MINI REVIEW ARTICLE
published: 21 January 2013
Reactive oxidants and myeloperoxidase and their
involvement in neutrophil extracellular traps
Heather Parker* and Christine C.Winterbourn
Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch, New Zealand
Marko Radic, University ofTennessee,
Nadine Varin-Blank, Institut National
de la Santé et de Recherche
Mariana J. Kaplan, University of
Heather Parker, Centre for Free
Radical Research, Department of
Pathology, University of Otago
Christchurch, P .O. Box 4345,
Christchurch 8140, New Zealand.
Neutrophils release extracellular traps (NETs) in response to a variety of inflammatory
stimuli. These structures are composed of a network of chromatin strands associated
with a variety of neutrophil-derived proteins including the enzyme myeloperoxidase (MPO).
Studies into the mechanisms leading to the formation of NETs indicate a complex process
that differs according to the stimulus. With some stimuli an active nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase is required. However, assigning specific reactive
oxygen species involved downstream of the oxidase is a difficult task and definitive proof
for any single oxidant is still lacking. Pharmacological inhibition of MPO and the use of
MPO-deficient neutrophils indicate active MPO is required with phorbol myristate acetate
as a stimulus but not necessarily with bacteria. Reactive oxidants and MPO may also play
a role in NET-mediated microbial killing. MPO is present on NETs and maintains activity at
this site.Therefore, MPO has the potential to generate reactive oxidants in close proximity
to trapped microorganisms and thus effect microbial killing. This brief review discusses
current evidence for the involvement of reactive oxidants and MPO in NET formation and
their potential contribution to NET antimicrobial activity.
Keywords: superoxide, hydrogen peroxide, hypochlorous acid
Neutrophils release extracellular traps (NETs) in response to a
diverse range of stimuli including a variety of microorganisms,
microbial products, and chemokines (refer to the review by
Guimaraes-Costa etal., 2012 for a more detailed list). NETs are
host defense, supplementary to neutrophil phagocytosis, by trap-
2004). However, extended exposure of self-DNA and damaging
neutrophil granule proteins may be detrimental to the host and
NETs have been linked with autoimmunity (Kessenbrock etal.,
2009; Lande etal.,2011) and other pathological conditions (Clark
etal., 2007; Fuchs etal., 2010; Narasaraju etal., 2011; Caudrillier
Activated neutrophils produce large amounts of superoxide
2) via their nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase. O•−
dismutates to hydrogen peroxide (H2O2)
leading to the formation of a variety of toxic oxygen derivatives,
especially those formed by MPO-catalyzed reactions. Both the
NADPH oxidase and MPO have been implicated in the regulation
of NET formation. However, the specific reactive oxygen species
(ROS) required remains to be clarified.
Myeloperoxidase catalyses the oxidation of chloride by H2O2
forming the strong oxidant hypochlorous acid (HOCl),the prime
Kettle, 2012). MPO is present on NETs (Urban etal., 2009) and
has the potential (given a supply of H2O2) to generate HOCl in
close proximity to trapped bacteria, thus providing a prospective
mechanism for oxidative NET-mediated killing. In this short
review, we summarize experimental evidence for the involvement
of ROS and MPO in the regulation of NET formation and discuss
their potential contribution to NET antimicrobial activity.
ROS AND MPO IN NET FORMATION
a complex process that differs depending on the stimulus. Given
the variability in NET inducers (Guimaraes-Costa etal.,2012) the
term NETosis is sometimes used to describe only those forms of
NET formation associated with cell death (Steinberg and Grin-
stein,2007),but NETs can be released from living cells (Yipp etal.,
2012), and here we use NETosis to describe any form of NET
formation. NETs differ with respect to composition, timing, the
involvement of cell death and dependency on reactive oxidants
(Clark etal., 2007; Fuchs etal., 2007; Yousefi etal., 2009; Pilsczek
etal., 2010). To date, the majority of inducers examined show
dependency on an active NADPH oxidase and there is evidence
that with some stimuli MPO is also involved.
NADPH OXIDASE DEPENDENCY
Evidence that an active NADPH oxidase is required for NET
formation has come from studies using inhibitors of the oxi-
dase, knockout mice, or neutrophils from patients with chronic
granulomatous disease (CGD) whose NADPH oxidase is non-
functional (Stasia and Li, 2008). Inhibition of the oxidase
with diphenyleneiodonium chloride (DPI) prevents NETosis in
response to several factors, including phorbol myristate acetate
(PMA; Fuchs etal., 2007), an nitric oxide (NO) donor (Keshari
etal., 2012), bacteria (Parker etal., 2012b), lipopolysaccharide
(LPS; Yost etal., 2009), and complement factor 5a (C5a) after
January 2013| Volume 3|Article 424|1
Parker and WinterbournNeutrophil extracellular traps and oxidants
priming with granulocyte/macrophage colony-stimulating factor
(GM-CSF; Yousefi etal., 2009). Interestingly with Staphylococcus
aureus, an early phase of NET release induced by secreted bac-
terial products is independent of the oxidase and of cell death,
with dependency on these increasing over time (Pilsczek etal.,
2010). The later release of NETs was possibly induced by bacterial
phagocytosis, which would have been slow under the conditions
employedinthisstudy. Thus,twodifferentformsof NETstimula-
this study it might be assumed that activation of the oxidase leads
for release from viable cells. However, oxidase-dependent NET
release from living cells has been reported (Yousefi etal., 2009).
Strong evidence for NADPH oxidase-dependent NETosis
comes from the finding that CGD neutrophils do not form NETs
when stimulated with PMA, bacteria (Fuchs etal., 2007), or GM-
the ability of CGD neutrophils to produce NETs (Fuchs etal.,
2007), as does gene therapy to reconstitute NADPH oxidase
function (Bianchi etal., 2009). Using a mouse model of CGD,
Ermert etal. (2009) found that gp91−/−mice neutrophils do not
make NETs when stimulated with PMA or Candida albicans. Fur-
thermore, using genetically different inbred mouse strains these
investigators observed that the level of NET formation correlated
with the amount of ROS produced.
NET formation can also occur independently of oxidase activ-
ity. Not all stimulants activate the oxidase (Farley etal., 2012)
and some that do may induce NETs independent of this. For
example,the calcium ionophore ionomycin activates the NADPH
oxidase yet induces NETs similarly in the presence or absence
of DPI (Parker etal., 2012b). S. aureus leukocidins also induce
NETs when oxidase activity is inhibited (Pilsczek etal.,2010). The
oxidative burst was not measured in this study; however, similar
concentrations of purified leukocidin combinations can induce
ROS production (Colin and Monteil, 2003).
explanation for its effect on NETosis is that it inhibits the NADPH
oxidase, and this is supported by the CGD neutrophil and knock-
of mitochondrial complex I and inducible nitric oxide synthase
(iNOS). However, even though an NO donor has been shown
to induce NETs (Keshari etal., 2012), the low levels of iNOS in
isolated human neutrophils make it unlikely that DPI prevents
NETosis by inhibiting iNOS. Of note, a recent report describes
DPI-sensitive NET induction by platelet activating factor, which
does not activate the oxidase (Farley etal., 2012).
THE ROLE OF MPO
There is growing evidence that MPO is necessary for PMA-
stimulated NETosis and the majority of studies indicate that an
active enzyme is required. Inhibition of MPO decreases PMA-
stimulated NETs (Akong-Moore etal., 2012; Palmer etal., 2012;
Metzler etal. (2011) found the level of NETs produced correlated
deficient in MPO could not make NETs. We observed just 3%
of normal MPO activity was sufficient to allow PMA-induced
NETosis (Parker etal., 2012b). Inhibition of this residual activity
abrogated NET formation (Figure 1A).
Myeloperoxidase may not be required with all stimuli. We
found inhibiting MPO in control donor neutrophils had no effect
on Pseudomonas aeruginosa, S. aureus, or Escherichia coli NET
induction (Parker etal., 2012b). MPO-deficient neutrophils also
made NETs as efficiently as those from control donors when stim-
ulated with P. aeruginosa and inhibition of residual MPO activity
had no effect (Figure 1B; Parker etal., 2012b). In contrast to our
observations,Akong-Moore etal. (2012) prevented Pseudomonas-
induced NETosis with MPO inhibition. Our conditions favored
phagocytosis (Parker etal.,2012b) and may account for the differ-
sis in mouse neutrophils (Akong-Moore etal.,2012) indicating an
apparent species-specific difference in NET formation. Of note,
mouse neutrophils contain less MPO than human (Rausch and
Myeloperoxidase is reported to contribute toward NETosis,
independent of its activity, by aiding chromatin decondensa-
tion (Papayannopoulos etal., 2010). Purified MPO increased
nuclear decondensation in a cell-free system but the most dra-
matic increase occurred when MPO was added in conjunction
with neutrophil elastase. In PMA-stimulated neutrophils, elastase
translocated to the nucleus early in NETosis while MPO localized
there later, when NET release was occurring (Papayannopoulos
etal., 2010). Therefore, in neutrophils MPO may not play a direct
role in chromatin decondensation.
To sum up, there is good evidence that MPO is important
for PMA induction of NETs. From our studies, it would appear
that this is not the case with bacteria. However, there are incon-
sistencies in the results from different laboratories that require
explanation. Whether MPO is required with other physiological
NET inducers is currently unknown. Nevertheless when MPO is
needed, it appears that very little is actually required to facilitate
ASSIGNING THE SPECIFIC ROS REQUIRED
Activation of the neutrophil NADPH oxidase leads to the produc-
tionof avarietyof ROS.AssigningwhicharerequiredforNETosis
is not simple. The site of oxidase activation and degree of degran-
ulation, which vary depending on the stimulus, affect the relative
amounts of the different ROS produced as well as access to dif-
ferent cell constituents. With soluble stimuli, such as PMA, and
non-phagocytosed particulate stimuli,activation largely occurs at
the plasma membrane although some occurs at intracellular sites
(reviewed in Bylund etal., 2010; Figure 1C). As yet these are not
well characterized. During phagocytosis,activation mainly occurs
at the phagosomal membrane (Winterbourn and Kettle, 2012),
but electron microscope evidence shows that some also occurs
elsewhere in the cell (Robinson, 2008; Figure 1D).
The NADPH oxidase removes electrons from cellular NADPH
and transfers them across a membrane to oxygen, forming O•−
in the extracellular environment,phagosome or a currently unde-
fined intracellular compartment. O•−
but rapidly dismutates to membrane permeable H2O2. Some of
is membrane impermeable
Frontiers in Immunology |Molecular Innate Immunity
January 2013| Volume 3|Article 424|2
Parker and Winterbourn Neutrophil extracellular traps and oxidants
FIGURE 1 | Myeloperoxidase (MPO) is required for PMA but not bacterial
induction of NETs. (A,B)The release of NETs from control (filled bars) and
MPO-deficient (open bars) neutrophils measured over 4 h. MPO-deficient
neutrophils formed NETs less efficiently with PMA, but not with P .
aeruginosa, than neutrophils from control donors.To inhibit MPO, samples
were incubated in the presence of 100 μM of the MPO inhibitor
4-aminobenzoic acid hydrazide (ABAH). Results are means ± SEM of two to
three independent experiments. For PMA, p = 0.02 at 180 min; p = 0.071 at
240 min by t-test. Data obtained with permission from Parker etal. (2012b).
(C,D) Schematic representations of the intra- and extracellular locations of
oxidant production in response to (C) soluble and non-phagocytic stimuli, or
(D) phagocytosis (reviewed in Bylund etal., 2010 and Robinson, 2008). Details
are given in the text. With PMA, oxidant production is predominately
extracellular while phagocytosis induces largely intracellular production.
the H2O2produced extracellularly may diffuse into the cell while
some may react with MPO outside the cell (Figures 1C,D). The
production of HOCl in the extracellular environment requires
MPO release, the timing or level of which varies with stimulus.
In the phagosome, due to high MPO concentrations, essentially
all of the H2O2should react with MPO before it can diffuse out
(Winterbourn and Kettle, 2012). H2O2 can also react to form
hydroxyl radicals and singlet oxygen (1O2). However, the gener-
ation of these oxidants by neutrophils is considered to be very
low (Winterbourn and Kettle, 2012). PMA gives a larger, more
sustained oxidative burst than other stimulants that induce NETs.
are released. O•−
is produced within a minute of stimulation and
continues for at least an hour but with the rate decreasing over
this time (Decoursey and Ligeti, 2005). Similarly, oxidase activity
Dahlgren,2001). Therefore,ROS produced must influence earlier
rather than later events in NETosis.
By the nature of NADPH oxidase activation, it would seem it
is likely that both the site of oxidant production and the nature
of the oxidants produced are important in NET formation. Sev-
eral groups have attempted to identify the specific ROS involved,
January 2013| Volume 3|Article 424|3
Parker and WinterbournNeutrophil extracellular traps and oxidants
primarily by using enzyme inhibitors or oxidant scavengers. One
of the difficulties with this approach is targeting these to the
appropriate compartment. It is straightforward to scavenge oxi-
dants that are generated extracellularly. However, where there
is intracellular oxidant production, as with PMA (Bylund etal.,
2010), this is much more difficult to intercept. Consequently,
there are still many uncertainties about what specific ROS gen-
erated by the NADPH oxidase or MPO are required in NETosis.
Several studies have shown that exogenously added H2O2is suf-
ficient to induce NETs (Fuchs etal., 2007; Neeli etal., 2009;
Lim etal., 2011). However, addition of an oxidant and obser-
vation of NETs does not necessarily mean that this oxidant is
responsible with physiological stimuli. With PMA, addition of
catalase to scavenge extracellular H2O2has little or no effect on
NETosis (Fuchs etal., 2007; Parker etal., 2012b). It is plausi-
ble sufficient H2O2is generated intracellularly to induce NETs
so that extracellular scavenging would have minimal effect. This
was examined using polyethylene glycol-catalase (PEG-catalase)
which is taken up by endocytosis (Beckman etal., 1988), though
its intracellular compartment is unknown. PEG-catalase reduced
but did not completely inhibit PMA-NETosis while bacterial
induction of NETs was unaffected (Parker etal., 2012b). Most
likely PEG-catalase did not gain access to the appropriate intra-
cellular sites to exert a full effect. Use of catalase inhibitors,
such as azide or amino-triazole, has given inconsistent results
(Fuchs etal., 2007; Palmer etal., 2012; Parker etal., 2012b). How-
ever, these also inhibit MPO, which complicates interpretation of
Addition of superoxide dismutase (SOD) to neutrophils has been
shown to modestly increase PMA-induced NETs (Palmer etal.,
but have little effect on any generated intracellularly.
Because most of the superoxide generated by neutrophils dismu-
we have no explanation for the SOD effect.
Hypochlorous acid and other MPO products
candidate for the oxidant responsible for MPO-dependent NET
formation. Indeed, addition of HOCl to neutrophils has been
reported to induce NETosis (Akong-Moore etal., 2012; Palmer
etal., 2012). However, there are issues with interpreting these
results. First, in our experience HOCl concentrations >50 μM
are rapidly toxic to neutrophils (Carr and Winterbourn, 1997),
whereas the concentrations used to induce NETs were several
millimolar. Second, HOCl was added to RPMI which contains
numerous scavengers, including >10 mM amino acids, which
would consume the HOCl within seconds (Pattison and Davies,
2006). Although this would overcome toxicity, it would mean
that very little HOCl would reach the neutrophils. Many prod-
ucts including amino acid chloramines would be formed, but it
FIGURE 2 |Addition of H2O2to NETs induces MPO-dependent killing.
Neutrophils were stimulated with PMA to form NETs then incubated with
S. aureus in the presence or absence of (A) varying concentrations of
H2O2or (B) 100 μM H2O2(added in 20 μM aliquots every 5 min to
facilitate MPO turnover). At the examined concentrations, H2O2in the
absence of NETs had no significant effect on S. aureus viability. (A)
Bacterial numbers significantly decreased with ≥40 μM H2O2(p < 0.05,
t-test on normalized data, n = 3). (B) Bacterial viability decreased with
H2O2(p < 0.001), and inhibition of MPO with ABAH and scavenging of
HOCl with methionine (Met) prevented killing (p < 0.01; one-way ANOVA
with Holm–Sidak pairwise comparison, n = 5). Results are presented as
percent of control cells (Con) incubated with NETs alone. Data obtained
with permission from Parker etal. (2012a).
is unclear which would be responsible for NET formation. Third,
addition of catalase to prevent extracellular HOCl formation, or
removing HOCl with the potent scavenger methionine, did not
inhibit PMA-stimulated NET formation (Parker etal., 2012b).
Inhibition by >50 mM taurine was seen (Palmer etal., 2012), but
interpretation of this observation depends on the specificity of
these high concentrations. It is still possible that HOCl generated
intracellularly could be involved, but more definitive evidence is
needed before drawing this conclusion.
Frontiers in Immunology |Molecular Innate Immunity
January 2013| Volume 3|Article 424|4
Parker and WinterbournNeutrophil extracellular traps and oxidants
Alternative MPO products could be involved in NETosis. One
example, singlet oxygen (1O2) has been implicated on the basis
that NETs were observed after1O2was generated using irradiated
Photofrin (Nishinaka etal., 2011). However, while it is theoret-
ically possible for neutrophils to generate1O2from H2O2and
HOCl (Kiryu etal., 1999), it is a minor product (Hurst, 2012)
and an unlikely candidate for NET regulation with other stimuli.
MPO also catalyzes radical reactions, including lipid peroxida-
tion. Interestingly, the radical scavenger Trolox inhibited PMA
This raises the possibility that a radical mechanism such as lipid
peroxidation could be involved in the formation of NETs.
Summary of ROS required
In most cases, NADPH oxidase activity is needed for NET for-
mation but the oxidants involved and their mechanisms of action
are still unknown. The best, but not definitive, evidence is for
H2O2involvement,and with PMA a picture is emerging in which
intracellularly generated MPO-derived ROS are important.
INVOLVEMENT OF ROS AND MPO IN NET-MEDIATED
It has been postulated that the role of NETs in vivo is to trap
and kill microorganisms and there are some excellent scanning
electron micrographs of NETs entrapping both bacteria and fungi
evidence for direct killing by NETs is less convincing (Nauseef,
2012). Most studies have examined NET killing by incubating
instances, failure to release bacteria from NETs may have been
interpreted as killing, a problem we encountered but overcame
with DNase treatment to degrade NETs (Parker etal., 2012a).
Using this method, several groups (Bruns etal., 2010; Menegazzi
etal., 2012; Parker etal., 2012a) have observed that NETs on their
own do not kill S. aureus, Aspergillus fumigatus conidia, or C.
EVIDENCE FOR MPO-MEDIATED NET KILLING
Myeloperoxidase is present on NETs (Brinkmann etal., 2004;
Urban etal., 2009; Parker etal., 2012a) placing it in close prox-
imity to ensnared bacteria. NET-bound MPO is active and
able to generate HOCl (Parker etal., 2012a). In our study,
incubation of S. aureus with isolated NETs had no effect on
bacterial viability. However, killing was observed when H2O2
was added as a substrate for MPO (Figure 2A). MPO inhibi-
tion and a potent HOCl scavenger prevented killing (Figure 2B).
Therefore, NET-MPO has the potential to generate HOCl and
effect microbial killing. At a site of inflammation, neutrophils
that have formed NETs will no longer be producing ROS.
However, during inflammation there is continued infiltration
and activation of neutrophils which should provide the H2O2
required. The close proximity of NET-MPO to trapped microor-
ganisms would be expected to facilitate exposure of microbes
to lethal concentrations of HOCl and avoid all the oxidant
being scavenged by the surrounding media. In vivo imaging
using HOCl sensitive probes and differential fluorescent detec-
tion of live/dead bacteria would confirm if this occurs in living
oxidase and MPO are important in NETosis but elucidation of
the specific ROS and their reactions that regulate NET formation
requires further investigation. While the use of scavengers and
inhibitors is a useful aid to the study of ROS in NET formation,
interpretationof resultsisconfoundedbylimitationsof specificity
the critical oxidant generation may occur. The intracellular path-
ways leading to chromatin decondensation and NET release are
still being worked out. Once this information becomes available,
and a clearer picture should emerge.
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Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest.
Received: 10 October 2012; accepted: 23
December 2012; published online: 21
Citation: Parker H and Winterbourn CC
(2013) Reactive oxidants and myeloper-
oxidase and their involvement in neu-
3:424. doi: 10.3389/fimmu.2012.00424
This article was submitted to Frontiers in
Molecular Innate Immunity, a specialty
of Frontiers in Immunology.
Copyright © 2013 Parker and Winter-
bourn. This is an open-access article dis-
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Frontiers in Immunology |Molecular Innate Immunity
January 2013| Volume 3|Article 424|6