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Macrophage Clearance of Neutrophil
Extracellular Traps Is a Silent Process
Consol Farrera and Bengt Fadeel
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of August 1, 2017.
J Immunol published online 31 July 2013
http://www.jimmunol.org/content/early/2013/07/30/jimmun
ol.1300436
http://www.jimmunol.org/content/suppl/2013/07/31/jimmunol.130043
6v1.DC1
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Supplementary
Material
Published July 31, 2013, doi:10.4049/jimmunol.1300436
The Journal of Immunology
Macrophage Clearance of Neutrophil Extracellular Traps Is
a Silent Process
Consol Farrera and Bengt Fadeel
N
eutrophils act as sentinels of the innate immune system,
ready to be rapidly recruited to the site of infection. These
cells are equipped with various strategies to combat
a broad range of pathogens. Phagocytosis of pathogens and intracellular destruction has traditionally been thought of as the main
weapon of neutrophils, but a novel strategy was described a few
years ago, that is, the formation of so-called neutrophil extracellular traps (NETs) (1, 2). NETs are formed on exposure to pathogens including bacteria, fungi, and parasites, and consist of a
backbone of nuclear chromatin decorated with granule proteins
such as neutrophil elastase (NE) and myeloperoxidase (MPO).
Recent studies show that neutrophils stay alive and retain the
ability to combat bacteria after expulsion of their DNA (3). NETs
are believed to be an effective defense system against infection, as
the entrapment of pathogens in NETs may aid in the local confinement of the infection, whereas the granule-derived enzymes
facilitate in the extracellular killing of the “netted” pathogens (4).
Several recent reports have implicated the excessive formation
of NETs in sepsis, atherosclerosis, or autoimmune diseases such
as systemic lupus erythematosus (SLE) or psoriasis (5), suggesting that NET formation can be related to exacerbated immune
responses and tissue damage. In fact, Abs against dsDNA or
histones are a hallmark of some autoimmune diseases. Defective
macrophage clearance of apoptotic cell debris has been suggested
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska
Institutet, 171 77 Stockholm, Sweden
Received for publication February 13, 2013. Accepted for publication June 26, 2013.
This work was supported by the Swedish Research Council (B.F.).
Address correspondence and reprint requests to Prof. Bengt Fadeel, Division of
Molecular Toxicology, Institute of Environmental Medicine, Nobels väg 13, Karolinska Institutet, 171 77 Stockholm, Sweden. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CQ, chloroquine; HMDM, human monocyte–derived macrophage; HMGB1, high mobility group box 1; MPO, myeloperoxidase; NE,
neutrophil elastase; NET, neutrophil extracellular trap; RT, room temperature; siRNA,
small interfering RNA; SLE, systemic lupus erythematosus.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300436
to be a source of immunogens (6), but excessive production of
NETs and/or inefficient dismantling of these structures may potentially serve as yet another source of immunogens (7). In the case
of apoptotic cells, several key steps in the clearance process have
been identified, many of which appear to be conserved through
evolution, starting with the exposition of “eat-me” signals on the
surface of apoptotic cells leading to the specific recognition by the
phagocyte, followed by engulfment of the apoptotic prey and its
processing in the lysosomal compartment of the engulfing cell, and
finally, the modulation of proinflammatory and anti-inflammatory
cytokine secretion by macrophages to preclude or resolve inflammation (8). In contrast, NET removal is thought to be accomplished
mainly by extracellular DNase I degradation (7). DNase I has been
shown to be of vital importance for the clearance of necrotic cell–
derived chromatin; deficiencies in this enzyme have been linked to
SLE (9). Recent studies have shown that NETs derived from SLE
patients contain antibacterial peptides and that SLE NETs activate
dendritic cells to produce high levels of IFN-a (10). SLE patients
were also found to develop autoantibodies to both the DNA and
antimicrobial peptide component of NETs (11). This would support
the notion that an active clearance of NETs might be required in
addition to the extracellular degradation of these structures to prevent inadvertent immunological responses.
The complement system is yet another effector mechanism of
the innate immune system. Circulating complement components
can sense “danger” signals, opsonize pathogens or dying cells, and
induce the recruitment and activation of the immune system (12).
Depending on the sensing mechanism, the complement system
can trigger phagocytosis without inflammation, as in the case of
apoptotic cell clearance, or its activation may lead to an inflammatory response. C1q is a component of the complement system
that is involved in the recognition and clearance of apoptotic cells
and cell debris, and defects in C1q have been linked to the development of SLE (13, 14). C1q is able to bind DNA and histones,
both of which are present in NETs (1). Hakkim et al. (7) reported
that DNase I is required for disassembly of NETs, and that a subset of SLE patients degraded NETs poorly. More recent studies
suggested that NETs are potent complement activators and that
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Neutrophil extracellular traps (NETs) facilitate the extracellular killing of pathogens. However, in recent years, excessive NET
formation has been implicated in several pathological conditions. Indeed, NETs that are not removed from tissues or from the
circulation might serve to trigger autoimmune responses. We observed that physiological amounts of DNase I do not suffice to
completely degrade NETs in vitro, suggesting that additional mechanisms are required for the removal of these extracellular structures. We show in this article that human monocyte–derived macrophages are able to engulf NETs in a cytochalasin D–dependent
manner, indicating that this is an active, endocytic process. Furthermore, preprocessing of NETs by DNase I facilitated their
clearance by macrophages. In addition, both recombinant C1q and endogenous C1q derived from human serum were found to
opsonize NETs, and this facilitated NET clearance. Upon internalization, NETs were apparently degraded in lysosomes, as
treatment with chloroquine led to accumulation of extranuclear DNA in human monocyte–derived macrophages. Finally, uptake
of NETs alone did not induce proinflammatory cytokine secretion, whereas LPS-induced production of IL-1b, IL-6, and TNF-a
was promoted by the uptake of NETs. In summary, we show that macrophages are capable of clearance of NETs and that this
occurs in an immunologically silent manner. The Journal of Immunology, 2013, 191: 000–000.
2
deposited C1q inhibits NET degradation in part through a direct
inhibition of DNase I (15).
In this study, we sought to determine whether macrophages are
capable of clearance of NETs and to identify the possible roles of
DNase I and C1q in this process. In addition, we asked whether
clearance of NETs would elicit the production of proinflammatory
or anti-inflammatory cytokines.
Materials and Methods
Isolation and culture of primary human neutrophils and
macrophages
NET induction and isolation
To induce the production of NETs, we incubated freshly isolated neutrophils
from healthy blood donors with 25 nM PMA (Sigma) for 2 h at 37˚C in RPMI
1640 medium. Cell culture supernatants were removed and fresh media was
added to isolate NETs. Then cells and NETs were removed through extensive pipetting. The mixture was centrifuged at 1500 rpm for 5 min to separate neutrophils and NETs containing supernatants were retrieved. For
quantification of NET DNA, Hoechst 33342 solution at a concentration of
5 mg/ml (Sigma) was added to the supernatant and fluorescence (excitation
wavelength 360 nm, emission wavelength 465 nm) was measured using a
Tecan plate reader (Tecan Group, Männedorf, Switzerland).
Immunofluorescence
Neutrophils were seeded in 24-well plates on poly-L-lysine–coated cover
slips. After 1 h of attachment, 25 nM PMA was added and neutrophils were
cultured for 2 h at 37˚C and allowed to produce NETs. After incubation,
slides were fixed with 4% paraformaldehyde solution for 30 min at room
temperature (RT). Blocking was performed with a 2% BSA PBS solution
for 30 min at RT. Staining with primary Abs or the corresponding negative
control Abs was performed for 1 h at RT at 1:300 in 2% BSA PBS.
Secondary staining with FITC-labeled anti-rabbit Abs (Sigma) or goat
anti-mouse Alexa 594–labeled Abs (Invitrogen) was performed for 1 h at
RT at 1:500 in 2% BSA-PBS. The following primary Abs were used:
neutrophil elastase (NE), C1q (Santa Cruz Biotechnology, Santa Cruz,
CA), MPO (DAKO, Stockholm, Sweden), and high mobility group box 1
(HMGB1; generous gift of Dr. Ulf Andersson, Karolinska Institutet).
Mounting medium containing DAPI (Vector Laboratories, Burlingame,
CA) was added, and slides were visualized in an inverted Nikon ECLIPSE
TE2000-S fluorescence microscope (Nikon, Tokyo, Japan). For visualization of macrophage engulfment of NETs, HMDMs were scraped off the
plastic using a sterile cell scraper, reseeded in 24-well plates containing
cover slips, and allowed to attach for at least 3 h. Supernatants were removed and NETs were added for the indicated times. After incubation,
cells were fixed with 4% paraformaldehyde and immunostaining was performed as described earlier with Abs against NE. HMDMs were counterstained with 5 mg/ml phalloidin–tetramethylrhodamine isothiocyanate
(Sigma), and DAPI containing mounting medium was then added. Slides
were visualized in an inverted Nikon ECLIPSE TE2000-S fluorescence
microscope.
Scanning electron microscopy
Neutrophils were induced to produce NETs and processed as indicated
earlier and then fixed with glutaraldehyde solution at 2% in PBS. After
fixation, cells were dehydrated by increasing concentrations of ethanol.
The slides were then immersed in 100% hexamethyldisilazane (Sigma)
for 3 min and dried in a moist-free environment. Finally, the samples were
coated with gold using a sputter coater and examined using a JEOL JSM7000F (Jeol, Akishima, Japan).
NET degradation assay
One milliliter of purified NETs was incubated for 1 h at 37˚C with DNase I
(Sigma) at the indicated concentrations or with indicated percentages of
human serum obtained from healthy human blood donors (Karolinska
University Hospital). After incubation, degradation was stopped by addition of 2.4 mM EDTA, and phenol-chloroform extraction of NET DNA
was performed as indicated later (see NET transfection section). Quantitation of the amount of DNA was performed using the Nanodrop 1000
(Thermo Scientific, Waltham, MA), and equal amounts of NET DNA were
loaded in 1% agarose gels and run at 100 V. Visualization was performed
in a Molecular Imager (Bio-Rad, Hercules, CA).
NE activity
For analysis of NET clearance, decrease in NE activity in cell culture
supernatants was used as an indirect readout as described previously (18).
HMDMs were seeded in 96-well plates, allowed to attach for at least 3 h, and
washed with phenol red free RPMI 1640 media. Purified NETs were then
added to the macrophages and incubated for the indicated times. Incubation
was performed in the presence of 5 mg/ml DNase I (Sigma), 10 mg/ml cytochalasin D (Sigma), 100 mg/ml C1q protein (Abcam, Cambridge, U.K.), or
a mixture of endocytosis inhibitors containing nystatin (25 mg/ml), genistein
(200 mM), and brefeldin A (10 mg/ml; Sigma). After incubation, supernatants containing nonengulfed NETs were recovered through repetitive
pipetting and added to a new plate. The plate was then centrifuged for 6 min
at 1600 rpm and supernatant was recovered. Micrococcal Nuclease (Worthington Biochemical, Lakewood, NJ) was added to NETs at a final concentration of 500 mU/ml followed by incubation for 10 min at 37˚C. After
NE release, N-(methoxysuccinyl)-Ala-Ala-Pro-Val 4-nitroanilide (Sigma)
was added at a concentration of 200 mM in assay buffer (0.1 M HEPES,
0.5 M NaCl, 0,1% NaN3, 1% BSA, pH 7.5). After incubation for 3 h at
RT, absorbance was measured at 405 nm in a Tecan plate reader.
Western blot
After incubation with NETs, DNase I treatment (5 mg/ml) for 10 min was
performed. Samples were washed twice in PBS; then cells were lysed with
RIPA buffer and protein was quantified by BCA assay. Equal amounts of
proteins were loaded, separated by SDS-PAGE and transferred to a nitrocellulose membrane, and the membrane was incubated with NE Ab (Santa
Cruz Biotechnology) or with b-actin Ab (Sigma). Then incubation with
secondary Abs conjugated with HRP (Dako) followed, and bands were
detected with ECL (GE Healthcare Europe) on x-ray film (Fujifilm Europe,
Düsseldorf, Germany).
Confocal microscopy
Confocal microscopy was used for the visualization and localization of
NETs within macrophages. To this end, HMDMs were seeded in 24-well
plates containing cover slips and allowed to attach for at least 3 h, and were
then incubated in the presence of 10 mM chloroquine (CQ; Sigma) for 30
min to block lysosomal activity. Thereafter, medium was replaced and
purified NETs were incubated with HMDMs for 2 h in the presence of 10
mM CQ. Thirty minutes before fixation, 1 mM Lysotracker (Invitrogen)
was added, and 10 min before fixation, 5 mg/ml DNase I was added. For
detection of Eea-1 and Lamp-1, Abs from Santa Cruz Technology were
used and staining was performed as described earlier (see Immunofluorescence section). HMDMs were fixed with 4% formaldehyde, and slides
were mounted with DAPI-containing mounting medium and visualized
with a ZEISS LSM510META confocal microscope (Carl Zeiss, Oberkochen, Germany).
DNase II gene silencing
Silencing of DNase II was performed using the Amaxa Nucleofector
(Lonza, Basel, Switzerland) according to the manufacturer’s instructions. In
brief, HMDMs were transfected 2 d after their purification with 3 mM of
either a Negative Universal Control Stealth RNAi (Invitrogen, San Diego,
CA) or DNase II–specific silence RNA (59-UGGUCACAGUGAACUAUGA(dTdT)-39; Eurofins MWG Operon, Ebersberg, Germany). After transfection, macrophages were cultured for 2 more days in complete cell culture
medium including recombinant human M-CSF. Efficiency of silencing
of DNase II was monitored by RT-PCR as described later.
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Peripheral blood neutrophils were isolated from buffy coats from healthy
adult blood donors (Karolinska University Hospital, Stockholm, Sweden)
by density gradient centrifugation using Lymphoprep (Nycomed Pharma,
Oslo, Norway) as described previously (16). Neutrophils were further
separated from erythrocytes by gradient sedimentation in a 5% dextran
solution, and residual erythrocytes were removed by hypotonic lysis. Purified neutrophils were cultured in phenol red free RPMI 1640 medium
(Sigma), supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and
100 mg/ml streptomycin. Human monocyte–derived macrophages (HMDMs)
were prepared as previously described (17). After density gradient centrifugation, the white blood fraction was separated and further incubated
with CD14 MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany).
After magnetic bead separation, purified CD14+ monocytes were plated in
24-well plates at a density of 106 cells/ml and differentiated into macrophages by culture in RPMI 1640 (Sigma) medium in the presence of 50 ng/ml
recombinant human M-CSF (R&D Systems, Minneapolis, MN) and supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml
penicillin, and 100 mg/ml streptomycin for 3 d.
NET CLEARANCE BY MACROPHAGES
The Journal of Immunology
Cytokine production
To analyze the production of cytokines, we used the Luminex system (BioRad) according to the manufacturer’s instructions. In brief, HMDMs were
seeded in 96-well plates, allowed to attach for at least 3 h, and washed with
phenol red free RPMI 1640 medium, and then purified NETs were added.
In some cases, stimulation was performed with 100 ng/ml LPS (Sigma).
After 1 h, supernatant was removed and kept at 280˚C. Then fresh medium was provided and supernatants were collected again after 24 h and
saved at 280˚C. Experiments were performed with macrophages from
three different blood donors. Detection limits for the cytokines were 1.5
ng/ml for IFN-a, 2.05 pg/ml for IL-1b, 1.66 pg/ml for IL-6, 5.44 pg/ml for
TNF-a, and 1.51 pg/ml for IL-10. IFN-a, IFN-b, and TNF-a were also
detected by ELISA with detection limits of 7 pg/ml, 2.5 IU/ml, and 13 pg/
ml, respectively. ELISA kits were from Mabtech (IFN-a and TNF-a)
(Stockholm, Sweden) and Invitrogen (IFN-b).
NET transfection
Quantitative real-time PCR analysis
HMDMs were lysed with TRIzol (Invitrogen) and mRNA was purified
following the manufacturer’s instructions. Samples were then quantified
with Nanodrop (Thermo Scientific, Waltham, MA), and cDNA was ob-
FIGURE 1. NET production by
neutrophils. Neutrophils obtained from
healthy adult blood donors were incubated in the presence of 25 nM
PMA for 2 h at 37˚C. After fixation,
NETs were detected by the presence
of NE (A) in green or MPO (B) in
red. DAPI staining was included in
the mounting medium for visualization of cell nuclei. The merged images
illustrate the characteristic network
of the extracellular traps. Original
magnification 340. (C) Scanning electron microscopy images of neutrophils
fixed for imaging after treatment with
25 nM PMA for 2 h at 37˚C. NET
formation is clearly seen. Scale bars,
10 mm (left panel); 2 mm (right panel).
tained by retrotranscription of 1 mg RNA using the Revert Aid-H Minus
First Strand cDNA Synthesis Kit (Thermo Scientific). For determination of
the amount of DNase II after transfection with control or DNase II–specific
small interfering RNA (siRNA), RT-PCR using the Power SYBR Green
Reagent Kit (Applied Biosystems, Foster City, CA) was performed. Data
are reported as relative mRNA levels normalized to the expression values
of the gapdh housekeeping gene. Primers used were as follows: dnaseII
forward, 59-TTCCTGCTCTACAATGACCAAC-39, dnaseII reverse, 59GGAAGTTAGGTACACTGTGGACC-39; gapdh forward, 59-GGGTCTTTGCAGTCGTATGG-39, gapdh reverse, 59-ACCTCCTGTTTCTGGGGACT-39. To monitor IFN responses, we used the following primers:
IFN-a forward, 59-GCCTCGCCCTTTGCTTTACT-39; IFN-a reverse,
59-CTGTGGGTCTCAGGGAGATCA-39; IFN-b forward, 59-ATGACCAACAAGTGTCTCCTCC-39; IFN b reverse, 59-GGAATCCAAGCAAGTTGTAGCTC-39. Data are normalized to the expression values of the gapdh
gene.
Statistics
Statistical significance was tested with unpaired two-tailed Student t test
or one-way ANOVA with Tukey correction using GraphPad Prism version
5.02 for Windows (GraphPad Software, San Diego, CA). The level of significance for rejecting the null hypothesis of zero treatment effect was p =
0.05. Data are reported as mean 6 SD.
Results
NET production by primary human neutrophils
Neutrophils can die through several mechanisms. First, in the
absence of inflammatory stimuli, they have a tendency to undergo
apoptosis within 24–48 h (16). Moreover, neutrophils have been
described to die during the production of NETs, a process referred
to as NETosis (19). Special care should thus be taken so that the
study of NETs is not interfered with by DNA from other origins
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Phenol-chloroform extraction was performed to obtain DNA from purified
NETs. Phenol-chloroform solution (Invitrogen) was thus added 1:1to purified NETs. After centrifugation, DNA was precipitated with 100% ethanol,
further washed with 75% ethanol, and resuspended in RNase and DNase
free water. Quantitation of DNA purified was performed using the Nanodrop, and 1 mg purified NET DNA or the corresponding amounts of purified NETs were mixed with the transfection reagent, Lipofectin (Life
Technologies), and incubated at RT for 15 min. HMDMs were then
transfected with this solution or incubated with the same amount of purified NETs as described earlier. HMDMs pretreated with CQ (10 mM)
were also stimulated with the same amount of NETs. After 24 h, total RNA
was collected for PCR analysis as described later.
3
4
NET CLEARANCE BY MACROPHAGES
unrelated to the release of NETs. We therefore tested a range of
concentrations of the NET-inducing stimulus, PMA, as well as a
range of time points, and found that incubation with 25 nM PMA
for 2 h resulted in the induction of NETs without apoptosis or
necrosis of neutrophils. The production of NETs was determined
by immunofluorescence staining of the NET components, NE and
MPO (Fig. 1A, 1B), and scanning electron microscopy further
confirmed the formation of NETs (Fig. 1C). Purified NETs were
obtained and quantified using Hoechst 33342 labeling, and NE
activity was measured and shown to correlate with the quantification by Hoechst 33342 (Supplemental Fig. 1).
Partial NET processing by DNase I and human serum
NET clearance by monocyte-derived macrophages
During inflammation, macrophages are recruited shortly after the
initial recruitment of neutrophils to assist in the clearance of
pathogens and cellular debris. We hypothesized that macrophages
might aid in the clearance of NETs in such a scenario. To test this
hypothesis in our in vitro system, we incubated HMDMs with
NETs purified from neutrophils in serum-free media. After incubation for 1 h, NETs internalized by macrophages were identified
using Abs against NE and macrophages were counterstained with
DAPI and phalloidin (Fig. 3A). Confocal microscopy was used to
confirm macrophage ingestion of NETs (Supplemental Fig. 4).
Notably, an aggregation of actin filaments can be seen at the point
of interaction of NETs and the engulfing cell. About 15% of
macrophages were found to be engaged in clearance of NETs
under these conditions. Moreover, it was common to find several
macrophages collaborating in the engulfment of the same NET
(Fig. 3A). Macrophage clearance of NETs was further quantified
using the measurement of NE activity as an indirect readout, given
that NE present in NETs has been shown to be active and to
FIGURE 2. Digestion of NETs by DNase I. (A) Purified NETs from
PMA-treated neutrophils were incubated for 1 h at 37˚C in the absence or
presence of DNase I at the indicated concentration. Purified NETs alone
were also included as a control. Equal amounts of NET DNA were loaded
in an agarose gel for visualization. The left lane corresponds to m.w.
marker, with a higher band of 10,000 bp and a lower 500-bp band. Disappearance of the high m.w. band, corresponding to nondegraded NETs
DNA, indicated degradation of NETs at the 5 mg/ml concentration. Experiments were performed in triplicates with NETs derived from three different
donors with similar results. (B) NET degradation was assessed as in (A), but
incubation of NETs was performed in the presence of 10% serum derived
from four different human donors (D1–D4). DNase I (5 mg/ml) was included
as a positive control. (C) NET degradation was assessed in the presence of
DNase I at 5 mg/ml and in combination with 10% human serum. Results
show that human serum does not prevent NET degradation by DNase I.
correlate to the amount of NETs (Supplemental Fig. 1) (18). Incubation with HMDMs resulted in a decrease in the amount of NE
activity in cell culture supernatants, and this was shown to reach
a plateau at 2 h (Fig. 3B).
NETs are typically larger structures as compared with macrophages (or neutrophils), and we hypothesized that the dismantlement of NETs through the actions of DNase I might facilitate their
subsequent clearance by macrophages. To test this, we monitored
NET clearance in the presence of DNase I. Indeed, the addition of
this endonuclease accelerated the clearance of NETs by macrophages (Fig. 3C). Furthermore, to determine whether macrophage
clearance of NETs was an active process, cytochalasin D, an agent
that blocks the rearrangement of actin filaments, was added to
HMDM cultures. Incubation with cytochalasin D prevented NET
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DNase I is mainly produced in the pancreas and kidneys, and
hydrolyzes dsDNA under physiological conditions. Several reports
have shown that this enzyme can dismantle NETs, and certain
bacteria express DNases to escape from NETs (7, 20–22). Therefore, we tested the ability of this enzyme to degrade NETs. As
expected, incubation of NETs with DNase I at a high concentration (5 mg/ml) rendered fully degraded NETs (Fig. 2A). Nonetheless, incubation of NETs with a physiological concentration of
DNase I (20 ng/ml) (23) did not lead to degradation of NETs (Fig.
2A), thus demonstrating that under physiological circumstances,
the activity of this enzyme might not be sufficient for the complete
degradation of NETs. To further test this hypothesis, we incubated
NETs for 1 h with 10% serum from normal healthy donors and
tested its ability to degrade NETs (Fig. 2B). In addition, we also
increased the amount of serum to 20% and the incubation time up
to 12 h (Supplemental Fig. 2). Serum from healthy donors (10 or
20%) triggered only partial degradation of NETs after incubation
up to 12 h, implying that other mechanisms might be required for
the effective dismantling and clearance of NETs.
In addition to DNase I, serum may contain inhibitors of this
endonuclease. Moreover, proteins such as LL-37 and HMGB1 have
been shown to bind to NETs and protect them from degradation
(10, 11). However, we could not find any HMGB1 bound to NETs
produced under the conditions used in this study (Supplemental
Fig. 3), indicating that the presence of this protein in NETs might
occur only under certain conditions, for example, during NETosis.
We also tested the capacity of serum to prevent NET degradation
and found that incubation of NETs with 10% serum did not protect from DNase I when applied at a concentration of 5 mg/ml
(Fig. 2C).
The Journal of Immunology
5
clearance by macrophages, showing that this is an active, endocyticphagocytic mechanism (Fig. 3C). Preincubation with a mixture of
endocytosis inhibitors (nystatin, genistein, and brefeldin A) also
suppressed uptake of NETs (Fig. 3D). The inhibitors alone were
noncytotoxic (data not shown). To confirm the presence of NET
components within macrophages, we performed immunoblot analysis on macrophage cell lysates retrieved on incubation with NETs.
As shown in Fig. 3E, NE was detected in macrophages incubated
with NETs, but not in macrophages alone. The presence of residual
amounts of PMA in the NET suspension could not be excluded;
therefore, a possible influence of PMA on macrophage clearance
of NETs was assessed. Increasing amounts of PMA (up to 25 nM)
were added to macrophages in the NETs clearance assay, showing
no influence of PMA on the clearance of NETs by macrophages
(data not shown).
shown the ability of recombinant C1q to bind NETs (15). Indeed,
as shown in Fig. 4A, recombinant C1q is able to bind to NETs.
Furthermore, brief (10-min) incubation of NETs with serum derived from healthy human donors loaded the NETs with C1q (Fig.
4B). C1q is the recognition subunit of the C1 complex, which is
the initiator of the classical pathway of the complement. Binding
of C1q to its targets can lead either directly to the engulfment of
the target or to conformational changes within the C1 complex,
leading to the activation of the complement cascade (24). We
therefore tested the ability of recombinant C1q to facilitate the
clearance of NETs. Using the NE assay, we determined macrophage clearance in the presence or absence of 100 mg/ml recombinant C1q (Fig. 4C). C1q promoted the clearance of NETs, indicating that the binding of C1q to these structures targets them
for recognition by HMDMs.
NET opsonization by complement factor C1q
Evidence for lysosomal degradation of NETs
As reported earlier, HMDMs are able to clear NETs under serumfree conditions, but it remains possible that serum-derived factors
could facilitate this process. C1q has been shown to bind DNA and
histones, and we hypothesized that C1q could also opsonize NETs
and target them for clearance. In fact, a recent publication has
To provide further evidence of macrophage clearance of NETs, we
sought to visualize NET DNA in macrophages. To this end, DNA
was stained using DAPI. However, we could not detect any accumulation of extranuclear DNA in macrophages after coincubation with NETs for 1 h (Fig. 5B), suggesting that once NETs are
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FIGURE 3. Macrophages engulf NETs. (A) Immunofluorescence image showing HMDMs in the process of engulfing NETs. HMDMs were incubated for
60 min with purified NETs and after fixation, NETs were stained for NE (green) and macrophages were visualized with DAPI (blue) and phalloidin (red).
Original magnification 340. (B) Time course of NET clearance by macrophages as determined using the NE assay. HMDMs were incubated for different
time points in the presence of purified NETs. Supernatants were then recovered and NE activity was determined. For each independent experiment,
triplicates were performed for each time point. Results shown are the mean values 6 SD of three independent experiments. One-way ANOVA analysis was
performed with Tukey correction, and significant differences in reference to initial point are indicated (**p , 0.01, ***p , 0.001). (C) NE activity was
measured as in (B), but incubation was performed also in the absence or presence of either 5 mg/ml DNase I or 10 mg/ml cytochalasin D. Results shown are
the mean values 6 SD of three independent experiments. Unpaired, two-tailed Student t test analysis was performed, and significant differences between
the indicated points and the corresponding control (**p , 0.01) are indicated. (D) NE activity was measured at different time points in the presence of
a mixture of endocytosis inhibitors containing nystatin (25 mg/ml), genistein (200 mM), and brefeldin A (10 mg/ml). Results shown are the mean values of
three independent experiments 6 SD. One-way ANOVA analysis was performed with Tukey correction, and significant differences in reference to the
corresponding value without the mixture are depicted (*p , 0.05). (E) The presence of NE inside macrophages was assessed by Western blot after 2 h of
incubation with NETs in the presence of 10 mM CQ. b-actin was determined as a loading control.
6
NET CLEARANCE BY MACROPHAGES
No proinflammatory cytokine production upon NET clearance
FIGURE 4. C1q opsonizes NETs and facilitates their clearance. (A)
Human neutrophils were induced to form NETs by incubation with 25 nM
PMA for 2 h at 37˚C. A total of 100 mg/ml human recombinant C1q was
added 30 min before fixation. NETs were detected by immunofluorescence
staining of MPO in red and C1q in green. DAPI was included in the
mounting medium for the visualization of cells. (B) Human neutrophils
were induced to form NETs as in (A), and 10% human serum was added 10
min before fixation. Staining for MPO and C1q was performed as indicated
earlier. Original magnification 340. (C) C1q facilitates the clearance of
NETs. HMDMs were incubated for different time points in a 96-well plate
with 200 ml purified NETs in the absence or presence of 100 mg/ml
recombinant C1q. At the indicated time points, supernatants were recovered and NE activity was determined as an indicator of NET clearance. For
each independent experiment, triplicates were performed for each time
point. Results shown are the mean values 6 SD of three independent
experiments. One-way ANOVA analysis was performed with Tukey correction and significant differences in reference to the same time-point
without C1q are depicted. *p , 0.05.
ingested, these structures are efficiently processed within the engulfing cell. Therefore, we prestimulated macrophages with CQ to
block lysosomal activity (25) before the addition of purified NETs.
DNase I (5 mg/ml) was also added during the final 10 min of
coculture of HMDMs and NETs to eliminate any extracellular
DNA. After this treatment, an accumulation of extranuclear DNA
dots was seen in macrophages (Fig. 5C, 5D). In an attempt to
define the localization of these extranuclear dots, cells were stained
with the early endosomal marker Eea-1, but we found no evidence
To further decipher macrophage responses after NET clearance,
we analyzed a panel of cytokines in cell culture supernatants of
macrophages after ingestion of NETs. We could only detect a very
minor induction of IFN-a at 24 h postingestion, but not after 1 h
(Fig. 6A). No induction of the proinflammatory cytokines, IL-1b,
IL-6, and TNF-a, was detected (Fig. 6B–D), and no induction of
IL-10 (data not shown). These results indicate that NET clearance
by macrophages does not lead to an inflammatory response. Nonetheless, under physiological conditions, the production of NETs
is likely to be triggered by bacteria or other pathogens, and we
thus attempted to mimic this situation by stimulating macrophages
with bacterial LPS (100 ng/ml) + NETs. As expected, the induction of the proinflammatory cytokines IL-1b, IL-6, and TNF-a
was noted for LPS-stimulated macrophages (Fig. 6B–D). Moreover, a marked potentiation of the LPS-triggered cytokine response was seen when macrophages were cocultured with NETs
for 1 h. As shown in Fig. 5F, silencing of DNase II in macrophages
reduced the production of TNF-a in response to bacterial LPS +
NETs, but did not affect responses to NETs alone.
As noted earlier, clearance of NETs by macrophages does not
lead to type I IFN production. These observations are in contrast
with several previous results showing the capacity of mammalian
DNA to stimulate IFN production (25, 29). To further investigate
the capacity of NETs to induce type I IFNs, we transfected NETs
or purified NET DNA into HMDMs and analyzed type I IFN
expression at 24 h using RT-PCR. By this approach, we could
observe the induction of expression of both IFN-a and IFN-b after
transfection of NETs or NET DNA into macrophages (Fig. 7A),
and these results were further confirmed by ELISA (Fig. 7B).
However, incubation of macrophages with NETs, leading to their
engulfment, did not trigger the induction of these cytokines, nor
did cotreatment with CQ, leading to an accumulation of extranuclear DNA in macrophages that are engaged in NET clearance.
This experiment demonstrates that the DNA component of NETs
can potentially induce a type I IFN response and suggests that
macrophages are able to process NETs in an immunologically
silent manner when NETs enter via the endocytosis-phagocytosis
route.
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of extranuclear dots being localized in the early endosomal compartment (data not shown). Staining with the late endosomal
marker Lamp-1 showed a redistribution of this protein in macrophages after ingestion of NETs with the appearance of vesicular
structures (Fig. 5A, 5B). After treatment with CQ, some of the
extranuclear DNA dots were localized within Lamp-1+ vesicles
(Fig. 5C). Furthermore, using Lysotracker to identify lysosomes,
we noted that the majority of accumulated DNA dots colocalized
with lysosomes (Fig. 5D). The latter results suggest that lysosomal
enzymes are responsible for the intracellular degradation of NETs
in macrophages. DNase II is a key enzyme in lysosomes that is
responsible for digestion of DNA derived from apoptotic cells and
erythroid precursors (26, 27). To test its involvement in the
clearance of NET DNA, we silenced DNase II expression in
HMDMs using specific siRNA (Fig. 5E) and then asked whether
this would lead to an accumulation of extranuclear (nondegraded)
DNA in macrophages after ingestion of NETs. However, we
were unable to visualize an increase in undigested DNA under
these conditions (data not shown). Kawane et al. (28) reported that
DNase II–deficient macrophages that cannot degrade DNA from
apoptotic cells produce TNF-a. However, the production of proinflammatory TNF-a in response to NET internalization was not
increased in macrophages that lack expression of DNase II
(Fig. 5F).
The Journal of Immunology
Discussion
The mechanisms that drive NET formation have been studied in
detail (30–32), but not much attention has been paid to their ultimate fate once these structures have been released into extracellular space. We report for the first time, to our knowledge, that
macrophages actively engulf NETs, and that this event is facili-
7
tated by the preprocessing of NETs by DNase I. Our studies underscore the importance of using physiological concentrations of
DNase I when assessing the capacity of this enzyme to degrade
NETs. Hence, high concentrations of DNase I, which are typically
used in most studies (7, 18, 33), can completely digest NETs, but
physiological concentrations of the enzyme do not. This obser-
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FIGURE 5. NETs are processed in lysosomes. HMDMs were incubated with NETs for 2 h, and Lamp-1 staining (red) together with DNA (DAPI, blue)
was performed in control macrophages (A) or macrophages incubated with NETs (B). Note the redistribution of Lamp-1 upon ingestion of NETs (B). (C and
D) HMDMs were pretreated with or without 10 mM CQ for 30 min to block lysosomal function. Then purified NETs were added to macrophages in the
presence of CQ (10 mM). Ten minutes before fixation, DNase I (5 mg/ml) was added to avoid interference from extracellular DNA. (C) Lamp-1 staining
(red) was performed and visualized by confocal microscopy together with DNA (DAPI, blue). White arrows mark extranuclear DNA dots contained within
Lamp-1+ vesicular structures. (D) Cells were stained to visualize lysosomes (Lysotracker) in red and DNA (DAPI) in blue, and visualized using a confocal
microscope. The merged image is shown to facilitate visualization of extranuclear dots indicating the colocalization (white arrows) of DNA and lysosomes.
Original magnification 360. (E) DNaseII mRNA expression in macrophages transfected with specific DNase II siRNA or control siRNA as described in
Materials and Methods was determined using RT-PCR. Data are normalized to the expression values of the gapdh gene. One-way ANOVA analysis was
performed with Tukey correction and significant differences in reference to the corresponding control (***p , 0.001). (F) TNF-a secretion after incubation
of macrophages with or without a reduction of DNase II expression with 100 ng/ml LPS, purified NETs, or LPS + NETs. Cell culture supernatants were
harvested after 24 h, and cytokine levels were determined by ELISA. Results shown are the mean values 6 SD of triplicate values from one representative
experiment. Unpaired, two-tailed Student t test analysis was performed, and significant differences between the indicated samples and the corresponding
control are indicated (***p , 0.001).
8
NET CLEARANCE BY MACROPHAGES
vation is potentially relevant in an in vivo setting as the rapid and
complete digestion of NETs might prevent NETs from performing
their actual task, that is, to ensnare and kill pathogens, whereas
a lower or slower rate of degradation may be required to facilitate
NET clearance by macrophages.
The complement factor, C1q, has been shown to act in cooperation with DNase I in the clearance of chromatin from necrotic
cells (34). Moreover, C1q is known to act as an opsonin for apoptotic cells (14). Our data are in accordance with these findings
as we have been able to show opsonization of NETs with C1q and
the facilitation of macrophage clearance of NETs decorated with
C1q. Leffler et al. (15) reported that NETs are a potent complement activator and suggested that this may play an important role
in SLE insofar as sera from a subset of patients with active SLE
had a reduced ability to degrade in vitro–generated NETs and
displayed lower levels of complement proteins C4 and C3. In our
study, serum from healthy blood donors could only partially degrade NETs, as determined by agarose gel electrophoresis. In
previous studies from Leffler et al. (15) and Hakkim et al. (7),
degradation was monitored by fluorescence spectrometry of DNA
content in cell culture supernatants; the latter assay may not report
complete degradation of NETs.
Macrophages are endowed with several means to ensure effective clearance of nucleic acids from various origins. Our results
indicate that NETs are internalized through an endocytic mechanism leading to lysosomal degradation, as shown by blockage of
NET clearance by a mixture of endocytosis inhibitors, accumulation of extranuclear DNA on CQ treatment, and colocalization
of these extranuclear DNA dots within Lamp-1 vesicles and lysosomes. DNase II resides in the lysosomal compartment and has
been shown to digest DNA from apoptotic cells, and Nagata and
coworkers have reported that its deficiency leads to activation of
the innate immune system (25, 35). Macrophages from DNase II–
deficient mice accumulate undigested DNA, resulting in the production of TNF-a and polyarthritis (28). DNase II also forms
a barrier against the propagation of horizontally transferred DNA
in normal cells (36). In this study, silencing of DNase II did not
lead to any discernible accumulation of extranuclear DNA in
macrophages nor did this lead to the secretion of proinflammatory
TNF-a by macrophages after the ingestion of NETs. Nonetheless,
it is noteworthy that TNF-a secretion in response to LPS + NETs
was significantly reduced in HMDMs with reduced DNase II expression, suggesting a role for this nuclease in the sensing of NET
DNA in cells costimulated through TLRs. Moreover, transfection
of NETs or NET DNA inside macrophages leads to the production
of type I IFNs, whereas the normal uptake of NETs by macrophages is immunologically silent. Further studies of intracellular
DNA-sensing systems, as well as DNA-degrading systems in macrophages that are engaged in the clearance of NETs, are warranted.
In the present model of PMA-triggered NET formation, macrophage internalization of NETs alone did not give rise to a proinflammatory response. PMA is a potent activator of the NADPH
Downloaded from http://www.jimmunol.org/ by guest on August 1, 2017
FIGURE 6. NET clearance by macrophages is noninflammatory. (A) Purified NETs were administered to HMDMs for their ingestion, and macrophage
supernatants were recovered after 1 or 24 h and analyzed for the presence of IFN-a. Complete medium (see Materials and Methods) and supernatant from
neutrophils nonstimulated with PMA were included as negative controls. Results shown are the mean values 6 SD of three independent experiments. Oneway ANOVA analysis was performed with Tukey correction, and significant differences in reference to the medium control are indicated (***p , 0.001).
Note the very low levels of IFN-a production. (B–D) HMDMs were treated as in (A) with additional stimulations in the presence of 100 ng/ml LPS as
indicated, and supernatants were harvested for the analysis of IL-1b (B), TNF-a (C), and IL-6 (D). Results shown are the mean values 6 SD of three
independent experiments. One-way ANOVA analysis was performed with Tukey correction, and significant differences in reference to the corresponding
LPS control (*p , 0.05, ***p , 0.001) are indicated.
The Journal of Immunology
9
oxidase (17), and NET formation is largely NADPH oxidase dependent (2, 37), although examples do exist of NADPH oxidase–
independent NET induction (38). Of note, the very recent observation that NADPH oxidase (Nox2)–deficient mice display exacerbated lupus-like symptoms implies that NET formation per se
does not contribute to SLE (39). Indeed, based on the latter animal
study and the current in vitro studies using primary human neutrophils and macrophages, NETosis may be viewed as an inherently
noninflammatory form of cell death, in cases of NADPH oxidase–
dependent generation of NETs. This is in accordance with recent
studies of mice immunized with NETs, which have shown that
isolated exposure to NETs prepared using hydrogen peroxide stimulation is insufficient to break immune tolerance (40). How may
these observations be reconciled with other recent reports (10, 11)
demonstrating that the delivery of NETs to dendritic cells has a
proinflammatory effect, resulting in the production of type I IFNs
through a TLR-dependent pathway? It is conceivable that the presence of additional microbial and/or cellular factors such as HMGB1
(not present in NETs in our study) is needed. In fact, we noted
that the LPS-triggered cytokine response was enhanced when
macrophages were cocultured with NETs as compared with LPS
stimulation only. Based on these observations, one may speculate
that in an inflammatory milieu, NETs loaded with bacteria elicit
a stronger proinflammatory response in neighboring monocytes/
macrophages than bacteria alone, thus enabling the immune system
to mount a more robust and prolonged response against invading
pathogens.
In summary, we report that monocyte-derived macrophages are
capable of efficient clearance of NETs that have been extruded
from neutrophils, and that this process is facilitated by extracellular
preprocessing of NETs by DNase I, as well as by the opsonization
of NETs with C1q. Following their uptake, NETs are apparently
shuttled via phagosomes to lysosomes, and experiments using CQ
suggested that degradation occurred in the lysosomal compartment;
however, we could not conclusively demonstrate a role for DNase II
in the degradation process and we cannot exclude that degradation
may be taking place in other compartment(s). Further studies are
thus needed to identify the nuclease(s) responsible for NET degradation in macrophages. Importantly, the process of NET clearance is immunologically silent insofar as macrophages do not
produce proinflammatory cytokines after ingestion of NETs alone.
In this respect, the clearance of NETs resembles the clearance of
apoptotic cells, a process that is also “silent” and thus serves to
preserve homeostasis in tissues (41). Rapid clearance of NETs
by macrophages could act to prevent the induction of autoimmune
responses toward NET DNA or other NET components and, consequently, a deficiency in macrophage clearance of NETs may
therefore lead to autoimmune disease.
Acknowledgments
We thank Dr. Kjell Hultenby (Electron Microscopy Unit, Karolinska University Hospital Huddinge, Stockholm, Sweden) for assistance with scanning
electron microscopy. We also thank Ingrid Delin (Institute of Environmental
Medicine, Karolinska Institutet) and Dr. Florian Salomons (Department of
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FIGURE 7. NETs and NET DNA can be sensed by macrophages. (A) Expression of IFN-a and IFN-b mRNA in HMDMs incubated for 24 h with NETs
in the presence or absence of CQ (10 mM) was monitored using RT-PCR. The same amount of NETs or corresponding amounts of DNA purified from NETs
were transfected into HMDMs using Lipofectin. HMDMs incubated with Lipofectin alone were included as a negative control. Gene expression values were
normalized to gapdh expression levels. Results shown are the mean values 6 SD of three independent experiments. Unpaired, two-tailed Student t test
analysis was performed, and significant differences between the indicated samples and the corresponding Lipofectin control are indicated (*p , 0.05,
***p , 0.001). (B) The same experimental conditions as in (A) were used to transfect NETs or NET DNA into macrophages, and IFN-a and IFN-b
protein levels were assessed by ELISA. One-way ANOVA analysis was performed with Tukey correction, and significant differences in reference to the
corresponding Lipofectin control (*p , 0.05, ***p , 0.001) are indicated.
10
Cell and Molecular Biology, Karolinska Institutet) for technical advice on
cytokine analysis and confocal microscopy, respectively.
Disclosures
The authors have no financial conflicts of interest.
References
20. Thomas, G. M., C. Carbo, B. R. Curtis, K. Martinod, I. B. Mazo, D. Schatzberg,
S. M. Cifuni, T. A. Fuchs, U. H. von Andrian, J. H. Hartwig, et al. 2012. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans
and mice. Blood 119: 6335–6343.
21. Buchanan, J. T., A. J. Simpson, R. K. Aziz, G. Y. Liu, S. A. Kristian, M. Kotb,
J. Feramisco, and V. Nizet. 2006. DNase expression allows the pathogen group A
Streptococcus to escape killing in neutrophil extracellular traps. Curr. Biol. 16:
396–400.
22. Berends, E. T., A. R. Horswill, N. M. Haste, M. Monestier, V. Nizet, and
M. von Köckritz-Blickwede. 2010. Nuclease expression by Staphylococcus
aureus facilitates escape from neutrophil extracellular traps. J. Innate Immun. 2:
576–586.
23. Tinazzi, E., A. Puccetti, R. Gerli, A. Rigo, P. Migliorini, S. Simeoni, R. Beri,
M. Dolcino, N. Martinelli, R. Corrocher, and C. Lunardi. 2009. Serum DNase I,
soluble Fas/FasL levels and cell surface Fas expression in patients with SLE:
a possible explanation for the lack of efficacy of hrDNase I treatment. Int.
Immunol. 21: 237–243.
24. Sjöberg, A. P., L. A. Trouw, and A. M. Blom. 2009. Complement activation and
inhibition: a delicate balance. Trends Immunol. 30: 83–90.
25. Okabe, Y., K. Kawane, S. Akira, T. Taniguchi, and S. Nagata. 2005. Toll-like
receptor-independent gene induction program activated by mammalian DNA
escaped from apoptotic DNA degradation. J. Exp. Med. 202: 1333–1339.
26. Kawane, K., H. Fukuyama, G. Kondoh, J. Takeda, Y. Ohsawa, Y. Uchiyama, and
S. Nagata. 2001. Requirement of DNase II for definitive erythropoiesis in the
mouse fetal liver. Science 292: 1546–1549.
27. Kawane, K., H. Fukuyama, H. Yoshida, H. Nagase, Y. Ohsawa, Y. Uchiyama,
K. Okada, T. Iida, and S. Nagata. 2003. Impaired thymic development in mouse
embryos deficient in apoptotic DNA degradation. Nat. Immunol. 4: 138–144.
28. Kawane, K., M. Ohtani, K. Miwa, T. Kizawa, Y. Kanbara, Y. Yoshioka,
H. Yoshikawa, and S. Nagata. 2006. Chronic polyarthritis caused by mammalian
DNA that escapes from degradation in macrophages. Nature 443: 998–1002.
29. Yoshida, H., Y. Okabe, K. Kawane, H. Fukuyama, and S. Nagata. 2005. Lethal
anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA. Nat. Immunol. 6: 49–56.
30. Clark, S. R., A. C. Ma, S. A. Tavener, B. McDonald, Z. Goodarzi, M. M. Kelly,
K. D. Patel, S. Chakrabarti, E. McAvoy, G. D. Sinclair, et al. 2007. Platelet TLR4
activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat.
Med. 13: 463–469.
31. Papayannopoulos, V., K. D. Metzler, A. Hakkim, and A. Zychlinsky. 2010.
Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil
extracellular traps. J. Cell Biol. 191: 677–691.
32. Hakkim, A., T. A. Fuchs, N. E. Martinez, S. Hess, H. Prinz, A. Zychlinsky, and
H. Waldmann. 2011. Activation of the Raf-MEK-ERK pathway is required for
neutrophil extracellular trap formation. Nat. Chem. Biol. 7: 75–77.
33. Menegazzi, R., E. Decleva, and P. Dri. 2012. Killing by neutrophil extracellular
traps: fact or folklore? Blood 119: 1214–1216.
34. Gaipl, U. S., T. D. Beyer, P. Heyder, S. Kuenkele, A. Böttcher, R. E. Voll,
J. R. Kalden, and M. Herrmann. 2004. Cooperation between C1q and DNase I in
the clearance of necrotic cell-derived chromatin. Arthritis Rheum. 50: 640–649.
35. Kawane, K., H. Tanaka, Y. Kitahara, S. Shimaoka, and S. Nagata. 2010.
Cytokine-dependent but acquired immunity-independent arthritis caused by
DNA escaped from degradation. Proc. Natl. Acad. Sci. USA 107: 19432–19437.
36. Bergsmedh, A., J. Ehnfors, K. Kawane, N. Motoyama, S. Nagata, and
L. Holmgren. 2006. DNase II and the Chk2 DNA damage pathway form a genetic barrier blocking replication of horizontally transferred DNA. Mol. Cancer
Res. 4: 187–195.
37. Keshari, R. S., A. Verma, M. K. Barthwal, and M. Dikshit. 2013. Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced
NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540.
38. Farley, K., J. M. Stolley, P. Zhao, J. Cooley, and E. Remold-O’Donnell. 2012. A
serpinB1 regulatory mechanism is essential for restricting neutrophil extracellular trap generation. J. Immunol. 189: 4574–4581.
39. Campbell, A. M., M. Kashgarian, M. J. Shlomchik. 2012. NADPH oxidase
inhibits the pathogenesis of systemic lupus erythematosus. Sci. Transl. Med. 4:
157ra141.
40. Liu, C. L., S. Tangsombatvisit, J. M. Rosenberg, G. Mandelbaum,
E. C. Gillespie, O. P. Gozani, A. A. Alizadeh, and P. J. Utz. 2012. Specific posttranslational histone modifications of neutrophil extracellular traps as immunogens
and potential targets of lupus autoantibodies. Arthritis Res. Ther. 14: R25.
41. Fadeel, B., D. Xue, and V. Kagan. 2010. Programmed cell clearance: molecular
regulation of the elimination of apoptotic cell corpses and its role in the resolution of inflammation. Biochem. Biophys. Res. Commun. 396: 7–10.
Downloaded from http://www.jimmunol.org/ by guest on August 1, 2017
1. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, D. S. Weiss,
Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil extracellular traps kill
bacteria. Science 303: 1532–1535.
2. Fuchs, T. A., U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn,
Y. Weinrauch, V. Brinkmann, and A. Zychlinsky. 2007. Novel cell death program
leads to neutrophil extracellular traps. J. Cell Biol. 176: 231–241.
3. Yipp, B. G., B. Petri, D. Salina, C. N. Jenne, B. N. Scott, L. D. Zbytnuik,
K. Pittman, M. Asaduzzaman, K. Wu, H. C. Meijndert, et al. 2012. Infectioninduced NETosis is a dynamic process involving neutrophil multitasking in vivo.
Nat. Med. 18: 1386–1393.
4. Papayannopoulos, V., and A. Zychlinsky. 2009. NETs: a new strategy for using
old weapons. Trends Immunol. 30: 513–521.
5. Saffarzadeh, M., and K. T. Preissner. 2013. Fighting against the dark side of
neutrophil extracellular traps in disease: manoeuvres for host protection. Curr.
Opin. Hematol. 20: 3–9.
6. Muñoz, L. E., K. Lauber, M. Schiller, A. A. Manfredi, and M. Herrmann. 2010.
The role of defective clearance of apoptotic cells in systemic autoimmunity. Nat.
Rev. Rheumatol. 6: 280–289.
7. Hakkim, A., B. G. Fürnrohr, K. Amann, B. Laube, U. A. Abed, V. Brinkmann,
M. Herrmann, R. E. Voll, and A. Zychlinsky. 2010. Impairment of neutrophil
extracellular trap degradation is associated with lupus nephritis. Proc. Natl.
Acad. Sci. USA 107: 9813–9818.
8. Lauber, K., S. G. Blumenthal, M. Waibel, and S. Wesselborg. 2004. Clearance of
apoptotic cells: getting rid of the corpses. Mol. Cell 14: 277–287.
9. Napirei, M., H. Karsunky, B. Zevnik, H. Stephan, H. G. Mannherz, and
T. Möröy. 2000. Features of systemic lupus erythematosus in Dnase1-deficient
mice. Nat. Genet. 25: 177–181.
10. Garcia-Romo, G. S., S. Caielli, B. Vega, J. Connolly, F. Allantaz, Z. Xu,
M. Punaro, J. Baisch, C. Guiducci, R. L. Coffman, et al. 2011. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus
erythematosus. Sci. Transl. Med. 3: 73ra20.
11. Lande, R., D. Ganguly, V. Facchinetti, L. Frasca, C. Conrad, J. Gregorio,
S. Meller, G. Chamilos, R. Sebasigari, V. Riccieri, et al. 2011. Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in
systemic lupus erythematosus. Sci. Transl. Med. 3: 73ra19.
12. Ricklin, D., G. Hajishengallis, K. Yang, and J. D. Lambris. 2010. Complement: a
key system for immune surveillance and homeostasis. Nat. Immunol. 11: 785–797.
13. Botto, M., C. Dell’Agnola, A. E. Bygrave, E. M. Thompson, H. T. Cook,
F. Petry, M. Loos, P. P. Pandolfi, and M. J. Walport. 1998. Homozygous C1q
deficiency causes glomerulonephritis associated with multiple apoptotic bodies.
Nat. Genet. 19: 56–59.
14. Taylor, P. R., A. Carugati, V. A. Fadok, H. T. Cook, M. Andrews, M. C. Carroll,
J. S. Savill, P. M. Henson, M. Botto, and M. J. Walport. 2000. A hierarchical role
for classical pathway complement proteins in the clearance of apoptotic cells
in vivo. J. Exp. Med. 192: 359–366.
15. Leffler, J., M. Martin, B. Gullstrand, H. Tydén, C. Lood, L. Truedsson,
A. A. Bengtsson, and A. M. Blom. 2012. Neutrophil extracellular traps that are
not degraded in systemic lupus erythematosus activate complement exacerbating
the disease. J. Immunol. 188: 3522–3531.
16. Fadeel, B., A. Åhlin, J. I. Henter, S. Orrenius, and M. B. Hampton. 1998. Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen
species. Blood 92: 4808–4818.
17. Kagan, V. E., B. Gleiss, Y. Y. Tyurina, V. A. Tyurin, C. Elenström-Magnusson,
S. X. Liu, F. B. Serinkan, A. Arroyo, J. Chandra, S. Orrenius, and B. Fadeel.
2002. A role for oxidative stress in apoptosis: oxidation and externalization of
phosphatidylserine is required for macrophage clearance of cells undergoing
Fas-mediated apoptosis. J. Immunol. 169: 487–499.
18. Beiter, K., F. Wartha, B. Albiger, S. Normark, A. Zychlinsky, and B. HenriquesNormark. 2006. An endonuclease allows Streptococcus pneumoniae to escape
from neutrophil extracellular traps. Curr. Biol. 16: 401–407.
19. Remijsen, Q., T. W. Kuijpers, E. Wirawan, S. Lippens, P. Vandenabeele, and
T. Vanden Berghe. 2011. Dying for a cause: NETosis, mechanisms behind an
antimicrobial cell death modality. Cell Death Differ. 18: 581–588.
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