TLR4 Signaling Is Involved in Brain Vascular Toxicity of
PCB153 Bound to Nanoparticles
Bei Zhang1,2, Jeong June Choi2, Sung Yong Eum2, Sylvia Daunert2, Michal Toborek2*
1Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Kentucky, United States of America, 2Department of Biochemistry and Molecular Biology,
University of Miami School of Medicine, Miami, Florida, United States of America
PCBs bind to environmental particles; however, potential toxicity exhibited by such complexes is not well understood. The
aim of the present study is to study the hypothesis that assembling onto nanoparticles can influence the PCB153-induced
brain endothelial toxicity via interaction with the toll-like receptor 4 (TLR4). To address this hypothesis, TLR4-deficient and
wild type control mice (males, 10 week old) were exposed to PCB153 (5 ng/g body weight) bound to chemically inert silica
nanoparticles (PCB153-NPs), PCB153 alone, silica nanoparticles (NPs; diameter, 20 nm), or vehicle. Selected animals were
also subjected to 40 min ischemia, followed by a 24 h reperfusion. As compared to exposure to PCB153 alone, treatment
with PCB153-NP potentiated the brain infarct volume in control mice. Importantly, this effect was attenuated in TLR4-
deficient mice. Similarly, PCB153-NP-induced proinflammatory responses and disruption of tight junction integrity were less
pronounced in TLR4-deficient mice as compared to control animals. Additional in vitro experiments revealed that TLR4
mediates toxicity of PCB153-NP via recruitment of tumor necrosis factor-associated factor 6 (TRAF6). The results of current
study indicate that binding to seemingly inert nanoparticles increase cerebrovascular toxicity of PCBs and suggest that
targeting the TLR4/TRAF6 signaling may protect against these effects.
Citation: Zhang B, Choi JJ, Eum SY, Daunert S, Toborek M (2013) TLR4 Signaling Is Involved in Brain Vascular Toxicity of PCB153 Bound to Nanoparticles. PLoS
ONE 8(5): e63159. doi:10.1371/journal.pone.0063159
Editor: Michelle L. Block, Virginia Commonwealth University, United States of America
Received January 21, 2013; Accepted March 29, 2013; Published May 14, 2013
Copyright: ? 2013 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the grants from the National Institutes of Health (NIH) ES07380, CA133257, MH63022, MH072567, and DA027569. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Dr. Toborek is a member of the Editorial Board. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data
* E-mail: email@example.com
Environmental exposure to polychlorinated biphenyls (PCBs) is
an ongoing environmental problem. Because of their chemical
stability, slow degradation rate, and high tendency to bioaccumu-
late in the food chain, PCBs are among the most persistent and
widespread organic pollutants . The fate and transport of PCBs
are associated with the specific structure of individual PCB
congeners. PCBs are readily adsorbed onto particles, such as
atmospheric particulates, soil, and sediments. Recent publications
have reported PCBs bound to particles ranging from 0.95 mm to
1.5 mm in ambient air [2–4].
Exposure to PCBs has been linked to various adverse health
effects in humans. Recent reports from a PCB contaminated site
located in Anniston, AL indicated that increased serum PCB levels
are highly associated with increased prevalence of diabetes  and
hypertension . These diseases are considered to be risk factors
for stroke. In fact, an increase in the incidence of stroke was
observed in people exposed to PCBs [7,8] and living in proximity
to PCB hazardous wastes . These observations are important
because stroke is one of the leading causes of death worldwide.
A functional blood-brain barrier (BBB) is a the key element for
the homeostasis of the central nervous system (CNS). The BBB
consists of highly specialized brain endothelial cells which are
characterized by the unique phenotype of intercellular tight
junctions (TJs) and numerous polarized transport systems .
Disruption of TJ proteins is often observed during acute and
chronic diseases of the CNS, including stroke . Our research
group reported that oral administration of selective PCB
congeners resulted in accumulation of PCBs in brain tissue and
increased permeability of the BBB [12–14]. Highly chlorinated
ortho-PCBs preferentially accumulate in brain tissue and are
associated with several CNS diseases, such as Parkinson’s disease
 or developmental alterations . The most representative
(PCB153), which is commonly detected in human [17,18] and in
[19,20]. We hypothesize that binding of PCB153 to nanoparticles
(NPs) can influence their toxic properties. However, the mecha-
nisms by which PCB153-NP complexes are sensed and transduced
via cellular signaling are largely unknown.
Biological systems universally respond to various stimuli of
environmental signals by using evolutionarily conserved mecha-
nisms [21,22]. One such example is toll-like receptors (TLRs),
which recognize and respond to an expansive variety of
[23,24]. TLRs are widely expressed in various cell types in the
brain, including microglia, astrocytes, neurons, and endothelial
cells [25,26]. Recent evidence indicates that TLR4, the first
characterized of mammalian TLRs, may play a vital role in
ischemia/reperfusion injury [27,28].
In the present study, we hypothesize that exposure to PCB153
assembled onto nanoparticles contributes to the development of
stroke by disruption of the integrity of the cerebral endothelium
PLOS ONE | www.plosone.org1 May 2013 | Volume 8 | Issue 5 | e63159
and induction of proinflammatory responses through stimulation
of TLR4 signaling. The results of the present study support this
notion and indicate that targeting of the TLR4/tumor necrosis
factor-associated factor 6 (TRAF6) signaling can protect against
cerebrovascular toxicity of PCB153-NP complexes.
Materials and Methods
2,29,4,49,5,59-hexachlorobiphenyl (PCB153) congener was pur-
chased from AccuStandard (New Haven, CT) and silica NPs from
NanoAmor (Houston, TX). Characterization of NPs and con-
struction of silica NPs coated with PCB153 were described in our
previous study . Briefly, silica NPs (80 mg) and PCB153
(10 mg) were dispersed in acetone and sonicated to prevent
aggregation. The highly hydrophobic surface character of PCB153
and silica NPs allows them to interact with each other based on
electrostatic attraction. After evaporation of acetone, the particles
were resuspended in phosphate buffered saline (PBS) or cell
culture medium, sonicated, and centrifuged at 12,000 rpm for
5 min. The supernatant containing PCB153-NPs was then
collected to analyze PCB153 levels by gas chromatography/mass
spectrometry (GC/MS). The hydrodynamic size distribution and
the amounts of PCB153-NPs were monitored by dynamic light
scattering (DLS) and atomic force microscope (AFM), respectively.
Control NPs were prepared using the same procedure, without
adding PCB153. All treatment factors were tested for possible
endotoxin contamination using the LAL chromogenic endotoxin
quantitation kit (Thermo Scientific Pierce, Rockford, IL). The
levels of endotoxin in all preparations were below the detection
limit, indicating no contamination.
Experimental Groups and Surgical Procedures
All experimental procedures and protocols were approved by
the National Institutes of Health Guide for the Care and Use of
Laboratory Animals. C3H/HeJ mice contain a point mutation in
the TLR4 gene and are TLR4-deficient, whereas C3H/HeouJ
mice express normal TLR4 activity and were used as controls.
Mice (males, 10–12 weeks old; Jackson Laboratories) were infused
with a) PCB153 bound to nanoparticles (PCB153-NPs), b)
PCB153 dissolved in 0.01% DMSO (PCB153), c) nanoparticles
(NPs) alone, or d) vehicle (PBS). PCB153 was administered in the
amount of 5 ng/g body weight. All infusions were performed
through the internal carotid artery (ICA) using a surgical
technique standardized by our research group  for selective
drug delivery into the brain vasculature.
Transient focal cerebral ischemia was induced by a 40 min
occlusion of the middle cerebral artery (MCA), following a 24 h
reperfusion as described previously . This procedure is
frequently used to induce experimental stroke model. During
Figure 1. PCB153-NP-induced enhancement of infarct volume following ischemia/reperfusion is reduced in TLR4-deficient mice.
Mice were exposed to PCB153-NPs (5 ng PCB153/g body weight bound to 1.046105silica NPs) by infusion into the internal carotid artery (ICA).
Control mice were infused with the same amounts of NPs, PCB153 or vehicle (PBS). After 24 h, all animals were subjected to a 40 min occlusion of the
middle cerebral artery (MCA), followed by a 24 h reperfusion. The infarct area was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining and
the image illustrates the representative results. The loss of tissue viability is reflected by unstained (white) areas. Quantified results are depicted in a
bar graph. Results are means 6 SEM, n=5. Significantly different as compared to vehicle treatment in mice with normal TLR4 expression at
***p,0.001. Results in the TLR4-deficient mice treated with PCB153-NPs are statistically different from those in control animals exposed to PCB153-
TLR4 Mediates PCB153-NP Toxicity
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standardization, we established that a 40 min MCA occlusion was
relatively well-tolerated and did not induce mortality as assessed
24 h after the procedure. Briefly, in anesthetized mice, a 6–0
surgical nylon suture coated with silicon (Doccol, Redlands, CA)
was advanced through the left common carotid artery and up to
the ICA to block the origin of the MCA. After occlusion for
40 min, reperfusion was initiated by removing the suture to restore
the blood flow.
Assessment of the Infarct Volume
The mouse brain was removed and sectioned into 7 coronal
slices with 1 mm thickness from the frontal pole to the occipital
pole using a coronal acrylic matrice (Braintree Sci., Braintree,
MA). The brain slices were then stained with 2% 2,3,5-
triphenyltetrazolium chloride (TTC) at 37uC for 20 min. The
viable brain tissue was stained in red, whereas the infarcted area
appeared unstained. The infarct size and volume were calculated
using ImageJ software as previously described .
Brain Microvessel Isolation
Isolation of brain microvessels was performed as described
previously . After removing meninges and choroids plexus,
brain tissue was homogenized in ice-cold buffer containing
103 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4,
1.2 mM MgSO4, 15 mM HEPES, 25 mM NaHCO3, 10 mM
glucose, 1 mM Na pyruvate, 10 g/L dextran and protease
inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN).
The homogenates were mixed with 26% dextran and centrifuged
at 5,8006 g at 4uC for 20 min. The collected pellets were
resuspended in ice-cold buffer and filtered through a 70 mm cell
strainer (BD Biosciences, San Jose, CA). Filtered samples were re-
pelleted by centrifugation, followed by either resuspension in
150 mL of 6 M urea lysis buffer for Western blot analyses, or
resuspension in 200 ml of TRIZOL (Invitrogen, Carlsbad, CA) for
total RNA extraction.
Cell Cultures, Treatment Factors, and Gene Silencing
Human brain endothelial cells (hCMEC/D3 cell line) were
developed by Weksler et al. . They represent a stable, well
characterized, and differentiated cell line. Cells were cultured as
previously described . Confluent cultures were exposed to
PCB153-NPs, NPs, PCB153 alone, or vehicle for 24 h. In cell
culture experiments, PCB153 was used in subtoxic concentration
of 1.6 mM, which is lower than the levels reported in humans
acutely exposed to PCBs [35,36]. In selected experiments, cultured
cells were treated with 10 mM CLI095, a pharmacological
inhibitor of TLR4, which blocks the signaling mediated by the
intracellular domain of TLR4.
Cultured cells at 70–80% confluency were transfected with
60 nM of control or TRAF6 specific siRNA (Applied Biosystems,
Figure 2. TLR4 deficiency protects against PCB153-NP-induced TJ protein disruption. Mice were infused with PCB153-NPs or control
treatments as in Figure 1. Expression of ZO-1 and claudin-5 was analyzed in brain microvessels isolated from TLR4-deficient or control mice by
immunoblotting. Results are means 6 SEM, n=5. Significantly different as compared to control treatments in normal mice at *p,0.05. Results in the
TLR4-deficient mice treated with PCB153-NPs are statistically different from those in control animals exposed to PCB153-NPs at#p,0.05.
TLR4 Mediates PCB153-NP Toxicity
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Carlsbad, CA) using GeneSilencer (Genlantis, San Diego, CA).
The cells were incubated with transfection mixtures for 24 h and
allowed to recover in complete medium for 48 h before exposure
to PCB153 and/or NPs.
Immunoblotting and Immunoprecipitation
Immunoblotting was performed with either whole cell lysates
(30 mg protein per sample) prepared in RIPA lysis buffer (50 mM
Tris–HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate,
150 mM NaCl, and 1 mM EDTA) or lysed mouse brain
microvessels (50 mg protein per sample). Protein samples were
separated on SDS-polyacrylamide gel, blotted onto polyvinyl
difluoride membranes (Bio-Rad Laboratories, Hercules, CA), and
incubated with the respective antibodies. Anti-occludin and anti-
claudin-5 antibodies were from Invitrogen, anti-TLR4 antibody
from Santa Cruz Biotechnology (Santa Cruz, CA), anti-actin
antibody from Sigma, and all secondary antibodies from Cell
Signaling Technology (Danvers, MA). For visualization of detected
proteins, immunoblots were analyzed using an ECL Western blot
detection kit (GE Healthcare Life Sciences, Piscataway, NJ) and
proteins of interest were semi-quantitated with ImageJ software.
Immunoprecipitation of TRAF6 was performed using 800 mg of
protein extracted from whole cell lysate. Samples were incubated
with 1 mg of anti-TRAF6 antibody (Cell Signaling Technology)
overnight at 4uC. Next day, 30 mL of Protein A/G Plus Agarose
(Thermo Scientific Pierce, Rockford, IL) was added to each
sample and immunoprecipitation was performed for 2 h at 4uC.
Bound proteins were eluted by boiling in SDS sample buffer for
5 min and analyzed on SDS-polyacrylamide gel.
Total RNA was extracted from freshly isolated microvessels
using TRIZOL reagent (Invitrogen) according to the manufac-
turer’s instructions with an additional chloroform extraction,
phase separation, and an extra wash in 70% ethanol. Then, 1 mg
of RNA was reverse-transcribed using the Reverse Transcription
System (Promega, Madison, WI) and 3 mL of final RT product
was used for PCR amplification. Taqman Universal PCR Master
Mix, pre-developed primer pairs and probes were purchased from
Figure 3. Inhibition of TLR4 prevents PCB153-NP-induced TJ protein disruption in human brain endothelial cells. Conuent brain
endothelial cell cultures were treated with PCB153-NPs (PCB153, 1.6 mM; NPs, 2.086105), or the same amounts of PCB153, NPs, or vehicle for 24 h.
Selective cultures were pretreated with TLR4 inhibitor CLI095 (10 mM) or vehicle (DMSO, 0.01%) for 1 h, followed by co-exposure to PCB153 and/or
NPs for 24 h. TLR4 inhibitor was retained in media throughout PCB153 and/or NPs treatment. Occludin and claudin-5 levels were measured by
immunoblotting. Results are means 6 SD, n=5. Significantly different as compared to vehicle at *p,0.05 or ***p,0.001. Results in cultures
pretreated with CLI095 are statistically different from those in the corresponding cultures without added CLI095 at#p,0.05 or###p,0.001.
TLR4 Mediates PCB153-NP Toxicity
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Applied Biosystesms (Foster City, CA). The following thermo-
cycling conditions were employed: 95uC for 10 min, followed by
95uC for 15 sec, and 60uC for 60 sec (for up to 40 cycles).
Expression of mRNA was calculated and analyzed by the
comparative CTmethod as described .
The levels of cytokines and chemokines in cell culture media
were determined using Multi-Analyte ELISArray kit (Qiagen,
Valencia, CA). Briefly, 50 mL aliquots of culture media were
added into individual wells of the ELISArray kit and incubated at
room temperature for 2 h. After the plate was washed three times
with washing buffer, 50 mL of biotin-conjugated anti-IL-6, anti-
CXCL-8, anti-CCL-2, and anti-CCL-5 antibodies were added
into indicated wells and incubated at temperature for 1 h. Then,
the plate was washed three times and avidin-conjugated horse-
radish peroxidase was added to each well for 30 min incubation at
room temperature, followed by four washings with the washing
buffer. After 15 min incubation with development solution, stop
solution was added to each well and the absorbance was measured
at 450 nm using SpectraMax 190 absorbance microplate reader
(Molecular Devices, Sunnyvale, CA). The standard curve was
generated using antigen standard of each target protein at the
concentrations between 0 to 200 pg/mL.
Statistical analysis was completed using SigmaPlot 12.0 (Systat
Software, San Jose, CA). One-way or Two-way ANOVA followed
by Holm-Sidak post hoc test was used to compare mean responses
among the treatments. A statistical probability of p,0.05 was
TLR4 Deficiency Diminishes PCB153-NP-induced
Enhancement of Infarct Volume and Disruption of the
To test the hypothesis that PCB153-NPs potentiate the ischemic
injury through activation of TLR4, mice with a point mutation in
Figure 4. TLR4 deficiency protects against PCB153-NP-induced expression of proinflammatory genes in brain microvessels.
were treated as in Figure 1. mRNA levels of IL-6, ICAM-1, CCL-2 (MCP-1) and CCL-5 (RANTES) were determined in isolated brain microvessels by real-
time PCR. Results are means 6 SEM, n=5. Significantly different as compared to vehicle treatments in normal mice at *p,0.05, or ***p,0.001.
Results in the TLR4-deficient mice treated with PCB153-NPs are statistically different from those in control animals exposed to PCB153-NPs at
TLR4 Mediates PCB153-NP Toxicity
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the TLR4 gene (C3H/HeJ) and mice expressing normal TLR4
activity (C3H/HeouJ) were employed. As indicated in Figure 1,
exposure to PCB153-NPs significantly increased the infarct
volume in the control mice as compared to treatment with vehicle
(PBS), NPs, or PCB153 alone. However, TLR4-deficient mice
infused with PCB153-NPs showed significantly smaller infarct
volume as compared to control mice.
Disruption of TJs is a typical event during cerebral ischemia.
Therefore, we evaluated the effects of PCB153 and/or NPs on
expression of transmembrane TJ proteins, such as occludin and
claudin-5 as well as TJ-associated protein ZO-1 both in animals
and cell cultures of brain endothelial cells. Exposure to PCB153-
NPs but not to PCB153 or NPs alone resulted in a decrease in
claudin-5 and ZO-1 levels in mice with normal TLR4 expression,
whereas deficiency of TLR4 diminished the effect (Figure 2).
Consistent with these results, levels of occludin and claudin-5 were
also markedly reduced following exposure to PCB153-NPs in
brain endothelial cells. Importantly, inhibition of TLR4 activity
with CLI095 attenuated these effects (Figure 3), further indicating
that TLR4 pathways is involved in PCB153-NP-induced alteration
of TJ expression.
Overexpression of proinflammatory cytokines, chemokines, and
adhesion molecules in the brain is hallmark of neuroinflammation
. Therefore, we evaluated the expression levels of proin-
flammatory cytokines (IL-6 and CXCL-8 [IL-8]), chemokines
(CCL-2 and CCL-5 [RANTES]) and adhesion molecule ICAM-1
following exposure to PCB153 and/or NPs in brain microvessels
and cultured brain endothelial cells. Working in concert, these
proinflammatory mediators target the subsequent critical steps of
neuroinflammatory responses, such as inflammatory cell attraction
to the proximity of the endothelium, adhesion, and transendothe-
lial migration. As shown in Figure 4, mRNA levels of IL-6, CCL-2,
CCL-5, and ICAM-1 were significantly elevated in brain
capillaries of wild type mice exposed to PCB153-NPs but not to
PCB153 or NPs alone. Importantly, deficiency of TLR4 effectively
protected against these effects. Inhibition of TLR4 signaling by
CLI095 also attenuated PCB153-NP-induced overproduction of
IL-6, CXCL-8, CCL-2 and CCL-5 protein levels in cultured
human brain endothelial cells (Figure 5).
Exposure to PCB153-NPs Induces TRAF6 Interaction with
Upon activation, TLRs recruit adaptor molecules, such as
MyD88, which then activate a series of downstream signaling
molecules, including TRAF6 . To investigate these events,
brain endothelial cells were treated with PCB153-NPs for up to
4 h. Cell lysates were then immunoprecipitated with anti-TRAF6
antibody and probed for TLR4. Figure 6A indicates that PCB153-
Figure 5. Inhibition of TLR4 prevents PCB153-NP-induced proinfl m
brain endothelial cells were treated as in Figure 3 for 24 h. Protein levels of IL-6, CXCL-8 (IL-8), CCL-2 and CCL-5 were determined by ELISA in
cell culture supernatants. Significantly different a s compared to vehicle at ***p
sponses in human brain endothelial cells. Confluent
,0.001. Results in cultures pretreated with CLI095 are statistically
TLR4 Mediates PCB153-NP Toxicity
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a matory re
different from those in the corresponding cultures without added CLI095 at
NPs induced a rapid (10 min) but transient recruitment of TRAF6
to TLR4. Treatment with PCB153 alone for 10 min also resulted
in binding of TRAF6 to TLR4; however, this effect was less
prominent as compared to PCB153-NPs (Figure 6B).
TRAF6 Mediates PCB153-NP-induced Alterations in TJ
Protein Expression and Proinflammatory Responses
To investigate the involvement of TRAF6 in PCB153-NP-
mediated TJ disruption, expression of TRAF6 in brain endothelial
cells was silenced with TRAF6 siRNA (Figure 7A), followed by
exposure to PCB153 and/or NPs for 24 h. The results indicated
that silencing of TRAF6 markedly protected against PCB153-NP-
mediated reduction in occludin (Figure 7B) and claudin-5
(Figure 7C) protein levels.
In the last series of experiments, we investigated the role of
TRAF6 in PCB153-NP-stimulated production of inflammatory
mediators. Consistent with the results in Figure 5, exposure to
PCB153-NPs significantly increased the production of IL-6,
CXCL-8, CCL-2 and CCL-5 in brain endothelial cells transfected
with scrambled (control) siRNA. Importantly, silencing of TRAF6
effectively reduced the production of these inflammatory media-
tors in response to PCB153-NPs (Figure 8).
While the cellular effects of dioxin-like PCBs are linked to
activation of the aryl hydrocarbon receptor (AhR), signal
transduction mechanisms induced by ortho-PCBs are complex
and include more diverse number of receptors and signaling
pathways. Non-coplanar PCBs, such as PCB153 used in the
present study, possess at least two ortho chlorines on the biphenyl
ring, which generate steric forces that rotate the ring structure
away from a single plane. Such a structure precludes interactions
with the AhR; however, ortho-PCB congeners can act as ligands for
the constitutive andorstane receptor (CAR) and/or the pregnane-
X receptor (PXR), and activate genes targeted by these receptors
. In addition to the nuclear receptors, ryanodine receptors
(RyRs) have also been identified as candidates to mediate ortho-
PCB-induced perturbations in cellular Ca2+signaling, which plays
a pivotal role in metabolism, proliferation, gene transcription, and
protein translation in almost all cell types . For example,
PCB95 and PCB153 at concentrations lower than 1 mM were
shown to significantly enhance activity of RyR1 and RyR2 .
Furthermore, ortho-PCBs can activate several signaling cascades
including Janus kinase (JAK), epidermal growth factor receptor
(EGFR), Src kinase, and mitogen-activated protein kinase (MAPK)
[42–44]. We demonstrated that PCB153 upregulates expression of
ICAM-1 and VCAM-1 through the Src/JAK/EGFR redox
Figure 6. Exposure to PCB153-NPs induces TLR4 interaction with TRAF6. (A) Confluent brain endothelial cells were treated with PCB153-NPs
for the indicated time. Cellular extracts were immunoprecipitated using anti-TRAF6 antibody, followed by immunoblotting with anti-TLR4 antibody.
(B) Confluent brain endothelial cells were exposed to PCB153 and/or NPs for 10 min, followed by determination of TLR4 interaction with TRAF6 as in
(A). The blot illustrates the representative data of four independent experiments and the bar graph shows quantified results. Results are the mean 6
SD, n=4. Significantly different as compared to vehicle treatments in normal mice at *p,0.05.
TLR4 Mediates PCB153-NP Toxicity
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signaling, which is triggered by the NADPH oxidase-mediated
increase of superoxide generation .
In the current study, we present evidence that TLR4 is yet
another cellular receptor that is involved in ortho-PCB-mediated
vascular toxicity . While it is generally accepted that TLRs are
sensors of a variety of biological molecules, like polysaccharides,
proteins and nucleic acids , our observations that a non-
biological material, such as PCB153-NPs, can act via the TLR4
signaling pathway are novel. By inhibition of TLR4 activity via
pharmacological inhibitors and by using TLR4 deficient mice, we
demonstrated that proinflammatory effects of PCB153-NPs are
sensed via TLR4 in both brain microvessels and brain endothelial
cells. These proinflammatory mediators are actively involved in
the development of cerebrovascular and neurovascular alterations.
ICAM-1 is an adhesion molecule which stimulates firm adhesion
of leukocytes to the vascular endothelium and plays a critical role
in the pathology of numerous proinflammatory vascular diseases,
including atherosclerosis . CXCL-8 is one of the CXC
chemokine members that has potent chemotactic activity for
neutrophils . It has also been shown that CXCL-8 can induce
generation of superoxide and hydrogen peroxide  as well as
increase expression of adhesion molecules [50,51]. CC chemo-
kines, such as CCL-2 and CCL-5, are implicated in the activation
of monocytes, macrophages and lymphocytes . Additionally,
CCL-2 stimulates monocytes to produce tissue factor and
proinflammatory cytokines, including IL-6 . An elevated IL-
6 level is associated with an increased infarct volume and severity
of stroke outcome [53,54].
Activation of TLR4 results in interaction of its intracellular TIR
domain with MyD88, whose amino-terminal death domain (DD)
associates with the serine kinase IL-1 receptor-associated kinase
(IRAK). These events subsequently recruit TRAF6 , followed
by nuclear translocation of proinflammatory transcription factors
NF-kB and AP-1. In agreement with this general pathway, we
observed that treatment of brain endothelial cells with PCB153-
NPs resulted in binding of TRAF6 to TLR4. While these
interactions were transient, their importance was evident as
silencing of TRAF6 significantly attenuated PCB153-NP-induced
overproduction of inflammatory mediators.
Although the involvement of TLR4 in modulating BBB
disruption has been reported , the precise mechanisms
involved are not fully understood. Therefore, our observation
that TLR4 signaling modulates PCB153-NP-induced disruption of
TJ protein expression is another novel finding in the current study.
Figure 7. TRAF6 mediates PCB153-NP-induced a decrease in TJ protein expression. (A) Immunoblotting illustrating the efficiency of TRAF6
silencing. (B and C) Human brain endothelial cells were transfected with TRAF6 siRNA, followed by treatment with PCB153 and/or NPs as in Figure 3
for 24 h. Expression of occludin (B) and claudin-5 (C) was assessed by immunoblotting. The blot illustrates the representative data of four
independent experiments and the bar graph shows quantified results. Results are means 6 SEM. Significantly different as compared to vehicle at
*p,0.05 or **p,0.01. Results in cultures with silenced TRAF6 are statistically different from those in the corresponding cultures transfected with
control (scrambled) siRNA at#p,0.05 or##p,0.01.
TLR4 Mediates PCB153-NP Toxicity
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We propose that TLR4-mediated an increase in inflammatory
mediators may be responsible, at least in part, for these effects.
Indeed, CCL-2 has been reported to induce occludin phosphor-
ylation on both serine/threonine residues, resulting in increased
BBB permeability. Furthermore, CCL-2 targets ZO-1 and
claudin-5 phosphorylation through a signaling pathway involving
Rho and protein kinase C (PKC) [56,57]. Evidence for the
phosphorylation and ubiquitin-mediated proteasomal degradation
of TJ proteins has been demonstrated previously [58,59]. In
addition, TLR4/TRAF6 signaling can stimulate activation of
matrix metalloproteinase-9 (MMP-9) , an enzyme which is
responsible for proteolytic degradation of TJ proteins . It was
also reported that TLR4/TRAF6 signaling is involved in
nanomaterial-induced autophagy formation . While autoph-
agy is a highly conserved pathway of intracellular protein
degradation [63,64], our laboratory provided evidence that
stimulation of autophagy in brain endothelial cells is associated
with decreased expression of the TJ proteins .
Dysregulation of TLR4 signaling appears to be involved in
several disorders, including cerebral ischemia and stroke [28,66].
Consistent with these reports, we observed that the infarct volume
in TLR4-deficient mice treated with PCB153-NPs was signifi-
cantly decreased as compared to mice with normal expression
TLR4. While a variety of factors can contribute to the
development of stroke, the pathology of ischemia/reperfusion
has a very strong inflammatory component . Therefore,
inflammatory responses induced by PCB153-NPs in cerebral
vessels are likely to be responsible for the development of
enhanced brain infarct. BBB breakdown, due to disruption of
TJs and infiltration with inflammatory cells, may be another
contributing factor to the progress of the brain injury following
ischemia/reperfusion and exposure to PCB153-NPs.
In summary, our study demonstrates that exposure to PCB153
bound onto silica nanoparticles triggers TLR4/TRAF6-regulated
inflammatory responses and alterations of TJ protein expression,
which then contribute to enhanced brain injury following
ischemia/reperfusion (Figure 9). These results indicate an impor-
tant role for TLR4 signaling in PCB-mediated cerebrovascular
toxicity, suggesting that this signaling pathway may be a potential
target for therapeutic intervention in cerebrovascular disorders.
Conceived and designed the experiments: BZ SE SD MT. Performed the
experiments: BZ JC. Analyzed the data: BZ MT. Contributed reagents/
materials/analysis tools: BZ JC SE SD MT. Wrote the paper: BZ JC SE
Figure 8. TRAF6 mediates PCB153-NP-induced production of inflammatory mediators. Human brain endothelial cells were treated as in
Figures 7B and C, followed by the assessment of IL-6, CXCL-8, CCL-2 and CCL-5 in culture medium by ELISA. Results are means 6 SD, n=4.
Significantly different as compared to vehicle at ***p,0.001. Results in cultures with silenced TRAF6 are statistically different from those in the
corresponding cultures transfected with control (scrambled) siRNA at###p,0.001.
TLR4 Mediates PCB153-NP Toxicity
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