Pyocyanin-Enhanced Neutrophil Extracellular Trap
Formation Requires the NADPH Oxidase
Bala ´zs Rada1,2*, Meghan A. Jendrysik1, Lan Pang2, Craig P. Hayes2, Dae-goon Yoo2, Jonathan J. Park1,
Samuel M. Moskowitz3,4, Harry L. Malech1, Thomas L. Leto1*
1Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America,
2Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America, 3Department of Pediatrics,
Massachusetts General Hospital, Boston, Massachusetts, United States of America, 4Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United
States of America
Beyond intracellular killing, a novel neutrophil-based antimicrobial mechanism has been recently discovered: entrapment
and killing by neutrophil extracellular traps (NETs). NETs consist of extruded nuclear DNA webs decorated with granule
proteins. Although NET formation is an important innate immune mechanism, uncontrolled NET release damages host
tissues and has been linked to several diseases including cystic fibrosis (CF). The major CF airway pathogen Pseudomonas
aeruginosa establishes chronic infection. Pseudomonas imbedded within biofilms is protected against the immune system,
but maintains chronic inflammation that worsens disease symptoms. Aberrant NET release from recruited neutrophils was
found in CF, but the underlying mechanisms remain unclear. One of the most important Pseudomonas virulence factors is
pyocyanin, a redox-active pigment that has been associated with diminished lung function in CF. Here we show that
pyocyanin promotes NET formation in a time- and dose-dependent manner. Most CF Pseudomonas clinical isolates tested
produce pyocyanin in vitro. Pyocyanin-derived reactive oxygen species are required for its NET release. Inhibitor
experiments demonstrated involvement of Jun N-terminal Kinase (JNK) and phosphatidylinositol 3-Kinase (PI3K) in
pyocyanin-induced NET formation. Pyocyanin-induced NETs also require the NADPH oxidase because NET release in chronic
granulomatous disease neutrophils was greatly reduced. Comparison of neutrophils from gp91phox- and p47phox-deficient
patients revealed that pyocyanin-triggered NET formation is proportional to their residual superoxide production. Our
studies identify pyocyanin as the first secreted bacterial toxin that enhances NET formation. The involvement of NADPH
oxidase in pyocyanin-induced NET formation represents a novel mechanism of pyocyanin toxicity.
Citation: Rada B, Jendrysik MA, Pang L, Hayes CP, Yoo D-g, et al. (2013) Pyocyanin-Enhanced Neutrophil Extracellular Trap Formation Requires the NADPH
Oxidase. PLoS ONE 8(1): e54205. doi:10.1371/journal.pone.0054205
Editor: Amit Gaggar, University of Alabama-Birmingham, United States of America
Received August 10, 2012; Accepted November 26, 2012; Published January 14, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by funds to TLL from the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and
Infectious Diseases (ZO1-AI-000614), by Public Health Service grants K08HL067903 to SMM from the National Heart Lung and Blood Institute and R01AI067653 to
SMM from the National Institute of Allergy and Infectious Diseases and by grant MOSKOW01A0 to SMM from the CF Foundation. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (TLL); firstname.lastname@example.org (BR)
Polymorphonuclear neutrophil granulocytes (PMN) provide the
first line of defense against bacteria. Neutrophils migrate to the site
of infection, engulf and kill the invaders by exposing them to
a variety of antimicrobial peptides, proteins and reactive oxygen
species. Neutrophils also combat pathogens by a recently de-
scribed novel mechanism, formation of neutrophil extracellular
traps (NETs). NETs are composed of a DNA backbone decorated
with histones and several antimicrobial neutrophil granule
components: myeloperoxidase (MPO), lactoferrin, elastase and
bactericidal/permeability-increasing protein.  NETs kill bacte-
ria in vitro by ensnaring the microbes within high local concentra-
tions of the neutrophils’ weaponry. Recent live imaging of in vivo
NET formation in an acute bacterial skin infection model
provided further evidence for the importance of NETs in
immunity.  NET formation (NETosis) is a novel form of
neutrophil cell death different from apoptosis or necrosis. [3,4]
The mechanisms triggering NETs are poorly understood and are
under investigation. Bacteria (whole cells, LPS, pilus) or in-
flammatory mediators (IL-8, IFN I+II, C5a) have been reported to
induce NETs.  Reactive oxygen species (ROS) produced by the
phagocytic NADPH oxidase are essential for NET formation,
since neutrophils of chronic granulomatous disease (CGD) patients
are unable to release NETs in response to a variety of stimuli. 
CGD neutrophils produce very little or no superoxide due to
genetic deficiencies in any one of several components of the
superoxide-producing NADPH oxidase enzyme complex .
NETs are a crucial part of antimicrobial innate immunity;
however, accumulating evidence suggests that uncontrolled NET
release can also correlate with disease severity.  Aberrant NET
formation has already been implicated in a variety of diseases
including systemic lupus erythematosus, autoimmune small-vessel
vasculitis and cystic fibrosis (CF). [8–10] Cystic fibrosis is
a common inherited life shortening disease among Caucasians.
 The primary cause of the disease is a genetic defect in the
cystic fibrosis transmembrane conductance regulator (CFTR)
protein, a cAMP-regulated anion channel expressed in several
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organs including the airways, pancreas and sweat glands.  The
absence of normal CFTR in airways leads to altered ion transport
across epithelial cells, thicker mucus and hindered microbial
mucociliary clearance giving rise to chronic bacterial infections.
 The most common Gram-negative pathogen infecting the
airways of CF patients is Pseudomonas aeruginosa.  The bacterium
colonizes CF airways early in life and establishes chronic infection,
which is the major cause of death in CF.  In healthy airways
Pseudomonas aeruginosa hardly ever causes problems and neutrophils
play an important role in clearance of Pseudomonas.  In CF
airways, however, persistent Pseudomonas infections are charac-
terized by biofilm growth, which protects bacteria from both
opsonization and access of neutrophils to phagocytose and kill
them. The established presence of bacteria maintains chronic
inflammation resulting in mucus hypersecretion and robust
neutrophil infiltration through production of virulence factors
such as pyocyanin .
The exotoxin pyocyanin is an important virulence factor of
Pseudomonas aeruginosa; its induction through quorum signaling
correlates with the biofilm growth stage of the bacterium
accounting for the high concentrations found in CF patients’
airways. [16–19] Pseudomonas virulence is seriously diminished in
the absence of pyocyanin production in mouse models. 
Pyocyanin concentrations in CF sputum samples correlate with
decreased lung function and its rate of decline.  Although
pyocyanin has a wide range of toxic effects, the proposed basis for
its toxicity is production of superoxide anions and downstream
ROS inside of cells by oxidizing NAD(P)H. [21–23] This depletion
of intracellular NADPH reserves supporting intracellular oxidant
production imposes oxidative stress on host cells. In neutrophils,
pyocyanin has been shown to lower NADPH levels and inhibit
killing of Staphylococcus aureus, but its effect on NET formation has
never been studied [24,25].
Pseudomonas embedded within biofilms in CF airways is well-
protected against the attack of recruited neutrophils, which can
release their antimicrobial load into the airway lumen and
contribute to tissue damage.  Pulmonary function of CF
patients (FEV1) has been negatively correlated with sputum
concentrations of DNA, myeloperoxidase (MPO) and autoanti-
bodies against bactericidal-permeability increasing protein, all of
which are neutrophil components that could be derived from
NETs. [27,28] Human recombinant DNAse treatment has been
shown to improve mucociliary clearance within CF patients’
airways.  Pyocyanin has been shown to promote DNA release
from Pseudomonas aeruginosa.  NETs were detected in CF
patients’ sputa.  However, induction of NETs by Pseudomonas
aeruginosa has not been characterized to date and nothing is known
about signaling mechanisms or virulence factors participating in
Pseudomonas-stimulated NET formation.
Here, we aimed at characterizing pyocyanin-elicited NET
formation in adherent neutrophils. We investigated the effects of
pyocyanin at concentrations reported in CF airways [18,19]. Our
data identify pyocyanin as a novel NET inducer that requires the
NADPH oxidase for its action. Our findings suggest that NET
induction by pyocyanin contributes to the inflammatory condi-
tions found in CF airways.
Pyocyanin Induces NET Formation
NET formation induced by bacteria requires ROS but it is
unknown which microbial factor(s) mediate this process.  Since
ROS originating from NADPH oxidase-independent sources
(bolus H2O2, glucose oxidase (GO)) are capable of inducing
NETs, we tested whether pyocyanin, a redox-active exotoxin of
Pseudomonas, could affect NET formation.  Pyocyanin is
essential for full virulence in a variety of Pseudomonas infection
models.  One study found that sputa of three out of 4 CF
patients contained pyocyanin at levels equal to or higher than
those used in our study.  Another recent study reported high
pyocyanin levels in CF patients’ airways which negatively
correlated with lung function, clearly indicating that pyocyanin
is an important contributor to CF airway pathology.  To assess
the importance of pyocyanin production in Pseudomonas airway
infection in CF we examined in vitro pyocyanin production by
40 CF clinical isolates of Pseudomonas aeruginosa. Pyocyanin
concentrations were measured in the culture supernatants of
stationary phase cultures and compared to the laboratory control
strain PA14. (Fig. 1). Most of the clinical isolates produced
pyocyanin, only six of the forty isolates did not produce the toxin
(Fig. 1). Clinical isolates obtained from CF patients with mild or
severe disease and obtained from early or late phases of the disease
(from the same 17 patients) were compared. A wide range of
production capabilities was detected from these isolates and no
trends in pyocyanin production were observed. Average pyocyanin
concentrations were (mean +/2 SD, mM): mild CF (17.5+/
227.26), severe CF (24.2+/231.01), early isolates (22.1+/
231.52), late isolates (19.7+/226.57).
Next, we examined whether the purified toxin itself is capable of
inducing NETs. Adherent neutrophils were exposed to 20 mM
pyocyanin for 3 hrs and cells were stained simultaneously with the
membrane impermeable DNA dye Sytox Orange or the
membrane-permeable DNA stain Sytox Green. We found that
PMNs released NETs in response to pyocyanin (Fig.2A).
Extracellular DNAse treatment degraded pyocyanin-induced
NETs (Fig. 2A). A negative black and white image of DAPI-
stained NETs in pyocyanin-treated PMNs reveals fine, detailed
DNA structure (Fig. 2B). In a concentration range characteristic
for CF patients’ airways (0–30 mM), pyocyanin induces NETs in
a time- and dose-dependent manner (Fig. 2C). [18,19] To
appreciate the contribution of pyocyanin to NET formation
induced by Pseudomonas aeruginosa, we compared NET release by
the pyocyanin-secreting PA14 wild type strain and its pyocyanin
(PhzM)-deficient mutant. Pyocyanin deficiency results in a 58.6+/
234.3% reduction in NET release induced by Pseudomonas
aeruginosa (Fig. 2D). Furthermore, addition of the purified toxin
to wild-type Pseudomonas increases NET release and superoxide
production in a dose-dependent manner (Fig. 2E). In NETs
granule components are attached to DNA fibers.  Deimination
of certain arginine residues into citrullin in histones by
peptidylarginine deiminase 4 (PAD4) has been shown to be
required for NET formation. [31–33] To show co-localization of
granule components and histones with DNA, we co-stained
pyocyanin-treated neutrophils for myeloperoxidase (MPO) or
citrullinated histone H4 (citH4) and DNA (DAPI). Both, MPO
and citH4 staining co-localized with DNA staining in pyocyanin-
treated PMNs (Fig. 3A–B).
ROS are Required for Pyocyanin-triggered NETs
Although pyocyanin has been shown to cause a broad range of
toxic effects in different host cells, its diverse toxicity originates
from production of ROS.  Pyocyanin lowers intracellular
NADPH levels in neutrophils as it generates intracellular ROS.
 We therefore asked if NETs induced by pyocyanin are
mediated by ROS. Using the Diogenes-based superoxide detection
method we found that pyocyanin enhanced superoxide release by
adherent neutrophils in a dose-dependent manner (Fig. 4A, B). We
next found that pretreatment of neutrophils with the ROS
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scavengers N-acetyl-cysteine (NAC, 10 mM in HBSS) blocked
both spontaneous and toxin-elicited NET release (Fig. 4C, D).
Both basal and pyocyanin-elicited superoxide productions were
abolished by NAC pre-treatment (Fig. 4E). We conclude that ROS
are required for pyocyanin to induce NETs.
Jun N-terminal Kinase (JNK) and Phosphatidylinositol 3-
kinase (PI3K) Mediate NET Release Induced by Pyocyanin
The signaling mechanisms behind NETosis are still largely
unexplored although the Raf-MEK-ERK signaling pathway has
already been implicated.  To gain insight into the signaling
steps of pyocyanin-elicited NET formation, we surveyed different
pathway inhibitors. We found that the JNK inhibitor (SB600125)
and the PI3K inhibitor (wortmannin) exhibited strong inhibition
(SB600125: 71.7%, wortmannin: 51.5%; mean, n=4) on
pyocyanin-stimulated NET release whereas inhibition of MEK1
and p38 had no significant effects (Fig. 4F).
Pyocyanin Enhances Superoxide Production in
Neutrophils by Activating the NADPH Oxidase
Pyocyanin oxidizes reduced NADH or NADPH and produces
superoxide under aerobic conditions (Fig. 5A). NAD(P)H oxida-
tion by pyocyanin is insensitive to the NADPH oxidase inhibitor
DPI in a cell-free in vitro system (Fig. 5A). In host cells, pyocyanin
crosses the plasma membrane, oxidizes intracellular NAD(P)H
pools and produces ROS. We therefore hypothesized that
pyocyanin produces superoxide in PMNs in an NADPH
oxidase-independent manner and that DPI would have no effect.
The PMA-stimulated superoxide response was blocked by DPI as
expected, but to our surprise DPI also entirely inhibited the
pyocyanin-elicited response (Fig. 5B). This indicates that pyocya-
nin is acting through a flavoenzyme in intact PMNs, most
probably the NADPH oxidase. To test if pyocyanin-enhanced
superoxide production is NADPH oxidase-dependent, we com-
pared normal and X-CGD neutrophils by using three different
indicators. DCF-DA oxidation measures low amounts of in-
tracellular ROS, the insensitive lucigenin-based chemilumines-
cence detects mainly extracellular superoxide, whereas the
fluorescent dye MitoSoxRed detects mitochondrial superoxide.
Both DCF-DA and MitoSoxRed detected a small but significant
intracellular ROS signal in both normal and X-CGD PMNs
(Fig. 5C, D). These data are consistent with the fact that pyocyanin
reacts with intracellular NAD(P)H directly. Using lucigenin the
detected basal superoxide production in normal PMNs was further
increased by pyocyanin (Fig. 5E). In contrast, in X-CGD PMNs
basal superoxide release detected with lucigenin was completely
absent and pyocyanin failed to increase it (Fig. 5E). To gain insight
into the requirement of intracellular and/or extracellular ROS
production for pyocyanin-induced NETs, we next measured
superoxide production and NET release in the presence of the
extracellular ROS scavengers, catalase and superoxide dismutase
(SOD). Catalase- and SOD-treatment entirely blocked pyocyanin-
stimulated (but not basal) ROS production and NET release
(Fig. 5F). These data indicate that although pyocyanin produces
superoxide by direct oxidation of NAD(P)H, extracellular super-
oxide mediating NET release in pyocyanin-exposed neutrophils
comes from the activated NADPH oxidase.
CGD Neutrophils Show Impaired NET Formation in
Response to Pyocyanin
Next we studied the question of whether the NADPH oxidase
mediates pyocyanin-stimulated NET formation as well. To address
this we exposed adherent PMNs obtained from four different
CGD patients (patient 1,3,4, and 5) and healthy individuals to 0–
30 mM pyocyanin, PMA or GO for 3.5 hrs and measured NET
release (Fig. 6A–C). A longer time interval was chosen to detect
spontaneous NET formation in CGD PMNs. Normal PMNs
released 28.4% NETs without stimulation, which was further
increased up to 44.2% by 30 mM pyocyanin and 46.3% by PMA
(Fig. 6A). Fig. 6B–C show corresponding images of Sytox Orange-
stained normal PMNs at the end of the assay. Three subjects
studied are X-CGD patients while the fourth one (patient 5) is
p47phox-deficient. Although all patients suffer from CGD, their
residual superoxide production is different ranging between 0.42–
1.05% of that of normal controls.  We found that NET
Figure 1. Majority of CF clinical isolates of Pseudomonas aeruginosa produce pyocyanin in vitro. Cystic fibrosis clinical isolates of
Pseudomonas aeruginosa were grown in LB medium for 48 hrs and pyocyanin concentrations in the culture supernatants were determined. Data are
organized according to disease severity of CF patients (mild/severe) or early/late phase origin of the isolates (for details see methods). Data show
mean +/2 S.E.M. of three independent experiments.
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formation among the four patients was different. NET release was
completely undetectable in patient 1 whose cells have the lowest
superoxide production (0.42%) (Fig. 6A). PMNs from patient 4
show minor increase in superoxide production (0.57%) and in
basal (0.9%), PMA-stimulated (1.1%) and pyocyanin-elicited
(2.8%) NET release, as well (Fig. 6A). Strikingly, patient 3 and 5
who have higher residual superoxide production (1.05%, 1.04%)
showed enhanced levels of basal (9.7%, 7.6%) and PMA-
stimulated NET formation (17.9%, 14.6%) (Fig. 6A, C). Patients
1 and 4 did not release NETs in response to any pyocyanin
concentration tested (Fig. 6A). In the case of patient 3, lower
concentrations of pyocyanin did not induce NETs, but the highest
dose (30 mM) made PMNs form NETs (41.1%) (Fig. 6A, B). PMNs
of patient 5 showed increased sensitivity towards pyocyanin, in
which not only 30 mM but also 10 mM pyocyanin induced NETs
(Fig. 6A,). These results indicate that basal, pyocyanin- and PMA-
stimulated NET release in CGD neutrophils is increasing in
relation to residual NADPH oxidase activity.
Figure 2. Pyocyanin induces extracellular DNA release in human neutrophils. A) Detection of NET formation in human neutrophils
stimulated by pyocyanin. Adherent neutrophils were exposed to 20 mM pyocyanin or 100 nM PMA for 3 hours, stained with 2.5 mM Sytox Orange and
5 mM Sytox Green, and NETs were visualized by fluorescence microscopy. Samples in the lower panels were treated with 1 U/mL DNase1 (20 min
37C). Two other experiments resulted in similar data. B) Negative black and white image of pyocyanin-stimulated neutrophils reveals fine structural
details of pyocyanin-triggered NETs. Adherent neutrophils were stimulated with 20 mM pyocyanin in vitro for 3 hrs, stained with DAPI and details of
the NET structures were visualized by fluorescence microscopy. Results were converted to negative black and white images to achieve better contrast
between DNA network and the background. This picture is representative of data obtained on 4 different donors. C) Quantification of NETs induced
by pyocyanin. Neutrophils were incubated for 2.5 hrs with different concentrations of pyocyanin (0–30 mM) in the presence of Sytox Orange (2.5 mM).
Release of extracellular DNA (increase in fluorescence) was followed in time with the fluorescence microplate assay (one representative experiment of
six) (left panel). NET release was (increase in fluorescence in 2.5 hours) quantitated as percentage of maximal. Data represent mean +/2S.E.M. (n=6)
(right panel). D) Human neutrophils were exposed to 26106wild-type (PA14 WT, pyocyanin-producing) and PhzM-deficient (PA14 PhzM, pyocyanin-
deficient) Pseudomonas aeruginosa PA14 for 2.5 hours in the presence of 2.5 mM Sytox Orange. Increase in fluorescence induced by bacteria over
baseline was calculated and expressed as increases in extracellular DNA (% of maximal) (mean+/2S.E.M., n=4). E) Extracellular DNA release was
initiated in adherent neutrophils by co-addition of wild-type Pseudomonas aeruginosa PA14 (26106/well) and increasing concentrations of purified
pyocyanin (0,3,10,30 mM). Sytox Orange fluorescence (extracellular DNA release, 2.5 mM) and Diogenes chemiluminescence (superoxide production,
integrated RLU/60 min) were increased in a dose-dependent manner by pyocyanin (mean+/2S.E.M., n=4). PMA, phorbol 12-myristate-13-acetate.
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Figure 3. Myeloperoxidase and citrullinated histone H4 co-localize with extracellular DNA in pyocyanin-stimulated NETs. A) Human
neutrophils were seeded on glass coverslips and incubated in the presence of 20 mM pyocyanin for 3 hours. Cells were fixed and stained for MPO
(FITC-labeled anti-MPO Ab) and DNA (DAPI). Two independent fields show co-localization of MPO and DNA (one representative experiment, n=3). B)
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Neutrophil extracellular trap formation is a fascinating recently
discovered mechanism by which neutrophils entrap bacteria. 
NETs capture Aspergillus nidulans, Candida albicans, Shigella flexneri,
Staphylococcus aureus, Salmonella typhimurium, nontypeable Heamophilus
influenzae and Group A Streptococci. Recently, Pseudomonas
aeruginosa, the major CF pathogen, has also been shown to trigger
NETs. [35,36] However, Pseudomonas-triggered NET formation
Citrullinated histone H4 co-localizes with NET DNA structures in pyocyanin-stimulated neutrophils. Adherent neutrophils were treated with 20 mM
pyocyanin for 3 hours, then fixed, washed and subjected to anti-citH4 immunostaining and DAPI staining. Two other experiments gave similar data.
Figure 4. Pyocyanin-triggered NET formation involves reactive oxygen species, Jun N-terminal Kinase and Phosphatidylinositol 3-
kinase. A) Representative kinetics of superoxide production in adherent human neutrophils exposed to 1, 3, 10 mM pyocyanin (Diogenes, 60 min,
another donor gave similar data). B) Extracellular superoxide production stimulated by pyocyanin is dose-dependent and abolished by 12.5 mg/ml
superoxide dismutase (sod) (mean+/2S.D. of two different donors, ‘‘–‘‘=untreated). C) NAC blocks pyocyanin-induced NET formation. Human
neutrophils were pretreated with 10 mM NAC for 10 min followed by exposure to 20 mM pyocyanin for 3 hrs. Images of DAPI-stained NETs were
taken with fluorescence microscopy. Similar results were obtained with another donor. D) Quantification of the inhibitory effect of NAC (10 mM) on
pyocyanin-stimulated NET formation by the fluorescence (Sytox Orange) microplate assay. Data are mean+/2 S.E.M. (n=3). E) NAC (10 mM) blocks
both basal and pyocyanin-elicited superoxide production in neutrophils (Lucigenin, Diogenes, mean+/2S.E.M., n=3) F) Inhibitors of JNK (SB600125,
10 mM, p=0.0124)) and PI3K (wortmannin, 100 nM, p=0.0374) suppress pyocyanin-stimulated NET formation whereas p38 and MEK1 pathway
inhibitors were without any effect (10mM PD980589, 10 mM SB203580). Data show mean+/2 S.E.M. (n=4). NAC, N-acetyl-cysteine; RLU, relative
luminescence unit; JNK, Jun N-terminal Kinase; PI3K, Phosphatidylinositol 3-kinase; sod, superoxide dismutase; * marks significant changes were
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Figure 5. The NADPH oxidase is the source of extracellular superoxide mediating NET release in pyocyanin-stimulated human
neutrophils. A) Pyocyanin (50 mM) oxidizes b-NADH (20 mM) and b-NADPH (20 mM) in a cell-free system and produces superoxide anions as
detected by the Diogenes assay. DPI (10 mM) and DMSO (solvent of DPI) have no effect (data are mean+/2 S.E.M., n=3). B) DPI inhibits basal, PMA-
and pyocyanin-stimulated superoxide production in human neutrophils. PMNs were pretreated with 10 mM DPI, stimulated by 20 mM pyocyanin and
superoxide production was measured by the Diogenes assay for 60 min. Concentrations of PMNs were: 106/mL for PMA stimulation and 56106/mL
for pyocyanin. C) Pyocyanin produces low-level intracellular ROS in the absence of the NADPH oxidase. DCFDA-loaded healthy and X-CGD neutrophils
(patient 1) were exposed to 20 mM pyocyanin or 100 nM PMA and intracellular production of reactive oxygen species was measured by flow
cytometry. Similar results were obtained with patient 29s cells. D) Low mitochondrial superoxide levels are detected in both, healthy and X-CGD
neutrophils stimulated by pyocyanin. Healthy or CGD neutrophils (patient #2) loaded with MitoSox Red were exposed to 30 mM pyocyanin for
30 min and mitochondrial superoxide production was measured by flow cytometry. Data were only obtained from one CGD patient. Antimycin A was
used as positive control. E) Dose-dependence of pyocyanin-stimulated superoxide production in normal and X-CGD neutrophils (Lucigenin) (patient 2
and 3). F) Scavenging extracellular ROS (catalase and SOD) eliminates pyocyanin-induced ROS production and NET formation in human neutrophils.
Human neutrophils were exposed to 30 mM pyocyanin in the presence or absence of 1500 U/mL catalase and 12.5 mg/mL SOD. Superoxide
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by adherent neutrophils has not been characterized and the
mechanisms involved have been unclear.
Most of the CF clinical isolates of Pseudomonas we surveyed
produced pyocyanin (Fig. 1). Most of CF patients’ sputum samples
contain pyocyanin levels higher than 10 mM, a concentration that
increases spontaneous NET formation by 120% (Fig. 2C)
[18,19,24]. Pyocyanin is essential for full Pseudomonas virulence
in a variety of animal models.  Pyocyanin is a phenazine
exotoxin  with toxic effects in a broad range of target cells that
are based on its ability to induce oxidative stress.  Although
pyocyanin has been studied in several laboratories worldwide, and
increased levels of both pyocyanin and neutrophil components in
the CF lung have been associated with poor lung function, no
reports to date have described any direct effects of pyocyanin on
NET formation. Contrary to earlier reports showing that
pyocyanin induces apoptosis in neutrophils, we found that
pyocyanin in shorter time frames and at lower concentrations
(typical for CF airways) induces NET formation (Fig. 2 and 3). 
Antioxidant and inhibitor treatments revealed roles for ROS,
JNK, and PI3K in pyocyanin-elicited NET formation (Fig. 4A–F).
We are the first to highlight a role of JNK in NET formation,
whereas PI3Ks play an important role in regulating neutrophil
functions such as NADPH oxidase assembly and affect NET
induction by other agonists such as PMA.  The PI3K/Akt/
mTOR pathway regulates autophagy in several cell types and
autophagy has been implicated in NET formation. [4,39] ROS
produced by NADPH oxidases have been associated with
antibacterial autophagy. [40,41] Since our data show PI3K
activation and ROS production by pyocyanin, it is possible that
the toxin induces NET formation through autophagy.
Pyocyanin is a redox-active toxin that shuttles electrons between
donors and acceptors, thereby catalyzing redox reactions in the
host cell. Under aerobic conditions, molecular oxygen is the
primary electron acceptor  and its reduction by pyocyanin
results in production of superoxide anions by one-electron transfer.
 The main electron donor is NADPH (Fig. 5A). Pyocyanin is
a non-enzymatic NAD(P)H oxidase (Fig. 5G) and thus executes the
same chemical reaction as the members of the Nox NADPH
oxidase enzyme family (Nox 1–5 and Duox 1 and 2).  This
could be explained by the similarity in chemical structures of
pyocyanin and the core of the electron transport chain of every
NADPH oxidase, their essential redox cofactor flavin adenine
dinucleotide (FAD) (Fig. 5G). In PMNs, the Nox2-based NADPH
oxidase is by far the most abundant and most important oxidase
involved in crucial cell functions (respiratory burst, intracellular
killing, NET formation). Under non-physiological conditions
(maximal NADPH oxidase activation by PMA-stimulation), high
concentrations of pyocyanin (50–100 mM) inhibit oxidase activity
by consuming the substrate, NADPH; in contrast, our current
study demonstrates that lower concentrations of pyocyanin (1–
30 mM) stimulate NADPH oxidase activity in PMNs (Fig. 2E,
4A, B, E, 5B, E, F).  This pyocyanin-mediated superoxide
release by PMNs is entirely DPI-sensitive and is absent in CGD
PMNs (Fig. 5B, E).  We also found that basal, pyocyanin- or
PMA-stimulated NET formation in CGD patients was greatly
impaired (Fig. 6). Most of the toxic effects of pyocyanin described
to date are attributed to the oxidative stress resulting from direct
consumption of NADPH. We are the first to show here a new
consequence of exposure to pyocyanin (NET formation) that is
caused by oxidative stress not derived from direct NADPH
oxidation but by activation of the phagocytic NADPH oxidase.
Previously we have shown that higher doses of pyocyanin inhibit
activation of another NADPH oxidase, Duox in airway epithelial
cells.  The reason for the opposite effects of pyocyanin on two
different NADPH oxidases could be related to the different cell
types studied, different toxin doses used, or the relative amounts of
pyocyanin and the NADPH oxidases compared to cellular
NADPH pools. Further studies are required to unravel the exact
Most of the ROS data presented in our study measured
superoxide production in neutrophils because superoxide anions
are the primary products of both NADPH oxidase activity and
direct oxidation of NAD(P)H by pyocyanin. However, the exact
molecular identity of ROS directly responsible to initiate NET
formation remains to be studied. MPO-derived hypochlorous acid
was found to be involved in NET release but no clear evidence for
the involvement of hydrogen peroxide, superoxide or mitochon-
drial ROS has been presented yet. Most likely hydrogen peroxide
plays an important role in NET initiation and superoxide is only
the primary short-lived ROS product since hydrogen peroxide is
relatively long-lived among ROS, can penetrate biological
membranes, is readily formed from superoxide by dismutation
and when added exogenously (by the glucose/GO system) it
triggers maximal NET release .
By comparing neutrophils from 4 different CGD patients, we
found that basal and PMA-stimulated NET formation was
dependent on residual NADPH oxidase activity (Fig. 6). In-
terestingly, NETs induced by an external ROS source (glucose/
GO system) were also dependent on the residual capacity of CGD
neutrophils to produce ROS (Fig. 6A). We also showed that higher
residual oxidase activity results in lower pyocyanin levels required
to induce NETs in CGD PMNs (Fig. 6). These exogenous sources
of ROS can trigger NET formation but they are not equivalent to
and cannot compensate for some critical level of NADPH oxidase-
derived ROS. These observations can be explained by a direct
stimulatory effect of ROS on NADPH oxidase activation.
NADPH oxidase activation triggered by exogenous hydrogen
peroxide was described recently by two groups; one study showed
H2O2promotes membrane translocation of p40phox, whereas the
other showed that H2O2affects Ca2+influx and cAbl kinase acting
upstream of PKC-delta. [44,45] Thus, there appears to be some
critical ‘‘threshold’’ for NADPH oxidase-derived ROS required to
induce NETs and this can be reached more readily in cells already
exposed to oxidative stress through basal NADPH oxidase
activation. This is the first study to show that NET formation
among CGD patients can differ and depends on their residual
respiratory burst activity. In the past we have shown the
importance of residual superoxide production in neutrophil
bacterial killing against Staphylococcus aureus.  Another recent
study linked residual respiratory burst activity and patient survival
in a large cohort of CGD patients.  According to our findings,
the increased survival of CGD patients with higher residual
oxidative responses could in part relate to a higher capacity for
NET release. .
production was followed with Lucigenin for 60 min; NET formation was followed for 4 hrs. Data represent mean+/2 S.E.M. of two independent
experiments. G) Similarity of chemical structures of pyocyanin and flavin adenine dinucleotide (FAD) and their redox reactions involved in superoxide
generation. DMSO, dimethyl sulfoxide; DPI, diphenylene iodonium; RLU, relative luminescence unit; DCF-DA, 29-79-Dichlorodihydrofluorescein
diacetate; CGD, chronic granulomatous disease; PMA, phorbol 12-myristate-13-acetate; PMN, polymorphonuclear neutrophil; FAD, flavin adenine
dinucleotide; cat, catalase; SOD, superoxide dismutase.
NET Formation Induced by Pyocyanin
PLOS ONE | www.plosone.org8January 2013 | Volume 8 | Issue 1 | e54205
Figure 6. CGD neutrophils have impaired NET formation by pyocyanin. A) Comparison of pyocyanin-induced NET formation among three
X-CGD patients, one p47-deficient CGD patient and their normal controls. Adherent non-CGD or CGD neutrophils (patients 1,3,4, 5) were exposed to
0–30 mM pyocyanin, 100 nM PMA or 0.1 U/mL glucose oxidase (GO), Sytox Orange fluorescence was monitored for 3.5 hrs and NET formation was
quantified. The numbers in the boxes show residual superoxide productions of CGD neutrophils as % of the average of normal donors (100%). B)
Images of adherent neutrophils of CGD patient 3 and a non-CGD control (Sytox Orange fluorescence) after 3.5 hour-exposure to 10, 30 mM
pyocyanin, 0.1 U/mL glucose oxidase (GO) or 100 nM PMA. C) Images of NETs formed by p47-deficient CGD neutrophils and normal controls exposed
to the same stimuli as previously. Magnified micrograph in the upper right corner shows normal neutrophils in different stages of NET formation at
the end (3 hrs) of the Sytox Orange microplate assay. Examples for different stages are marked by white arrowheads and numbers. 1– These cells did
not produce NETs. At 3 hrs their plasma membrane became somewhat permeable and Sytox Orange stains their resting multilobular nucleus. 2-
These cells are in an intermediate phase. The plasma membrane is permeable, the typical lobulated nuclear morphology is lost but the integrity of
the cell is still observed. 3– PMNs in the final stage of NETosis. The nuclear and cell morphology is completely lost, the DNA was released around the
cell and it covers a larger area than the original cell size. Due to the weak fluorescent signal of NETs a longer exposure time had to be chosen and
NET Formation Induced by Pyocyanin
PLOS ONE | www.plosone.org9 January 2013 | Volume 8 | Issue 1 | e54205
CGD patients, whose neutrophils are unable to make NETs, are
not susceptible to infections by Pseudomonas; this may relate to
the importance of other oxidases (i.e. airway epithelial Duox) or
non-oxidative mechanisms in controlling airway Pseudomonas
infections. [22,23,43] In cystic fibrosis airways Pseudomonas
resides in biofilms protected from neutrophil phagocytic attack or
from becoming ensnared in NETs. Thus NET formation may not
reflect a critical mechanism for combating Pseudomonas. Rather, we
speculate that enhanced ROS-dependent NET release by
Pseudomonas and pyocyanin contributes to the inflammatory
conditions observed in CF airways that are chronically infected.
Human Subjects/ethics Statement
Human subjects included normal volunteers and X-linked
(gp91phox-deficient) and autosomal (p47phox-deficient) chronic
granulomatous disease patients participating in a study titled
‘‘Evaluation of Patients with Immune Function Abnormalities’’
(National Institute of Allergy and Infectious Diseases/NIH in-
stitution review board approved protocol NIH#05-I-0213), and
also cystic fibrosis patients participating in an observational study
of CF lung disease severity, ‘‘Genetics of CF Lung Disease’’
(Seattle Children’s Hospital institutional review board approved
protocol 10855 and Partners Healthcare Systems/Massachusetts
General Hospital institutional review board approved protocol
2011P000544). The protocols and informed consent procedures
were approved by the Institutional Review Boards of the NIAID/
NIH, Seattle Children’s Hospital, and Partners Healthcare
Systems/Massachusetts General Hospital, and the University of
Georgia, and the studies were conducted in accordance with the
ethical guidelines of Declaration of Helsinki. Human subjects
recruited under the guidelines of IRB-approved protocols (NIAID,
NIH#05-I-0213 and University of Georgia, UGA# 2012-10769-
4) provided written informed consent for participation in the
specific studies described below (i.e., neutrophil NADPH oxidase-
related functional assays). Human subjects recruited under the
guidelines of the Seattle Children’s Hospital IRB-approved
protocol 10855 provided written informed consent for storage of
specimens and data for use in future cystic fibrosis research (i.e.,
specimen and data banking). Written informed consent for storage
of specimens and data for future research use was received from
parents or legal guardians of those patients who were minors. The
Partners Healthcare Systems/Massachusetts General Hospital
IRB assumed regulatory responsibility for a portion of the
‘‘Genetics of CF Lung Disease’’ study after the principal
investigator relocated from Seattle Children’s Hospital to
Massachusetts General Hospital, and reviewed and approved the
use of previously stored specimens and data for the specific studies
described below, under 45 CFR46.110 and 21 CFR56.110
(expedited review of minimal risk human subjects research). All
CF patients in this study were homozygous for the F508del allele
of the cystic fibrosis transmembrane conductance regulator
(CFTR) gene, and were categorized as having ‘‘mild’’ or ‘‘severe’’
lung disease if they were in the highest or lowest quintile for age of
airway obstruction, as assessed by their median forced expiratory
volume during the initial second of exhalation. Anticoagulated
whole blood (10 mL) was drawn from CGD patients and was
processed in parallel with healthy volunteer’s blood. Four X-CGD
patients (Patient #1-4) and one p47phox-deficient CGD patient
(#5) participated in our study; all subjects were characterized in
previous studies with regards to their genetic defects and residual
NADPH oxidase activities.  Patient #1 (gp91-91a) has the
lowest residual superoxide production of 0.94 nmol/106cells/hr
followed by patient #4 (1.29 nmol/106cells/hr, gp91-22a,
0.57%), patient #2 (1.7 nmol/106cells/hr, gp-146, 0.75%) and
patient #3 (2.38 nmol/106cells/hr (1.05%).  The p47phox2/
2 patient (Patient #5; p47-16) had NADPH oxidase activity of
2.34 nmol/106cells/hr (1.04%) and superoxide production in
healthy neutrophils was 226 nmol/106cells/hr (100%). .
Isolation of Human Neutrophils
Neutrophils were purified as described with some modifications.
 Whole blood was drawn at the Transfusion Medicine Branch
of the National Institutes of Health or at the Health Center of
University of Georgia. 50 mL blood was anticoagulated by ACD
(anticoagulant citrate dextrose) or heparin. 6 mL aliquots of blood
were carefully layered on top of 6 mL Histopaque 1119 (Sigma)
and centrifuged (800 g 30 min RT). The upper plasma layer was
aspirated, and the middle phase containing white blood cells was
collected and washed in PBS. Pellets were resuspended in 4 mL
PBS and 2 mL were layered on top of a 5-step Percoll gradient
(65, 70, 75, 80 and 85%, Sigma) in 15 mL conical tubes. After
centrifugation the 70–75–80% Percoll layers containing neutro-
phils were collected and washed in PBS. Pelleted neutrophils were
resuspended in autologous serum, cell concentrations were de-
termined and cells were kept at room temperature until use.
Viability of the cells was determined by Trypan Blue dye extrusion
and resulted in .98% viable neutrophils. The purity of the
preparations was determined by Wright-Giemsa staining and
yielded .95% neutrophil granulocytes.
Pseudomonas Aeruginosa Strains
PA14 wild-type and pyocyanin-deficient mutants PhzM were
provided by Frederick M. Ausubel (Harvard Medical School,
Boston). Clinical isolates of Pseudomonas aeruginosa were cultured
from sputum or oropharyngeal swabs obtained from CF patients
participating in the ‘‘Genetics of CF Lung Disease’’ observational
study. Isolates were categorized as ‘‘early’’ or ‘‘late’’ relative to the
course of each patient’s onset of infection. Early isolates were
obtained at 3 months to 11 years of age; late isolates were obtained
at 5–20 years later than early isolates. All clinical isolates were
stored in Luria-Bertani broth with 16% glycerol at 280uC.
Determination of Pyocyanin Concentration
CF clinical isolates of Pseudomonas aeruginosa, the pyocyanin-
producing control strain PA14 WT (wild-type) and the pyocyanin-
deficient strain PA14 PhzM were grown in LB liquid medium for
48 hrs with continuous shaking. Bacteria were removed by high-
speed centrifugation and pyocyanin concentrations in the culture
supernatants were determined by subsequent chloroform/0.2 M
HCl extraction steps. Absorbance was measured at 520 nm and
concentration values (micromol/L) were calculated using a cali-
bration series with known pyocyanin concentrations. The exper-
iment was repeated three times and the mean+/2S.E.M. values
are presented (Fig. 1). The clinical isolates were deidentified during
the experiments and were only unblinded once the results were
obtained. Pyocyanin was purchased from Cayman Chemical and
dissolved in DMSO as 20 mM stock. Final concentrations were
diluted in HBSS and were free of bacterial LPS as measured by
the LAL endotoxin test kit.
PMNs in stages 1 and 2 are overexposed. PMA, phorbol-myristate-acetate; PMN, polymorphonuclear neutrophil.
NET Formation Induced by Pyocyanin
PLOS ONE | www.plosone.org 10January 2013 | Volume 8 | Issue 1 | e54205
Visualization of NETs by Fluorescence Microscopy
To visualize NETs released by pyocyanin-stimulated neutro-
phils, 56105neutrophils were allowed to adhere in 2 mL assay
medium to poly-d-lysine-coated 35 mm glass bottom culture
dishes (MatTEK Corp) (30 min, 37uC). Pyocyanin (1, 3, 10 or
30 mM) was added to stimulate NET formation. After three hours,
DAPI, Sytox Orange (2.5 mM) and/or Sytox Green (5 mM) were
gently added and the samples were immediately analyzed without
further disturbance with a Leica DM IRBE inverted fluorescence
microscope. Images were taken, analyzed and processed with
SPOT Advanced software (Spot Imaging Solutions, Sterling
Heights, MI). To visualize pyocyanin-triggered NET formation,
fluorescence images were also recorded on Sytox Orange-stained
neutrophils studied in the 96-well plate assay with the Leica
Quantification of Neutrophil Extracellular Traps
25,000 neutrophils/well were allowed to adhere on uncoated
96-well black transparent bottom plates at 37uC in 50 mL/well
assay medium (FBS- and phenol red-free RPMI 1640 medium
containing 1% HEPES and 0.1% human serum albumin (HSA)).
After 15 minutes, 50 mL/well HSA-free assay medium containing
5 mM Sytox Orange (Invitrogen) membrane-impermeable DNA
dye (final cc: 2.5 mM) and stimuli or inhibitors were added gently.
Fluorescence (excitation: 530 nm, emission: 590 nm) was recorded
in fluorescence plate reader (Labsystems, Fluoroskan) for 3 hours
at 2 min intervals (37uC, no shaking). The initial fluorescence in
samples containing neutrophils and 0.5 mg/mL saponin was taken
as maximal signal (100%). Relative fluorescence change in the
unknown samples over the 2.5–3.5 hr period was calculated and
referred to as NET formation (% of max). We used uncoated
plates to promote spontaneous NET formation. When bacteria
were used to induce NETs, PA14 wild-type and pyocyanin-
deficient PhzM strains were grown overnight in LB medium.
Bacteria were washed twice in HBSS and were added to adherent
neutrophils (26106PA14/well) in HBSS. When inhibitors were
applied the following concentrations were used: SB202474
(negative control for SB compounds, Sigma, 10 mM), SB600125
(JNK inhibitor, Sigma, 10 mM), wortmannin (PI3K inhibitor,
Sigma, 100 nM), PD98059 (MEK1 inhibitor, Sigma, 25 mM),
SB203580 (p38 inhibitor, Sigma, 10 mM). The ROS scavenger N-
acetyl-cysteine (NAC) was dissolved at 1 M concentration in
HBSS, the acidic pH was adjusted to 7.4 and NAC was used in the
experiments at a final concentration of 10 mM.
Immunostaining of Myeloperoxidase (MPO) and
Citrullinated Histone H4 (citH4)
26105neutrophils/well in 1 mL assay medium were allowed to
adhere to sterile 13 mm round glass cover slips placed in 12-well
plates for 30 min. Pyocyanin was added to the cells in 100 uL
assay medium resulting in 20 mM final concentration. Neutrophils
were incubated for 3 hours at 37uC, then fixed by 4% para-
formaldehyde dissolved in PBS for 10 min. Cells were permeabi-
lized in 0.1% Triton X-100 for 2 min RT, washed 3 times in PBS
for 5 min and blocked with 5% donkey serum in PBS for 30 min
37C. Neutrophils were incubated with monoclonal mouse anti-
human myeloperoxidase/FITC antibody (Dako, Clone MPO-7)
(2 hrs, RT, dark, 1:1000), or polyclonal rabbit anti-histone H4
(citrulline 3) (Millipore, 1:1000), washed 3 times in PBS. After
Alexa Fluor 488-labelled goat anti-rabbit secondary antibody was
added (1 hr, 1:1000), cells were stained with DAPI (2 min, RT,
1:10000) and washed 3 times in PBS. Preparations were mounted
with ProLong Antifade Kit (Molecular Probes) following the
manufacturer’s instructions and analyzed with a Leica fluores-
cence microscope. Fluorescence images were analyzed with SPOT
Advanced software. The original green fluorescence of the histone-
staining was converted in the imaging software to red to illustrate
the co-localization of DNA and citH4 (Fig. 3B).
Measurement of Superoxide Production
Superoxide production in neutrophils was measured either by
Lucigenin (9,99-Bis-N-methylacridinium nitrate)-amplified chemi-
luminescence or by the Diogenes cellular luminescence enhance-
ment system (National Diagnostics, Atlanta, GA, USA). Lucigenin
detects both, intracellular and extracellular superoxide but it is
insensitive, whereas Diogenes only measures extracellular super-
oxide but it is highly sensitive. In both assays the cells were
incubated in 96-well white plates and luminescence was followed
in a Luminoskan Ascent microplate luminometer (ThermoScien-
tific, Hudson, NH, USA). Superoxide production is either
presented as kinetics of luminescence or as integrated lumines-
cence units (int. RLU, area under the measured curve over
specified time intervals).
Neutrophils were incubated with 50 mg/mL Lucigenin in HBSS
for 10 min at 37uC before addition of any stimuli. To measure
pyocyanin- or PMA-stimulated superoxide production, neutro-
phils (106/mL) were treated with 1 mM PMA or 1–100 mM
pyocyanin and luminescence was recorded for 30 min at 37uC.
Superoxide production was also measured by the Diogenes
cellular luminescence enhancement system (National Diagnostics,
Atlanta, GA, USA). Neutrophils in suspension (106/mL, 10 min)
or attached (50,000/well, 1 hr) were stimulated with 1 mM PMA
or 1–30 mM pyocyanin and luminescence was recorded at 37uC.
Superoxide production by 50 mM pyocyanin in a cell-free
system in the presence of 20 mM b-NADH or b-NADPH was
measured for 30 min with the Diogenes assay.
Measurement of Intracellular ROS Production
Human neutrophils (normal and X-CGD) were incubated with
1 mM DCFH-DA (29-79-Dichlorodihydrofluorescein diacetate) for
10 min, washed twice in HBSS and exposed to 1 mM PMA or
20 mM pyocyanin. After 30 min ROS-mediated oxidation of
H2DCF into the highly fluorescent DCF was measured by flow
cytometry (AlexaFluor 488, 20.000 cells/sample). Data are shown
Measurement of Mitochondrial Superoxide
Neutrophils (normal and X-CGD) were incubated with 5 mM
MitoSox Red (15 min 37C), washed twice and stimulated either by
30 mM pyocyanin or 1 mM antimycin A. After 30 min mitochon-
drial superoxide production was detected by flow cytometry (exc/
em: 510/580 nm).
Statistical Analysis of Data
Data are shown as mean +/2 SEM. When independent
variables were studied significance was calculated with Student’s t-
test. When results of trends were compared data were analyzed by
ANOVA and post-hoc Dunnett’s test. Significant changes were
marked as * when p,0.05, ** when p,0.01 and *** when
We thank Sandra Anaya-O’Brien for recruiting the CGD patients, Douglas
Kuhns for archived patient data, and Helen Song for help with normal
neutrophil preparations. We are grateful for Kol Zarember for performing
the LAL endotoxin test on the pyocyanin solutions.
NET Formation Induced by Pyocyanin
PLOS ONE | www.plosone.org 11 January 2013 | Volume 8 | Issue 1 | e54205
Author Contributions Download full-text
Provided patient derived material and supportive clinical data/medical
histories: SMM HLM. Recruited patients and maintained clinical protocols
with institutional approval: HLM SMM BR. Conceived and designed the
experiments: BR TLL. Performed the experiments: BR MAJ LP CH DY
JJP TLL. Analyzed the data: BR TLL. Wrote the paper: BR TLL.
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