Surface area-dependence of gas-particle interactions influences pulmonary and neuroinflammatory outcomes
Tyler et al. Particle and Fibre Toxicology
Surface area-dependence of gas-particle interactions influences pulmonary and neuroinflammatory outcomes
Christina R. Tyler 0
Katherine E. Zychowski 0
Bethany N. Sanchez 0
Valeria Rivero 0
Selita Lucas 0
Guy Herbert 0
June Liu 2
Hammad Irshad 2
Jacob D. McDonald 2
Barry E. Bleske 1
Matthew J. Campen 0
0 Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy , Albuquerque, NM , USA
1 Department of Pharmacy Practice & Administrative Sciences, The University of New Mexico , Albuquerque, NM , USA
2 Lovelace Respiratory Research Institute , Albuquerque, NM , USA
Background: Deleterious consequences of exposure to traffic emissions may derive from interactions between carbonaceous particulate matter (PM) and gaseous components in a manner that is dependent on the surface area or complexity of the particles. To determine the validity of this hypothesis, we examined pulmonary and neurological inflammatory outcomes in C57BL/6 and apolipoprotein E knockout (ApoE−/−) male mice after acute and chronic exposure to vehicle engine-derived particulate matter, generated as ultrafine (UFP) and fine (FP) sizes, with additional exposures using UFP or FP combined with gaseous copollutants derived from fresh gasoline and diesel emissions, labeled as UFP + G and FP + G. Results: The UFP and UFP + G exposure groups resulted in the most profound pulmonary and neuroinflammatory effects. Phagocytosis of UFP + G particles via resident alveolar macrophages was substantial in both mouse strains, particularly after chronic exposure, with concurrent increased proinflammatory cytokine expression of CXCL1 and TNFα in the bronchial lavage fluid. In the acute exposure paradigm, only UFP and UFP + G induced significant changes in pulmonary inflammation and only in the ApoE−/− animals. Similarly, acute exposure to UFP and UFP + G increased the expression of several cytokines in the hippocampus of ApoE−/− mice including Il-1β, IL-6, Tgf-β and Tnf-α and in the hippocampus of C57BL/6 mice including Ccl5, Cxcl1, Il-1β, and Tnf-α. Interestingly, Il-6 and Tgf-β expression were decreased in the C57BL/6 hippocampus after acute exposure. Chronic exposure to UFP + G increased expression of Ccl5, Cxcl1, Il-6, and Tgf-β in the ApoE−/− hippocampus, but this effect was minimal in the C57BL/6 mice, suggesting compensatory mechanisms to manage neuroinflammation in this strain. Conclusions: Inflammatory responses the lung and brain were most substantial in ApoE−/− animals exposed to UFP + G, suggesting that the surface area-dependent interaction of gases and particles is an important determinant of toxic responses. As such, freshly generated UFP, in the presence of combustion-derived gas phase pollutants, may be a greater health hazard than would be predicted from PM concentration, alone, lending support for epidemiological findings of adverse neurological outcomes associated with roadway proximity.
Epidemiological studies have identified that traffic
emissions, or near-roadway exposures, are often associated
with greater risk for cardiopulmonary and neurological
morbidity than are other metrics of air pollution [1, 2].
While concentrations of exhaust components are highest
at the source and become dilute with transport away
from the roadway, there is also the likelihood that
freshly generated particulate matter (PM) may have a
greater toxicity than aged PM. Findings from recent
toxicological studies suggest that an interaction may
occur between particulate and gaseous components of
vehicle exhausts that potentiates cardiopulmonary
toxicity, although it was unclear if this effect was due to the
additive toxicity of the two components or if PM toxicity
was modified by the presence of adsorbed species .
Recent innovations in diesel exhaust reduction technology
© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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effectively lower PM emissions from vehicles by filtration,
but gaseous components may still be as high or higher.
These components can then interact with background PM
or resuspended road dust during operation. Thus,
improved understanding of gas-particle interactions is
important for effective protection of human health.
The cardiovascular effects of vehicle engine-derived
pollutants have been established in the literature,
especially as they relate to diesel emissions [4–8] and, to a
lesser extent, gasoline engine emissions . More
recently, however, compelling studies have noted a
correlation between PM exposure and the onset of
neurodegenerative disorders, such as Alzheimer’s disease
(AD) . Recent in vivo and in vitro studies have
reported exposure to particulate matter induces adverse
neurological outcomes, including neuroinflammation
assessed by oxidative stress and cytokine production,
associated with impaired cognitive function and
neuropathology reminiscent of neurodegenerative disorders
including AD and Parkinson’s disease [11–15]. The
mechanism by which this toxicity in the brain occurs is
still unknown; however, neuroinflammation, particularly
priming of the brain’s resident immune cells, microglia,
resulting in both detrimental and protective functions
under pathological conditions, as measured by cytokine
production, may underlie several cognitive and
neurodegenerative disorders . Additionally, chronic systemic
inflammation resulting from cardiovascular disease
(CVD) may be associated with an increased risk for
developing neuroinflammation potentially leading to
neurodegenerative disorders .
Inhaled particulate matter induces cardiovascular and
respiratory inflammation [18–22]. Interactions between
combustion-source particulate matter and associated
gaseous components may potentiate toxicity, resulting in
greater systemic inflammation and, potentially,
neuroinflammation, thus increasing the risk for development of
neurodegenerative disorders, like AD. We have
previously demonstrated interactions between gaseous and
particulate components in driving cardiovascular effects
[3, 23]; however, it was unclear whether such
interactions were influenced more by particulate morphology
and surface area or chemical composition. In the present
study, we hypothesize that smaller PM, with a higher
surface area per mass, will have a greater interaction
with gaseous co-pollutants and lead to exacerbated
pulmonary and systemic toxicity. To assess this, we
developed a complex exposure paradigm that allows for the
vapor phase of mixed engine emissions to be combined
with carbonaceous PM distinctly in ultrafine particle
(UFP) or fine particle (FP) modes, which were then used
for exposures in a sensitive model of vascular disease, the
apolipoprotein E-deficient mouse (ApoE−/−). To our
knowledge, this is the first report to provide toxicological
evidence demonstrating pulmonary and
neuroinflammatory outcomes that were determined by PM surface area
and presumably physicochemical interactions with
adsorptive gaseous species.
Development of atmosphere combinations
The exposure atmospheres are described in Fig. 1a,
which included UFP, UFP with gaseous copollutants
(UFP + G), FP, and FP with gaseous copollutants (FP +
G). These core atmospheres were derived from mixed
vehicle exhaust (MVE), which consisted of a
combination of both diesel engine exhausts (DEE) and gasoline
engine exhausts (GEE), as previously described [3, 24].
Target exposure mass concentration was 300 μg PM/m3
for all exposure groups. A description of the aerosol
generation technique for each of the individual
atmospheric components or mixtures is described below.
Exhausts from a gasoline engine (GEE) and a diesel engine
(DEE) were generated and characterized as previously
reported. The exhausts were extracted via eduction
pumps into a 2 m3 mixing chamber where they were
combined and subsequently routed to one of several
separate inhalation chambers.
Generation of the base MVE atmosphere
MVE is a model of combined DEE and GEE designed to
reflect natural mixtures in near-roadway conditions and
thus was used as the basis for the UFP and FP
permutations. Specifically, target levels for PM and gases were
derived from previous work with this fresh combustion
mixture, and more detailed methods and schematics of
the system are published elsewhere [3, 23]. DEE was
produced from a single-cylinder, 5500-watt, Yanmar
diesel-engine generator using a combination of heavy
sulfur fuel and Number 2 Diesel Certification Fuel
(Phillips Chemical Company) and 40 weight motor oil
(Rotella T, Shell) as previously described . Electrical
current was pulled from the engine to provide a
constant load (90%) during operation. The desired
concentrations were attained by diluting the direct exhaust with
filtered air. The air used for filtered air control exposures
and also for dilution of engine emissions was pre-treated
by passage through a carbon-impregnated filter to
remove volatile organics and through a HEPA filter to
remove PM. GEE was generated as previously described
, with the notable exception that one engine was
used during a 6 h exposure period instead of two
engines. In brief, exhaust was generated from a 1996
General Motors 4.3 L V6 gasoline engine equipped with a
stock exhaust system (including muffler and catalyst).
The engine was connected to an eddy current
dynamometer (Model Alpha 240, Zöllner, Kiel, West
Germany) linked to a dynamometer interface (Type
Fig. 1 Atmospheric characterization. (a) Visual representation of each exposure group demonstrating the relevant size and complexity of ultrafine
particulate matter (UFP), ultrafine particulate matter combined with vehicular gases (UFP + G), fine particulate matter (FP), and fine particulate
matter combined with gases (FP + G). The particulate matter target concentration (300 μg/m3) was reached for each exposure group in both the
(b) 1-day (acute) and (c) 30-day (chronic) studies. The distribution of the (d) ultrafine particle (UFP) group is in the nanometer range, similar to
the distribution of the (f) ultrafine particles recombined with vehicular gases (UFP + G) group. Characterization of the (e) fine particle (FP) and (g)
fine particles recombined with vehicular gases (FP + G) demonstrate that these exposures do not contain detectable PM in the nanometer ultrafine
range suggesting larger particulate matter size
DTC-1, Dyne Systems Co., LLC, Germantown, WI) that
was controlled by a custom software program (Cell
Assistant, Dyne Systems Co.). The engine was fueled with
gasoline obtained from a local station in Albuquerque,
NM. Crankcase oil (10 W-30, Pennzoil Products
Company, Houston, TX) and oil filter (Duraguard PF52, AC
Delco, Detroit, MI) were changed every 122 h
(equivalent to 3000 miles) of engine operation.
The MVE was then created by combining a dilution of
GEE (diluted approximately 10:1 with filtered air) with a
similar dilution of DEE to achieve a target of
300 μg PM/m3. Relative PM contributions to the MVE
atmosphere reflected a ratio of GEE to DEE of
approximately 1:5; however, GEE accounted for the
majority of carbon monoxide and non-methane volatile
organic compounds [3, 24]
Ultrafine particulate with gases (UFP + G) and ultrafine
particulate without gases (UFP)
UFP + G atmosphere was generated using the MVE
directly from a 1-m3 mixing chamber containing DEE and
GEE. UFP atmosphere was generated by passing the
MVE from 1-m3 mixing chamber through a Harvard
denuder . This denuder is a parallel plate opposite
flow diffusion denuder with gaseous removal efficiency
Fine Particulate with gases (FP + G) and fine particulate
without gases (FP)
FP + G atmosphere was generated using the deposits
from the exhaust line of the diesel engine. The deposits
were packed in the delivery cup of a Wright Dust Feeder
(WDF; CH Technologies, USA) and delivered to the
inhalation exposure chamber using HEPA-filtered
compressed air. The output from the WDF was passed
through a cyclone with a cut-point of 2.5 μm (URG, Inc.,
Chapel Hill, NC) to remove large particulates from the
aerosol. HEPA filtered gases from 1 m3 mixing chambers
containing MVE were pumped into the exposure
chamber. FP without gases atmosphere was generated using a
similar technique as described above for FP + G; no
HEPA filtered MVE gases were provided in the exposure
Atmosphere monitoring and characterization
Test atmosphere characterization was conducted as
previously described [25–27]. In brief, sample collection
strategies were developed to capture and measure gas,
semivolatile, and particle phases for a broad spectrum of
chemical classes. Gases were analyzed by
chemiluminescence (NOx) and infrared spectroscopy (CO). Particulate
mass concentration was measured via gravimetrical analysis
on 47-mm glass fiber filters (GE Whatman, Pittsburgh,
PA). Particle size distribution was measured with a Fast
Mobility Particle Sizer (FMPS, TSI, St. Paul, MN) for the
~10–500 nm size range and an Aerodynamic Particle Sizer
(TSI, St Paul, MN) to measure the 0.5–20 μm size range.
Whole-body inhalation exposures to atmospheres
C57BL/6 and ApoE−/− male mice obtained from the
Jackson Laboratory aged 6–8 weeks were pair-housed
and maintained in controlled environment (30–60%
relative humidity, 20–24 °C) on a 12-h light:dark cycle with
ad libitum access to water and standard chow (2016C
Harlan Global Certified Rodent Chow for C57BL/6
mice). ApoE−/− mice were fed high-fat chow (TD.88137
Harlan, 21% fat, 0.2% cholesterol) for the entire period
of study to model previously demonstrated vulnerability
to vascular inflammatory conditions [5, 28]. Exposure
chambers were also monitored for temperature and
humidity to ensure adherence to AAALAC standards.
Animals were randomized into the following groups:
Filtered air (FA, control), UFP, UFP + G, FP, FP + G,
using 8 mice per strain per group; in one group (ApoE
−/− mice in the FP + G group), four mice had to be
excluded from post-exposure biological analyses for
preexisting health reasons. Animals were placed in 2-m3
whole-body rodent inhalation chambers (Lab Products,
Inc., Maywood, NJ) and chow was removed during the
daily exposures. Animals received either one 6-h whole
body inhalation exposure (acute exposure) or 30 days of
6-h whole body inhalation exposure (chronic exposure)
to 300 μg/m3 of the atmospheres (described above). All
procedures were approved by the Lovelace Respiratory
Research Institute’s Animal Care and Use Committee
and conform to the Guide for the Care and Use of
Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85–23, revised
1996). Animal body weights were monitored weekly
throughout the study. All mice were fasted overnight
prior to necropsy. Animals were euthanized via
intraperitoneal injection of barbiturate-based sedative
(Euthasol™). The following tissues were rapidly dissected,
placed in liquid nitrogen, and stored at −80 °C until use:
cerebrum (meninges, brain stem, cerebellum, OB
removed), lung lobes, whole aorta (collected from
ascending aorta before abdominal bifurcation), liver, heart
(upper), heart (lower), and abdominal adipose tissue.
Bronchoalveolar lavage (BALF) collection
BALF was collected via whole lung lavage with 800 μl
Dulbecco’s PBS. After centrifugation, BALF supernatant
was snap frozen in liquid nitrogen and stored at −80 °C
until use. An aliquot of BALF supernatant was assayed
for lactate dehydrogenase (LDH) and total protein as
previously described .
BALF cells were used to prepare Cytospin slides for
cell differential counting and histological scoring of
particulate uptake. After fixation, the slides were stained
with a modified Wright-Giemsa method, and subjected
to qualitative scoring by a blind observer using the
following scoring system under light microscope: 0, no
particles; 1, scattered cells with intra-cellular particles; 3,
slightly increased numbers of cells with particles, may
not be easily detected on casual examination; 5, number
of cells with particles is substantially greater than normal
and can be easily noted upon examination; 7, the
abundance of cells with particles is marked, and the
intracellular particles are prominent or have severe cytosolic
characters. The representative cell images were taken
with an Olympus CCD camera through an oil
immersion objective (×100).
Cytokine analysis in BALF
Cytokine protein concentration was assessed in BALF
using the V-Plex Proinflammatory Panel 1 Mouse Kit
(Meso Scale Discovery, Gaithersburg, MD, USA)
according to the manufacturer’s instructions. Briefly, plates
were pre-coated with a capture antibody for the
following cytokines: IFN-γ (interferon gamma), IL-10
(interleukin 10), IL-12p70 (interleukin 12 active heterodimer),
IL-1β (interleukin 1 beta), IL-2 (interleukin 2), IL-4
(interleukin 4), IL-5 (interleukin 5), IL-6 (interleukin 6),
KC/GRO (CXCL1, chemokine C-X-C motif ligand 1),
and TNF-α (tumor necrosis factor alpha). BALF (50 μl/
well) was added and allowed to react for 2 h at RT.
Plates were washed 3× with buffer containing 1X PBS
and 0.05% Tween 20. Detection antibody was added to
each well and allowed to react for 1 h at RT. Plates were
washed as previously noted and Read Buffer was added
to each well. Plates were analyzed on MSD QuickPlex
SQ 120 instrument (MSD, AI0AA-0); Discovery
Workbench (v. 4.0) software calculated cytokine
concentrations using a linear regression analysis of the standard
Transcription profiling in brain
The hippocampus and rest of cortex were dissected from
the stored brains, placed in RNAlater (LifeTechnologies,
AM7020), and immediately homogenized for 3 consecutive
rounds using MagNA Lyzer beads (Roche, 03358941001)
and homogenizer set at 6000 rpm for 60 s increments.
RNA isolation and qPCR
Total RNA was isolated from homogenized hippocampal
tissue using the RNeasy Mini Kit (Qiagen, 74104). One
microgram of high quality (260/280 = ~2.0 and 260/230
= ~1.0) RNA was converted cDNA using Applied
Biosystem’s High Capacity cDNA Reverse Transcription Kit
(LifeTechnologies, 4368814). Primer validation was
performed for all TaqMan primers (LifeTechnologies) used
in this study; genes of interest were matched with
housekeeping genes of the same efficiency. Quantitative PCR
was performed with 25 ng cDNA from hippocampal
tissue; qPCR conditions were as follows: 2 min @ 50 °C,
10 min @ 95 °C, and 45 cycles of 15 s @ 95 °C and 60 s
@ 60 °C. No template controls and RT- controls for each
gene and each sample were used for each qPCR plate.
Genes of interest (GOI) include: Ccl2 (C-C motif
chemokine ligand 2; Mm00441242_m1), Ccl5 (C-C motif
chemokine ligand 5; Mm01302427_m1), Cxcl1
(chemokine C-X-C motif ligand 1; Mm04207460_m1), Et-1
(endothelin 1; Mm00438656_m1), Il-6 (interleukin 6;
Mm00446190_m1), Il-1β (interleukin 1 beta;
Mm00434228_m1), Tgf-β (transforming growth factor
beta 1; Mm01178820_m1), Tnf-α (tumor necrosis factor
alpha; Mm00443258_m1), Vcam-1 (vascular cell
adhesion molecule 1; Mm01320970_m1). Housekeeping
genes (HKG) included B2m (beta-2-microglobulin;
Mm00437762_m1), Hprt (hypoxanthine
phosphoribosyltransferase Mm03024075_m1), and Tbp (TATA-box
binding protein; Mm01277042_m1).
All data was run in duplicate; only samples with
threshold cycle values (CT) under 35 were used for analysis.
Average GOI CT values were normalized to average
HKG CT values for associated HKGs. Subsequent ΔCT
values were assessed using the comparative CT method
(ΔΔCT), and results are expressed as fold change.
One-way ANOVA (SPSS, v.19) with post-hoc analysis
was performed as needed and corrected for multiple
comparisons with Bonferroni, n = 6–8 per group for all
analysis, except where indicated in the results section.
Statistical significance was set at 95% confidence.
Quantification of accumulation of particulate matter within
resident alveolar macrophages was analyzed using the
Kruskal-Wallis test for nonparametrics for both
exposure paradigms in both strains of animals as each
distribution failed the normality assumption of the one-way
Characterization of inhalation exposure atmospheres
Concentrations of particulate matter, carbon monoxide,
and nitrogen oxide gases for each of the test atmospheres
during the acute and chronic exposures are provided in
Table 1 and Fig. 1b, c. Extensive characterization of gases
and particulate matter for this exposure paradigm is
provided in previous reports [3, 23]. PM distribution in the
ultrafine range is shown in Fig. 1d–g, with UFP atmospheres
exhibiting comparable distributions (UFP mmad = 147.1 ±
1.3 nm; UFP + G mmad = 142.1 ± 1.3 nm), while FP
atmospheres were largely devoid of detectable PM in the
ultrafine range, despite close matching of the overall mass
concentrations. Fine PM was approximately 10 times larger,
within the low micron range; typical number median
aerodynamic diameter (NMAD) and mass median aerodynamic
diameter (MMAD) for FP and FP + G atmospheres, as
measured by an Aerodynamic Particle Sizer, ranged from
1.5 to 3.0 μm ± 1.3–1.6 μm (geometric standard deviation).
Body weight changes, pulmonary inflammation and
macrophage uptake of PM
Mice in all groups exhibited significant increases in body
weight during the course of the chronic 30-day exposure
(Fig. 2a, b); however, when normalized to starting body
weights, the UFP + G exposed C57BL/6 and ApoE−/−
mice exhibited a clear reduction in this overall growth
compared to control (Fig. 2c, d). Assessment of BALF
for markers of inflammation, including total cell counts,
LDH activity, and total protein, indicated no significant
response to either acute (Fig. 3a–f ) or chronic (Fig. 4a–
f ) exposure to PM or PM + G in either mouse strain.
Additionally, we did not see significant influx of
neutrophils, lymphocytes or eosinophils in any group. All
oneway ANOVA analyses are provided in Table 2 for
bronchial lavage fluid assessments.
Despite the lack of cellular infiltration into the lungs,
macrophages in the BALF did reveal an interesting
Table 1 Test atmosphere concentrations of particulate matter (PM), CO, and NOx
aOnly one measurement was captured for gas concentrations in the FP chamber per day
pattern of PM uptake. Visualization of resident lung
macrophages after chronic exposure indicated significant
phagocytosis of PM in both strains of mice for all
atmospheres, which was qualitatively scored by a blinded
operator (Fig. 5a–c). Scoring of PM uptake after acute
exposures indicated moderate, but significant
accumulation of phagocytized PM in the UFP + G and FP + G
groups in C57BL/6 animals (Fig. 5d, p < .0001) and
significant accumulation of the UFP and UFP + G particles
in ApoE−/− animals (Fig. 5e, p < .0001). Chronic exposure
resulted in more dense accumulation of particulate
matter and an apparent potentiation of uptake in both
strains of mice exposed to UFP + G. Significant
accumulation of UFP (p < .0001), UFP + G (p < .0001), and FP (p
< .001) particles occurred in the C57BL/6 animals
(Fig. 5f ), while ApoE−/− animals had significant
accumulation of particles in all chronic exposure groups (Fig. 5g).
Table 3 provides all one-way ANOVA values and
posthoc analyses for the quantification of accumulation of
particulate matter. Thus, chronic co-exposure of gases
and UFP led to clearly potentiated phagocytosis of PM
in comparison to other exposures, despite identical mass
concentrations of PM and no measurable increase in
lavagable cells in any group.
Cytokine induction in lung lavage
A single exposure to the MVE-derived atmospheres in
C57BL/6 mice caused no obvious induction of cytokines
Fig. 2 Assessment of body weights in C57BL/6 and ApoE−/− mice. Body weight was determined weekly throughout the chronic exposure
(30day) paradigm. Body weight increased throughout the chronic exposure for (a) C57BL/6 male mice and (b) ApoE−/− male mice, aged 6–8 weeks.
Normalization of body weight to initial weight reveals significant reduction in growth due to UFP + G exposure in (c) C57BL/6 male mice at week
four and in (d) ApoE−/− male mice starting at week two. n = 7–8 per group, *p < .05; **p < .01; ***p < .001
Fig. 3 Assessment of bronchial lavage fluid for cellular infiltration after acute (1-day) exposure to atmospheres. Acute exposure to UFP, UFP + G,
FP, and FP + G does not induce cellular infiltration into the pulmonary system in the C57BL/6 mice as indicated by no change in bronchial lavage
fluid (BALF) assessment of (a) total cell counts, (b) lactate dehydrogenase (LDH) activity, and (c) total protein (albumin). A similar lack of effect was
observed in ApoE−/− mice for (d) total cell counts, (e) lactate dehydrogenase (LDH) activity, and (g) total protein (albumin). n = 7–8 per group, *p
< .05; **p < .01; ***p < .001
in the lung (Fig. 6a, b). Interestingly, interferon-γ
(IFNγ) was significantly reduced in the lavage of UFP +
G-exposed C57BL/6 mice (p < .05). Conversely, ApoE−/− mice
had increased expression of TNFα in the lung lavage
after exposure to either UFP (p < .01) or UFP + G (p
< .001) and no change in IFN-γ expression (Fig. 6c, d).
Numerous other assayed cytokines were unaltered by
acute exposure in either strain (data not shown). After
30 days of exposure, a clear picture emerged with both
strains exhibiting a robust and selective increase in
CXCL1 and TNFα protein expression only in the UFP +
G group, (Fig. 7a–c, p < .0001, d, p < .001). CXCL1
increased expression was significantly greater in C57BL/6
mice than in ApoE−/− mice (Fig. 7e, p < .001), with a
significant main effect of exposure (F(1,26) = 84.11, p
< .0001), main effect of animal strain (F(1,26) = 6.534, p
Fig. 4 Assessment of bronchial lavage fluid for cellular infiltration after chronic (30-day) exposure to atmospheres. Chronic exposure to UFP, UFP
+ G, FP, and FP + G does not induce cellular infiltration into the pulmonary system in the C57BL/6 mice as indicated by no changes in bronchial
lavage fluid (BALF) assessment of (a) total cell counts, (b) lactate dehydrogenase (LDH) activity, or (c) total protein (albumin). A similar lack of
effect was observed in ApoE−/− mice for (d) total cell counts, (e) lactate dehydrogenase (LDH) activity, and (g) total protein (albumin). n = 7–8 per
group, *p < .05; **p < .01; ***p < .001
< .0168), and a significant interaction between exposure
and strain, (F(1, 26) = 11.41, p < .0023). Table 4 provides
all one-way ANOVA values and posthoc analyses for
quantification of cytokine protein expression in the lung
lavage after acute and chronic exposures in both strains of
mice. Table 5 provides 2-way ANOVA values and posthoc
analyses for CXCL1 expression in the lung lavage after
chronic exposure to UFP+G in both strains of mice.
Acute exposure and neuroinflammatory effects in the
To determine the effect of particulate matter size and
composition on the potential for neuroinflammation,
quantification of mRNA expression of several cytokines in the
hippocampus in response to exposures was performed.
Despite minimal impacts on pulmonary outcomes, acute
exposure induced significant changes in the expression of
several cytokines in the hippocampus of both C57BL/6 and
ApoE−/− animals suggesting that particulate matter induces
neuroinflammation even after one day of exposure
(Fig. 8a–f ). In C57BL/6 mice, acute exposure to UFP alone
resulted in increased cytokine expression of Ccl5 (Fig. 8a, p
< .05), Cxcl1 (Fig. 8b, p < .01), Tgf-β (Fig. 8e, p < .05), and
Fig. 5 Particulate matter uptake in bronchial macrophages after acute and chronic exposure to atmospheres. Representative images of macrophages
in the bronchial lavage fluid for (a) animals exposed to filtered air, (b) animals exposed to UFP, and (c) animals exposed to UFP + G. Images
demonstrate the rubric for particulate matter uptake scoring on a scale of 0–5. Acute (1-day) exposure resulted in significant uptake of particulate
matter in the (d) UFP + G and FP + G exposure groups in C57BL/6 animals and in the (e) UFP and UFP + G exposure groups in in ApoE−/− animals.
Chronic (30-day) exposure resulted in significant uptake of particulate matter in the (f) UFP, UFP + G, and FP exposure groups in C57BL/6 animals, and
in (g) all exposure groups, UFP, UFP + G, FP, FP + G in ApoE−/− animals. n = 7–8 per group, *p < .05; **p < .01; ***p < .001; ****p < .0001
Table 3 One-way ANOVA statistical analysis of particulate matter uptake in lung
UFP, UFP + G (Dunn’s)
UFP, UFP + G, FP
UFP, UFP + G, FP, FP + G
Tnf-α (Fig. 8f, p < .05) in the hippocampus. UFP + G and
FP + G exposure, however, only increased hippocampal
mRNA expression of Il-1β (Fig. 8c, p < .05), while reducing
expression of Il-6 (Fig. 8d, p < .0001). Interestingly, acute
exposure to FP and FP + G also resulted in decreased
expression of Il-6 (Fig. 8d, p < .0001) and Tgf-β (Fig. 8e, p
< .05 and p < .0001, respectively) in the C57BL/6 mice.
Different results were found for acute exposures in
ApoE−/− mice, which appeared to be more prone to broad
inflammatory responses to most exposures. Ccl5 and
Cxcl1 expression did not appear to be increased by any
atmosphere after a single exposure (Fig. 9a, b). Acute
exposure to UFP alone resulted in significantly increased
expression of Il-1β (Fig. 9c, p < .05), Il-6 (Fig. 9d, p < .05),
Tgf-β (Fig. 9e, p < .05) and Tnf-α (Fig. 9e, p < .05). While
Il-6 and Tgf-β expression were decreased in the
hippocampus of C57BL/6 mice for most atmospheres,
ApoE−/− mice exhibited opposite trends with Il-6 elevated
in UFP (p < .05), UFP + G (p < .05) and FP (p < .001)
groups (with no difference in FP + G, Fig. 9d) and Tgf-β
elevated in UFP (p < .05) and FP (p < .05) exposures and
unchanged by addition of gases (Fig. 9e). Hippocampal Tnf-α
was uniformly elevated by all single exposures in ApoE−/−
mice, approximately 2-fold (Fig. 9f, p < .05).
Chronic exposure and neuroinflammatory effects in the
After 30 days of exposure to the four atmospheres, few
inflammatory effects were noted in C57BL/6 mice. Chronic
exposure to UFP resulted in significantly increased
expression of Ccl5 (Fig. 10a, p < .05), with no changes in Cxcl1
nor IL-6 cytokine expression (Fig. 10b–c). UFP + G and FP
Fig. 6 Cytokine protein expression profile in bronchial lavage fluid after acute (1-day) exposure to atmospheres. After 1-day of exposure to the
atmospheres, bronchial lavage fluid (BALF) was obtained from animals and assessed for cytokine protein expression for (a) TNF-α in C57BL/76
BALF, (b) IFN-γ in C57BL/6 BALF, (c) TNF-α in ApoE−/− BALF, and (d) IFN-γ in ApoE−/− BALF. No change in (a) TNF-α expression was observed in
C57BL/6 animals, while (b) IFN-γ expression was significantly reduced in the UFP + G exposure group in the same animals. In ApoE−/− animals, (c)
TNF-α protein expression was increased in the UFP and UFP + G groups with (d) no significant change in IFN- γ expression. n = 7–8 per group, *p
< .05; **p < .01; ***p < .001; ****p < .0001
Fig. 7 Cytokine protein expression profile in bronchial lavage fluid after chronic (30-day) exposure to atmospheres. After 30-days of exposure to the
atmospheres, bronchial lavage fluid (BALF) was obtained from animals and assessed for cytokine protein expression for the (a) CXCL1 in C57BL/6 BALF,
(b) TNF-α in C57BL/6 BALF, (c) CXCL1 in ApoE−/− BALF and (d) TNF-α in ApoE−/− BALF. The most significant changes were induced by exposure to UFP
+ G: (a) CXCL1 expression in C57BL/6 animals, (b) TNF-α expression in C57BL/6 animals, (c) CXCL1 expression in ApoE−/− animals, and (d) TNF-α
expression in ApoE−/− were all significantly increased in after chronic exposure to UFP + G. (e) CXCL1 expression was significantly increased in C57BL/6
animals in response to UFP + G compared to ApoE−/− animals. n = 7–8 per group, *p < .05; **p < .01; ***p < .001; ****p < .0001
exposures led to significant increased expression of
hippocampal Tgf-β (Fig. 10d, p < .05). No other inflammatory
gene changes, including Ccl1 and Et1 (data not shown),
were observed in hippocampal tissues from the WT mice.
In contrast, the 30-day chronic exposure paradigm
induced a greater and more consistent pattern of
inflammatory responses in the hippocampus of ApoE−/−
animals. Assessment of the same cytokine profiles
revealed significantly increased expression of Ccl5 (Fig. 11a,
p < .05), Cxcl1 (Fig. 11b, p < .05, p < .01), Il-6 (Fig. 11c, p
< .05, p < .001) and Tgf-β (Fig. 11d, p < .01, p < .0001),
with the greatest changes in cytokine expression
resulting from the UFP + G exposure atmospheres, and a
smaller but notable increase from exposure to UFP
alone. Similar to C57BL/6 animals, no change in mRNA
expression of Ccl2 was determined (data not shown).
Table 6 includes all one-way ANOVA data values for the
quantification of mRNA expression for all cytokines
measured in the hippocampus of C57BL/6 and ApoE−/−
mice after acute and chronic exposures. These results
suggest that C57BL/6 mice exhibit healthy adaptive
responses to minimize neuroinflammatory effects of
vehicle-derived pollutant exposure, while the
hypercholesterolemic mice are impaired in such homeostatic
compensatory mechanisms, leading to measureable
Table 4 One-way ANOVA statistical analysis of protein expression in BALF
Chronic exposure to MVE and MVE + gases alters
expression of genes involved in cerebral vasculature
To determine if the size and composition of particulate
matter impacted the expression of vascular
inflammatory response genes in the brain, mRNA expression of
Vcam1 and Et1 was assessed. Acute exposures to
vehicle-derived pollutants did not alter the expression
of Vcam1 in the hippocampus of C57BL/6 mice
(Fig. 12a). In ApoE−/− mice, acute exposure to FP
only caused a 50% increase in Vcam1 (Fig. 12b, p
< .0001). Chronic exposure to UFP + G and FP
increased Vcam1 expression in C57BL/6 mice (Fig. 12c,
p < .05) but had no effect on Vcam1 expression in the
hippocampus of ApoE−/− mice (Fig. 12d). No effect of
chronic exposure was determined on the mRNA
expression of Et1 in either strain of animal (Additional
file 1: Figure S1). See Table 6 for all ANOVA and F
values for Vcam1 and Et1 expression after acute and
chronic exposure in both animal strains.
Recent epidemiological studies have linked traffic-related
air pollution to adverse neurological outcomes [2, 30]
with several toxicological reports demonstrating that PM
exposure has the capacity to alter neural development
[31, 32], induce neuroinflammation , impair
cognition , and potentially induce neurodegeneration .
However, there is still much debate regarding such
findings owing to inconsistent results and a lack of
understanding as to how inhalation exposures lead to
Table 5 Two-way ANOVA statistical analysis of CXCL1 protein
expression in BALF
Exposure Protein F-value
p = .0023
p = 0.0168
p = .0035
neurological deficits. The present study adds
substantially to the biological plausibility of this phenomenon
by providing evidence that neuroinflammatory outcomes
of exposure, as assessed via cytokine production, are
most pronounced with smaller PM, particularly in the
presence of vehicle engine co-pollutants, a likely
consequence of a high PM surface area for semivolatile
compound adsorption. Furthermore, ApoE−/− mice exhibited
clear vulnerability to long-term exposure to ultrafine
PM recombined with gases (UFP + G exposure group),
suggesting impairment of homeostatic compensatory
mechanisms fully intact in C57BL/6 mice with potential
neuroprotective function. Which specific aspects of the
ApoE−/− mouse model on a high fat diet that contributed
to this neuroinflammatory vulnerability remain uncertain.
Lastly, data presented here provide an intriguing link to
pulmonary effects, with clearly enhanced PM phagocytosis
by alveolar macrophages and induction of
proinflammatory cytokines in the airways without any indication of
neutrophil influx or lung injury. Thus, this study suggests
a potential crosstalk between pulmonary and neurological
systems in response to inhaled toxicants with clear
demonstration of exacerbated toxicological effects when PM is
combined with gaseous co-pollutants.
Ample evidence supports a unique and enhanced toxicity
from UFP compared to larger PM . Vehicle-derived
UFP are generated in the presence of gaseous
copollutants, including carbon monoxide, oxides of nitrogen,
and semivolatile and volatile hydrocarbons [25, 26, 37].
Numerous epidemiological findings highlight a role for
roadway proximity or vehicle pollution-specific components as
potent drivers of health effects [38, 39], strongly suggesting
that freshly-generated PM may have enhanced toxicity. We
recently found that, indeed, combined gas and PM
mixtures drove more profound vascular toxicity in ApoE−/−
mice . As the role of PM surface interactions with
gaseous species remained uncertain, we developed the present
exposure paradigm to compare different sizes of PM of the
same chemical composition in a head-to-head manner.
While our findings suggest that certain biological outcomes
can be induced by all respirable PM, the most consistent
Fig. 8 Cytokine expression profile in the C57BL/6 hippocampus after acute (1-day) exposure to atmospheres. After 1-day of exposure to
atmospheres, cytokine mRNA expression was assessed in the hippocampus of the C57BL/6 mouse brain for the following: (a) Ccl5, (b) Cxcl1, (c)
IL-1β, (d) IL-6, (e) Tgf-β, and (f) Tnf-α. Exposure to UFP significantly altered mRNA expression of (a) Ccl5, (b) Cxcl1, (c) IL-1β, (e) Tgf-β, and (f) Tnf-α.
Exposure to UFP+G or FP+G significantly increased mRNA expression of (c) IL-1β. Exposure to UFP + G, FP and FP + G significantly decreased
mRNA expression of (d) IL-6 and (e) Tgf-β. n = 7–8 per group, *p < .05; **p < .01; ***p < .001; ****p < .0001
and severe pulmonary effects and neuroinflammatory
consequences were clearly manifested in the UFP + G
group, consistent with the hypothesis that co-pollutant
gases adsorb onto the surface of PM, and greater PM
surface area permits enhanced adsorption, leading to
more deleterious outcomes. Indeed, the size of UFPs
results in deeper deposition in the lung after
inhalation and subsequent location within and beyond the
lung epithelial barrier with potential uptake into cells
resulting in greater potential toxicity .
Fig. 9 Cytokine expression profile in the ApoE−/− hippocampus after acute (1-day) exposure to atmospheres. After 1-day of exposure to
atmospheres, cytokine mRNA expression was assessed in the hippocampus of the ApoE−/− mouse brain for the following: (a) Ccl5, (b) Cxcl1, (c)
IL-1β, (d) IL-6, (e) Tgf-β, and (f) Tnf-α. None of the exposures significantly altered mRNA expression of (a) Ccl5 or (b) Cxcl1. Exposure to UFP and
UFP + G significantly altered mRNA expression of (c) IL-1β, (d) IL-6, (e) Tgf-β, and (f) Tnf-α, with exposures having the greatest impact on (f) Tnf-α
expression. n = 7–8 per group, *p < .05; **p < .01; ***p < .001
Fig. 10 Cytokine expression profile in the C57BL/6 hippocampus after chronic (30-day) exposure to atmospheres. After 30 days of exposure to
atmospheres, cytokine mRNA expression was assessed in the hippocampus of the C57BL/6 mouse brain for the following: (a) Ccl5, (b) Cxcl1, (c)
IL-6, and (d) Tgf-β. UFP exposure only increased (a) Ccl5 mRNA expression after thirty days, while UFP + G expression increased (d) Tgf-β. n = 7–8
per group, *p < .05; **p < .01; ***p < .001
Among the more interesting outcomes from this novel
study design, we observed dramatic differences in
macrophage phagocytosis of PM in the UFP + G chronic (30 day)
exposure group, concurrent with increased TNFα and
CXCL1 protein expression in the lavage but not increased
cellularity or total protein. Severe accumulation of PM did
not occur after a single day of exposure; however, UFP + G
exposure led to as much PM uptake as any of the other
exposures. Macrophage activation resulting from diesel PM
uptake has been shown to cause release of highly toxic
factors that activate endothelial cells more so than PM
exposure can induce directly, though these factors have yet to be
characterized . Mutlu and colleagues demonstrated a
role for macrophage-derived cytokines, specifically IL-6, in
driving systemic platelet activation . Thus, such
phagocytosis and macrophage activation appears mechanistically
linked to systemic outcomes. Why the combination of UFP
and co-pollutant gases leads to greater phagocytosis that
the same amount of denuded UFP or same mass
concentration of FP with gases is unclear. However, freshly
generated elemental carbon-based PM has been shown to have
greater potential to induced oxidative stress in
macrophages in vitro ; this study further indicated that total
surface area of different PM samples (all largely comprised
of elemental carbon) was a key driver of all oxidative
outcomes. A major challenge for understanding the
mechanistic basis of the enhanced phagocytosis relates to the
need for freshly-generated UFP from the MVE system.
Collection of PM from fresh exhaust for later exposure
leads to the loss of volatile components that may well be
the causative factors. Further study of this phenomenon
will, however, provide important information regarding
the potency of near-roadway pollution and the
pathogenesis of PM-induced systemic disease.
In addition to pulmonary effects, we found the most
severe neuroinflammatory consequences were induced by
the UFP + G exposure group after chronic exposure in the
ApoE−/− animals. Air pollution is correlated with adverse
neurodevelopmental outcomes, for which epidemiological
evidence suggests a link to autism [44, 45], and
neurodegeneration  with a potential neuroinflammatory and
oxidative stress mechanism of action . The
hippocampus, a region of the brain important for learning and
memory, is particularly sensitive to environmental toxin
exposure leading to disruption of function, neuronal loss,
and cognitive decline ; therefore, we chose to focus
the cytokine profile in this region. While C57BL/6 mice
had increased proinflammatory cytokine expression after
the acute exposure paradigm, these effects were abolished
after chronic exposure. Interestingly, decreased expression
Fig. 11 Cytokine expression profile in the ApoE−/− hippocampus after chronic (30-day) exposure to atmospheres. After 30 days of exposure to
atmospheres, cytokine mRNA expression was assessed in the hippocampus of the ApoE−/− mouse brain for the following: (a) Ccl5, (b) Cxcl1, (c)
IL-6, and (d) Tgf-β. UFP exposure increased (b) Cxcl1, (c) IL-6, and (d) Tgf-β mRNA expression after thirty days, while UFP + G expression increased
expression of all cytokines assessed after chronic exposure (a) Ccl5, (b) Cxcl1, (c) IL-6, and (d) Tgf-β. n = 7–8 per group, *p < .05; **p < .01;
***p < .001; ****p < .0001
of Il-6 and Tgf-β after acute exposures in the hippocampus
of C57BL/6 mice was observed. As the balance between
pro- and anti-inflammatory processes in the brain is
highly dynamic and temporally regulated, especially with
IL-6 , compensatory mechanisms were potentially
already underway 24 h post-acute exposure. TGF-β has
been shown to be neuroprotective and reduce brain injury
in response to ischemia ; thus, lack of TGF-β signaling
could indicate that the exposure groups were altering the
balance between the pro- and anti-inflammatory cascades
in the C57BL/6 hippocampus. After chronic exposure,
increased Tgf-β expression in the C57BL/6 animals in
response to UFP + G may indicate anti-inflammatory
protective response, perhaps derived from microglia, to
control the pro-inflammatory cascade that occurs earlier
in the exposure paradigm. Thus, C57BL/6 mice exhibit
healthy adaptive responses to minimize
neuroinflammatory effects of vehicle-derived pollutant exposure. In sharp
contrast to the C57BL/6 animals, the
hypercholesterolemic ApoE−/− mice displayed measurable
neuroinflammation via increased cytokine expression, suggesting
impaired homeostatic compensatory mechanisms after
30 days of exposure. Anti-inflammatory responses
including increased Cxcl1 and Tgf-β expression, potentially from
microglia, in the hippocampus may have been combating
increased proinflammatory cytokine expression of Il-6 and
Ccl5, though IL-6 for its part has been implicated in both
pro and anti-inflammatory responses in the brain and may
be paramount for healthy aging of the brain . It should
be noted that “activation” of microglia, while not directly
measured in this study, results in the production of a
number of pro- and anti-inflammatory cytokines
depending on the environmental niche . Microglia can have
divergent responses depending on their localization, hence
increased pro-inflammatory and anti-inflammatory
cytokines, like IL-6 and TGF-β, in the same temporal range, as
in the hippocampus of ApoE−/− mice after chronic
exposure. While we cannot definitively state that this cytokine
profile is directly from microglia, it is likely that
neuroinflammation is occurring after chronic exposure in the
ApoE−/− mice, and this could underlie altered microglia
activity eventually culminating in neuronal death and
neurodegeneration. These effects will be determined in future
studies. For now, we can conclude that as observed after
acute exposure in the C57BL/6 animals, in ApoE−/−
animals, the greatest response in the cytokine profile was
elicited by the UFP and UFP + G groups, even after chronic
exposure, suggesting that particulate matter size and the
p = 0.0199
p = .0105
p = 0.0066
p = 0.05
p = .0102
p = .0054
p = .0448
p = .0452
p = .0465
p = .0241
p = .0209
ns (p = .0560)
UFP + G, FP + G
UFP, UFP + G, FP
UFP, UFP + G, FP, FP + G
Table 6 One-way ANOVA statistical analysis of qPCR gene expression in hippocampus
Strain Exposure Gene F-value
recombination of smaller particulate matter with gases
induces a greater inflammatory response than larger particles
(even when larger particles are combined with gases).
The small, highly complex exposure groups generated
for this study appropriately model the type of
nearroadway air pollution to which many people are exposed.
While several studies have assessed the
neuroinflammatory potential of exposure to PM, these studies have
focused on larger sized particles, PM2.5 and PM10, without
assessment of particulate matter recombined with gases
for modeling typical exposure paradigms in humans. For
example, long-term, chronic exposure to PM2.5 increases
proinflammatory cytokine expression of TNF-α and IL-1β
in the rat hippocampus that is concurrent with decreased
dendritic arborization, deficits in learning and memory,
and depressive-like behaviors . Studies assessing UFP
exposure have shown various outcomes including
oxidative stress, IL-1α and TNF- α expression in the brain ,
altered MAPK signaling, and elevated GFAP indicative of
astrocyte activation in ApoE−/− mice . Early postnatal
exposure to UFP results in differential microglial
responses dependent on sex and region of the brain, with
some areas, such as the hippocampus, responding to UFP
via increased microglial activation [31, 54], suggesting
enhanced vulnerability to UFP exposure during
neurodevelopment. UFP exposure exacerbates natural aging
mechanisms in the brain via translocation to brain regions
resulting in chronic neuroinflammation concurrent with
neurodegenerative outcomes [55–57]. While further
characterization of the specific microglial response, the
Fig. 12 Vasculature mRNA expression in the hippocampus of C57BL/6 and ApoE−/− mice after acute and chronic exposure to atmospheres.
Expression of Vcam-1 mRNA was determined after acute exposures in the (a) hippocampus of C57BL/6 mice and (b) hippocampus of ApoE−/−
mice. The only significant effect was that of acute exposure to FP on Vcam-1 in the ApoE−/− hippocampus. Expression of Vcam-1 mRNA was
determined after chronic exposures in the (c) hippocampus of C57BL/6 mice and (d) hippocampus of ApoE−/− mice. UFP + G and FP significantly
increased Vcam-1 expression in the C57BL/6 hippocampus after 30 days of exposure. n = 7–8 per group, *p < .05; **p < .01; ***p < .001
potential for neurodegeneration, and the mechanism by
which the exposures induce neuroinflammation
(potentially via translocation of the particles) is required, this
study is the first to demonstrate neuroinflammatory
outcomes using exposure models that include recombination
with gases to most accurately model traffic-related air
pollution. Furthermore, ApoE−/− mice fed high fat chow were
used to model individuals with increased cardiovascular
disease (CVD) risk; our results suggest that common
traffic emission exposures, whether acute or chronic, could
result in more profound neuroinflammation in at-risk
populations, such as those with CVD, who may lack the
appropriate compensatory mechanisms to combat
proinflammatory cascades in the brain. Based on these data,
we can speculate that long-term exposure to common
traffic emissions (UFP + G) would likely result in even
greater exacerbation of neuroinflammation, potentially
resulting in behavioral manifestations and
neurodegeneration, possibly modeling neurological deficits observed in
We developed an exposure model to determine the
contribution of particle size and particle complexity to
pulmonary and potential neuroinflammatory outcomes
after acute and chronic exposures in C57BL/6 and ApoE
−/− animals. Our results showed that chronic exposure
to ultrafine carbonaceous particles mixed with vehicular
emissions exerted greater effects than UFP alone, or fine
particles mixed with the gas phase mixture, suggesting
that the surface area of particulates and the interaction
of surface-adhering gaseous components enhances
pulmonary and systemic toxicity. This outcome implicates
freshly generated UFP as inherently more toxic than PM
that has aged and lost surface-adhered volatile gases,
and further suggests that near-roadway PM may have
greater health effects than would be predicted from
concentration alone. Future analysis into the potential
pathogenesis of neuroinflammatory outcomes by inhaled
pollutants and the role of cardiovascular disease as a
contributing factor will help resolve important
mechanistic questions not addressed by the present study.
Additional file 1: Figure S1. Et-1 mRNA expression in the
hippocampus of C57BL/6 and ApoE−/− mice after acute and chronic
exposure to atmospheres. Expression of Et-1 mRNA was determined after
chronic exposures in the (a) hippocampus of C57BL/6 mice and (b)
hippocampus of ApoE−/− mice, with no significant effects induced by
any atmospheres. (TIFF 754 kb)
The work was supported by the National Institutes of Health (NIH ES014639
to MJC) and by the Environmental Protection Agency, Assistance Agreement
RD-83479601-0 (Clean Air Research Centers). The views expressed in this
document are those of the authors. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use by
the United States EPA.
Availability of data and materials
The datasets supporting the conclusions of this article are included within
the article and its supplementary files.
CRT, KEZ, BEB, and MJC designed experiments and interpreted data. CRT and
MJC analyzed data and wrote the manuscript. CRT and GH conducted
experiments. BNS and VR aided in preparation of samples. JL, HI, and JDM
designed and performed the exposures and the analysis of particulate
matter size and distribution. All authors approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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