ectosome marker CD11b. Some of these structures are indicated with arrows. The lower panels show the magnification of the area highlighted on the middle panels. Scale bars = 10 μm. c Control antibodies do not recognize structures on NETs when used under the same experimental conditions. Scale bar = 10 μm. d Immunofluorescence analysis shows that the cytosolic subunit p47 phox and the cytochrome b558 component p22phox colocalize on punctate structures distributed on NETs. Some of these structures are indicated with arrows. Neither NETs nor NADPH oxidase subunits were detected extracellularly in the absence of stimuli. Scale bars = 10 μm. e Immunofluorescence analysis of the distribution of NADPH oxidase subunits on NETs in neutrophils stimulated with 100 ng/ml LPS for 90 min in the absence (upper panels) or presence (lower panels) of 100 U/ml DNase I. Scale bars = 10 μm.
Fig. 4
Fig. 4
Localization of NADPH oxidase subunits on NETs. Neutrophils were stimulated with PMA (100 ng/ml), LPS (100 ng/ml) or HKLM for 90 min, and the NADPH oxidase subunits were detected on NETs by immunofluorescence confocal microscopy analysis as described in Materials and Methods. a Immunofluorescence analysis showing that p22phox is present on extracellular DNA fibers in fields where there is significant NET formation in neutrophils stimulated with phorbol ester (upper panels). The lower panels show a higher magnification of a field where p22phox can be detected in punctate structures on NETs. Arrows indicate p22phox-specific staining on NETs; arrowheads indicate p22phox-specific staining on intact neutrophils. Scale bars = 10 μm. b The upper and middle panels show localization of p67phox and p22phox, respectively, on NETs detected using DAPI. NADPH oxidase subunits were detected in close proximity to the ectosome marker CD11b. Some of these structures are indicated with arrows. The lower panels show the magnification of the area highlighted on the middle panels. Scale bars = 10 μm. c Control antibodies do not recognize structures on NETs when used under the same experimental conditions. Scale bar = 10 μm. d Immunofluorescence analysis shows that the cytosolic subunit p47 phox and the cytochrome b558 component p22phox colocalize on punctate structures distributed on NETs. Some of these structures are indicated with arrows. Neither NETs nor NADPH oxidase subunits were detected extracellularly in the absence of stimuli. Scale bars = 10 μm. e Immunofluorescence analysis of the distribution of NADPH oxidase subunits on NETs in neutrophils stimulated with 100 ng/ml LPS for 90 min in the absence (upper panels) or presence (lower panels) of 100 U/ml DNase I. Scale bars = 10 μm.
Fig. 5
Fig. 5
Lack of inhibition of NADPH oxidase and MPO by DNase I treatment. a The NADPH oxidase total recombinant system was used to evaluate a possible effect of DNase I on the oxidase activity. To this end, recombinant proteins were incubated for 5 min to allow the assembly of the oxidase and were subsequently incubated in the presence of 100 U/ml DNase IA (New England Biolab) and DNase IB (Worthington) in their corresponding reaction buffers, in the absence of DNase I and DNase buffer (control) or in the presence of superoxide dismutase (SOD) for 10 min at 37°C. The production of superoxide anion was continuously monitored by cytochrome c reduction at 550 nm. The results (mean ± SEM) are representative of 2 independent experiments performed in triplicates. b Human MPO (60 ng/ml) was incubated in the presence or absence of DNase I (100 U/ml) for 1 h at 37°C. MPO activity was measured as described in Materials and Methods. The results are the mean ± SEM of 3 independent experiments performed in triplicates. No significant differences were found between DNase-I-treated and –untreated samples.
Fig. 6
Fig. 6
Transmission electron microscopy analysis of NETs. Human neutrophils were seeded in untreated 35-mm culture dishes and treated with 100 ng/ml LPS (a–f) or PMA (g, h) for 90 min at 37°C and then processed for transmission electron microscopy as described in Materials and Methods. a The arrows indicate NETforming cells with undetectable nuclear envelope or condensed chromatin. b Magnification of the area selected in a. The arrow shows partial preservation of the plasma membrane. c–f NETs usually contain vesicular membranes (arrowhead in e) and granular structures (arrow in e). d–f Magnifications of the areas selected in c–e, respectively. f Extracellular DNA fibers appear as a filamentous material (arrowheads) containing granular structures (arrows). g, h Analysis of cross-sections of NETs showing that the characteristic filamentous material adjacent to a PMAstimulated neutrophil (g) is completely dismantled by treatment with DNase I (h).
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