Article

Reactive oxidants and myeloperoxidase and their involvement in neutrophil extracellular traps

Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch Christchurch, New Zealand.
Frontiers in Immunology 11/2012; 3:424. DOI: 10.3389/fimmu.2012.00424
Source: PubMed
ABSTRACT
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.

Full-text

Available from: Heather Parker, Jun 30, 2015
“fimmu-03-00424” 2013/1/21 10:46 page1—#1
MINI REVIEW ARTICLE
published: 21 January 2013
doi: 10.3389/fimmu.2012.00424
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
Edited by:
Marko Radic, University of Tennessee,
USA
Reviewed by:
Nadine Varin-Blank, Institut National
de la Santé et de Recherche
Médicale, France
Mariana J. Kaplan, University of
Michigan, USA
*Correspondence:
Heather Parker, Centre for Free
Radical Research, Department of
Pathology, University of Otago
Christchurch, P.O. Box 4345,
Christchurch 8140, New Zealand.
e-mail: heather.parker@otago.ac.nz
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 maint ains 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
INTRODUCTION
Neutrophils release extracellular traps (NETs) in response to a
diverse r ange of stimuli including a variety of microorganisms,
microbial products, and chemokines (refer to the review by
Guimaraes-Costa et al., 2012 for a more detailed list). NETs are
composed of a scaffold of chromatin decorated with an assortment
of neutrophil-derived proteins, including the enzyme myeloperox-
idase (MPO; Ur ban et al., 2009). NETs are believed to contribute to
host defense, supplementary to neutrophil phagocytosis, by trap-
ping and potentially killing invading pathogens (Brinkmann et al.,
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 et al.,
2009; Lande et al., 2011) and other pathological conditions (Clark
et al., 2007; Fuchs et al., 2010; Narasaraju et al., 2011; Caudrillier
et al., 2012).
Activated neutrophils produce large amounts of superoxide
(O
•−
2
) v ia their nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase. O
•−
2
dismutates to hydrogen peroxide (H
2
O
2
)
leading to the formation of a variety of toxic oxygen der ivatives,
especially those for med 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 H
2
O
2
forming the strong oxidant hypochlorous acid (HOCl), the prime
mediator of oxidative killing in the phagosome (Winterbourn and
Kettle, 2012). MPO is present on NETs (Urban et al., 2009) and
has the potential (given a supply of H
2
O
2
)togenerateHOClin
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
Studies into the mechanisms of NET formation (NETosis) indicate
a complex process that differs depending on the stimulus. Given
the variability in NET inducers (Guimaraes-Costa et al., 2012) the
existence of more than one pathway is perhaps not surprising. 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 et al.,
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 et al., 2007; Fuchs et al., 2007; Yousefi et al., 2009; Pilsczek
et al., 2010). To date, the majority of inducers examined show
dependency on an active NADPH oxidase and there is evidence
that w ith 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 et al., 2007), an nitri c oxide (NO) donor (Keshari
et al., 2012), bacteria (Parker et al., 2012b), lipopolysaccharide
(LPS; Yost et al., 2009), and complement factor 5a (C5a) after
www.frontiersin.org January 2013
|
Volume 3
|
Article 424
|
1
Page 1
“fimmu-03-00424” 2013/1/21 10:46 page2—#2
Parker and Winterbourn Neutrophil extracellular traps and oxidants
priming with granulocyte/macrophage colony-stimulating factor
(GM-CSF; Yousefi et al., 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 et al.,
2010). The later release of NETs was possibly induced by bacterial
phagocytosis, which would have been slow under the conditions
employed in this study. Thus, two different forms of NET stimula-
tion could have operated over the course of the experiments. From
this study it might be assumed that activation of the oxidase leads
to NET expulsion by cell death and that the oxidase is not required
for release from viable cells. However, oxidase-dependent NET
release from living cells has been reported (Yousefi et al., 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 et al., 2007), or GM-
CSF + C5a (Yousefi et al., 2009). Exogenously added H
2
O
2
restores
the ability of CGD neutrophils to produce NETs (Fuchs et al.,
2007), as does gene therapy to reconstitute NADPH oxidase
function (Bianchi et al., 2009). Using a mouse model of CGD,
Ermert et al. (2009) found that gp91
/
mice neutrophils do not
make NETs when stimulated with PMA or Candida albicans. Fur-
thermore, using genetically different inbred mouse str ains 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 et al., 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 et al., 2012b). S. aureus leukocidins also induce
NETs when oxidase activity is inhibited (Pilsczek et al., 2010). The
oxidative burst was not measured in this study; however, similar
concentrations of purified leukocidin combinations can induce
ROSproduction(Colin and Monteil, 2003).
Although DPI is a general flavoenzyme inhibitor, the most likely
explanation for its effect on NETosis is that it inhibits the NADPH
oxidase, and this is supported by the CGD neutrophil and knock-
out mice studies. DPI does have other effects, including inhibition
of mitochondrial complex I and inducible nitric oxide synthase
(iNOS). However, even though an NO donor has been shown
to induce NETs (Keshari et al., 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 et al., 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 et al., 2012; Palmer et al., 2012;
Parker et al., 2012b) and neutrophils from MPO-deficient patients
have reduced ability to produce NETs when stimulated with PMA.
Metzler et al. (2011) found the level of NETs produced correlated
with the degree of MPO deficiency and that neutrophils completely
deficient in MPO could not make NETs. We observed just 3%
of normal MPO activity was sufficient to allow PMA-induced
NETosis (Parker et al., 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,orEscherichia coli NET
induction (Parker et al., 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
hadnoeffect(Figure 1B; Parker et al., 2012b). In contrast to our
observations, Akong-Moore et al. (2012) prevented Pseudomonas-
induced NETosis with MPO inhibition. Our conditions favored
phagocytosis (Parker et al., 2012b) and may account for the differ-
ences observed between the studies but this remains to be explored.
Interestingly, MPO inhibition or knock out had no effect on NETo-
sis in mouse neutrophils (Akong-Moore et al., 2012) indicating an
apparent species-specific difference in NET formation. Of note,
mouse neutrophils contain less MPO than human (Rausch and
Moore, 1975).
Myeloperoxidase is reported to contribute toward NETosis,
independent of its activity, by aiding chromatin decondensa-
tion (Papayannopoulos et al., 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
et al., 2010). Therefore, in neutrophils MPO may not play a direct
role in chromatin decondensation.
To sum up, there is good ev idence 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
NETosis.
ASSIGNING THE SPECIFIC ROS REQUIRED
Activation of the neutrophil NADPH oxidase leads to the produc-
tion of a variety of ROS. Assigning which are required for NETosis
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 et al., 2010; Figure 1C). As yet these are not
well characterized. During phagocytosis, activation mainly occurs
at the phagosomal membrane (Winterbourn and Kettle, 2012),
but elect ron 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
•−
2
in the extracellular environment, phagosome or a currently unde-
fined intra cellular compartment. O
•−
2
is membrane impermeable
but rapidly dismutates to membrane permeable H
2
O
2
.Someof
Frontiers in Immunology
|
Molecular Innate Immunity January 2013
|
Volume 3
|
Article 424
|
2
Page 2
“fimmu-03-00424” 2013/1/21 10:46 page3—#3
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 et al. (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 et al., 2010 and Robinson, 2008). Details
are given in the text. With PMA, oxidant production is predominately
extracellular while phagocytosis induces largely intracellular production.
the H
2
O
2
produced 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 H
2
O
2
should react with MPO before it can diffuse out
(Winterbourn and Kettle, 2012). H
2
O
2
can also react to form
hydroxyl radicals and singlet oxygen (
1
O
2
). 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.
However, even with PMA, oxidase activity is over well before NETs
are released. O
•−
2
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
continues for about 30 min following phagocytosis (Granfeldt and
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,
www.frontiersin.org January 2013
|
Volume 3
|
Article 424
|
3
Page 3
“fimmu-03-00424” 2013/1/21 10:46 page4—#4
Parker and Winterbourn Neutrophil 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 et al.,
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.
The following sections discuss the evidence available for individual
species.
Hydrogen peroxide
Several studies have shown that exogenously added H
2
O
2
is suf-
ficient to induce NETs (Fuchs et al., 2007; Neeli et al., 2009;
Lim et al., 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 H
2
O
2
has little or no effect on
NETosis (Fuchs et al., 2007; Parker et al., 2012b). It is plausi-
ble sufficient H
2
O
2
is 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 et al., 1988), though
its intr acellular compartment is unknown. PEG-catalase reduced
but did not completely inhibit PMA-NETosis while bacterial
induction of NETs was unaffected (Parker et al., 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 et al., 2007; Palmer et al., 2012; Parker et al., 2012b). How-
ever, these also inhibit MPO, which complicates interpretation of
effects.
Superoxide
Addition of superoxide dismutase (SOD) to neutrophils has been
shown to modestly increase PMA-induced NETs (Palmer et al.,
2012; Parker et al., 2012b). This would accelerate removal of extra-
cellular O
•−
2
but have little effect on any generated intracellularly.
Because most of the superoxide generated by neutrophils dismu-
tates anyway, the presence of SOD would also make little difference
to the amount of H
2
O
2
produced (Winterbourn, 2008). At present
we have no explanation for the SOD effect.
Hypochlorous acid and other MPO products
As the major strong oxidant produced by MPO, HOCl is a potential
candidate for the oxidant responsible for MPO-dependent NET
formation. Indeed, addition of HOCl to neutrophils has been
reported to induce NETosis (Akong-Moore et al., 2012; Palmer
et al., 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 H
2
O
2
to 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
H
2
O
2
or (B) 100 μMH
2
O
2
(added in 20 μM aliquots every 5 min to
facilitate MPO turnover). At the examined concentrations, H
2
O
2
in the
absence of NETs had no significant effect on S. aureus viability. (A)
Bacterial numbers significantly decreased with
40 μMH
2
O
2
(p < 0.05,
t-test on normalized data, n
= 3). (B) Bacterial viability decreased with
H
2
O
2
(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 et al. (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 et al., 2012b).
Inhibition by >50 mM taurine was seen (Palmer et al., 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
Page 4
“fimmu-03-00424” 2013/1/21 10:46 page5—#5
Parker and Winterbourn Neutrophil extracellular traps and oxidants
Alternative MPO products could be involved in NETosis. One
example, singlet oxygen (
1
O
2
) has been implicated on the basis
that NETs were observed after
1
O
2
was generated using irradiated
Photofrin (Nishinaka et al., 2011). However, while it is theoret-
ically possible for neutrophils to generate
1
O
2
from H
2
O
2
and
HOCl (Kiryu et al., 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 r adical scavenger Trolox inhibited PMA
and LPS-induced NETosis in mouse neutrophils (Lim et al., 2011).
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
H
2
O
2
involvement, and with PMA a picture is emerging in which
intracellularly generated MPO-derived ROS are important.
INVOLVEMENT OF ROS AND MPO IN NET-MEDIATED
MICROBIAL KILLING
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
(Brinkmann et al., 2004; Beiter et al., 2006; Bruns et al., 2010). The
evidence for direct killing by NETs is less convincing (Nauseef,
2012). Most studies have examined NET killing by incubating
pre-formed NETs with bacteria then diluting and plating. In some
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 et al., 2012a).
Using this method, several groups (Bruns et al., 2010; Menegazzi
et al., 2012; Parker et al., 2012a) have observed that NETs on their
own do not kill S. aureus, Aspergillus fumigatus conidia, or C.
albicans blastospores.
EVIDENCE FOR MPO-MEDIATED NET KILLING
Myeloperoxidase is present on NETs (Brinkmann et al., 2004;
Urban et al., 2009; Parker et al., 2012a) placing it in close prox-
imity to ensnared bacteria. NET-bound MPO is active and
able to generate HOCl (Parker et al., 2012a). In our study,
incubation of S. aureus with isolated NETs had no effect on
bacterial viability. However, killing was observed when H
2
O
2
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 H
2
O
2
required. The close proximity of NET-MPO to trapped microor-
ganisms would be expected to facilitate exposure of microbes
to lethal concent rations 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
organisms.
SUMMARY
There is good evidence that the enzymatic processes of the NADPH
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,
interpretation of results is confounded by limitations of specificity
and getting sufficient concentrations to intracellular locales where
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,
the involvement of oxidants in individual steps can be investigated
and a clearer picture should emerge.
REFERENCES
Akong-Moore, K., Chow, O. A.,
Von Kockritz-Blickwede, M., and
Nizet, V. (2012). Influences of chlo-
ride and hypochlorite on neutrophil
extracellular trap formation. PLoS
ONE 7:e42984. doi: 10.1371/jour-
nal.pone.0042984
Beckman, J. S., Minor, R. L. JR., White,
C. W., Repine, J. E., Rosen, G. M.,
and Freeman, B. A. (1988). Super-
oxide dismutase and catalase conju-
gated to polyethylene glycol increases
endothelial enzyme activity and oxi-
dant resistance. J. Biol. Chem. 263,
6884–6892.
Beiter, K., Wartha, F., Albiger, B.,
Normark, S., Zychlinsky, A., and
Henriques-Normark, B. (2006). An
endonuclease allows Streptococcus
pneumoniae to escape from neu-
trophil extracellular tr aps. Curr. Biol.
16, 401–407.
Bianchi, M., Hakkim, A., Brinkmann,
V., Siler, U., Seger, R. A., Zychlinsky,
A., and Reichenbach, J. (2009).
Restoration of NET formation by
gene therapy in CGD controls
aspergillosis. Blood 114, 2619–2622.
Brinkmann, V., Reichard, U., Goos-
mann, C., Fauler, B., Uhlemann, Y.,
Weiss, D. S., et al. (2004). Neutrophil
extracellular traps kill bacteria. Sci-
ence 303, 1532–1535.
Bruns, S., Kniemeyer, O., Hasenberg,
M., Aimanianda, V., Nietzsche, S.,
Thywissen, A., et al. (2010). Pro-
duction of extracellular traps against
Aspergillus fumigatus in vitro and
in infected lung tissue is depen-
dent on invading neutrophils and
influenced by hydrophobin RodA.
PLoS Pathog. 6:e1000873. doi:
10.1371/journal.ppat.1000873
Bylund, J., Brown, K. L., Movitz,
C., Dahlgren, C., and Karlsson, A.
(2010). Intracellular generation of
superoxide by the phagocyte NADPH
oxidase: how, where, and what for?
Free Radic. Biol. Med. 49, 1834–1845.
Carr, A. C., and Winterbourn, C.
C. (1997). Oxidation of neutrophil
glutathione and protein thiols by
myeloperoxidase-derived hypochlor-
ous acid. Biochem. J. 327(Pt 1),
275–281.
Caudrillier, A., Kessenbrock, K., Gilliss,
B. M., Nguyen, J. X., Marques, M.
B., Monestier, M., et al. (2012).
Platelets induce neutrophil extra-
cellular traps in transfusion-related
acute lung injury. J. Clin. Invest. 122,
2661–2671.
Clark, S. R., Ma, A. C., Tavener,
S. A., Mcdonald, B., Goodarzi,
Z., Kelly, M. M., et al. (2007).
Platelet TLR4 activates neutrophil
extracellular traps to ensnare bacte-
riainsepticblood. Nat. Med. 13,
463–469.
Colin, D. A., and Monteil, H. (2003).
Control of the oxidative burst of
human neutrophils by staphylococ-
cal leukotoxins. Infect. Immun. 71,
3724–3729.
Decoursey, T. E., and Ligeti, E.
(2005). Regulation and termination
of NADPH oxidase activity. Cell. Mol.
Life Sc i. 62, 2173–2193.
Ermert, D., Urban, C. F., Laube, B.,
Goosmann, C., Zychlinsky, A., and
Brinkmann, V. (2009). Mouse neu-
trophil extracellular traps in micro-
bial infections. J. Innate Immun. 1,
181–193.
Farley, K., Stolley, J. M., Zhao, P., Coo-
ley, J., and Remold-O’Donnell, E.
(2012). A Ser pinB1 regulatory mech-
anism is essential for restricting neu-
trophil extracellular trap generation.
J. Immunol. 189, 4574–4581.
Fuchs, T. A., Abed, U., Goosmann, C.,
Hurwitz, R., Schulze, I., Wahn, V.,
et al. (2007). Novel cell death pro-
gram leads to neutrophil extracellular
traps. J. Cell Biol. 176, 231–241.
Fuchs, T. A., Brill, A., Duerschmied, D.,
Schatzberg, D., Monestier, M., Myers,
D. D. JR., et al. (2010). Extracellu-
lar DNA traps promote thrombosis.
www.frontiersin.org January 2013
|
Volume 3
|
Article 424
|
5
Page 5
“fimmu-03-00424” 2013/1/21 10:46 page6—#6
Parker and Winterbourn Neutrophil extracellular traps and oxidants
Proc. Natl. Acad. Sci. U.S.A. 107,
15880–15885.
Granfeldt, D., and Dahlgren, C. (2001).
An intact cytoskeleton is required
for prolonged respiratory burst activ-
ity during neutrophil phagocytosis.
Inflammation 25, 165–169.
Guimaraes-Costa, A. B., Nascimento,
M. T., Wardini, A. B., Pinto-Da-Silva,
L. H., and Saraiva, E. M. (2012).
ETosis: a microbicidal mechanism
beyond cell death. J. Parasitol. Res.
2012, 929743.
Hurst, J. K. (2012). What really hap-
pens in the neutrophil phagosome?
Free Radic. Biol. Med. 53, 508–520.
Keshari, R. S., Jyoti, A., Kumar, S.,
Dubey, M., Verma, A., Srinag, B. S.,
et al. (2012). Neutrophil extracellular
traps contain mitochondrial as well
as nuclear DNA and exhibit inflam-
matory potential. Cytometry A 81,
238–247.
Kessenbrock, K., Krumbholz, M.,
Schonermarck, U., Back, W., Gross,
W. L., Werb, Z., et al. (2009).
Netting neutrophils in autoimmune
small-vessel vasculitis. Nat. Med. 15,
623–625.
Kiryu, C., Makiuchi, M., Miyazaki,
J., Fujinaga, T., and Kakinuma,
K. (1999). Physiological produc-
tion of singlet molecular oxygen in
the myeloperoxidase-H
2
O
2
-chloride
system. FEBS Lett. 443, 154–158.
Lande, R., Ganguly, D., Facchinetti,
V., Frasca, L., Conrad, C., Gre-
gorio, J., et al. (2011). Neutrophils
activate plasmacytoid dendritic cells
by releasing self-DNA-peptide com-
plexes in systemic lupus erythemato-
sus. Sci. Transl. Med. 3, 73ra19.
Lim, M. B., Kuiper, J. W., Katchky,
A., Goldberg, H., and Glogauer, M.
(2011). Rac2 is required for the for-
mation of neutrophil extracellular
traps. J. Leukoc. Biol. 90, 771–776.
Menegazzi, R., Decleva, E., and Dri, P.
(2012). Killing by neutrophil extra-
cellular traps: fact or folklore? Blood
119, 1214–1216.
Metzler, K. D., Fuchs, T. A., Nauseef,
W. M., Reumaux, D., Roesler, J.,
Schulze, I., et al. (2011). Myeloper-
oxidase is required for neutrophil
extracellular trap formation: implica-
tions for innate immunity. Blood 117,
953–959.
Narasaraju, T., Yang, E., Samy, R.
P., Ng, H. H., Poh, W. P., Liew,
A. A., et al. (2011). Excessive neu-
trophils and neutrophil extracellular
traps contribute to acute lung injury
of influenza pneumonitis. Am. J.
Pathol. 179, 199–210.
Nauseef, W. M. (2012). Editorial: Nyet
to NETs? A pause for healthy skepti-
cism. J. Leukoc. Biol. 91, 353–355.
Neeli, I., Dwivedi, N., Khan, S., and
Radic, M. (2009). Regulation of
extracellular chromatin release from
neutrophils. J. Innate Immun. 1,
194–201.
Nishinaka, Y., Arai, T., Adachi, S.,
Takaori-Kondo, A., and Yamashita,
K. (2011). Singlet oxygen is essen-
tial for neutrophil extracellular trap
formation. Biochem. Biophys. Res.
Commun. 413, 75–79.
Palmer, L. J., Cooper, P. R., Ling, M.
R., Wright, H. J., Huissoon, A., and
Chapple, I. L. (2012). Hypochlorous
acid regulates neutrophil extracellu-
lar trap release in humans. Clin. Exp.
Immunol. 167, 261–268.
Papayannopoulos, V., Metzler, K.
D., Hakkim, A., and Zychlinsky,
A. (2010). Neutrophil elastase and
myeloperoxidase regulate the forma-
tion of neutrophil extracellular traps.
J. Cell Biol. 191, 677–691.
Parker, H., Albrett, A. M., Ket-
tle, A. J., and Winterbourn, C. C.
(2012a). Myeloperoxidase associated
with neutrophil extracellular traps is
active and mediates bacterial killing
in the presence of hydrogen peroxide.
J. Leukoc. Biol. 91, 369–376.
Parker, H., Dragunow, M., Hamp-
ton, M. B., Kettle, A. J., and Win-
terbourn, C. C. (2012b). Require-
ments for NADPH oxidase and
myeloperoxidase in neutrophil extra-
cellular trap formation differ depend-
ing on the stimulus. J. Leukoc. Biol.
92, 841–849.
Pattison, D. I., and Davies, M. J. (2006).
Reactions of myeloperoxidase-
derived oxidants with biological
substrates: gaining chemical insight
into human inflammatory diseases.
Curr. Med. Chem. 13, 3271–3290.
Pilsczek, F. H., Salina, D., Poon, K.
K., Fahey, C., Yi pp, B. G., Sibley, C.
D., et al. (2010). A novel mechanism
of rapid nuclear neutrophil extracel-
lular trap formation in response to
Staphylococcus aureus. J. Immunol.
185, 7413–7425.
Rausch, P. G., and Moore, T. G.
(1975). Granule enzymes of poly-
morphonuclear neutrophils: a phy-
logenetic comparison. Blood 46,
913–919.
Robinson, J. M. (2008). Reactive oxy-
gen species in phagocytic leukocytes.
Histochem. Cell Biol. 130, 281–297.
Stasia, M. J., and Li, X. J. (2008).
Genetics and immunopathology
of chronic granulomatous disease.
Semin. Immunopathol. 30, 209–235.
Steinberg, B. E., and Grinstein, S.
(2007). Unconventional roles of
the NADPH oxidase: signaling, ion
homeostasis, and cell death. Sci.
STKE 2007, pe11.
Urban, C. F., Ermert, D., Schmid,
M., Abu-Abed, U., Goosmann, C.,
Nacken, W., et al. (2009). Neu-
trophil extracellular traps contain
calprotectin, a cytosolic protein com-
plex involved in host defense against
Candida albicans. PLoS Pathog.
5:e1000639. doi: 10.1371/journal.
ppat.1000639
Winterbourn, C. C. (2008). Reconciling
the chemistry and biology of reactive
oxygen species. Nat. Chem. Biol. 4,
278–286.
Winterbourn, C. C., and Kettle, A.
J. (2012). Redox reactions and
microbial killing in the neutrophil
phagosome. Antioxid. Redox Signal.
doi:10.1089/ars.2012.4827 [Epub
ahead of print].
Yipp, B. G., Petri, B., Salina, D., Jenne,
C. N., Scott, B. N., Zbytnuik, L.
D., et al. (2012). Infection-induced
NETosis is a dynamic process involv-
ing neutrophil multitasking in vivo.
Nat. Med. 18, 1386–1393.
Yost, C. C., Cody, M. J., Harris,
E. S., Thornton, N. L., Mcin-
turff, A. M., Martinez, M. L., et al.
(2009). Impaired neutrophil extra-
cellular trap (NET ) formation: a
novel innate immune deficiency of
human neonates. Blood 113, 6419–
6427.
Yousefi, S., Mihalache, C., Kozlowski, E.,
Schmid, I., and Simon, H. U. (2009).
Viable neutrophils release mitochon-
drial DNA to form neutrophil extra-
cellular traps. Cell Death Differ. 16,
1438–1444.
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
January 2013.
Citation: Par ker H and Winterbourn CC
(2013) Reactive oxidants and myeloper-
oxidase and their involvement in neu-
trophil extracellular traps. Front. Immun.
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-
tributed under the terms of the Creative
Commons Attribution License, which
permits use, distribution and reproduc-
tion in other forums, provided the origi-
nal authors and source are credited and
subject to any copyright notices concern-
ing any third-party graphics etc.
Frontiers in Immunology
|
Molecular Innate Immunity January 2013
|
Volume 3
|
Article 424
|
6
Page 6
  • Source
    • "This discrepancy may be explained by the limited bacteria-neutrophil interactions under in vitro conditions. In vivo, however, neutrophils and pathogens come together in contact in an environmental milieu influenced by overlapping factors, such as proinflammatory chemokines and ROS, both of which stimulate neutrophils to induce NETosis via multiple receptors [9, 31, 32]. In this study, we have investigated three relatively prevalent serotypes of pneumococcus that are included in the pneumococcal polysaccharide vaccines [33]. "
    [Show abstract] [Hide abstract] ABSTRACT: Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza. In this study, we assessed the correlation of pneumococcal capsule size with pulmonary NETs formation and disease severity. We compared NETs formation in the lungs of mice infected with three pneumococcal strains of varying virulence namely serotypes 3, 4 and 19F, as well as a capsule-deficient mutant of serotype 4. In primary pneumonia, NETs generation was strongly associated with the pneumococcal capsule thickness, and was proportional to the disease severity. Interestingly, during secondary pneumonia after primary influenza infection, intense pulmonary NETs generation together with elevated myeloperoxidase activity and cytokine dysregulation determined the disease severity. These findings highlight the crucial role played by the size of pneumococcal capsule in determining the extent of innate immune responses such as NETs formation that may contribute to the severity of pneumonia.
    Preview · Article · Mar 2016 · Oncotarget
  • Source
    • "NET formation was not affected by the thiol reagents, NAC and GSH, and although uric acid was partially inhibitory, its ability to react via complex mechanisms with numerous oxidants, including peroxynitrite, complicates interpretation of this result. With many stimulants, NET formation is dependent on NOX activation [28, 40]. Despite our observation that plasma treatment led to a small but significant activation of the NOX, this activity was not required to induce NETs, as it was not inhibited by DPI. "
    [Show abstract] [Hide abstract] ABSTRACT: Cold physical plasma is an ionized gas with a multitude of components, including hydrogen peroxide and other reactive oxygen and nitrogen species. Recent studies suggest that exposure of wounds to cold plasma may accelerate healing. Upon wounding, neutrophils are the first line of defense against invading microorganisms but have also been identified to play a role in delayed healing. In this study, we examined how plasma treatment affects the functions of peripheral blood neutrophils. Plasma treatment induced oxidative stress, as assessed by the oxidation of intracellular fluorescent redox probes; reduced metabolic activity; but did not induce early apoptosis. Neutrophil oxidative burst was only modestly affected after plasma treatment, and the killing of Pseudomonas aeruginosa and Staphylococcus aureus was not significantly affected. Intriguingly, we found that plasma induced profound extracellular trap formation. This was inhibited by the presence of catalase during plasma treatment but was not replicated by adding an equivalent concentration of hydrogen peroxide. Plasma-induced neutrophil extracellular trap formation was not dependent on the activity of myeloperoxidase or NADPH oxidase 2 but seemed to involve short-lived molecules. The amount of DNA release and the time course after plasma treatment were similar to that with the common neutrophil extracellular trap inducer PMA. After neutrophil extracellular traps had formed, concentrations of IL-8 were also significantly increased in supernatants of plasma-treated neutrophils. Both neutrophil extracellular traps and IL-8 release may aid antimicrobial activity and spur inflammation at the wound site. Whether this aids or exacerbates wound healing needs to be tested.
    Preview · Article · Mar 2016 · Journal of Leukocyte Biology
    • "One of the most well-recognized aspects of NET formation is the involvement of the oxidative burst and ROS production, and NADPH oxidase and MPO play a key role in regulating and generating these products [81,115116117118119, as illustrated by findings that neutrophils isolated from NADPH oxidase-deficient human chronic granulomatous disease patients have impaired NET-forming capabilities [4]. The ETosis process is delayed by enzymatic inhibitors and in patients with genetic deficiencies in MPO activity, although minimal MPO function is still able to drive NET formation [117, 120]. The small GTPase Ras-related protein Rab27a, an important component of the azurophilic granule secretion system, is also involved in the process of ROSdependent NETosis and activation of associated enzymes [121], along with the kinase mammalian target of rapamycin, via its regulation of the transcription factor hypoxia-inducible factor 1 a and autophagy pathways [122, 123]. "
    [Show abstract] [Hide abstract] ABSTRACT: Alcohol use disorders (AUDs) are associated with increased susceptibility to pulmonary diseases, including bacterial pneumonia and acute respiratory distress syndrome (ARDS). Alveolar macrophages (AMs) play a vital role in the clearance of pathogens and regulation of inflammation, but these functions may be impaired in the setting of alcohol exposure. We examined the effect of AUDs on profiles of cytokines, chemokines, and growth factors in human AMs isolated from bronchoalveolar lavage (BAL) samples from 19 AUD subjects and 20 age-, sex-, and smoking-matched control subjects. By multiplex bead array, the lysates of AMs from subjects with AUDs had significant elevation in the cytokine tumor necrosis factor α (TNF-α), as well as chemokine (C-X-C motif) ligand 8 (CXCL8), CXCL10, and chemokine (C-C motif) ligand 5 (CCL5) (p < 0.05). Additionally, a 1.8-fold increase in IL-1β, 2.0-fold increase in IL-6, 2.3-fold increase in interferon gamma (IFN-γ), 1.4-fold increase in CCL3, and a 2.3-fold increase in CCL4 was observed in the AUD group as compared to the control group. We also observed compensatory increases in the anti-inflammatory cytokine IL-1RA (p < 0.05). AUD subjects had 5-fold higher levels of CXCL11 mRNA expression (p < 0.05) and a 2.4-fold increase in IL-6 mRNA expression by RT-PCR as well. In these investigations, alcohol use disorders were associated with functional changes in human AMs, suggesting that chronic alcohol exposure portends a chronically pro-inflammatory profile in these cells.
    No preview · Article · Nov 2015 · Alcohol (Fayetteville, N.Y.)
Show more