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The FASEB Journal article fj.201700410R. Published online October 23, 2017.
THE
JOURNAL
• RESEARCH •
www.fasebj.org
Endothelial nitric oxide synthase modulates Toll-like
receptor 4–mediated IL-6 production and permeability
via nitric oxide–independent signaling
Ryan J. Stark,*,1 Stephen R. Koch,* Hyehun Choi,* Eric H. Mace,* Sergey I. Dikalov,† Edward R. Sherwood,‡
and Fred S. Lamb*
*Department of Pediatrics, †Department of Medicine, and ‡Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville,
Tennessee, USA
Endothelial dysfunction, characterized by changes in eNOS, is a common finding in chronic
inflammatory vascular diseases. These states are associated with increased infectious complications. We hypothesized
that alterations in eNOS would enhance the response to LPS-mediated TLR4 inflammation. Human microvascular
endothelial cells were treated with sepiapterin or N-nitro-L-arginine methylester (L-NAME) to alter endogenous NO
production, and small interfering RNA to knockdown eNOS. Alterations of endogenous NO by sepiapterin, and
L-NAME provided no significant changes to LPS inflammation. In contrast, eNOS knockdown greatly enhanced
endothelial IL-6 production and permeability in response to LPS. Knockdown of eNOS enhanced LPS-induced p38.
Inhibition of p38 with SB203580 prevented IL-6 production, without altering permeability. Knockdown of p38
impaired NF-kB activation. Physical interaction between p38 and eNOS was demonstrated by immunoprecipitation,
suggesting a novel, NO-independent mechanism for eNOS regulation of TLR4. In correlation, biopsy samples
in patients with systemic lupus erythematous showed reduced eNOS expression with associated elevations in TLR4
and p38, suggesting an in vivo link. Thus, reduced expression of eNOS, as seen in chronic inflammatory disease, was
associated with enhanced TLR4 signaling through p38. This may enhance the response to infection in patients with
chronic inflammatory conditions.—Stark, R. J., Koch, S. R., Choi, H., Mace, E. H., Dikalov, S. I., Sherwood, E. R.,
Lamb, F. S. Endothelial nitric oxide synthase modulates Toll-like receptor 4–mediated IL-6 production and permeability via nitric oxide-independent signaling. FASEB J. 32, 000–000 (2018). www.fasebj.org
ABSTRACT:
KEY WORDS:
inflammation
•
eNOS
•
endothelial cells
In periods of sustained systemic inflammation, the endothelium, which serves as an interface between the blood
and tissues, becomes injured (1). The chronic inflammation
that induces that injury comes in many forms, from pure
vascular pathologies, such as hypertension and atherosclerosis, to more systemic autoimmune diseases
(1, 2). That injury disturbs the normal physiologic
function of the endothelium and is termed endothelial
dysfunction. Endothelial dysfunction is characterized
by a change in eNOS function, which is associated with
ABBREVIATIONS: BH4, tetrahydrobiopterin; DAF-FM DA, 4-amino-5-
methylamino-29,79-difluorofluorescein diacetate; FBS, fetal bovine serum;
G-CSF, granulocyte colony-stimulating factor; HMVEC, human dermal
microvascular endothelial cell; L-NAME, N-nitro-L-arginine methylester;
RA, rheumatoid arthritis; siRNA, small interfering RNA; SLE, systemic
lupus erythematosus
1
Correspondence: Vanderbilt University School of Medicine, 2200
Children’s Way, 5121 Doctors’ Office Tower, Nashville, TN 37232-9075,
USA. E-mail: ryan.stark@vanderbilt.edu
doi: 10.1096/fj.201700410R
This article includes supplemental data. Please visit http://www.fasebj.org to
obtain this information.
0892-6638/18/0032-0001 © FASEB
•
TLR-4
•
p38
reduced production of NO and increased formation of
reactive oxygen species (3, 4). Additional postulated
mechanisms by which alterations in eNOS biology may
lead to endothelial dysfunction include a reduction in the
bioavailability of tetrahydrobiopterin (BH4), a cofactor
that helps maintain dimerization of eNOS; altered phosphorylation of eNOS; and reduction in eNOS expression
(5–7). An underlying premise of endothelial dysfunction is
that there is a correlation between disruption of eNOS
function and endothelial injury, such as that seen in chronic
inflammation. Although that correlation is well established, the contribution of eNOS and endothelial injury
to acute systemic inflammation is poorly understood.
Sepsis, which is an infection-mediated, systemic, inflammatory response, carries a high burden of morbidity
and mortality (8, 9). The invading pathogen sets off a
cascade of normal and abnormal physiologic responses
within the host’s immune system, causing the clinical
features of sepsis. Integral to that innate host response is
the endothelium, which can enhance coagulation and
leukocyte trafficking, produce cytokines, and become
more permeable, leading to barrier dysfunction (10).
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1
Although injury to the endothelium can occur via numerous mechanisms, a critical pathway is activation
of the TLR (11). Those receptors recognize exogenous
ligands expressed by bacteria, such as LPS, produced by
gram-negative bacteria. They propagate the inflammatory response via activation of a variety of intracellular
signaling pathways, including enhancement of NO
production (12). It has long been postulated that the
increased NO production associated with sepsis leads
to worse outcomes and, potentially, adversely effects
endothelial function (13, 14). However, the role of NO in
sepsis remains controversial, as demonstrated in worsened
outcomes in animal and clinical trials with NO inhibition
(15). Consistent with that, animals overexpressing eNOS
were protected in an LPS-mediated model of septic shock
(16).
Specifically how eNOS, NO, and TLR modulate
vascular inflammation remains unclear, but an interrelationship has been suggested (17). LPS is known
to strongly activate MAPKs, and eNOS is known to
possess MAPK docking sites (18, 19). Based on those
studies, we sought to independently define the effect
of eNOS protein expression and NO on TLR4 signaling.
We hypothesized that eNOS-dependent modulation
of TLR4 signaling would be mediated by changes in
MAPK activation, which would lead to an altered inflammatory phenotype. By understanding those relationships, we hoped to discover new potential targets to
limit the deleterious effects that alterations in eNOS
biology have on infectious inflammation.
MATERIALS AND METHODS
incubated with Dharmafect (Dharmacon) in serum-free medium
for 20 min. The resultant complex of siRNA–Dharmafect was
added to the cells in 5% FBS medium without antibiotics for 6 h.
Afterward, the transfection medium was replaced with complete
medium, including antibiotics, for another 18 or 66 h before
exposure to the agonist. Percentage of protein knockdown was
determined by Western blot analysis.
Intracellular NO assay
Glass-bottom 96-well plates were seeded with HMVECs at
30,000 cells/well 1 d before assay. Cells were exposed to LPS,
with or without sepiapterin or L-NAME, for 1 or 16 h in phenolred–free, 5% FBS medium. Afterward, supernatants were
removed, cells were washed with phenol-red–free medium and
exposed to 2 mM DAF-FM DA (Thermo Fisher Scientific) for 40
min in the dark at 24°C. The medium containing DAF-FM DA
was then removed and replaced with fresh phenol-red–free
medium, and the cells remained in the dark for an additional 10
min at 24°C. Cells were then placed in a fluorescence plate
reader (FluoStar Omega; BMG Labtech, Ortenberg, Germany)
and read for retained intracellular probe [excitation (ex)/
emission (em) 485/520 nm], and blank wells were subtracted
from the treated wells to obtain normalized well fluorescence
intensity.
Cytokine and chemokine production
Culture supernatants were collected at the completion of the
agonist exposure (6 or 16 h). Collected supernatants were stored
at 280°C. Supernatant IL-6 (eBioscience, San Diego, CA, USA),
IL-8, and granulocyte colony-stimulating factor (G-CSF; R&D
Systems, Minneapolis, MN, USA) concentrations were assessed
with a commercially available ELISA kit, according to the manufacturer’s specifications.
Cells and culture
Pooled, neonatal, dermal human microvascular endothelial cells (HMVECs) were purchased from Lonza (Basel,
Switzerland) and grown in Endothelial Growth Media-2 (Lonza),
supplemented with 5% fetal bovine serum (FBS). HMVECs
were plated at a density of approximately 30,000 cells/cm2
and grown to confluence. Experiments were conducted between the second and fifth passages. Medium was exchanged
every 3 d.
Reagents
The following reagents and concentrations were used in experiments: 100 ng/ml Ultra-Pure LPS (List Biological Laboratories,
Campbell, CA, USA), 100 mM L-sepiapterin (Cayman Chemicals,
Ann Arbor, MI, USA), 10 mM N-nitro-L-arginine methylester
(L-NAME) (MilliporeSigma, Billerica, MA, USA), 10 mM 1400W
dihydrochloride (Thermo Fisher Scientific, Waltham, MA, USA),
10 mM SB203580 (Cell Signaling Technology, Danvers, MA,
USA), 100 nM SCH772984 (ApexBio Technology, Houston, TX,
USA).
Small interfering RNA transfection
HMVECs were treated with small interfering RNA (siRNA;
scrambled siControl, sieNOS, siiNOS, or sip38), according to the
manufacturer’s recommendations. In brief, siRNA were procured
from Dharmacon (Lafayette, CO, USA). siRNA (25 nM) was
2
Vol. 32
February 2018
Intercellular space assay
Glass-bottom 96-well plates were used for an in vitro vascular
permeability imaging assay (MilliporeSigma). Wells were treated
with poly-L-lysine, glutaraldehyde, and biotinylated gelatin, per
the manufacturer’s recommendations. HMVECs were seeded at
a density of 30,000 cells/well and incubated for 1 d to achieve a
confluent monolayer. In some experiments, the cells were then
exposed to siRNA. In a later study, cells will be treated with LPS
(100 ng/ml) or vehicle in the presence of sepiapterin, L-NAME,
SB203580, or SCH772984, as described in Results. At 6 or 16 h
after LPS exposure, supernatants were collected for ELISA, and
cells were washed with 120 ml of Live Cell imaging solution
(Thermo Fisher Scientific). Afterward, cells were exposed to
streptavidin–fluorescein (1:2000 dilution) and stained with
NucBlue (1 drop/well; Thermo Fisher Scientific) for 20 min to
test for total cell count or, in some instances, ethidium homodimer (10 mM; AdipoGen, Liestal, Switzerland) for 15 min to
examine cell death. Cells were then washed again with imaging
solution to remove excess probe, and images were acquired on a
fluorescence inverted microscope (Leica DM IRB; Leica Microsystems, Wetzlar, Germany) using filters appropriate for DAPI
(ex/em 360/460 nm), ethidium (ex/em 528/617 nm), or fluorescein (ex/em 490/520 nm). Cell count was obtained via
quantification of nuclear staining using ImageJ (National Institutes of Health, Bethesda, MD, USA) software, which ensured a
consistent density of the endothelial layers, and monolayer
integrity was assessed via quantification of the area of fluorescein
staining (ImageJ), as an indicator of intercellular gap size.
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STARK ET AL.
Western blot and electrophoresis
Cell lysates were collected at 1 and 16 h after agonist exposure,
as described in Results. For eNOS dimer and monomer determination, cells lysates were kept between 0 and 4°C and were
maintained in 2-mercaptoethanol–free sample buffer. Protein
extracts (50 mg/sample) were separated by SDS electrophoresis
on a polyacrylamide gel (10%) and transferred to nitrocellulose
membranes. Membranes were blocked with Odyssey Blocking
Buffer (Li-Cor Biosciences, Lincoln, NE, USA) for 1 h at roomtemperature. Membranes were incubated with primary antibodies overnight at 4°C on a rocker. Antibodies were as follows:
IKKab, phospho-IKKb, p-p38, p38, p-ERK, ERK, p-JNK, JNK,
p-Akt, Akt, eNOS, p-eNOS (serine 1177), p-eNOS (threonine 495)
(Cell Signaling Technology), iNOS (R&D Systems), tubulin
(Vanderbilt Antibody Core; Vanderbilt Antibody and Protein
Resource, Nashville, TN, USA). Afterward, membranes were
incubated with fluorescent secondary antibodies and analyzed
using the Odyssey Imaging System (Li-Cor Biosciences). Protein
quantification was performed via densitometry and normalized
as a ratio of expressed protein to tubulin, phosphorylated protein
to respective total protein, or dimer to monomer ratio.
Immunoprecipitation
Cells were exposed to siRNA for 72 h, then lysed in buffer (0.02 M
Tris base, 1 mM EDTA, 20 mM NaCl, 1% nonidet P40, protease
inhibitor cocktail, and PMSF at pH 7.4). Lysed cells were nutated
for 1 h at 4°C, then centrifuged at 20,000 g for 30 min. Supernatants were precleared with protein-G sepharose beads for 30
min at 4°C. After centrifugation, the supernatants (500 mg) were
incubated with anti-eNOS antibody (2 mg, clone 49G3; Cell
Signaling Technology) for 1.5 h, then incubated with protein-G
sepharose for an additional 1 h. Beads were then washed with
lysis buffer, resuspended in sample buffer containing SDS, and
boiled. Associated proteins were then analyzed by Western blot
using the following antibodies: p38 and eNOS (6H2; Cell
Signaling Technology).
Gene profiles of synovial biopsies
Gene array profiles of synovial biopsies were obtained from the
publically available National Centers for Biotechnology Information (NCBI; Bethesda, MD, USA) Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) database. Values
were obtained from GEO DataSet (GSE36700), initially collected
by Nzeusseu Toukap et al. (20), “Systemic lupus erythematosus
and arthritides: synovial biopsies.” The database was queried for
NOS3 (eNOS, ID: NM_000603), MAPK14 (p38, ID: L35253.1),
and TLR4 (TLR4, ID: NM_003266) from samples run on an
Affymetrix Human Genome U133 Plus 2.0 Array (Thermo Fisher
Scientific).
Statistical analysis
For endothelial cell-culture experiments, data are expressed as
means 6 SE of multiple, individual experiments. Comparisons of
treatment groups and conditions were performed via an unpaired Student’s t test for single comparisons and 1-way
ANOVA, with Bonferroni correction, for multiple-group comparisons. For gene profile expression of biopsy samples, data are
expressed as medians (5–95%) 6 SE of individual samples.
Comparisons of groups were performed via the Kruskal-Wallis
test, with the Dunn correction for multiple-group comparisons,
and linear regression for direct expression comparison. All
analysis was performed with GraphPad Prism 5.03 statistical
ENOS ALTERS TLR4-MEDIATED INFLAMMATION
software (GraphPad Software, La Jolla, CA, USA). A value of
P , 0.05 was considered statistically significant.
RESULTS
Sepiapterin enhanced eNOS dimerization and
NO production in endothelial cells
Alterations in eNOS are one of the hallmarks of endothelial dysfunction and can occur via multiple mechanisms,
including dimerization, altered phosphorylation or
dephosphorylation, and reduction in total protein. To
examine the relationship between LPS and NO production
by eNOS, we exposed HMVECs to LPS for 16 h in the
presence or absence of sepiapterin, which promotes
production of BH4 and associated eNOS dimerization. In
that conformation, eNOS produces NO (21). In resting
HMVECs, eNOS was present in both dimer and monomer forms (Fig. 1A). When exposed to LPS, the dimer to
monomer ratio increased; however, that appeared to be
due more to the loss of the monomer band. The addition of
sepiapterin for 16 h significantly increased the amount of
dimerized eNOS, independent of LPS. Exposure of endothelial cells to LPS significantly suppressed the total
amount of eNOS in the cells, and that effect was not altered
by the presence of sepiapterin (Fig. 1B). LPS did not induce
changes in eNOS phosphorylation at serine 1177 or threonine 495 at 1 h of exposure (Supplemental Fig. 1A). To
examine the effect of eNOS dimerization on NO production, cells were exposed to LPS under control conditions or in the presence of sepiapterin for either 1 or 16 h. At
both times, sepiapterin significantly increased the amount
of NO detected by DAF-FM DA, and that effect was suppressed by L-NAME (Fig. 1C). Those experiments demonstrated that, as expected, sepiapterin enhanced eNOS
dimerization and NO production. The primary effect of
LPS on eNOS appeared to be a reduction in total protein.
Sepiapterin and L-NAME had limited effects
on LPS-induced IL-6 production and
intercellular space
NO has been postulated as having an important role in
regulating the inflammatory response of endothelial cells,
including permeability and cytokine production (22, 23).
To test whether eNOS dimerization and the associated
generation of endogenous NO altered the endothelial response to LPS, HMVECs were exposed to sepiapterin,
L-NAME, or a combination in the presence of LPS. The
addition of LPS for 16 h significantly increased the amount
of exposed biotin-labeled gelatin, a predicted indicator of
increased permeability (Fig. 2A). Despite the increase in
endothelial NO production induced by sepiapterin, there
was no difference in endothelial intercellular space compared with cells treated with LPS alone. Likewise, L-NAME
failed to induce any significant alteration in intercellular
space, with or without the presence of sepiapterin. When
examining IL-6 production 16 h after LPS exposure, once
again, there was little effect of altering eNOS-derived NO.
Although there was a statistically significant difference
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3
A
B
eNOS
Dimer
Monomer
250 kd
130 kd
Tubulin
eNOS
130 kd
Tubulin
55 kd
55 kd
Control
LPS
Sepiapterin
LPS + Sepiapterin
Control
*
LPS
1.5
NS
eNOS/Tubulin
(densitometry units)
eNOS dimer:monomer
(densitometry units)
15
10
5
*
LPS + Sepiapterin
*
*
1.0
0.5
0.0
0
C
Control
[NO]
Fluorescence Insensity (AU)
LPS + Sepiapterin
LPS
LPS + Sepiapterin + L-NAME
100
*
*
*
80
60
NS
40
20
0
1 hour
16 hours
Figure 1. Sepiapterin-induced eNOS dimerization and NO production. A) HMVECs were treated with LPS (100 ng/ml) or
sepiapterin (100 mM), alone or in combination, for 16 h. Ratios of the density of the dimer band (260 kDa) to the monomer
band (130 kDa) were determined by Western blot (n = 4/group). B) Total eNOS normalized to a-tubulin for HMVECs exposed
to LPS or LPS with sepiapterin for 16 h (n = 4/group). C ) Relative fluorescence intensity of DAF FM DA (2 mM) as an indication
of intracellular NO production after 1 or 16 h in the presence of LPS with or without sepiapterin and L-NAME (10 mM) (n = 12/
group). NS, nonsignificant. *P , 0.05 between compared groups.
between the response to LPS and sepiapterin compared
with LPS and L-NAME, that effect was small. Because
changes in endothelial intercellular space in response to
endotoxin can occur in the first hours after exposure, we
also tested an earlier time, 6 h after LPS (24). Similar to
the later time, 6 h of exposure to LPS increased intercellular space (Fig. 2B, C). At that earlier point, both
sepiapterin and L-NAME alone had no significant effects on intercellular space or IL-6 production; however,
in combination, there was a subtle increase in endothelial intercellular space in the presence of LPS. Despite that,
these results suggested that enhanced endogenous NO
production by sepiapterin-mediated eNOS dimerization
and inhibition of NO by L-NAME had no prominent effects
on intercellular space or cytokine production during
exposure to LPS.
4
Vol. 32
February 2018
Knockdown of eNOS-enhanced, LPS-induced
IL-6 production, and intercellular space
Given that eNOS-derived NO did not significantly alter
the global endothelial response to LPS, we next examined
the effect of siRNA knockdown on total eNOS protein, as
would be seen in chronic inflammatory conditions (25).
After 24 h of initial exposure to siRNA against eNOS
(sieNOS), there were no notable changes to intercellular
space in the knockdown groups after LPS exposure (Fig. 3A).
However, the amount of IL-6 produced in the sieNOS
group at 6 h after LPS was significantly increased compared with those cells treated with scrambled siRNA
(siControl). Documentation of effective eNOS knockdown is provided in Supplemental Fig. 1B. To examine
whether knockdown of iNOS would affect LPS-induced
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STARK ET AL.
A
LPS
LPS + Sepiapterin
LPS + Sepiapterin + L-NAME
2500
NS
50
40
†
†
†
†
30
20
*
2000
IL-6 (pg/ml)
Intercellular Space (%)
Control
LPS + L-NAME
1500
1000
10
500
0
0
16 hours
16 hours
B
C
Control
LPS + L-NAME
1500
NS
†
†
†
†
20
NS
IL-6 (pg/ml)
Intercellular Space (%)
LPS + Sepiapterin
LPS + Sepiapterin + L-NAME
*
40
30
LPS
1000
500
10
0
0
6 hours
6 hours
Figure 2. Neither sepiapterin nor L-NAME significantly altered LPS-induced intercellular space or IL-6 production. A) HMVECs
were exposed to combinations of LPS (100 ng/ml), sepiapterin (100 mM), or L-NAME (10 mM) for 16 h. Representative images
of intercellular space under control conditions or with LPS (white, intercellular space; black, cells) (left). Percentage of exposed
intercellular space among treatment groups (middle). Amount of IL-6 produced among treatment groups as measured by ELISA
(right); dashed line indicates control group IL-6 levels (n = 4/group). B) Representative images of HMVECs exposed to LPS,
sepiapterin, or L-NAME for 6 h. C ) Percentage of exposed intercellular space (left) and IL-6 production (right) among groups
after 6 h of treatment (n = 4/group). Dashed line indicates control condition IL-6 levels. NS, nonsignificant. *P , 0.05 between
compared groups, †P , 0.05 between group compared with control conditions.
inflammation, cells were exposed to siRNA targeting
iNOS (siiNOS) and then to LPS. Although knockdown of
iNOS did not alter LPS-induced intercellular space, it did
increase baseline intercellular space, albeit minimally,
despite difficulty in detecting iNOS in the endothelial
cell lysates (Supplemental Fig. 1C). Exposure to siiNOS
did not significantly affect the IL-6 produced after LPS.
The absence of an important role for iNOS was confirmed using a selective iNOS inhibitor, 1400W (Supplemental Fig. 1D), which showed no alteration in IL-6
production. Next, we extended the exposure time to
siRNA to 72 h (Fig. 3B, C). Again, reduction of iNOS
increased baseline intercellular space to a small, but
statistically significant, degree but did not alter IL-6
ENOS ALTERS TLR4-MEDIATED INFLAMMATION
production. In contrast, reduction of eNOS-enhanced,
LPS-induced IL-6, and that effect was not altered by the
addition of sepiapterin or L-NAME (Supplemental Fig.
2C, D). In addition, exposure to sieNOS significantly
increased baseline intercellular space, which was further
enhanced by LPS. Together, these result show that
reduced eNOS enhanced inflammatory cytokine
production and intercellular space by LPS.
Reduction of eNOS protein abundance
enhanced p38 phosphorylation
To further examine how eNOS temporally altered cytokine production and intercellular space, we focused on
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5
A
*
*
50
*
*
*
6000
40
IL-6 (pg/ml)
Intercellular Space (%)
LPS
Control
†
30
20
4000
2000
10
0
0
siControl
sieNOS
siiNOS
siControl
24 hours siRNA
B
sieNOS
siiNOS
24 hours siRNA
siControl
sieNOS
siiNOS
Control
LPS
Control
‡
*
60
Intercellular Space (%)
LPS
10000
†
*
50
40
*
*
†
30
†
20
*
8000
†
IL-6 (pg/ml)
C
6000
4000
2000
10
0
0
siControl
sieNOS
siiNOS
siControl
72 hours siRNA
sieNOS
siiNOS
72 hours siRNA
Figure 3. eNOS knockdown enhanced LPS-induced intercellular space and IL-6 production. A) HMVECs were exposed to siRNA
for 24 h (siControl, sieNOS, siiNOS) and then treated with LPS (100 ng/ml) for 6 h. Percentage of exposed intercellular space
(left) and IL-6 production (right) among groups (n = 4/group). B) Representative images of intercellular space under control
conditions or with LPS after exposure to siControl, sieNOS, or siiNOS for 72 h (white, intercellular space; black, cells). C )
Percentage of exposed intercellular space (left) and IL-6 production (right) among siRNA groups exposed for 72 h (n = 4/
group). Dashed line indicates IL-6 levels for control conditions. *P , 0.05 between compared groups, †P , 0.05 between group
compared with siControl, ‡P , 0.05 between sieNOS group compared with siiNOS.
MAPK signaling because of the presence of postulated
MAPK docking sites on eNOS (19). Despite a significant
reduction in eNOS protein 24 h after exposure to sieNOS, 1
h of LPS exposure produced no significant changes in
MAPK phosphorylation among the siRNA groups, with
the exception of ERK1/2, which was reduced after exposure to eNOS siRNA (Fig. 4A). When siRNA exposure was
extended to 72 h, followed by 1 h of LPS, an increase in p38
phosphorylation was seen in the sieNOS group compared
with the siControl group (Fig. 4B). Again, JNK phosphorylation was unaffected, and ERK 1/2 phosphorylation in
6
Vol. 32
February 2018
the sieNOS group was no longer inhibited. There was a
trend toward an increase in phosphorylation of IKK, an
upstream component of NF-kB activation, which was not
statistically significant. There were no detectable changes in
Akt phosphorylation (Supplemental Fig. 2A, B). Thus,
these data suggested that reduced eNOS allowed for enhanced p38 activity, which could contribute to enhanced
inflammation.
To explore that, we tested intercellular space, analyzed
cytokine and chemokine production in HMVECs treated
with sieNOS, and examined the effect of the selective p38
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STARK ET AL.
Figure 4. p38 phosphorylation in response to
LPS was enhanced by eNOS knockdown. A)
Representative Western blot images of HMVECs
exposed to siRNA (siControl or sieNOS) for 24 h
and then treated with LPS (100 ng/ml) for 1 h
(left). Images show band intensity for p-p38, p38,
p-JNK, JNK, p-ERK1/2, ERK1/2, or a-tubulin
with ratios of phosphorylated protein to respective total protein (right) B) Representative
Western blot images of HMVECs exposed to
siRNA (siControl or sieNOS) for 72 h (left) with
1 h of LPS and respective ratios of phosphorylated protein to associated total protein (right)
(n = 6/group). *P , 0.05 between compared
groups, †P , 0.05 between group compared to
siControl.
inhibitor SB203580 on the response to LPS. SB203580 did
not alter the effect of LPS on intercellular space in the
siControl group (Fig. 5A). Likewise, SB203580 did not
prevent the enhancement of intercellular space caused by
sieNOS and, instead, increased intercellular space in both
vehicle and LPS treated conditions. However, p38 inhibition was associated with a significant reduction in IL-6
production in the LPS-treated siControl group (Fig. 5B).
That reduction in LPS-mediated IL-6 production was also
observed in the more reactive sieNOS group, where inhibition of p38 completely reduced the response to LPS to
those of siControl-treated cells. Contrary to that, eNOS
reduction did not affect LPS-mediated release of the
chemokines IL-8 or G-CSF. Chemokine release was less
dependent on p38 activity compared with IL-6 release.
We also tested the effect of the selective ERK1/2 inhibitor
SCH772984 because ERK1/2 has been shown previously
ENOS ALTERS TLR4-MEDIATED INFLAMMATION
to modulate endothelial intercellular space (26). However, inhibition of ERK1/2 did not alter IL-6 production
or intercellular space (Supplemental Fig. 3). Together,
these experiments suggest that eNOS protein reduction
enhanced p38 MAPK activation by LPS, contributing to
the greater IL-6 production in eNOS knockdown cells.
However, those changes in p38 activity did not account
for the increase in intercellular space in sieNOS-treated
cells and had no relation to chemokine production, suggesting independent mechanisms of regulation.
p38 had direct interactions with eNOS and
knockdown impaired IKK activation by LPS
Given the apparent p38-dependence of eNOS-mediated
IL-6 production, we next examined whether there was a
reciprocal relationship between p38 and eNOS that
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7
A
siControl
sieNOS
SB203580
(p38 inhibitor)
DSMO
(Vehicle)
SB203580
(p38 inhibitor)
Control
LPS
‡
50
Intercellular Space (%)
DSMO
(Vehicle)
*†
40
*
*
30
†
20
10
0
DMSO
B
*
‡
1500
1000
*
*
0
DMSO
SB203580
sieNOS - LPS
8000
2000
500
sieNOS - Control
SB203580
DMSO
SB203580
‡
†
IL-8 (pg/ml)
IL-6 (pg/ml)
2500
siControl - Control
siControl - LPS
DMSO
SB203580
*
‡
*
5000
‡
6000
4000
*
*
2000
0
G-CSF (pg/ml)
*
3000
*
*
4000
‡
‡
*
*
3000
*
2000
1000
0
DMSO
SB203580
DMSO
SB203580
DMSO
SB203580
DMSO
SB203580
Figure 5. Inhibition of p38 prevented eNOS knockdown from enhancing IL-6 production but not intercellular space. A)
Representative images of intercellular space under control conditions or 6 h of LPS (100 ng/ml), with or without the p38
inhibitor, SB203580 (10 mM), after 72 h exposure to siControl or sieNOS (white, intercellular space; black, cells) (left) and
graphical representation of the percentage of exposed intercellular space (right). B) IL-6, IL-8, and G-CSF production among
siRNA groups exposed with LPS and SB203580, as measured by ELISA (n = 4/group). *P , 0.05 between control or LPS-treated
conditions, †P , 0.05 between siControl and respective sieNOS group, ‡P , 0.05 between vehicle control and SB203580-treated
group as measured by ANOVA with the Bonferroni correction.
affected proinflammatory activation by LPS. Cells exposed to siRNA against p38 had a significant reduction in
p38 protein that correlated with an increase in total eNOS
(Fig. 6A). In addition, using immunoprecipitation, eNOS
protein pulled down with anti-eNOS antibody, stained for
p38, and that staining was dramatically reduced in the
presence of siRNA to p38, suggesting a physical interaction between the 2 proteins. Furthermore, cells in the
sip38 group demonstrated a significant decrease in IKK
phosphorylation after LPS compared with the siControl
group (Fig. 6B).
Because the current data were derived from cultured
endothelial cells, we were interested in seeing whether
there was evidence of eNOS–TLR4–p38 interactions in
samples derived from patients with systemic, vascular
autoimmune disease. To examine that relationship in vivo,
we queried the publicly available NCBI GEO database for
chronic inflammatory conditions. We used DataSet
GSE36700, obtained by Nzeusseu Toukap et al. (20), from
synovial biopsy samples collected from patients with
rheumatoid arthritis (RA) or systemic lupus erythematosus
(SLE). We focused on SLE, as well as RA, because of a
postulated correlation between increased disease
severity and reduced eNOS expression, similar to our
in vitro model (25). Samples from patients with seropositive disease had significantly reduced eNOS expression compared with those with osteoarthritis (Fig.
8
Vol. 32
February 2018
6C). Concurrently, the reduction of eNOS expression in
systemic lupus correlated with a trend toward increased
p38 and a significant increase in TLR4 expression compared with patients in the other groups. Together, these
data suggest a reciprocal relationship between eNOS
and TLR4/p38 signaling in chronic, systemic inflammatory diseases.
DISCUSSION
The role of NO and its associated synthases during severe
infections have been a source of much interest and debate
since its discovery (13, 27). This confusion has been enhanced by animal studies showing detrimental effects of
exaggerated NO-production by iNOS and potentially
protective effects of eNOS, with subsequent clinical studies showing increased mortality in septic patients given
nonselective NOS inhibitors (15, 16). To more specifically
examine the role of eNOS in TLR4-mediated inflammation, we used several different techniques involving
functional activation and inhibition of the eNOS enzyme
as well as protein knockdown. Sepiapterin enhanced
eNOS dimerization and thus NO production, whereas
L-NAME reduced NO production. However, neither
treatment conferred any significant protective or detrimental effects with regard to LPS-mediated inflammation.
The FASEB Journal x www.fasebj.org
Downloaded from www.fasebj.org to IP 61.129.42.30. The FASEB Journal Vol., No. , pp:, October, 2017
STARK ET AL.
A
B
siControl
sip38
IP (eNOS)
Lysates
38 kd
p38
p38
38 kd
38 kd
p38
eNOS
p-IKK
100 kd
IKK
100 kd
130 kd
eNOS
Control
5
0
0
10
20
30
40
Relative eNOS Expression
(AU)
pu
s
is
rit
is
ic
Ar
Lu
th
rit
th
ar
eo
em
st
st
O
Sy
he
R
r = -0.146
p = 0.673
200
150
100
50
0
0
0
Systemic Lupus
10
20
30
40
Relative eNOS Expression
(AU)
Relative TLR4 Expression
(AU)
50
100
s
Sy
he
R
100
Relative TLR4 Expression
(AU)
Relative p38 Expression
(AU)
Rheumatoid Arthritis
r = 0.473
p = 0.146
*
p = 0.927
200
Lu
th
Ar
d
st
O
R
D
*
300
pu
rit
is
rit
th
ar
eo
ic
em
st
Sy
he
Relative TLR4 Expression
(AU)
0
Lu
th
Ar
d
oi
at
um
50
pu
is
rit
is
rit
th
ar
eo
st
O
100
s
0
p = 0.412
is
10
*
150
sip38
400
ic
20
p = 0.057
em
p = 0.527
siControl
st
30
200
oi
*
Relative p38 Expression
(AU)
*
40
1
sip38
at
C
NS
2
d
siControl
sip38
um
siControl
3
0
0.0
0
Relative eNOS Expression
(AU)
0.5
*
4
at
1
1.0
LPS
um
2
p-IKK/IKK
(Fold Change)
p38/eNOS
(Fold Change)
eNOS/Tubulin
(Fold Change)
*
150
55 kd
Tubulin
1.5
*
3
130 kd
oi
55 kd
200
r = -0.762
p = 0.037
300
Relative p38 Expression
(AU)
Tubulin
r = -0.643
p = 0.096
150
200
100
100
0
0
10
20
30
40
Relative eNOS Expression
(AU)
50
0
0
10
20
30
40
Relative eNOS Expression
(AU)
Figure 6. p38 and eNOS had a direct reciprocal relationship in vitro and in vivo. A) Representative images of p38, eNOS, and
a-tubulin Western blots from whole-cell lysates (top, left), and of p38 and eNOS after immunoprecipitation with anti-eNOS (top,
right). HMVECs were exposed to p38 siRNA for 72 h. Ratios of eNOS or p38 to a-tubulin are provided below. B) Representative
Western blot images of HMVEC exposed to 72 h of p38 siRNA then LPS (100 ng/ml) or control medium for 1 h with antibodies
against phosphorylated IKK, total IKK, p38, and a-tubulin (top). Ratios of phosphorylated IKK to total IKK are displayed below
(n = 4/group). C ) Relative gene expression for eNOS, p38, and TLR4 from synovial biopsies obtained from patients with arthritis,
seronegative arthritis (control), rheumatoid arthritis, or systemic lupus erythematous. D) Linear regression analysis of eNOS
expression compared with TLR4 or p38 expression within respective sample populations of seronegative arthritis compared with
rheumatoid arthritis or systemic lupus erythematous (n = 4–7 patients/group). *P , 0.05 between designated groups.
ENOS ALTERS TLR4-MEDIATED INFLAMMATION
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9
In contrast, knockdown of eNOS greatly enhanced both
LPS-mediated IL-6 production and intercellular space, a
surrogate of permeability. Knockdown of eNOS was
associated with increased p38 activation by LPS. Inhibition
of p38 with a pharmacologic inhibitor attenuated the
increased IL-6 production induced by eNOS knockdown
but had no effect on intercellular space, suggesting a
divergence in the regulatory mechanisms. In congruence,
p38 associated with eNOS in a manner that could be
reduced with siRNA to p38 and, in turn, reduced LPSmediated IKK phosphorylation. A reciprocal relationship
between eNOS and TLR4/p38 signaling was further
supported in synovial biopsies from patients with chronic,
inflammatory arthritides that are associated with systemic
vascular disease and endothelial dysfunction. Together,
these data suggest an important role for eNOS in regulating TLR4-mediated endothelial inflammation.
NO has been suggested to have opposing roles during
sepsis (13, 27). The role of eNOS-mediated NO production
in the endothelial dysfunction associated with cardiovascular disease has been extensively investigated (5).
Mechanisms by which eNOS-dependent NO production
can be altered include BH4-mediated subunit dimerization, phosphorylation, and changes in eNOS expression
(5–7). In the current study, sepiapterin greatly enhanced
eNOS dimerization and NO production in response to
LPS, and as expected, L-NAME effectively inhibited NO
(Fig. 1). The increase in NO production after sepiapterin
was likely related to an increase in de novo BH4 synthesis;
however, that appears to be offset by a reduction of eNOS
protein abundance after LPS (7, 21). Furthermore, despite
increased eNOS dimerization and NO production, alterations in endogenous NO failed to either enhance or reduce LPS IL-6 production or permeability (Fig. 2). These
findings are contrary to the postulated concept that NO
can induce permeability and suppress cytokines (22, 23,
28). The reason for this discrepancy is likely multifactorial,
including the use of a different agonist (LPS vs. VEGF or
platelet-activating factor) and the testing of endogenous
NO vs. an exogenous NO donor. Our findings are more
consistent with the observation that inhibition of endogenous NO with L-NAME enhanced baseline endothelial
permeability and could be restored through the NO donor,
sodium nitroprusside (29). Additionally, acetylcholine,
which is known to induce eNOS phosphorylation and NO
production, was shown not to induce permeability (30).
Thus, it appears that the role of endogenous NO in regulating endothelial permeability depends on the mechanism of the stimulus, and regarding TLR4, NO does not
appear to have a significant role.
Because endogenous, eNOS-derived NO did not appear to have a significant role in TLR4-mediated inflammation, we transitioned to investigating an alternative
mechanism of endothelial dysfunction seen in chronic inflammatory conditions: reduced eNOS expression (25, 31).
Using siRNA, we were able to show that reduced eNOS
expression enhanced IL-6 production and altered baseline
permeability after LPS in a time-dependent fashion (Figs. 3
and 4). Furthermore, that effect was specific to eNOS
because siRNA to iNOS had no prominent effects, likely
related the low abundance and poor induction of iNOS
10
Vol. 32 February 2018
in endothelial cells (32). In contrast, the effect of eNOS
knockdown on vascular inflammation was striking. Our
data concurs with observations from eNOS knockout
mice, which had higher TNF-a expression, suggestive of a
link between reduced eNOS and acute-phase cytokine
production (33). Similarly, transgenic mice made to overexpress eNOS were protected in a model of endotoxemia,
with an associated reduction in pulmonary capillary leak,
suggesting a protective role of eNOS abundance (16).
Despite a potential protective role for eNOS in endothelial
permeability, there are other studies demonstrating that,
for VEGF-mediated permeability, the loss of eNOS, protected against permeability (34). Given these discrepant
data, it is likely that eNOS regulation of permeability and
cytokine production is ligand and stimulus specific. For
infectious stimuli, it appears that the role of eNOS in TLR4mediated inflammation is independent of eNOS-mediated
NO production and is instead related to another functional
role of eNOS protein.
To explore that possibility, we quantified MAPK
phosphorylation in cells with reduced eNOS expression
and observed enhanced activation of p38 after LPS (Fig. 4).
This relationship with p38 was responsible for the increased cytokine production because a p38 inhibitor
blocked nearly all IL-6 production in response to LPS and
completely abrogated the increment in IL-6 production
observed in sieNOS cells. However, sieNOS had no effect
on chemokine production, as eNOS knockdown did not
impair LPS-mediated IL-8 or G-CSF production. Furthermore, IL-8 and G-CSF were less affected by p38 inhibition,
suggesting eNOS and p38 have both synergistic and independent roles in mediating endothelial inflammation.
This was further seen in examining the intercellular space
in which p38 inhibition had no effect on LPS-mediated
intercellular space, despite eNOS knockdown (Fig. 5).
Although it has been suggested previously that p38 is involved in the regulation of LPS-induced permeability, the
effect was shown to be small and thus, p38 was not likely
to be a conserved mediator of both cytokine production
and permeability (35). In further experiments, we found
that eNOS had a direct association with p38 that could be
altered by p38 knockdown. Furthermore, p38 knockdown was associated with reduced NF-kB activation by
LPS (Fig. 6). Knockdown of p38 reduced NF-kB activation to a greater degree than eNOS knockdown had
enhanced signaling NF-kB and was an unexpected
finding. Although a p38–NF-kB pathway has been
established, little is known about how eNOS reduction,
or NO, directly affects NF-kB (36, 37). Given that NF-kB
is further downstream and subject to multiple regulatory pathways, it is likely that, although p38 regulates
IL-6 production through NF-kB, eNOS has differential
or counter-regulatory effects on NF-kB, which explains
how it regulates cytokine production and permeability
independently. In the case of mechanisms regulating LPSmediated endothelial inflammation, both IL-6 production
and intercellular space were enhanced by eNOS knockdown, but the eNOS–p38 interaction only appeared to
regulate cytokine production, whereas eNOS enhanced
permeability either synergistically or additively with TLR4
via an unknown mechanism that is independent of p38.
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STARK ET AL.
That interaction between p38 and eNOS driving IL-6 response is consistent with a previous study demonstrating
that eNOS contains a MAPK docking site and direct
binding between recombinant p38 and eNOS proteins (19).
To our knowledge, the current work represents the first
demonstration of that interaction in living cells. The observation that eNOS deficiency adversely affects TLR4mediated inflammation in animal models, coupled with
the current observation that eNOS reduction enhances LPS
responses, points to an ability of eNOS to modulate TLR4
signaling (17, 18).
The clinical relevance of that relationship was demonstrated in biopsy samples from patients with SLE and RA.
Both diseases were associated with dramatically suppressed eNOS mRNA levels. Further, patients with SLE
had an associated reciprocal relationship between eNOS
and TLR4 and p38 expression (Fig. 6C). Interestingly, the
relationships of reduced eNOS and increased TLR4 individually have been postulated to correlate with disease
severity in SLE (25, 38). Although a disease-specific reciprocal interaction has not been demonstrated, it has been
noted that endothelial dysfunction is more prevalent in
patients with SLE compared with those with RA (39).
Further, it has been demonstrated that patients with SLE
have higher circulating levels of LPS and that those higher
levels of LPS regulate inflammatory gene expression in a
p38-dependent manner (39, 40). What role eNOS expression and its associated alterations in TLR4-p38 signaling
have in these clinical outcomes is unknown, but our data
support the concept of a direct relationship among eNOS,
TLR4, and p38, which may regulate the endothelial
response to infectious challenge in chronic vascular
inflammation.
In summary, the current study demonstrates that LPSmediated endothelial injury was enhanced by the reduction of eNOS protein abundance in a NO-independent
manner. The potentiated response of eNOS knockdown
cells was p38 dependent regarding IL-6 production but not
endothelial permeability, suggesting independent mechanisms of LPS-induced and eNOS-regulated IL-6 production and permeability. The relationship between eNOS
and p38 abundance was reciprocal, and the ability of
eNOS to modulate p38 activity appeared to involve a direct, binding relationship. That reciprocal relationship
among eNOS, p38, and TLR4 was observed in biopsy
samples from patients with SLE. Although the direct
implications of that relationship have not been fully explored, this work provides a foundation for elucidating
the mechanisms of endothelial dysfunction in chronic
inflammation and its relationship to sepsis outcomes in
these patients.
ACKNOWLEDGMENTS
This work was supported by U.S. National Institutes of
Health (NIH) National Institute of General Medical Sciences
Grants K08-GM117367 (to R.J.S.) and R01-GM104306 (to E.R.S.)
and NIH National Heart, Lung, and Blood Institute Grant R01HL128386 (to F.S.L.), and by American Heart Association Grant
16SDG30610002 (to H.C.). The authors declare no conflicts of
interest.
ENOS ALTERS TLR4-MEDIATED INFLAMMATION
AUTHOR CONTRIBUTIONS
R. J. Stark and F. S. Lamb designed the research; R. J.
Stark, S. R. Koch, H. Choi, E. H. Mace, S. I. Dikalov, E. R.
Sherwood, and F. S. Lamb analyzed the data; and R. J.
Stark, S. R. Koch, H. Choi, E. H. Mace, S. I. Dikalov, E. R.
Sherwood, and F. S. Lamb wrote the paper.
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The FASEB Journal x www.fasebj.org
Received for publication May 4, 2017.
Accepted for publication October 10, 2017.
Downloaded from www.fasebj.org to IP 61.129.42.30. The FASEB Journal Vol., No. , pp:, October, 2017
STARK ET AL.
Endothelial nitric oxide synthase modulates Toll-like receptor 4−
mediated IL-6 production and permeability via nitric oxide−
independent signaling
Ryan J. Stark, Stephen R. Koch, Hyehun Choi, et al.
FASEB J published online October 23, 2017
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