Effects of urban coarse particles inhalation on oxidative and inflammatory parameters in the mouse lung and colon
Vignal et al. Particle and Fibre Toxicology
Effects of urban coarse particles inhalation on oxidative and inflammatory parameters in the mouse lung and colon
Cécile Vignal 0
Muriel Pichavant 2
Laurent Y. Alleman 1
Madjid Djouina 0
Florian Dingreville 0
Esperanza Perdrix 1
Christophe Waxin 0
Adil Ouali Alami 2
Corinne Gower-Rousseau 0
Pierre Desreumaux 0
Mathilde Body-Malapel 0
0 Inserm, CHU Lille, U995-LIRIC-Lille Inflammation Research International Center , Univ. Lille, F-59000 Lille , France
1 SAGE - Département Sciences de l'Atmosphère et Génie de l'Environnement, IMT Lille Douai , Univ. Lille, 59000 Lille , France
2 Inserm U1019, CNRS UMR 8204, Institut Pasteur de Lille- CIIL - Center for Infection and Immunity of Lille, Univ. Lille , F-59000 Lille , France
Background: Air pollution is a recognized aggravating factor for pulmonary diseases and has notably deleterious effects on asthma, bronchitis and pneumonia. Recent studies suggest that air pollution may also cause adverse effects in the gastrointestinal tract. Accumulating experimental evidence shows that immune responses in the pulmonary and intestinal mucosae are closely interrelated, and that gut-lung crosstalk controls pathophysiological processes such as responses to cigarette smoke and influenza virus infection. Our first aim was to collect urban coarse particulate matter (PM) and to characterize them for elemental content, gastric bioaccessibility, and oxidative potential; our second aim was to determine the short-term effects of urban coarse PM inhalation on pulmonary and colonic mucosae in mice, and to test the hypothesis that the well-known antioxidant N-acetyl-L-cysteine (NAC) reverses the effects of PM inhalation. Results: The collected PM had classical features of urban particles and possessed oxidative potential partly attributable to their metal fraction. Bioaccessibility study confirmed the high solubility of some metals at the gastric level. Male mice were exposed to urban coarse PM in a ventilated inhalation chamber for 15 days at a concentration relevant to episodic elevation peak of air pollution. Coarse PM inhalation induced systemic oxidative stress, recruited immune cells to the lung, and increased cytokine levels in the lung and colon. Concomitant oral administration of NAC reversed all the observed effects relative to the inhalation of coarse PM. Conclusions: Coarse PM-induced low-grade inflammation in the lung and colon is mediated by oxidative stress and deserves more investigation as potentiating factor for inflammatory diseases.
Particulate matter; Coarse PM; Oxidative stress; Inflammation; Gut-lung axis; N-acetyl-L-cysteine
Episodic increases in ambient air contaminant levels
have been demonstrated to modulate the pathogenesis
of an increasing number of chronic diseases, from
asthma to cancer and stroke [
]. Exposure to ambient
air pollution, especially to particulate matter (PM), is a
major risk factor for pulmonary diseases such as asthma,
chronic bronchitis, and pneumonia [
epidemiological studies indicate that long- and short-term
exposure to coarse PM is associated with adverse health
effects on the human respiratory system [
Although research on airborne pollutants has focused
mostly on cardiovascular and respiratory effects, emerging
epidemiological and experimental evidence suggests that
air pollutants can also cause adverse effects on the
gastrointestinal tract. Recent epidemiological studies have
reported that exposure to air pollution may be associated
with various gastrointestinal diseases including
inflammatory bowel diseases [
], appendicitis , irritable bowel
], and enteric infections in children [
Notably, a correlation has been reported between ambient air
pollution and hospitalizations for inflammatory bowel
diseases in Wisconsin [
To date, only one study has assessed the effects of PM
inhalation on the gastrointestinal tract in an animal
]. Li et al. reported that in Ldlr−/− mice,
inhalation of ultrafine PM led to shortened villus length,
which was accompanied by prominent macrophage and
neutrophil infiltration into the ileum. Ultrafine PM
exposure also increased the concentrations of intestinal
free oxidative fatty acids and lysophosphatidic acids.
This study was the first to report that inhaled particles
can trigger a deleterious effect at the intestinal level,
which justifies the value of exploring this topic further.
Investigations of the mechanisms responsible for air
pollution exposure-induced toxicity are challenging
because of the complexity of air pollutants. Reactive
gases such as ozone, nitrogen oxides, carbon
monoxide, sulfur dioxide, volatile organic compounds and
PM of varying size are part of the air pollutant
mixture. The physicochemical characteristics of this
complex matrix of gases and PM are also highly variable
depending on the generation mode and sources (e.g.,
point industrial sources, automotive combustion, natural
processes such as wildfires and volcanic eruptions, and
atmospheric conditions) [
]. Air particles are known
sinks for various organics molecules and a number of
inorganic chemicals including physiologically active
Our study focused on coarse PM, which has an
aerodynamic diameter between 2.5 and 10 μm. It therefore
includes an extra-thoracic particulate fraction (particles
from 5 to 10 μm in size) and a thoracic particulate fraction
(particles from 2.5 to 5 μm in size) [
]. The particulate
fraction deposited into the extra-thoracic region becomes
trapped in the nasal cavity, mouth, and pharynx. The vast
majority of particules deposited in the extrathoracic region
are removed via a combination of nose-blowing, sneezing,
and mucociliary transport to the gastrointestinal tract
]. Ingestion is therefore the dominant exposure
pathway to particles deposited in the extra-thoracic region.
Meanwhile, the thoracic particulate fraction deposits in
the tracheobronchial region. This region consists of
trachea, bronchi and terminal bronchioles. These particles,
trapped in the mucus produced by the bronchial
epithelial cells are typically cleared by mucociliary
transport into the throat, and then expectored or swallowed
]. Moreover, for both fractions, soluble particles
can be absorbed directly via the airway epithelium and
cleared into the blood or lymphatic system [
Therefore, coarse PM appears to be a relevant PM
fraction to study, because it comes into direct contact with
the digestive tract and has several input pathways
allowing it to affect both the pulmonary and intestinal
The first objective of this study was to characterize
metal content of urban coarse PM collected in the French
city of Douai, hereafter called coarse PMD (cPMD) and to
assess its gastric metal bioaccessibility. Because oxidative
stress is a major mechanism of PM toxicity, the oxidative
potential of cPMD was quantified. The second aim was to
assess the effects of inhalation of cPMD at a concentration
relevant to episodic elevation peak of air pollution in mice.
The third aim was to evaluate the effect of the
administration of a well-known antioxidant, the N-acetyl-L-cysteine
(NAC), on the effects of cPMD inhalation in mice.
Aerosol samples were collected with a High-Vol (35 m3/h)
six- stages (10.2, 4.2, 2.1, 1.4, 0.73, 0.41 μm) cascade
impactor (Tisch Environmental Inc.). Particles were collected
during the warm season from July 13 to October 9, 2013,
in Douai, a small city in a densely urbanized region in the
north of France, located about 100 km from the English
Channel coast (Additional file 1: Figure S1). Daily
meteorological data during the sampling period were
retrieved from the nearest weather station of the French
meteorological service Météo-France, located 20 km north
of the sampling site at the airport of Lille-Lesquin. PM
was collected onto each impactor plate covered with
adhesive Teflon stripes, which allowed the particles to be easily
swiped with a Teflon tip, and then transferred directly into
acid-cleaned sterile tubes, weighed, homogenized, and
kept at 4 °C until later sampling for chemical and
biological assays. The particle fraction size of the cPMD used
in this study was between 2.1 and 10.2 μm.
Total metal concentration
Particulate trace element mineralization and analysis were
performed in triplicate, following a previously published
]. About 3 mg of cPMD was acid digested in a
microwave oven (Milestone ETHOS) at 220 °C. Digests
were diluted to 50 mL with ultrapure water and analyzed
by inductively coupled plasma mass spectrometry
(ICPMS) (NeXion 300XX, PerkinElmer) for trace elements
(As, Ba, Bi, Cd, Ce, Co, Cr, Cs, Cu, Hg, La, Li, Mn, Mo,
Ni, Pb, Rb, Sb, Sc, Se, Sn, Sr, Th, Ti, Tl, U, V, Zn) and
major elements (Al, Ca, Fe, K, Mg, Na). An internal mixed
standard (69Ga, 103Rh) was added (1 μg/L) to all analyzed
solutions to correct the drift of the ICP-MS signal.
Reagent blanks, quality controls and standard reference
materials (NIST 1648a and ERM CZ-120) were also
analyzed repeatedly to validate the entire analytical
procedure, as previously described [
]. The total metal
concentration is expressed in micrograms of metal per
gram of cPMD.
Gastric bioaccessible fractions of cPMD were
characterized after in vitro extraction using a synthetic gastric
juice (SGJ), according to the previously described
]. The gastric extractions were performed using
three aliquots of 1–5 mg of cPMD by agitating the PM
suspensions for 2 h at 37 °C in accordance with the
estimated physiological residence time. The separation of
the particles from the synthetic fluid was performed by
centrifugation for 10 min at 14,600 rpm at 6 °C. The
supernatant was analyzed after a HNO3 digestion on a
hot plate, evaporation to dryness, and dilution to 10 mL
(1% HNO3). The results are expressed in micrograms of
solubilized metal in the supernatant per gram of cPMD.
To check the consistency of the measured concentrations,
the residual fraction was acid digested in a microwave
oven at 220 °C according to the method of Alleman et al.
]. Elemental analyses were performed in triplicate.
Trace and major elements were validated through
repeated analysis of reagent blanks, quality controls and
standard reference materials (NIST 1648a and 2584).
The bioaccessible fractions are presented as the ratio
(expressed as a percentage) of metal concentration
measured in the gastric leaching solutions to the total
Oxidative potential of cPMD measured in ascorbic acid
(AA) depletion assays
Oxidative potential is defined as a measure of the capacity
of PM to oxidize target molecules, here AA, by generating
reactive oxygen species in an acellular assay [
]. The AA
depletion assay was performed under physiological
conditions: at 37 °C and pH 7.4 in potassium
Phosphatebuffered saline (PBS) at 10−2 mol/L, containing 30%
KH2PO4 and 70% K2HPO4 on a molar basis, that had
been pretreated with Chelex resin to remove all potential
metallic contaminants. Ten milligrams of cPMD was
solubilized in 300 mL of PBS 10−2 mol/L in an ultrasonic bath
for 30 min and then agitated at 37 °C for 24 h. The cPMD
suspension was prefiltered through a 0.45 μm syringe filter
and divided into three cPMD extracts of 100 mL each.
Next, 0.5 mL of AA at 4.10−2 mol/L in PBS was added to
each cPMD extract and the sample was mixed. The
absorbance was measured at 265 nm for different times up
to 1 h for both the cPMD extract solution test and the
blank samples without cPMD. The molar concentration of
AA was then calculated from the absorbance at 265 nm
using a preestablished calibration curve. A faster AA
depletion rate in the presence of cPMD extract compared
with the blank indicates that cPMD promotes the
oxidation of AA. A similar assay using 10 mg of cPMD in
300 mL of PBS 10−2 mol/L was performed in the presence
of 87.5 mg of EDTA, a transition metal chelating agent,
before introducing the AA reactant to examine the effect
of metal redox activity on AA depletion (Zielinski et al.,
1999). All experiments were performed in triplicate.
Oxidative potential is expressed as the maximum AA
depletion rate in micromoles per liter per minute (i.e.,
the depletion rate calculated during the first hour)
calculated after subtracting the blank for a solid-to-liquid
ratio of 10 mg-to-300 mL.
The animal treatment protocol was approved by the
regional bioethics committee (committee no.75; authorization
no.CEEA2016072517274040) and all of the animals
received human care in accordance with the Guide for
the Care and the Use of Laboratory Animals (National
Research Council (US) Committee 2011).
Male C57BL/6 mice (aged 7 weeks) were purchased
from Janvier Labs and housed under standard conventional
conditions. The room relative humidity was 55% and the
temperature was 21 °C. Mice were randomly divided into
the different exposure groups (n = 10/group), as described
in Scheme 1 and Fig. 4a.
For inhalation experiments, mice were placed 4 h/day,
5 days/week for 2 weeks and for one additional day in a
ventilated whole-body inhalation chamber that allowed
free movement (InExpose, Scireq®) [
]. Nebulization was
achieved using an Aeroneb Lab™ ultrasonic nebulizer
directly connected to a 5 l–chamber and controlled through
the flexiWare software v.6 according to the following
parameters: bias flow of 2 l/min, nebulization rate of
0.083 ml/min, which were measured and controlled
throughout the experiment. The Aeroneb Lab™ ultrasonic
nebulizer produced droplets with a volume median
diameter of 11 μm characterized by laser diffraction using the
Spraytec system from Malvern Instruments. Mice were
exposed to a solution of 40 μg cPMD/ml or to the soluble or
insoluble fractions of cPMD. The dose concentration of the
aerosol achieved with these conditions was 1.66 μg/L.
Control mice were exposed to sterile water under the same
conditions. cPMD suspension was fractionated into soluble
and insoluble fractions by centrifugation for 5 min at
13000 g as previously described [
]. Soluble and
insoluble fractions were diluted in the same volume of sterile
water as the total fraction.
N-acetyl-L-cysteine (NAC) administration
Mice were administrated NAC (Sigma-Aldrich) in
drinking water (15 μg/kg/day), from the beginning of the
PM exposure until the day of sacrifice as described in
Scheme 1 Experimental design of the mouse model
Mice were euthanized the morning following the final
exposure day. The colon was dissected and measured.
Then, the colon was emptied by pushing the stool
outwards using a dissecting forceps, and weighed.
Bronchoalveolar lavage fluid (BALF) and samples of the lungs,
colon, and blood were collected and kept on ice for
FACS analysis or immediately frozen at −80 °C.
Cells harvested from BALF or extracted from lung
tissue were washed in PBS and incubated with antibodies
(BD, Franklin Lakes, NJ, USA) for 30 min in PBS and
then washed twice and suspended in PBS with 2% fetal
calf serum. The antibodies used were: fluorescein
isothiocyanate (FITC)-conjugated anti-I-A[b];
phycoerythrin (PE)-conjugated anti-F4/80; peridinin chlorophyll
protein complex (PerCP)/Cy5-conjugated anti-CD103;
PE/Cy7-conjugated anti-CD11c; allophycocyanin
(APC)conjugated anti-CCR2; Alexa 700-conjugated anti-CD86;
APC-H7-conjugated anti-Ly6G; V450-conjugated
antiCD11b; V500-conjugated anti-CD45; BV605-conjugated
anti-Ly6C; FITC-conjugated anti-CD5; PE-conjugated
anti-CD1d tetramer; PerCP/Cy5-conjugated anti-NK1.1;
APC-conjugated anti-CD25; Alexa 700-conjugated
antiCD69; APC-H7-conjugated anti-CD4; V450-conjugated
anti-T-cell receptor-β; V500-conjugated anti-CD8, and
BV605-conjugated CD45. Cells were analyzed on an LSR
Fortessa cell analyzer (BD). The generated data were
analyzed using FlowJo 8.7 (TreeStar, Stanford, CA, USA).
Gene expression in tissues
Total mRNA from lung and colon tissues was extracted
using a Nucleospin RNA II kit (Macherey Nagel).
Reverse transcription was performed using a High Capacity
cDNA Archive Kit and quantitative polymerase chain
reaction (PCR) with SYBR Green (Life Technologies). The
primer sequences were designed using Primer Express 3
(Life Technologies) and are available upon request.
Melting curve analyses were performed for each sample
and gene to confirm the specificity of the amplification.
Because the exposure to PM did not cause any
significant alterations in Polr2a mRNA expression, the
relative expression of each gene of interest was normalized
to the relative expression of this gene. The
quantification of the target gene expression was based on the
comparative cycle threshold (Ct) value. The fold
changes in the target genes were analyzed by the 2−ΔΔCt
Serum malondialdehyde (MDA) analysis
Serum samples (50 μL) were incubated with acetic acid
and SDS at 95 °C for 1 h, followed by centrifugation at
800 g for 10 min. Supernatants were transferred to a
96-well plate and the absorbance was measured at λex =
532 and λem = 553 nm. 1,1,3,3-Tetramethoxypropane
(Sigma-Aldrich) was used as a standard. Protein
concentration in samples was determined using a DC™ protein
assay (Bio-Rad Laboratories). Serum MDA concentration
was corrected by the sample protein concentration and is
expressed as nanomoles per milliliter of serum.
Myeloperoxidase activity assay
Neutrophil influx into colon was analyzed by measuring
the enzymatic activity of myeloperoxidase (MPO). Mice
colons were homogenized in 0.5%
hexadecyltrimethylammonium bromide (Sigma-Aldrich) in 50 mM PBS,
freeze-thawed three times, and centrifuged. MPO was
analyzed in the clear supernatant by adding 1 mg/mL of
dianisidine dihydrochloride (Sigma-Aldrich) and 5.10−4%
hydrogen peroxide (H2O2), and the change in optical
density was measured at 450 nm. Human neutrophil
MPO (Sigma-Aldrich) was used as a standard. One unit
of MPO activity was defined as the amount that
degraded 1 μmol of peroxide per min at 25 °C. Readings
from tissue samples were normalized to total protein
content as detected in the DC™ protein assay (Bio-Rad).
Results are expressed as mean ± SEM. The statistical
significance of differences between experimental groups was
calculated using the Mann–Whitney U test (GraphPad,
San Diego, CA).
Characteristics of cPMD
The meteorological conditions during the sampling period
were typical for summertime under the oceanic climate of
the northwestern European coast. The daily averages were
a temperature of 17.7 °C (Additional file 1: Figure S2A),
71% relative humidity (Additional file 1: Figure S2B),
1017 hPa atmospheric pressure and cumulated rain of
172 mm over the whole period (Additional file 1:
Figure S2C). These relatively high pressures and sparse
rainfall conditions were observed throughout the
period, and a continuing decrease in temperature and
increase in relative humidity were recorded from
summer to mid-autumn. The average wind speed was
3.3 m/s (Additional file 1: Figure S2D) and winds came
mostly (39%) from the west sector (marine air masses
from the English Channel and Atlantic Ocean) and
secondarily (28%) from the north sector (more continental
air masses toward the regional Scarpe-Escaut natural park
and the south of Belgium (Additional file 1: Figure S2E).
The concentrations of elements in cPMD varied from
1 to 57,312 μg/g in the following order: U < Tl < Cs < Th <
Sc < Hg < Bi < Se < La < Li < Cd < Co < As < Ce < Mo < Rb <
V < Ni < Sb < Sn < Cr < Sr < Pb < Ba < Cu < Mn < Ti < Zn for
trace elements and Mg < K < Al < Fe < Na < Ca for major
elements (Fig. 1a). Enrichment factors relative to the upper
continental crust with Th as the reference element [
were applied to assess the anthropogenic influence on
cPMD content (Fig. 1b). Enrichment factors >10 were
observed for As, Mo, Sn, Pb, Cu, Bi, Zn, Cd, and Sb. All of
these elements are commonly associated with traffic
nonexhaust emissions (i.e., brakes and tire wear), particularly Cu,
which is predominant in coarse PM in Europe [
Sb, which showed the highest enrichment factor, and
confirmed the urban typology of the sampling site. Cd,
Zn, Bi, and Pb are known tracers of nonferrous smelting
] and their enrichment may indicate the influence
of a nearby Zn smelter located 3 km north of our
sampling site. To characterize cPMD better, we sought to
assess their features in relation to other urban coarse
PM. For this purpose, the elemental concentrations in
cPMD were compared with those measured in other
European cities: an urban site in Helsinki, Finland [
and a traffic site in Budapest, Hungary [
] (Additional file 1:
Figure S3). Globally, the elemental concentrations were
very similar, except for the highest levels of Cd, Zn and Bi
in Douai, in accordance with a significant influence of the
nearby Zn smelter.
Gastric bioaccessibility of cPMD
Simulation of solubilization pathways was performed
using the SGF to approximate gastric conditions (Fig. 1c).
Metals showing the higher gastric bioaccessibility were
Pb > Sb > Cd > Co > Sr, with values ranging from 90% for
Pb to 70% for Sr. The lowest gastric bioaccessibility were
observed for Cr (9%) and Fe (11%).
Oxidative potential of cPMD
The oxidative potential of cPMD was assessed using an
AA depletion assay (Fig. 1d); AA is an antioxidant
naturally present in the human body. The rates of AA
depletion measured showed the effective oxidative potential
of cPMD. To examine the effects of metal redox activity
on AA depletion, the same experiment was performed
in the presence of EDTA, a transition metal chelating
agent. In the presence of EDTA, AA depletion was
significantly lower (− 42%) although not totally depleted,
which indicates a role of metals in the oxidative
potential of cPMD.
Effects of cPMD inhalation on oxidative stress and
inflammation in mice
We then exposed mice to this cPMD by inhalation in a
whole body inhalation chamber. By this physiological
way of exposure, as in real-life, coarse PM is expected to
deposit primarily in the upper respiratory tract, and then
to be transported from the conducting airways to the
gastrointestinal tract by mucociliary clearance [
We first aimed to assess the effects of an
environmentally relevant episodic increase in ambient PM exposure
on pulmonary and intestinal mucosal tissues. Following
14 days of exposure to inhaled cPMD, serum MDA
concentration was significantly higher in the PM mice
compared with CT mice that inhaled only water. This finding
reflects the appearance of systemic oxidative stress
(Fig. 2a). More cells were obtained in BALF from PM mice
0.1 Se Al ThRbSc U Ce Ti K La V CsSr FeBa LiMgCoNiMnCrCa Tl NaAsMoSnPbCuBi ZnCdSb
compared with CT mice (Fig. 2b). The numbers of
neutrophils and alveolar macrophages in the lung did not
differ significantly between PM and CT mice (Fig. 2c).
By contrast, more conventional T cells and invariant
natural killer (iNKT) cells were observed in the lung of
PM mice, and these increased cell numbers were
associated with higher Tnfα, Il5, Il22, and Il10 mRNA levels
(Fig. 2d). At the intestinal level, the colon weight size
ratio did not differ between PM and CT mice (Fig. 2e).
A trend toward an increase in MPO activity was
observed in the colons of PM mice (Fig. 2f ). The gene
expression levels of markers of neutrophils (Csf3r),
macrophages (Cd68), conventional T cells (Cd247), and
iNKT cells (Vα14) did not differ between PM and CT
mice (Fig. 2g). By contrast, significantly greater
expression of Tnfα, Ifnγ, Il10 and Cxcl10, and lower
expression of Il5 were found in the colons of PM mice
compared with CT mice (Fig. 2h). Together, these
results suggest that cPMD inhalation led to low-grade
inflammation in both the lungs and gut.
Effects of NAC administration on cPMD -induced deleterious
Oral administration of NAC has been shown to protect
against oxidative stress in the lung [
]. We evaluated
the consequences of NAC administration on the
previously observed PM-induced effects. NAC was added to
drinking water of mice that inhaled PM (PM + NAC
mice). A group of mice that inhaled PM and did not
receive NAC in drinking water was also included (PM mice).
Serum MDA concentration was lower in PM + NAC mice
than in PM mice (Fig. 3a). Fewer cells were found in BALF
from PM + NAC mice than in that from PM mice (Fig. 3b).
In the lungs, less iNKT recruitment (Fig. 3c) and lower
Ifnγ, Il5 and Cxcl10 expression levels were observed
(Fig. 3d). The mRNA levels of several oxidative stress
markers such as Nos2, COX2, Sod2, Cat, and Hmox1 were
markedly lower in the lungs of PM + NAC mice compared
with those from PM mice (Fig. 3e). The colons of PM +
NAC mice showed lower MPO activity (Fig. 3f ) and
reduced Tnfα, Ifnγ and Cxcl10 mRNA expression compared
with those from PM mice (Fig. 3g). Taken together,
these data show that NAC administration reversed the
deleterious effects induced by PM inhalation. NAC did
not alter the basal condition because NAC treatment
did not modify inflammatory and oxidative stress
parameters in serum, lung, or colon of CT mice that
inhaled water (Additional file 1: Figure S4).
Involvement of water-soluble and insoluble fractions on
cPMD-induced low-grade inflammation
To assess if the solubilization of PM in water during
nebulization is involved in the observed low-grade
inflammation, an experiment was performed including in
addition to the 2 previously described groups [mice
exposed to water (CT group), and mice exposed to cPMD
(total fraction, FT group)], a group exposed to the
soluble fraction of cPMD (Soluble Fraction, SF group) and a
group exposed to the insoluble fraction of cPMD
(Insoluble Fraction, IF group) (Fig. 4a). The increased number
of cells in BALF in TF-exposed mice compared to CT
mice was found also in IF-exposed mice, and not in
SFexposed mice (Fig. 4b). Similarly, the enhancement of
iNKT cells in lungs of TF-exposed mice compared to
CT mice was also found in IF-exposed mice, and not in
SF-exposed mice (Fig. 4c). In the colon, an unexpected
decrease of Tnfα levels was quantified in IF-exposed
mice compared to CT mice (Fig. 4d). Most interestingly,
no increase of Tnfα and Cxcl2 mRNA levels was detected
in the colons of SF-exposed mice, while the transcription
of both genes was increased in TF-exposed mice. Taken
together, these results show that the water-soluble fraction
of cPMD is not sufficient to induce the low-grade
inflammation associated to cPMD inhalation.
The cPMD used in this study comprised urban particles
collected over 4 months of the warm season in northern
France, and ranged in size from 2.1 to 10,2 μm. Fe, Cu,
and Zn were among the most concentrated metals
quantified in cPMD, and their high concentrations are of
concern because of their pro-oxidative potential [
most enriched element was Sb, which is consistent with
the main traffic-related origin of cPMD. Another metal
of concern is Pb, which is highly toxic and is enriched
by 86 times in cPMD [
]. Both Sb and Pb
concentrations were similar to those reported for other urban
30, 38, 39
]. The strong enrichment for Cd and As
in cPMD compared with the upper crust is likely to
contribute to their negative health impact. Cd exposure
is known to induce pulmonary damage such as
emphysema and lung cancer [
] and to cause intestinal
inflammation in mice [
]. Similarly, As exposure has
been repeatedly associated with lung carcinogenesis
] and has been shown to perturb the gut
microbiome in mice [
Furthermore, Pb, Sr, Sb, Co and Cd presented both
high concentration in cPMD and high gastric
bioaccessibility. A substantial proportion of these elements is
therefore likely to be found in the bloodstream during
air pollution peak in urban areas. The diffusion of these
metals in the bloodstream could lead to both directly
cytotoxic effects on circulatory monocytes [
], but also
to indirectly aggravating effects on inflammation in
peripheral tissues: notably, lead and cadmium levels in PM
have been found negatively associated with miR-146a
expression in blood leukocytes RNA from foundry workers
]. Mir-146a is involved in limiting inflammatory
responses triggered through the innate immune system
]. Moreover, miR-146a-mediated NOD2-SHH
signaling regulates gut inflammation in murine model of
inflammatory bowel diseases [
The main objective of the current study was to assess
the effects of cPMD inhalation at both the pulmonary
and colonic levels. Our in vivo study was conducted in
conditions as close as possible to real life, and aimed to
determine the effects of a realistic inhalation of coarse
PM. Exposure in an inhalation chamber was preferred
to oral or intratracheal administration. For their
aerosolization, the cPMD were solubilized in sterile water;
the impact of solubilization on the observed effects seems
negligible, since the mice that inhaled the water-soluble
fraction of the cPMD did not exhibit pulmonary and colon
low-grade inflammation. The dosage of PM nebulized in
the inhalation chamber is close to that breathed during
pollution peaks by inhabitants of megacities strongly
affected by particulate pollution. In urban areas, the mean
daily concentration of PM of ≤10 μm in diameter (PM10)
ranges from <10 μg/m3 to >200 μg/m3 [
]. In 2002,
the US Environmental Protection Agency reported a
range of maximal city PM concentrations of 26–534 μg/m3
]. Extreme hourly concentrations of PM10 reaching
800 μg/m3 have been measured at a traffic site in London
]. PM10 pollution peaks at >250 μg/m3 were measured
in megacities in India, Pakistan and China in 2010 [
Therefore, the dose used here is fairly representative of
high pollution episodes in the most affected megacities
The studies performed until now that assess the effects
of coarse PM at the pulmonary level were performed
using intratracheal instillation or oropharyngeal aspiration
]; these two modes of coarse PM administration
partially or totally excluded the physiological exposure of
the mouth, nose, larynx and pharynx. They revealed in
most of the cases pulmonary inflammation, which the
players were variable regarding the type of PM, the
timing and the dose of exposure. Some consistencies can
however be found with our study assessing the effects
of coarse PM following inhalation. Tumor necrosis
factor (TNFα) produced by activated alveolar macrophages
and by epithelial cells is a master cytokine of the
inflammation induced by PM in the lung [
]. As for
TNFα, increased levels of interleukin 5 (IL-5), IL-10,
and IL-22 are in agreement with previous studies.
Overexpression of both IL-5 and IL-10 in the lung has
been shown following early life exposure to
combustionderived PM [
]. IL-22 upregulation may be linked to
activation of the aryl hydrocarbon receptor (AHR), as
reported by previous work showing the ability of urban
dust PM to induce Th17 polarization and IL-22
production in an AHR-dependent manner [
]. The most
pronounced effects on the recruitment of immune cells
involved in pulmonary inflammation induced by cPMD
seemed to involve iNKT cells. Our report is the first to
show that this immune population is associated with
PM exposure, although its key role has been
demonstrated in the pulmonary response to ozone [
cigarette smoke [
Few studies have assessed the effects of PM inhalation
at the intestinal level. As expected because of the low
dose of cPMD used, we did not find evidence of colitis
clinical manifestation in the PM-exposed mice, as reflected
by the lack of effect on the colon weight size ratio.
Accordingly, some major intestinal immune populations, namely
neutrophils, macrophages, lymphocytes and iNKT cells, do
not seem to be significantly affected by PM inhalation.
However, the increase in colon MPO activity, which almost
reached significance, as well as the strong increase in the
colonic expression of Tnfα, Ifnγ and Cxcl10 argue in favor
of a low-grade inflammation generated in colon following
PM inhalation. The large production of Il10 may be
indicative of a regulatory response, although the decrease in Il5
level remains unexplained.
It is the first time that the effects of coarse PM
administration on mice are studied by natural ventilation in an
inhalation chamber. Nevertheless, when a high dose of
coarse PM was administrated to mice by gavage during
14 days, an increase of pro-inflammatory cytokines
(Il12a, Il17, and Il2) has been described [
increases in colon TNFα and IFNγ protein levels have
also been reported following 10 and 14 weeks of oral
coarse PM intoxication in mice starting from the
neonatal period [
]. Taken as a whole, the experimental
protocols used to explore the effects of coarse PM on
the gastrointestinal tract consistently describe low-grade
intestinal inflammation, but are too diverse to reveal
the common immune cells or molecular pathways
implicated in the colonic effects of PM. Notably, it remains
to be determined whether the observed effects on the
gastrointestinal tract are mediated through a topical effect
of PM, which comes into direct contact with intestinal
epithelium, or through a systemic mechanism.
The triggering of low-grade intestinal inflammation
could contribute in part to many health issues. Low-grade
intestinal inflammation is a feature of irritable bowel
]. It could also exacerbate Inflammatory Bowel
], and promote colon carcinogenesis [
Moreover, a huge body of evidence indicates that
lowgrade intestinal inflammation participates in whole-body
metabolism, and therefore to the metabolic syndrome
that embraces cardiovascular diseases, type 2 diabetes,
and non-alcoholic liver disease [
]. It has also been
speculated that intestinal low-grade inflammation
associated with dysbiosis may play a pathophysiological role
in human brain diseases, including autism spectrum
disorder, anxiety, depression, and chronic pain [
Intestinal low-grade inflammation has also been
associated with chronic obstructive pulmonary disease [
Because of its critical role in health, any disturbance of
intestinal immune homeostasis should be considered as
a serious health threat.
A prominent finding of our study is that oxidative
stress was a key mechanism for PM-induced deleterious
effects. The AA depletion assay showed the oxidative
potential of cPMD and the involvement of metal content
in this property. Consistently, PM metal chelation
prevents the systemic inflammatory response induced in mice
by repeated PM intratracheal instillations [
] and polycyclic aromatic hydrocarbons [
] present in
PM are likely to also contribute to the oxidative potential
of cPMD. The increase in serum MDA concentrations that
we observed in cPMD-exposed mice reflects systemic
oxidative stress, which is a well-known effect of PM in rats
] and in humans [
]. NAC has been used for several
years as an antioxidant agent [
]. In our study, NAC
administration led to a normalization of pulmonary and
systemic phenotypes. Our results lead us to hypothesize a
similar mechanism to that demonstrated in chronic
obstructive pulmonary disease induced in mice by
cigarette smoke exposure [
]: that the protective effects
of NAC are mediated through reduced accumulation and
activation of iNKT cells. Most noticeably, NAC
administration led to obvious protective effects in the colon, such
as downregulation of MPO activity and complete reversal
of the phenotype observed in PM-exposed mice compared
with control mice, i.e., extinction of Tnfα, Ifnγ, and Il10
upregulation. Because the concentration of NAC shown
to induce significant improvement in colitis following oral
administration (150 mg/kg body weight (bw)/day [
160 mg/kg bw/day [
]) is much higher than that used in
our study (15 μg/kg bw/day), these effects are not likely to
be attributed to a topical effect of NAC.
NAC has been also described as a metal-chelating agent,
able to increase urine gold excretion in rheumatoid
arthritis patients treated with gold sodium thiomalate [
Moreover, intraperitoneal administration of NAC was very
effective in increasing the urinary excretion of chromium
and borate, but not lead, in rats intoxicated with
potassium dichromate, boric acid and lead tetracetate
]. On the other hand, when NAC is administrated
orally, its anti-oxidative properties are not systematically
associated with chelating effects. By instance, in
leadexposed mice, oral administration of NAC (5.5 mmol/kg)
reduced several indices of oxidative stress in both brain
and liver samples, but not tissue lead levels [
NAC treatment of arsenic-exposed rats (1 mmoml/kg) did
not reduce significantly blood and liver arsenic levels, but
reversed the elevation of brain MDA levels observed in
untreated animals exposed to arsenic [
]. Especially, if
the effects observed are mediated by the bioaccessible
metal fraction of PM, it can be hypothesized that the
attenuating effects of NAC are partly due to its metal
chelating properties, but it is unlikely that this is the major
mode of action of NAC.
Taken together, our results suggest that PM inhalation
induces a succession of oxidative and inflammatory
reactions that involve immune populations in the blood and
in the pulmonary and gastrointestinal mucosae. Our work
did not allow us to determine how these populations
communicate but highlight an involvement of the gut-lung
axis. This concept is strengthened by several lines of
evidence showing the pathophysiological relevance of the
immune crosstalk between the gut and lung. For example,
house dust mite aeroallergens induce inflammation in the
respiratory mucosa and reduce the gut epithelial barrier
]. In allergic airway disease, gut microbiota
metabolism of dietary fibers influences the severity of
allergic inflammation [
]. The importance of the gut
microbiota has also been reported during respiratory
influenza virus infection [
] and pneumococcal pneumonia
]. The effects of cigarette smoke on the small intestine
and colon, as evidenced by epithelial barrier defects,
inflammatory cell recruitment, and microbial shifts, have
been well described [
62, 90, 91
]. Therefore, our data
demonstrating the deleterious effects of inhaled PM on
both the pulmonary and colonic mucosae constitute
additional evidence for the gut-lung axis, which
deserves further investigation.
In healthy mice, inhalation of urban coarse PM which
presented with significant oxidative potential induced a
lowgrade inflammation that affected both the pulmonary and
colonic mucosae. The development of this low-grade
inflammation was at least partly driven by an oxidative
stress mechanism, as evidenced by its reversal by
concomitant administration of the antioxidative compound
NAC. Our results provide further demonstration of a
gutlung axis, and highlight the importance of understanding
the mechanisms of crosstalk between the pulmonary and
colonic mucosae. Together with previous experimental
and epidemiological observations, our study strongly
suggests that coarse PM inhalation may trigger or accelerate
the development of both pulmonary and gastrointestinal
inflammatory diseases, particularly in genetically
Additional file 1: Figure S1. Location of the sampling site. Figure S2.
Meteorological conditions during the sampling period. A Temperature.
B Relative humidity. C Rain. D Wind speed. E Wind direction and
frequency. Figure S3. Comparison of trace element concentrations in
cPMD collected at Douai and coarse PM collected in Helsinki, Finland
] and Budapest, Hungary [
]. Figure S4. Effects of NAC in the
basal condition. All mice inhaled sterile water. Mice that received
NAC (15 μg/kg/day for 14 days) in drinking water were compared
with CT mice that did not receive NAC in drinking water. A Serum
MDA levels. B iNKT cell count measured by flow cytometry. C Quantitative
PCR (qPCR) analysis of cytokine and chemokine mRNA levels in the lung.
D qPCR analysis of mRNA levels of oxidative stress markers in the lung.
E MPO activity in the colon. F qPCR analysis of cytokine and chemokine
mRNA levels in the colon. Data are presented as mean ± SEM. *p < 0.05,
Mann-Whitney U test. (DOCX 1064 kb)
AA: Ascorbic acid; AHR: Aryl hydrocarbon receptor; AM: Alveolar macrophage;
APC: Allophycocyanin; BALF: Bronchoalveolar lavage fluid; Cat: Catalase; Cd: Cluster
of differentiation; ConvT: Conventional T cell; COX2: Cytochrome c oxidase
subunit II; cPMD: Coarse particulate matter from Douai; Csf3r: Colony stimulating
factor 3 receptor; CT: Control; Cxcl10: C-X-C motif chemokine 10; Cxcl2: C-X-C
motif chemokine 2; FITC: Fluorescein isothiocyanate; Gpx1: Glutathione peroxidase
1; Hmox1: heme oxygenase 1; ICP-MS: Inductively coupled plasma mass
spectrometry; Ifnγ: Interferon gamma; Il: Interleukin; iNKT: Invariant Natural Killer T
cell; MDA: Malondialdehyde; MPO: Myeloperoxidase; NAC: N-acetyl-L-cysteine;
Nos2: Nitric Oxide Synthase 2; PE: Phycoerythrin; PerCP: Peridinin chlorophyll
protein complex; PM: Particulate matter; PMN: Polymorphonuclear neutrophil;
SGJ: Synthetic gastric juice; Sod2: Superoxide dismutase 2; Tnfα: Tumor necrosis
factor alpha; Vα14: V alpha 14
The authors thank Bruno Mallet.
This work was supported by the Hauts-de-France Region and the Ministère
de l’Enseignement Supérieur et de la Recherche (CPER Climibio), the European
Fund for Regional Economic Development, and Digestscience (European
Research Foundation on Intestinal Diseases and Nutrition).
Availability of data and materials
Data supporting the findings are found within the manuscript. Raw data files
will be provided by the corresponding author upon request.
MD, FD, CW performed the animal exposure, acquired and analyzed the data.
CV, MP and MBM interpreted the results of animal experiments. LYA, EP, AOA
performed the particulate matter collect and analysis, and interpreted the
results of these experiments. MBM drafted the manuscript. CV and MBM shared
study supervision. CG and PD participated in manuscript writing. All authors
read and approved the final manuscript.
Ethics approval and consent to participate
The animal treatment protocol was approved by the regional bioethics
committee (committee no.75; authorization no.CEEA2016072517274040) and
all of the animals received human care in accordance with the Guide for the
Care and the Use of Laboratory Animals (National Research Council (US)
Consent for publication
The authors declare they have no competing financial interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Anderson JO , Thundiyil JG , Stolbach A . Clearing the air: a review of the effects of particulate matter air pollution on human health . J Med Toxicol . 2012 ; 8 : 166 - 75 .
2. Chen B , Kan H . Air pollution and population health: a global challenge . Environ Health Prev Med . 2008 ; 13 : 94 - 101 .
3. Badyda A , Gayer A , Czechowski PO , Majewski G , Dąbrowiecki P . Pulmonary function and incidence of selected respiratory diseases depending on the exposure to ambient PM10 . Int J Mol Sci . 2016 , 1954 ; 17
4. Beamish LA , Osornio-Vargas AR , Wine E. Air pollution: an environmental factor contributing to intestinal disease . J. Crohns Colitis . 2011 ; 5 : 279 - 86 .
5. Kaplan G. Air pollution and the inflammatory bowel diseases . Inflamm Bowel Dis . 2011 ; 17 : 1146 - 8 .
6. Kaplan GG , Dixon E , Panaccione R , Fong A , Chen L , Szyszkowicz M , et al. Effect of ambient air pollution on the incidence of appendicitis . Can Med Assoc J . 2009 ; 181 : 591 - 7 .
7. Kaplan GG , Szyszkowicz M , Fichna J , Rowe BH , Porada E , Vincent R , et al. Non-specific abdominal pain and air pollution: a novel association . PLoS One . 2012 ; 7 : e47669 .
8. Orazzo F , Nespoli L , Ito K , Tassinari D , Giardina D , Funis M , et al. Air pollution, aeroallergens, and emergency room visits for acute respiratory diseases and gastroenteric disorders among young children in six Italian cities . Environ Health Perspect . 2009 ; 117 : 1780 - 5 .
9. Ananthakrishnan AN , McGinley EL , Binion DG , Saeian K. Ambient air pollution correlates with hospitalizations for inflammatory bowel disease: an ecologic analysis . Inflamm Bowel Dis . 2011 ; 17 : 1138 - 45 .
10. Li R , Navab K , Hough G , Daher N , Zhang M , Mittelstein D , et al. Effect of exposure to atmospheric ultrafine particles on production of free fatty acids and lipid metabolites in the mouse small intestine . Environ Health Perspect . 2015 ; 123 : 34 - 41 .
11. Kumarathasan P , Blais E , Saravanamuthu A , Bielecki A , Mukherjee B , Bjarnason S , et al. Nitrative stress, oxidative stress and plasma endothelin levels after inhalation of particulate matter and ozone . Part. Fibre Toxicol . 2015 ; 12 : 28 .
12. Martin R , Dowling K , Pearce D , Sillitoe J , Florentine S. Health effects associated with inhalation of airborne arsenic arising from mining operations . Geosciences . 2014 ; 4 : 128 - 75 .
13. Smith JRH , Etherington G , Shutt AL , Youngman MJA . Study of aerosol deposition and clearance from the human nasal passage . Ann Occup Hyg . 2002 ; 46 : 309 - 13 .
14. Asgharian B , Hofmann W , Miller FJ . Mucociliary clearance of insoluble particles from the tracheobronchial airways of the human lung . J Aerosol Sci . 2001 ; 32 : 817 - 32 .
15. Smith JRH , Bailey MR , Etherington G , Shutt AL , Youngman MJ . Effect of particle size on slow particle clearance from the bronchial tree . Exp Lung Res . 2008 ; 34 : 287 - 312 .
16. Labiris NR , Dolovich MB . Pulmonary drug delivery. Part I: physiological factors affecting therapeutic effectiveness of aerosolized medications . Br J Clin Pharmacol . 2003 ; 56 : 588 - 99 .
17. Alleman LY , Lamaison L , Perdrix E , Robache A , Galloo J-C. PM10 metal concentrations and source identification using positive matrix factorization and wind sectoring in a French industrial zone . Atmospheric Res . 2010 ; 96 : 612 - 25 .
18. Mbengue S , Alleman LY , Flament P . Size-distributed metallic elements in submicronic and ultrafine atmospheric particles from urban and industrial areas in northern France . Atmospheric Res . 2014 ; 135 - 136 : 35 - 47 .
19. Hamel SC , Buckley B , Lioy PJ . Bioaccessibility of metals in soils for different liquid to solid ratios in synthetic gastric fluid . Environ. Sci. Technol . 1998 ; 32 : 358 - 62 .
20. Yang A , Jedynska A , Hellack B , Kooter I , Hoek G , Brunekreef B , et al. Measurement of the oxidative potential of PM 2.5 and its constituents: the effect of extraction solvent and filter type . Atmos Environ . 2014 ; 83 : 35 - 42 .
21. Jacobo-Estrada T , Cardenas-Gonzalez M , Santoyo-Sánchez M , Parada-Cruz B , Uria-Galicia E , Arreola-Mendoza L , et al. Evaluation of kidney injury biomarkers in rat amniotic fluid after gestational exposure to cadmium . J Appl Toxicol JAT . 2016 ; 36 : 1183 - 93 .
22. Knaapen AM , Shi T , Borm PJA , Schins RPF . Soluble metals as well as the insoluble particle fraction are involved in cellular DNA damage induced by particulate matter . Mol Cell Biochem . 2002 ; 234 - 235 : 317 - 26 .
23. Wegesser TC , Last JA . Lung response to coarse PM: bioassay in mice . Toxicol Appl Pharmacol . 2008 ; 230 : 159 - 66 .
24. Livak KJ , Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method . Methods . 2001 ; 25 : 402 - 8 .
25. McLennan SM . Relationships between the trace element composition of sedimentary rocks and upper continental crust . Geochem Geophys Geosystems . 2001 ; 2 : 1021 .
26. Tsai M-Y , Hoek G , Eeftens M , de Hoogh K , Beelen R , Beregszászi T , et al. Spatial variation of PM elemental composition between and within 20 European study areas - results of the ESCAPE project . Environ Int . 2015 ; 84 : 181 - 92 .
27. Sterckeman T , Douay F , Proix N , Fourrier H . Vertical distribution of cd, Pb and Zn in soils near smelters in the north of France . Environ Pollut . 2000 ; 107 : 377 - 89 .
28. Batonneau Y , Bremard C , Gengembre L , Laureyns J , Le Maguer A , Le Maguer D , et al. Speciation of PM10 sources of airborne nonferrous metals within the 3-km zone of lead/zinc smelters . Environ. Sci. Technol . 2004 ; 38 : 5281 - 9 .
29. Sobanska S , Ricq N , Laboudigue A , Guillermo R , Brémard C , Laureyns J , et al. Microchemical investigations of dust emitted by a lead smelter . Environ Sci Technol . 1999 ; 33 : 1334 - 9 .
30. Pakkanen TA , Loukkola K , Korhonen CH , Aurela M , Mäkelä T , Hillamo RE , et al. Sources and chemical composition of atmospheric fine and coarse particles in the Helsinki area . Atmos Environ . 2001 ; 35 : 5381 - 91 .
31. Maenhaut W , Raes N , Chi X , Cafmeyer J , Wang W , Salma I. Chemical composition and mass closure for fine and coarse aerosols at a kerbside in Budapest, Hungary , in spring 2002. X-Ray Spectrom . 2005 ; 34 : 290 - 6 .
32. Méndez LB , Gookin G , Phalen RF . Inhaled aerosol particle dosimetry in mice: a review . Inhal Toxicol . 2010 ; 22 : 1032 - 7 .
33. Blesa S , Cortijo J , Mata M , Serrano A , Closa D , Santangelo F , et al. Oral N-acetylcysteine attenuates the rat pulmonary inflammatory response to antigen . Eur Respir J . 2003 ; 21 : 394 - 400 .
34. Pichavant M , Rémy G , Bekaert S , Le Rouzic O , Kervoaze G , Vilain E , et al. Oxidative stress-mediated iNKT-cell activation is involved in COPD pathogenesis . Mucosal Immunol . 2014 ; 7 : 568 - 78 .
35. Charrier JG , McFall AS , Richards-Henderson NK , Anastasio C . Hydrogen peroxide formation in a surrogate lung fluid by transition metals and quinones present in particulate matter . Environ. Sci. Technol . 2014 ; 48 : 7010 - 7 .
36. Huang S , Hu H , Sánchez BN , Peterson KE , Ettinger AS , Lamadrid-Figueroa H , et al. Childhood blood lead levels and symptoms of attention deficit hyperactivity disorder (ADHD): a cross-sectional study of Mexican children . Environ Health Perspect . 2016 ; 124 : 868 - 74 .
37. Liao LM , Friesen MC , Xiang Y -B, Cai H , Koh D-H , Ji B-T , et al. Occupational lead exposure and associations with selected cancers: the shanghai Men's and Women's health study cohorts . Environ Health Perspect . 2016 ; 124 : 97 - 103 .
38. Querol X , Viana M , Alastuey A , Amato F , Moreno T , Castillo S , et al. Source origin of trace elements in PM from regional background, urban and industrial sites of Spain . Atmos Environ . 2007 ; 41 : 7219 - 31 .
39. Wiseman CLS , Zereini F . Characterizing metal(loid) solubility in airborne PM10, PM2.5 and PM1 in Frankfurt, Germany using simulated lung fluids . Atmos Environ . 2014 ; 89 : 282 - 9 .
40. Stayner L , Smith R , Thun M , Schnorr T , Lemen RA . Dose-response analysis and quantitative assessment of lung cancer risk and occupational cadmium exposure . Ann Epidemiol . 1992 ; 2 : 177 - 94 .
41. Dervan PA , Hayes JA . Peribronchiolar fibrosis following acute experimental lung damage by cadmium aerosol . J Pathol . 1979 ; 128 : 143 - 9 .
42. Breton J , Daniel C , Vignal C , Body-Malapel M , Garat A , Plé C , et al. Does oral exposure to cadmium and lead mediate susceptibility to colitis? The darkand-bright sides of heavy metals in gut ecology . Sci Rep . 2016 ; 6 : 19200 .
43. Celik I , Gallicchio L , Boyd K , Lam TK , Matanoski G , Tao X , et al. Arsenic in drinking water and lung cancer: a systematic review . Environ Res . 2008 ; 108 : 48 - 55 .
44. Lu K , Abo RP , Schlieper KA , Graffam ME , Levine S , Wishnok JS , et al. Arsenic exposure perturbs the gut microbiome and its metabolic profile in mice: an integrated metagenomics and metabolomics analysis . Environ Health Perspect . 2014 ; 122 : 284 - 91 .
45. Monn C , Becker S. Cytotoxicity and induction of Proinflammatory cytokines from human monocytes exposed to fine (PM2.5) and coarse particles (PM10-2.5) in outdoor and indoor air . Toxicol Appl Pharmacol . 1999 ; 155 : 245 - 52 .
46. Bollati V , Marinelli B , Apostoli P , Bonzini M , Nordio F , Hoxha M , et al. Exposure to metal-rich particulate matter modifies the expression of candidate MicroRNAs in peripheral blood leukocytes . Environ Health Perspect . 2010 ; 118 : 763 .
47. Williams AE , Perry MM , Moschos SA , Larner-Svensson HM , Lindsay MA . Role of miRNA-146a in the regulation of the innate immune response and cancer . Biochem Soc Trans . 2008 ; 36 : 1211 - 5 .
48. Ghorpade DS , Sinha AY , Holla S , Singh V , Balaji KN. NOD2-Nitric Oxideresponsive MicroRNA-146a Activates Sonic Hedgehog Signaling to Orchestrate Inflammatory Responses in Murine Model of Inflammatory Bowel Disease . J Biol Chem . 2013 ; 288 : 33037 - 48 .
49. World Health Organization. WHO | Exposure to ambient air pollution [Internet] . WHO. 2016 [cited 2016 Dec 29 ]. Available from: http://www.who. int/gho/phe/outdoor_air_pollution/exposure/en/
50. Brook RD , Franklin B , Cascio W , Hong Y , Howard G , Lipsett M , et al. Air pollution and cardiovascular disease a statement for healthcare professionals from the expert panel on population and prevention science of the American Heart Association . Circulation. 2004 ; 109 : 2655 - 71 .
51. Charron A , Harrison RM . Fine (PM2 . 5) and coarse (PM2.5-10) particulate matter on a heavily trafficked London highway: sources and processes . Environ. Sci. Technol . 2005 ; 39 : 7768 - 76 .
52. Gurjar BR , Ravindra K , Nagpure AS . Air pollution trends over Indian megacities and their local-to-global implications . Atmos Environ . 2016 ; 142 : 475 - 95 .
53. Huang K , Zhuang G , Lin Y , Wang Q , JS F , Fu Q , et al. How to improve the air quality over megacities in China: pollution characterization and source analysis in shanghai before, during, and after the 2010 world expo . Atmospheric. Chem Phys . 2013 ; 13 : 5927 - 42 .
54. Schins RPF , Lightbody JH , Borm PJA , Shi T , Donaldson K , Stone V . Inflammatory effects of coarse and fine particulate matter in relation to chemical and biological constituents . Toxicol Appl Pharmacol . 2004 ; 195 : 1 - 11 .
55. Cho S-H , Tong H , McGee JK , Baldauf RW , Krantz QT , Gilmour MI . Comparative toxicity of size-fractionated airborne particulate matter collected at different distances from an urban highway . Environ Health Perspect . 2009 ; 117 : 1682 - 9 .
56. Wegesser TC , Last JA . Mouse lung inflammation after instillation of particulate matter collected from a working dairy barn . Toxicol Appl Pharmacol . 2009 ; 236 : 348 .
57. Farina F , Sancini G , Mantecca P , Gallinotti D , Camatini M , Palestini P. The acute toxic effects of particulate matter in mouse lung are related to size and season of collection . Toxicol Lett . 2011 ; 202 : 209 - 17 .
58. Saravia J , You D , Thevenot P , Lee GI , Shrestha B , Lomnicki S , et al. Early-life exposure to combustion-derived particulate matter causes pulmonary immunosuppression . Mucosal Immunol . 2014 ; 7 : 694 - 704 .
59. Breznan D , Karthikeyan S , Phaneuf M , Kumarathasan P , Cakmak S , Denison MS , et al. Development of an integrated approach for comparison of in vitro and in vivo responses to particulate matter . Part Fibre Toxicol . 2016 ; 13 : 41 .
60. van Voorhis M , Knopp S , Julliard W , Fechner JH , Zhang X , Schauer JJ , et al. Exposure to atmospheric particulate matter enhances Th17 polarization through the aryl hydrocarbon receptor . PLoS One . 2013 ; 8 : e82545 .
61. Pichavant M , Goya S , Meyer EH , Johnston RA , Kim HY , Matangkasombut P , et al. Ozone exposure in a mouse model induces airway hyperreactivity that requires the presence of natural killer T cells and IL-17 . J Exp Med . 2008 ; 205 : 385 - 93 .
62. Montbarbon M , Pichavant M , Langlois A , Erdual E , Maggiotto F , Neut C , et al. Colonic inflammation in mice is improved by cigarette smoke through iNKT cells recruitment . PLoS One . 2013 ; 8 : e62208 .
63. Kish L , Hotte N , Kaplan GG , Vincent R , Tso R , Gänzle M , et al. Environmental particulate matter induces murine intestinal inflammatory responses and alters the gut microbiome . PLoS One . 2013 ; 8 : e62220 .
64. Salim SY , Kaplan GG , Madsen KL . Air pollution effects on the gut microbiota: a link between exposure and inflammatory disease . Gut Microbes . 2014 ; 5 : 215 - 9 .
65. Sinagra E , Pompei G , Tomasello G , Cappello F , Morreale GC , Amvrosiadis G , et al. Inflammation in irritable bowel syndrome: myth or new treatment target? World J Gastroenterol . 2016 ; 22 : 2242 .
66. Morris G , Berk M , Carvalho AF , Caso JR , Sanz Y , Maes M. The role of microbiota and intestinal permeability in the pathophysiology of autoimmune and Neuroimmune processes with an emphasis on inflammatory bowel disease type 1 diabetes and chronic fatigue syndrome . Curr Pharm Des . 2016 ; 22 : 6058 - 75 .
67. Viennois E , Merlin D , Gewirtz AT , Chassaing B. Dietary emulsifier -induced low-grade inflammation promotes colon carcinogenesis . Cancer res . 2016 :canres1359. 2016 .
68. Winer DA , Luck H , Tsai S , Winer S. The intestinal immune system in obesity and insulin resistance . Cell Metab . 2016 ; 23 : 413 - 26 .
69. Chassaing B , Gewirtz AT . Gut microbiota, low-grade inflammation, and metabolic syndrome . Toxicol Pathol . 2014 ; 42 : 49 - 53 .
70. Severance GE , Tveiten DH , Lindström LH , Yolken RL , Reichelt K. The gut microbiota and the emergence of autoimmunity: relevance to major psychiatric disorders . Curr Pharm Des . 2016 ; 22 : 6076 - 86 .
71. Xin X , Dai W , Wu J , Fang L , Zhao M , Zhang P , et al. Mechanism of intestinal mucosal barrier dysfunction in a rat model of chronic obstructive pulmonary disease: an observational study . Exp. Ther. Med . 2016 ; 12 : 1331 - 6 .
72. Pardo M , Porat Z , Rudich A , Schauer JJ , Rudich Y. Repeated exposures to roadside particulate matter extracts suppresses pulmonary defense mechanisms. Resulting in lipid and protein oxidative damage . Environ. Pollut. Barking Essex 1987 . 2015 ; 210 : 227 - 37 .
73. Shang Y , Zhang L , Jiang Y , Li Y , Airborne LP . Quinones induce cytotoxicity and DNA damage in human lung epithelial A549 cells: the role of reactive oxygen species . Chemosphere . 2014 ; 100 : 42 - 9 .
74. Lu S , Li Y , Zhang J , Zhang T , Liu G , Huang M , et al. Associations between polycyclic aromatic hydrocarbon (PAH) exposure and oxidative stress in people living near e-waste recycling facilities in China . Environ Int . 2016 ; 94 : 161 - 9 .
75. Dianat M , Radmanesh E , Badavi M , Goudarzi G , Mard SA . The effects of PM10 on electrocardiogram parameters, blood pressure and oxidative stress in healthy rats: the protective effects of vanillic acid . Environ Sci Pollut Res . 2016 ; 23 : 19551 - 60 .
76. Bertazzi PA , Cantone L , Pignatelli P , Angelici L , Bollati V , Bonzini M , et al. Does enhancement of oxidative stress markers mediate health effects of ambient air particles? Antioxid Redox Signal . 2013 ; 21 : 46 - 51 .
77. Samuni Y , Goldstein S , Dean OM , Berk M. The chemistry and biological activities of N-acetylcysteine . Biochim Biophys Acta . 2013 ; 1830 : 4117 - 29 .
78. Jin HM , Zhou DC , HF G , Qiao QY , SK F , Liu XL , et al. Antioxidant Nacetylcysteine protects pancreatic β-cells against aldosterone-induced oxidative stress and apoptosis in female db/db mice and insulin-producing MIN6 cells . Endocrinology . 2013 ; 154 : 4068 - 77 .
79. Lin H , Liu X , Yu J , Hua F , Antioxidant HZ . N-acetylcysteine attenuates hepatocarcinogenesis by inhibiting ROS/ER stress in TLR2 deficient mouse . PLoS One . 2013 ; 8 : e74130 .
80. Amrouche-Mekkioui I , Djerdjouri BN . Acetylcysteine improves redox status, mitochondrial dysfunction, mucin-depleted crypts and epithelial hyperplasia in dextran sulfate sodium-induced oxidative colitis in mice . Eur J Pharmacol . 2012 ; 691 : 209 - 17 .
81. Ebrahimi F , Esmaily H , Baeeri M , Mohammadirad A , Fallah S , Abdollahi M. Molecular evidences on the benefit of N-acetylcysteine in experimental colitis . Open. Life Sci . 2008 ; 3 : 135 - 42 .
82. Lorber A , Baumgartner WA , Bovy RA , Chang CC , Hollcraft R. Clinical application for heavy metal-complexing potential of N-acetylcysteine . J Clin Pharmacol . 1973 ; 13 : 332 - 6 .
83. Banner W , Koch M , Capin DM , Hopf SB , Chang S , Tong TG . Experimental chelation therapy in chromium, lead, and boron intoxication with Nacetylcysteine and other compounds . Toxicol Appl Pharmacol . 1986 ; 83 : 142 - 7 .
84. Ercal N , Treeratphan P , Hammond TC , Matthews RH , Grannemann NH , Spitz DR . Vivo indices of oxidative stress in lead-exposed C57BL/6 mice are reduced by treatment with meso-2,3-Dimercaptosuccinic acid or Nacetylcysteine . Free Radic Biol Med . 1996 ; 21 : 157 - 61 .
85. Flora SJ . Arsenic-induced oxidative stress and its reversibility following combined administration of N-acetylcysteine and meso 2,3- dimercaptosuccinic acid in rats . Clin Exp Pharmacol Physiol . 1999 ; 26 : 865 - 9 .
86. Tulic MK , Vivinus-Nébot M , Rekima A , Medeiros SR , Bonnart C , Shi H , et al. Presence of commensal house dust mite allergen in human gastrointestinal tract: a potential contributor to intestinal barrier dysfunction . Gut . 2016 ; 65 : 757 - 66 .
87. Trompette A , Gollwitzer ES , Yadava K , Sichelstiel AK , Sprenger N , Ngom-Bru C , et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis . Nat Med . 2014 ; 20 : 159 - 66 .
88. Wang J , Li F , Wei H , Lian Z-X , Sun R , Tian Z. Respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated Th17 cell-dependent inflammation . J Exp Med . 2014 ; 211 : 2397 - 410 .
89. Schuijt TJ , Lankelma JM , Scicluna BP , de Sousa E , Melo F , Roelofs JJTH , de Boer JD , et al. The gut microbiota plays a protective role in the host defence against pneumococcal pneumonia . Gut . 2016 ; 65 ( 4 ): 575 - 83 .
90. Allais L , Kerckhof F-M , Verschuere S , Bracke KR , De Smet R , Laukens D , et al. Chronic cigarette smoke exposure induces microbial and inflammatory shifts and mucin changes in the murine gut . Environ Microbiol . 2016 ; 18 : 1352 - 63 .
91. Zuo L , Li Y , Wang H , Wu R , Zhu W , Zhang W , et al. Cigarette smoking is associated with intestinal barrier dysfunction in the small intestine but not in the large intestine of mice . J Crohns Colitis . 2014 ; 8 : 1710 - 22 .