Original Article
Neutrophil Extracellular Traps Contain
Mitochondrial as well as Nuclear DNA and
Exhibit Inflammatory Potential
Ravi S. Keshari,1 Anupam Jyoti,1 Sachin Kumar,1 Megha Dubey,1 Anupam Verma,2
Bangalore S. Srinag,3 Hanumanthappa Krishnamurthy,3 Manoj K. Barthwal,1 Madhu Dikshit1*
1
Pharmacology Division, CSIR-Central
Drug Research Institute, Lucknow 226
001, India
2
Department of Transfusion Medicine,
Sanjay Gandhi Postgraduate Institute of
Medical Sciences, Lucknow, India
3
Central Image-Flow Facility, National
Centre for Biological Sciences,
Bangalore, India
Received 6 July 2011; Revision Received
4 October 2011; Accepted 9 November
2011
Additional Supporting Information may be
found in the online version of this article.
Grant sponsors: Department of Biotechnology, India; Council of Scientific and
Industrial Research, India; Carl Zeiss,
Germany; Olympus, Japan; Department of
Science and Technology, India; NCBS
imaging facility
CSIR-CDRI communication number: 8162
*Correspondence to: Madhu Dikshit,
Pharmacology Division, CSIR-Central
Drug Research Institute, Lucknow 226
001, India
Email: [email protected]
Published online 13 December 2011 in
Wiley Online Library
(wileyonlinelibrary.com)
DOI: 10.1002/cyto.a.21178
© 2011 International Society for
Advancement of Cytometry
Cytometry Part A 81A: 238 247, 2012
Abstract
Neutrophils expel extracellular traps (NETs) to entrap and exterminate the invaded
micro-organisms. Acute/chronic inflammatory disorders are often observed with aberrantly enhanced NETs formation and high nitric oxide (NO) availability. Recent study
from this laboratory demonstrated release of NETs from human neutrophils following
treatment with SNP or SNAP. This study is an extension of our previous finding to
explore the extracellular bacterial killing, source of DNA in the expelled NETs, their
ability to induce proinflammatory cytokines release from platelets/THP-1 cells, and
assessment of NO-mediated free radical formation by using a consistent NO donor,
DETA-NONOate. NO-mediated NETs exhibited extracellular bacterial killing as
determined by colony forming units. NO-mediated NETs formation was due to the
activation of NADPH oxidase and myeloperoxidase. NO- or PMA-mediated NETs were
positive for both nuclear and mitochondrial DNA as well as proteolytic enzymes. Incubation of NETs with human platelets enhanced the release of IL-1b and IL-8, while with
THP-1 cells, release of IL-1b, IL-8, and TNFa was observed. This study demonstrates
that NO by augmenting enzymatic free radical generation release NETs to promote
extracellular bacterial killing. These NETs were made up of mitochondrial and nuclear
DNA and potentiated release of proinflammatory cytokines. ' 2011 International Society
for Advancement of Cytometry
Key terms
nitric oxide; neutrophil extracellular traps; NADPH-oxidase; myeloperoxidase; inflammatory cytokine; mitochondrial and nuclear DNA
NEUTROPHILS, the most abundant phagocytic cells in human blood, are well recognized for their role in innate immune response and inflammation (1). In recent years,
many new facets of PMNs have been discovered such as antigen presentation (2),
neutrophil extracellular traps (NETs) formation (3), as well as their cross talk with
dendritic cells (DCs) (4), T cells (5), and macrophages (1). Neutrophils, besides
phagocytosis, also release NETs to extracellulary execute microbes (3), which was
defined as beneficial suicide (6) and was termed NETosis (7). PMA (3,6), LPS (6),
IL-8 (6), and nitric oxide (NO) (8) were found to promote NETs release by enhancing the formation of reactive oxygen species (ROS) (6). Since patients of chronic
granulomatous disease (CGD) were unable to form NETs, role of ROS in NETs
formation was confirmed (6). Conversely, NETs formation by CXCR2 in the cystic fibrosis airway inflammation was independent of NADPH oxidase activation (9).
NETs contain granular proteins such as elastase, cathepsin G, and myeloperoxidase,
which are embedded on the back bone of nuclear DNA and histones (3,6). Moreover,
Yousefi et al. (10) recently proposed that NETs release by GM-CSF and LPS contained mitochondrial DNA rather than nuclear DNA.
Neutrophils are recruited first to the site of infection, inflammation, or injury
(1). Consequently, NETs contents are also abundant at the site of infection and acute
ORIGINAL ARTICLE
inflammation (3,11,12). Numerous reports suggest high
amount of NO or its metabolites at these sites (13,14). NETs
formation has been documented in the patients of pre-eclampsia (15), sepsis (16), malaria (17), lupus nephritis (18), and
cystic fibrosis (9). Infective conditions are prevalent in these
pathologies, although pre-eclampsia is associated with sterile
inflammation. Augmented iNOS expression in the patients of
urinary tract infection, tuberculosis, malaria, and sepsis has also
been documented (14,19). NO, a key mediator of inflammation, also exhibits microbicidal, antifungal, and antiviral
properties (20). Neutrophils possess nNOS and iNOS isoforms
(21–23) and produce substantial amount of NO (24), which
modulate neutrophil free radical generation (25–29). Neutrophils use both ROS and RNS as armor against intruders
(20,24), and high amount of ascorbate pool in neutrophils
seems to support NO production by these cells (30).
We have recently reported that treatment of PMNs with
NO donors such as SNP and SNAP released NETs (8). There
are several concerns with these NO donors; SNP is not a spontaneous NO donor and also releases cyanide, which could
have potential side effects (31,32). Although SNAP is a spontaneous NO releaser, the amount of NO released is often not
related to its bioactivity and also exhibits differences in its
bioactivity and cytotoxicity (33). Moreover, previous study
did not ascertain capability of bacterial killing by SNP/SNAPinduced webs-like structure and the source of DNA that constituted NETs. This study was therefore undertaken by using a
more specific NO donor NONOates to further explore the
effect of NO-mediated NETs release on bacterial killing, source
of DNA, proinflammatory potential of NETs, and the confirmation of NO-mediated ROS generation in NETs release.
METHODS
Isolation of Human Neutrophils
Peripheral blood from adult volunteers was collected by
venipuncture using heparin/EDTA as anticoagulant. Blood
was layered on Histopaque 1119 and centrifuged for 20 min at
800g. The lower interphase having granulocytes was washed
with RPMI 1640 medium and was loaded on the discontinuous Percoll gradients as described earlier (34). Isolated PMNs
were suspended in RPMI 1640 medium containing 0.5% FBS.
The purity and viability of the isolated PMNs were ascertained
by CD15-PE and PI staining using flow cytometer (Becton
Dickinson, San Diego, CA, USA), which was never \95%.
The study was approved by the institutional ethics committee
and was conducted according to the Declaration of Helsinki.
NETs Formation
Neutrophils (1 3 106) were seeded on cover-slips (precoated with 0.001% poly L-lysine) and treated with NO donor
DETA-NONOate (100–500 lM), PMA (20 nM), or vehicle for
30–180 min at 378C in a CO2 incubator (RS Biotech, United
Kingdom). Effect of various interventions such as N-acetyl-Lcysteine (NAC, 5 mM), diphenyleneiodonium chloride (DPI,
10 lM), or 4-aminobenzoic acid hydrazide (ABAH, 100 lM)
was also monitored on NETs release by incubating with PMNs
Cytometry Part A 81A: 238 247, 2012
for 15 min at 378C and then treated with DETA-NONOate for
3 h. After fixation and blocking samples were stained overnight with 20 lg/ml of rabbit polyclonal elastase antibody
(Calbiochem, San Diego, CA, USA) and were visualized after
incubation with the secondary antibody (1:200, chicken antirabbit AF 488 antibody, Molecular Probes, Eugene, OR, USA)
by confocal microscope and assessed for the incidence of NETs
formation (12). DNA was stained with propidium iodide (PI)
(red, 10 lg/ml) or Sytox green (green, 5 lM). Mitochondrial
DNA was stained using MitoSox Red (red, 5 lM). High magnification and low magnification images were captured by
Carl Zeiss (Germany) or Olympus (Japan) confocal microscopes or Leica fluorescence microscope by using appropriate
lenses and filters (6).
Extracellular Bacterial Killing
1 3 106 cells/ml were seeded in 12-well tissue culture
plates and stimulated with DETA-NONOate (100 lM) or
PMA (20 nM) for 3 h at 378C in a 5% CO2 atmosphere to
form NETs. Supernatant from each well was removed, and
PMNs were treated with cytochalasin D (10 lg/ml) for 15 min
(3), then bacteria (E. coli DH5a) were added at a multiplicity
of infection (MOI) of 0.1 in 1.0 ml of RPMI per well and centrifuged at 700g for 10 min. Plates were incubated for 1 h at
378C in a 5% CO2 atmosphere. To dismantle the NETs, 100 U/
ml MNase was added into the respective well prior to the
addition of bacteria. After incubation each well was scraped
and mixed up properly, contents were collected, and bacterial
viability was assayed by assessing bacterial colony forming
units (CFUs). Briefly, 50 ll aliquot from each well was spread
onto agar plates and allowed to grow overnight at 378C. CFU
were counted, and results have been expressed as reported
earlier (3,35,36).
PCR of Mitochondrial and Nuclear Genes
DNA from the supernatants of DETA-NONOate or
PMA-treated PMNs was isolated by using the genomic DNA
isolation kit (Fermentas Life Sciences, Lithuania). The origin
of the extracellular DNA was determined by amplifying two
nuclear (gapdh, actin) and two mitochondrial (atp6, co1)
genes. PCR conditions and the primers used in the study have
been described earlier (10).
Quantification of DNA and Elastase Release
Neutrophils (1 3 106) suspended in RPMI 1640 medium
(phenol red free) containing 0.5% FBS were seeded on poly-Llysine precoated tissue culture plates (24 well) and subsequently treated with DETA-NONOate or PMA for 3 h. Plates
were centrifuged for 5 min at 500g, supernatant was collected,
and NETs associated elastase was released by incubating the
cells for 10 min in medium containing 500 mU/ml of MNase.
Total neutrophils elastase was quantified from resting neutrophils (1 3 106) lysed with 0.02% triton X-100 in 1 M NaCl.
Elastase activity was measured by using N-(Methoxysuccinyl)Ala-Ala-Pro-Val 4-nitroanilide (100 lM) (6), while NET
bound DNA in medium containing 500 mU/ml of MNase was
measured after inhibiting the nuclease activity by adding 5
239
ORIGINAL ARTICLE
mM EDTA. Total DNA was isolated from neutrophils with
DNazol supplemented with 1% polyacryl carrier (Molecular
Research Center) according to the manufacturer’s instructions
and solubilized in TE buffer. Supernatant was collected, and
released DNA was measured by using Sytox green (Invitrogen,
Carlsbad, CA, USA), as reported earlier (3,6).
Inflammatory Cytokine Release
PMNs (1 3 106 cells/ml) were seeded in 24-well tissue
culture plates and stimulated with DETA-NONOate (100 lM)
or PMA (20 nM) for 3 h at 378C in a 5% CO2 atmosphere to
form NETs. Supernatant from each well was removed, PMNs
were treated with cytochalasin D (10 lg/ml) for 15 min (3)
and incubated with platelet (1 3 107) or THP-1(1 3 106) cells
for 60 min. Plates were centrifuged, supernatant was collected
and stored at 2708C. IL-8, TNFa, and IL-1b content in the supernatant were determined by using ELISA kits (BD OptEIA;
BD Biosciences, San Diego, CA, USA).
Free Radical Generation
Dichlorofluorescein fluorescence. PMNs (2 3 106cells) were
incubated with vehicle or with various interventions [DPI
(diphenyleneiodonium chloride, 10 lM), ABAH (4-aminobenzoic acid hydrazide, 100 lM), and NAC (N-acetyl-L-cysteine, 5 mM)] for 15 min at 378C, loaded with DCF-DA
(dichlorofluorescein diacetate, 10 lM, Sigma Aldrich, St.
Louis, MO, USA; catalogue no. D6883) for 15 min, and then
treated with DETA-NONOate (1 lM- 1 mM) or PMA (20
nM). NEM (N-ethylmaleimide, 150 lM) were added either 5
min before the addition of stimulator designated as
(NEM1control) or 15 min after the addition of stimulator
(control1NEM). PMA was used as a positive control. PMNs
were initially characterized by CD15 (Log scale) and CD45
binding (Log scale) and in subsequent experiment, PMNs
were gated on the basis of FSC (Linear scale) and SSC (Linear
scale). Gate was designated as G1, and FL-1 fluorescence (Log
scale) of 10,000 cells were acquired to monitor free radical
generation by using FACS Calibur (Becton Dickinson, USA)
(28). Primary parameter (FSC) was used to set the threshold.
Histogram plot of the gated population (Count vs. FL-1) and
gate statistics were created, which includes mean fluorescence
of FL-1. Mean fluorescence intensity of all the samples was
noted, and results were expressed as mean stimulation index
[DCF (MSI)/free radical generation (MSI)]. MSI was calculated as mean fluorescence intensity of sample divided by the
mean fluorescence intensity of the control sample (Figs. 4C
and 4D). For the time kinetics experiments, MSI was calculated as mean fluorescence intensity of sample at a particular
time divided by the mean fluorescence intensity of control at
zero time (Fig. 4E).
Nitroblue tetrazolium reduction.. Neutrophils (1 3 106)
were preincubated with vehicle, DPI (10 lM), or apocynin
(100 lM) for 15 min, 10 lM nitroblue tetrazolium (NBT) was
added to all the tubes, subsequently vehicle, PMA (20 nM), or
DETA-NONOate (100 lM) was added, and samples were
240
incubated at 378C. After 60 min incubation, cells were washed
with PBS, lysed by adding 120 ll of 2 M KOH and 140 ll of
DMSO. Contents were properly mixed for 10 min by shaking
at room temperature, and absorbance was read at 620 nm
(BioTek, Winooski, VT, USA) (37).
Immunoblotting of p47 and DMPO-nitrone adduct.. NOmediated free radical generation was also measured by using
DMPO and DMPO-nitrone antibody. PMNs (5 3 107 cells)
were incubated at 378C for 60 min with DETA-NONOate (100
lM) or PMA (20 nM) and DMPO (50 mM) in the presence
or absence of NAC (5 mM), DPI (10 lM), ABAH (100 lM),
or apocynin (100 lM). Samples (30 lg protein) were loaded
on 10% SDS-PAGE and transferred to PVDF membrane,
which was blocked with 5% skimmed milk in TBST and then
incubated with rabbit anti-DMPO-nitrone antibody (1:3,000
in 1.5% skimmed milk TBST) for 2 h at room temperature
(38).
In another set of experiment, translocation of p47 phox
from the cytosol to neutrophil membranes was assessed.
Membrane fraction was prepared from cells (1 3 107cells) pretreated with PMA (20 nM) or DETA-NONOate (100 lM) at
378C for 60 min (39). Membrane fraction (20 lg protein) was
run on 10% SDS PAGE and subsequently transferred to PVDF
membrane. The membrane was blocked with 5% skimmed
milk in TBST, incubated with p47 phox antibody (1:500 in
1.5% skimmed milk TBST) for 2 h at room temperature. Both
proteins were detected after incubation with HRP-conjugated
anti-rabbit IgG (1:20,000) for 2 h by enhanced chemiluminescence detection reagents (Millipore, Bedford, MA, USA).
Statistical Analysis
Results have been represented as mean SEM of at least
three to five independent experiments in the each set as
described earlier. Student’s t-test or analysis of variance for
repeated measurements was used. When ‘‘F’’ value was significant, pair wise comparisons were performed by using Newman-Keul’s analysis. The alpha level of all the tests or P value
was set at 0.05.
RESULTS
DETA-NONOate- and PMA-Induced NETs Formation
and Bacterial Killing
Human neutrophils treated with DETA-NONOate (100–
500 lM) led to a time and concentration-dependent generation of NETs as characterized by elastase and DNA staining
(Figs. 1D–1F and 2A and 2B). PMA also induced the generation of NETs and was used as a positive control (Figs. 1B, 1G–
1I, and 2A and 2B). Resting neutrophils (Fig. 1A) stained with
elastase antibody and PI demonstrated punctated pattern of
elastase distribution and multilobed nuclei, indicating nuclear
and granular components of the cells. Cells treated with
DETA-NONOate however lost the normal morphology and
released NETs, which was evident from the colocalization of
elastase and DNA. DETA-NONOate-induced NETs were disintegrated by the MNase treatment confirming that extracellular
NETs Contain Mitochondrial and Nuclear DNA
ORIGINAL ARTICLE
Figure 1. DETA-NONOate- or PMA-induced NETs formation in neutrophils. (A) Resting neutrophils stained with elastase antibody conjugated with AF 488 (green) and PI (10 lg/ml, red) showing multilobed nuclei and punctate elastase. (B) Merge image of PMA and (C) DETA
NONOate treated neutrophils at lower magnification. (D, G) neutrophils stained with elastase antibody, (E, H) PI, and (F, I) merge image of
DETA-NONOate- (D-F) and PMA- (G-I) treated neutrophils for 3 h showing colocalization of elastase and DNA (NETs). (J) Resting neutrophils stained with sytox green. (K) DETA-NONOate-induced NETs stained with sytox green, (L) NETs dissolved after MNase treatment. (Bar
A-C and J-K 20 lm, D-I 10 lm). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
fibers/NETs were made up of DNA (Figs. 1K–1L). NETs have
been defined as a discrete area of bright fluorescence larger in
size than a neutrophil (12). Effect of DETA-NONOate on
extracellular killing of E. coli DH5a was also abolished by
MNase treatment, suggesting that the bacterial killing was
mediated by NETs (Fig. 2C).
PMNs stained with mitochondrial DNA binding dye,
MitoSox Red demonstrated the presence of mitochondrial
DNA in NETs following DETA-NONOate or PMA treatment
(Figs. 2F–2H).
DETA-NONOate- and PMA-Induced DNA
and Elastase Release
Since DNA constitutes the backbone of NETs, PCR was
thus performed to determine the source of DNA by using two
mitochondrial (atp6 and co1) and two nuclear (gapdh and
Cytometry Part A 81A: 238 247, 2012
actin) gene primers, which were amplified, confirming the
presence of both nuclear and mitochondrial DNA in NETs
(Fig. 2D). Release of DNA and elastase in the medium was
quantified following MNase (500 mU/ml) treatment. A significant increase in release of DNA and elastase was observed as
compared with control following DETA-NONOate or PMA
treatment, which also confirmed the formation of NETs by
DETA-NONOate (Fig. 2E).
NETs Enhanced Release of Proinflammatory Cytokines
Proinflammatory nature of NETs was explored by incubating the PMNs with DETA-NONOate or PMA to induce the
NETs formation as defined in the previous sections. After
removing the supernatant, NETs were incubated with platelet
or THP-1 cells. Release of proinflammatory cytokines was
observed from platelets (IL-1b and IL-8) and THP cells (IL241
ORIGINAL ARTICLE
Figure 2. DETA-NONOate- or PMA-induced NETs formation, bacterial killing, DNA, and elastase release (A) DETA-NONOate (100 lM) treated neutrophils released NETs in a time-dependent manner (*P \ 0.05, **P \ 0.01, ***P \ 0.001 vs. control). (B) NETs were counted after 2
h of DETA-NONOate or PMA treatment (***P \ 0.001 vs. control). Data presented as % release are mean count SEM of five transect from
three individual experiments, a NET has been defined as a discrete area of bright fluorescence larger in size than a neutrophil (12). (C) Bar
diagram representing NETs-mediated killing, which was substantially reduced by MNase treatment (*P \ 0.001 vs. MNase treatment). (D)
PCR amplification of mitochondrial and nuclear DNA fragments: ATP synthase subunit 6 (atp6), cytochrome oxidase c subunit 1 (co1),
Glyceraldehyde-3-phosphate dehydrogenase (gapdh), and b-actin, (Lane1-control, 2-DETA-NO, 3-PMA treated sample). (E) Quantification
of NET bound elastase and DNA following MNase (500 mU/ml) treatment. (***P \ 0.001 vs. control). (F) Resting PMNs stained with MitoSox Red. (G) PMA and (H) DETA-NONOate-treated PMNs stained with MitoSox Red. (Bar F-H 10 lm). [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
1b, TNFa and IL-8) using the specific ELISA kits. DETANONOate and PMA-induced NETs were capable of proinflammatory cytokines release from platelets and THP-1 cells,
which demonstrated the proinflammatory property of NETs
(Fig. 3). Interestingly, when the NETs were incubated with
fixed THP-1 cells or platelets (data not shown), the cytokines
release was similar to the control in the absence of platelets or
THP-1, suggesting that IL-1b, TNF, and IL-8 were released
from the viable platelets or THP-1 cells.
DETA-NONOate-Induced Free Radical
Generation and NETs Formation
It was reported that NETs formation was dependent on the
ROS generation from the activated NADPH oxidase. DETANONOate-induced NETs formation was also dependent on
ROS/RNS generation, which was determined by the various
methods (NBT reduction assay, migration of p47 phox to
plasma membrane, DCF oxidation, and DMPO nitrone adduct
formation). Treatment of PMNs with DETA-NONOate or
PMA led to the generation of superoxide radicals as monitored
242
by NBT reduction and was inhibited by NADPH oxidase inhibitors (DPI and apocynin) (Fig. 4A). NBT reduction assay also
suggested that DETA-NONOate-induced superoxide radical
generation was dependent on the activation of NADPH oxidase,
which was also confirmed by the migration of p47 phox from
cytosol to the plasma membrane. PMNs incubated with DETANONOate or PMA exhibited significant migration of p47 phox
to the membrane as compared with the control, suggesting activation and assembly of NADPH oxidase subunits (Fig. 4B).
Moreover, free radical generation was also determined by using
DCF-DA (dichlorofluorescein diacetate) in DETA-NONOateor PMA-treated cells (Fig. 4C). N-ethylmaleimide (NEM), an
inhibitor of the activation or assembly of NADPH oxidase,
when added 5 min before the addition of DETA-NONOate or
PMA, it almost completely inhibited the free radical generation.
However, when NEM was added 15 min after the addition of
DETA-NONOate or PMA, it reduced the free radical generation, suggesting that continuous replenishment of NADPH oxidase was necessary for free radical generation in both NO and
PMA treated cells (Fig. 4C). DCF fluorescence was increased
NETs Contain Mitochondrial and Nuclear DNA
ORIGINAL ARTICLE
Figure 3. NETs-induced IL-1b, TNF, and IL-8 release. PMNs were allowed to make NETs and then incubated with platelets and THP-1, supernatant were collected and (A) IL-1 b, (B) TNF and (C) IL-8 concentration were determined by ELISA (*P \ 0.05, **P \ 0.01, ***P \ 0.001
vs. control).
significantly following DETA-NONOate treatment in a concentration- (Fig. 4D, supplementary information) and time- (Fig.
4E) dependent manner. In the absence of PMNs, DETA-NONOate did not react with DCF to generate fluorescent adduct
(data not shown). Neutrophils pretreated with NAC, DPI
(NADPH oxidase inhibitor), or ABAH (myeloperoxidase inhibitor) significantly inhibited free radical generation (DCF fluorescence), suggesting the enzymatic free radical generation from
NADPH oxidase and myeloperoxidase (Fig. 4D, supplementary
information). DMPO (5, 5-dimethyl-1-pyrroline-N-oxide), a
spin trap, by reacting with radicals produces DMPO nitrone
adduct, which was formed in DETA-NONOate- or PMA-treated PMNs. NAC, a free radical scavenger, DPI, ABAH, and
apocynin prevented the formation of DMPO nitrone adduct,
suggesting generation of free radicals, and involvement of
NADPH oxidase as well as myeloperoxidase (Figs. 5A and 5B).
These experiments indicated that treatment of neutrophils with
DETA-NONOate induced free radical generation. Neutrophils
preincubated with NAC, DPI, and ABAH also inhibited the
release of NETs (Figs. 6A–6E), suggesting involvement of
NADPH oxidase and myeloperoxidase in DETA-NONOateinduced NETs formation. Experiments carried out with DETA
Figure 4. DETA-NONOate-mediated free radical generation and effect of interventions. (A) DETA- NONOate- or PMA-induced superoxide
production was inhibited by DPI or apocynin (*P \ 0.001 vs. control, $P \ 0.001 vs. DETA-NONOate treated cells). (B) DETA-NONOate- or
PMA-induced migration of p47 phox from cytosol to plasma membrane of neutrophils (representative blot of three similar experiments).
(C) DETA-NONOate- and PMA-induced free radical generation (*P \ 0.05, ***P \ 0.001 vs. control, $$$P \ 0.001 vs. DETA-NONOate treated
cells, ###P \ 0.001 vs. PMA treated cells. (D) Treatment of neutrophils with DETA-NONOate- (1 lM-1 mM) induced free radical generation
as assessed by DCF fluorescence, which was inhibited by NAC, DPI, and ABAH (*P \ 0.05, **P \ 0.001 vs. control, $P \ 0.01 vs. DETA-NONOate-treated cells). (E) Time-dependent free radical generation after stimulation with DETA-NONOate (100 lM).
Cytometry Part A 81A: 238 247, 2012
243
ORIGINAL ARTICLE
Figure 5. DETA-NONOate- and PMA-induced free radical generation as assessed by DMPO nitrone adduct antibody. (A) DETA-NONOateor PMA-induced DMPO nitrone adduct formation was inhibited by NAC (B) DETA-NONOate-induced DMPO nitrone adduct formation was
inhibited by DPI, ABAH, or apocynin (representative blot of three similar experiments).
alone did not lead to NBT reduction (Fig. 4A), DCF oxidation
(Fig. 4D), and NETs formation (Fig. 6A), and the results were
comparable to control, suggesting that effect of DETA-NONOate was due to NO release.
DISCUSSION
This study demonstrates that NO by augmenting free
radical generation induced NETs release from human neutrophils, which were capable of bacterial killing and induced the
release of proinflammatory cytokines form platelets and THP1 cells. iNOS induction and high circulating NO content have
been reported during sepsis and malaria (40–43), these
patients also have high level of circulating NET contents
(17,44), a possible correlation between NO and NETs release
was proposed by this laboratory (8), which has been further
explored in the present study.
Brinkmann et al. (3) described first the extracellular
microbial killing by human neutrophils through NETs formation, which was found to be mediated by the activation of
NADPH oxidase (6) and myeloperoxidase (8,45). Deposition
of NETs also initiated inflammatory response in the kidney,
while circulating MPO-DNA complexes triggered vasculitis
and promoted the autoimmune response against neutrophil
components in individuals with small-vessel Vasculitis (46).
Figure 6. Involvement of free radicals, NADPH oxidase, and myeloperoxidase in DETA-NONOate-induced NETs formation. (A) DETA-NONOate-induced NETs formation was inhibited by NAC, DPI, and ABAH (**P \ 0.001 vs. control, $P \ 0.001 vs. DETA-NONOate-treated cells).
(B) Bright field image of DETA-NONOate-treated neutrophils for 3 h formed NETs like structure, which was inhibited by the (C) NAC, (D)
DPI, and (E) ABAH (Bar B-E 20 lm).
244
NETs Contain Mitochondrial and Nuclear DNA
ORIGINAL ARTICLE
Aberrant NETs formation and lack of DNases to degrade NETs
in the patient’s might contribute to tissue damage and autoimmune diseases (47). Impairment of NETs degradation or
physical protection of NETs due to presence of serum DNase1
inhibitors is reported in systemic lupus erythematosus (SLE)
disease patients (18). Serum DNase1 inhibits the late phase of
ROS generation in neutrophils following E. coli or Listeria
monocytogenes treatments (48). Presence of antineutrophil
cytoplasmic autoantibodies (ANCA) against NETs components have been reported in SLE, primary vasculitides and
inflammatory bowel diseases (48,49). NETs release has also
been defined as an active process and was typically distinct
from neutrophil apoptosis and necrosis (6). NETs, the important components of innate defense system, limit the spreading
of microbial pathogens in vivo (3). The major constituent of
NETs were defined to be nuclear DNA and histones, which
trapped antimicrobial peptides, proteolytic enzymes (elastase,
cathepsin G), and myeloperoxidase (3).
PMNs, which reach first to the site of infection and
inflammation, get exposed to a large amount of NO, which
modulated PMNs free radical generation (27,50). Galkina
et al. (2009) by adding 1 mM DEA-NONOate (a short duration NO donor) demonstrated the formation of long tubulovesicular extensions (TVE, cytonemes, or membrane tethers),
which were found to extracellulary bind and aggregate Salmonella enterica serovar Typhimurium bacteria. Characterization
of TVE was done only on the basis of their dimensions (51).
We subsequently reported that treatment of adhered PMNs
with NO donors, such as SNP and SNAP, released NETs (8).
The present study ascertained the functional potential of NETs
for bactericidal action and inflammatory potential as well as
confirmed the role of free radicals in NETs release. DETANONOate was the donor of choice due to its long half life and
the ability of sustained NO release, 1 mM of DETA-NONOate
released 0.8 lM NO per minute with a half life of 20 h at 378C
(52). Large amount of NO production has been observed during various pathological conditions (14,53–55). Results
obtained from the present study demonstrated that neutrophils incubated with DETA-NONOate released NETs like
structures in a time- and concentration-dependent manner
(56), which labeled for DNA/elastase and were dissolved by
MNase. Biochemical quantification of elastase and DNA
release further confirmed NETs formation. Interestingly, both
nuclear and mitochondrial DNA was found to be released following treatment with DETA-NONOate or PMA, and these
webs-like structures were also capable of bacterial killing (Fig.
2C). Staining of PMNs with mitochondrial DNA binding dye
MitoSox Red demonstrated the presence of mitochondrial
DNA in NETs following DETA-NONOate or PMA treatment.
Neutrophils treated with NO donor, SNP, and SIN-1 were
found to exhibit more bactericidal activity in vitro (57). Fuchs
et al. (6) reported the release of nuclear components (as
demonstrated by histone labeling) after the stimulation of
PMNs with PMA. However, Yousefi et al. (10) recently
reported that only mitochondrial DNA rather than nuclear
DNA was released after treatment with GM-CSF and LPS.
This discrepancy might be due to the use of different stimulaCytometry Part A 81A: 238 247, 2012
tors to generate NETs. In the present study, release of both mitochondrial and nuclear DNA was seen following PMA/NO
treatment.
Inflammation is characterized by a multitude of interactions between monocytes, leukocytes, endothelial cells, and
platelets. Macrophages promote atherosclerosis via production
of various key biomediators including cytokines such as IL-1b,
TNF-a, and IL-8, concentrations of the cytokines released
from these cells appear to be a more sensitive indicator of
inflammatory status (58). Platelets distinctive armamentarium
with proinflammatory mediators as well as their surface receptors is predominantly known for their involvement in inflammatory or immune processes. Activated platelets release IL-1b
and IL-8 (59,60). Incubation of platelets or THP-1 cells with
NETs released significantly more TNFa, IL-1 b, and IL-8 (Fig.
3). When the NETs were incubated with fixed platelets or
THP-1 cells, the release of cytokines was similar to control
suggesting that cytokines were secreted from platelets and
THP-1. In the present study, we failed to detect TNFa secretion from platelets possibly platelets secrete very low amount
of TNFa. The results obtained thus suggest the proinflammatory potential of NETs for the first time.
NO-mediated free radical generation has been documented from this laboratory (27–29), and we extended further to
assess the involvement of free radicals in DETA-NONOatemediated NETs release from human neutrophils. Free radical
generation was assessed by DCF oxidation, NBT reduction,
DMPO nitrone adduct formation, and p47 translocation. Specificity of DCF and DMPO used in the present study has been
discussed earlier (8). NO donor-induced increase in DCF fluorescence and NBT reduction was inhibited by specific enzymatic
inhibitors and DETA alone did not induce NBT reduction, DCF
oxidation, as well as NETs formation suggesting that effect of
DETA-NONOate was specifically due to NO release. DETANONOate-induced NETs formation was inhibited by free radical scavenger (NAC), NADPH oxidase inhibitor (DPI), and
MPO inhibitor (ABAH), suggesting the role of ROS/RNS generation involving NADPH oxidase and MPO. Interaction of
NO with O2 or superoxide radicals (O22) also leads to the oxidative and nitrosative stress, which have been associated with
inflammatory pathologies (61–63). O22 and NO rapidly interact to produce potent cytotoxic oxidants, peroxynitrite
(ONOO2), and its conjugated acid ONOOH (64). Fully activated neutrophils produce 100–1,000 times more O22 than
NO, suggesting that PMNs would possibly produce very less
ONOO2/ONOOH. If NO production is more than superoxide,
NO2 will be formed, which reacts with NO to produce N2O3, a
nitrosating species (65,66). Moreover, peroxynitrite might mediate superoxide radical generation from human PMNs via ERKmediated activation of NADPH-oxidase (25). NO22 formed
from the oxidation of NO can be further oxidized by either
HOCl or MPO to generate reactive nitrogen species, NO2Cl,
and NO2 (67). NO22 dependent mechanisms might contribute
to tyrosine nitration observed in pathologies such as atherosclerosis (68), rheumatoid arthritis (69), and septic lung injury
(70). Formation of all these reactive species is plausible in the
experimental conditions used in the present study.
245
ORIGINAL ARTICLE
The results obtained have shown that NO-mediated generation of NETs were capable of bacterial killing, induced
release of proinflammatory cytokines, made up of nuclear and
mitochondrial DNA, and were dependent on free radical generation involving NADPH-oxidase and MPO. The present
study thus provides a convincing explanation for the earlier
findings of high NO availability, oxidative stress, and presence
of inflammatory cytokines along with the presence of NETs
contents in various pathological conditions.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical help
rendered by Mr. A.L. Vishwakarma and Mrs. M. Chaturvedi.
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