Sequential airâ€“liquid exposure of human respiratory cells to chemical and biological pollutants
Sequential air–liquid exposure of human respiratory cells to chemical andbiological pollutants
Charles Persoz , Christopher Leleu , Sophie Achard , Magali Fasseu , Jean Menotti ,Pascale Meneceur , Isabelle Momas , Francis Derouin , Nathalie Seta
a Université Paris Descartes, Laboratoire de Santé Publique et Environnement – EA 4064, Paris, Franceb Université Paris Diderot, Sorbonne Paris Cité, EA 3520, Paris, Francec Centre de recherche Bichat-Beaujon, U773, Inserm, Paris, Franced AP-HP, Hôpital Saint-Louis, Parasitologie-Mycologie, Paris, Francee AP-HP, Hôpital Bichat, Biochimie, Paris, France
Although indoor air has wide ranging effects on human health, the effects of environmental, chemical,
and biological pollutants on the respiratory system are not fully understood. In order to clarify the health
effects of airborne pollutant exposure, it would appear that toxicological evidence is needed to com-
plement epidemiological observations to support by providing biological plausibility. The aim of this
study is to manage air–liquid successive exposures to different pollutants such as a chemical pollutant(formaldehyde – FA), and a biological contaminant (Aspergillus fumigatus – Asp) using our in vitro model.
Human alveolar cells (A549) were exposed at the air–liquid interface in an exposure module, ﬁrstly
to an environmental level of FA (50 g/m3) (or air) for 30 min, and 14 h later to Asp (7 × 108 spores/m3)
(or air) for 30 min. After 10 h post-incubation, cellular viability was assessed. Inﬂammation biomarkers
(IL-8, MCP-1) were assayed by ELISA and by RT-PCR.
Whatever the conditions, no cytotoxic effect was observed. FA followed by air exposure did not induce
modiﬁcation of production and expression of cytokines, conﬁrming results with a unique FA exposure. Airfollowed by Asp exposure tended to induce IL-8 expression whereas IL-8 production tended to increaseafter FA and Asp exposure compared to FA and air exposure. The reaction of cells to sequential exposureto FA and Asp was moderate. These results show the feasibility of our model for sequential exposuresto different types of environmental pollutants, allowing using it for preliminary assessment of cellularactivity modiﬁcation induced by airborne contaminants.
2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction
The effects of environmental chemical and biological com-
pounds and their association with human respiratory health,
Indoor air has important and wide ranging effects on human
health and work efﬁciency, due to the fact that humans are perma-
For example, several studies have reported a relation
nently inhaling a complex mixture containing numerous pollutants
between formaldehyde (FA) exposure at environmental level and
asthma, asthma symptoms or airway inﬂammation (This association is notclearly established, however, with discrepancy in the conclusions(Similarly, as for
∗ Corresponding author at: Laboratoire de Santé Publique et Environnement
– EA4064, Faculté de Pharmacie Université Paris Descartes, 4, avenue de
are frequently found in indoor environment
l’Observatoire, 75006 Paris, France. Tel.: +33 1 53 73 97 28; fax: +33 1 43 25 38
numerous studies have reported an association between
mold exposure in the home and asthma symptoms
but some disagree on the consequences of exposure to fungal
spores. For instance, one current child case–control study has
reported asthma cases having signiﬁcantly higher exposures to
1 Both authors contributed equally to this work.
recently that children living in a farm and exposed
0378-4274/$ – see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:
C. Persoz et al. / Toxicology Letters 207 (2011) 53–59
to a wider range of fungi than the reference group, were partly
chambers which can hold three separate inserts. The module is equipped with a
protected against asthma. This demonstrates the complexity of
heated water jacket to keep cells and culture medium at a steady temperature of
interaction between these pollutant exposures and induced respi-
37 ◦C. For both FA and Asp experiments, the test atmosphere was delivered on thecellular apical side at a ﬂow rate of 5 ± 0.1 mL/min.
ratory health effects, and it would appear epidemiological studies
FA atmosphere was generated as previously described The
need to be complemented by toxicological studies in order to
chosen dose for these experiments (50 g/m3) has been formerly determined as the
fully understand the health effects of airborne pollutant exposure
For experiment with Aspergillus spores (Asp), Asp atmosphere (7 × 108
spores/m3) was generated as follows. A genetically modiﬁed strain of A. fumigatus
Beside experimental toxicological studies traditionally based on
expressing a red ﬂuorescent protein, DsRed (CBS 144.89/DsRed) (
complete animal tests, in vitro methods using cell culture technolo-
used, kindly provided by J.P. Latgé, Institut Pasteur, Paris, France.
gies are currently being developed and implemented in modern
This strain was grown on Sabouraud-Chloramphenicol agar Petri dishes at 37 ◦C for
toxicology (Indeed the 3Rs principles that
7 days. Then conidia were harvested by rinsing the culture with sterilized water
promote alternatives to animal experimentation are now always
containing 0.002% Tween 80. The collected suspension was centrifuged at 3000 × gfor 15 min, then the pellet was resuspended in water containing 0.002% Tween 80
encouraged, due notably to ethical reasons and cost
and ﬁltered through a sterile sieve to eliminate remaining agar debris. The puriﬁed
Classical in vitro exposure methods of airborne contaminants,
spore suspension was counted using a Malassez hemacytometer and adjusted to a
also called indirect or submerged exposure methods, consist of
ﬁnal concentration of 108 conidia/mL. The viability of collected conidia was checked
cell cultures exposed to test pollutants dissolved or suspended in
by subculture of serial dilutions of each conidial preparation.
The conidia suspension obtained was aerosolized using a Collison Nebulizer
culture medium, like particles collected by impaction or ﬁltration
(BGI, USA) provided by compressed air at 2-bars pressure. In preliminary experi-
methods, or gas dissolved by bubbling through buffered saline solu-
ments, using a particle counter we checked that such conditions allowed generating
an aerosol containing >70% of particles ranging between 1 and 5 m, i.e. compris-
however do not really reﬂect human exposure, and are not there-
ing the 2.5–3 m size of Aspergillus spores. Less than 5% of particles >5 m were
fore suitable for gaseous pollutant testing.
detected suggesting that few spores clustered during nebulization. The generatedaerosol was connected to the Vitrocell® exposure device, resulting in exposure of
Methods which better mimic the inhalation process and contact
the cellular apical side of cell culture.
between epithelial cells and air are considered to be more relevant
In order to check the conidial concentration at the cell culture level, a sample
than the in vitro exposure methods above. Two main direct expo-
of air was collected in the Vitrocell® inlet using a vacuum pump at a ﬂow rate of
sure approaches are available: the ﬁrst is intermittent exposure
10 L/min during 1 min then passed through a 0.8 micron size ﬁlter (AAWP, Millipore). The ﬁlter was then incubated into 1 mL of saponin 2% during 15 min and vortexed to
(cells are exposed to gaseous compounds and culture medium at
elute the collected conidia. These were enumerated in a Malassez hemacytometer.
regular intervals using rocker platforms or rolling bottles), and the
Three repeated measurements were performed for each exposure experiment.
second is continuous exposure. In this latter case, cells culturedon an insert made of microporous membranes are continuously
2.3. Cell line, culture conditions and cell exposures
exposed to airborne pollutants on their apical side for a period oftime, whilst their basolateral side is in contact with the culture
Human alveolar epithelial cells, A549 (CCL-185 purchased from ATCC, VA, USA)
were cultured as previously described (Cells (at the density of20,000 cells per insert) were grown on inserts (1.13 cm2). Passages of cells were
certain tools have recently been developed to perform air–liquid
between 3 and 15 for all experiments. Culture medium (DMEM:F12 supplemented
exposure to environmental pollutants, most have been used for sin-
with 10% FCS) was added on both sides: 300 L on the apical side, and 1000 L on
gle period exposure to an unique pollutant at levels far above the
the basolateral one, and incubated 72 h before experimentation at 37 ◦C and 5% CO2.
Just before exposure, culture medium was removed and replaced with DMEM:F12
medium supplemented with 1% FCS. Cells were then exposed at the air–liquidinterface for 30 min to either air or pollutants. the experimen-
tal protocols. Protocol A includes unique exposure to air or FA followed by 14 h
such an in vitro model for formaldehyde single period exposure of
post-incubation. Protocol B consisted of two sequential exposures of 30 min each
lung epithelial cells (However, in these exper-
(exposures 1 and 2): the ﬁrst to air or FA and the second to air or Asp. In this case, a
imental conditions, the pollutants studied were always chemical
10 h post-incubation followed the second exposure. Four experimental conditionswere thus obtained as shown in B).
rather than microbiological. If we are to mimic real atmospheric
Prior to each exposure the culture medium at the apical side was removed to
exposure then cells must be exposed for longer, or more frequently,
allow a direct contact between the cells and the tested pollutants. After each expo-
sure, 300 L of culture medium with 10% FCS were added at the apical side and
In this context, the aim of this study is to manage air–liquid
cells were post-incubated for 14 h (exposure 1) and 10 h (exposure 2) at 37 ◦C, 5%
successive exposures to different pollutants such as a chemi-
CO2. At the end of each post-incubation, culture medium was removed and storedat −20 ◦C for cytokine assay and at 4 ◦C for LDH assay. The cell monolayer was used
cal pollutant (formaldehyde – FA), and a biological contaminant
for RT-qPCR at the end of the experiment.
(Aspergillus fumigatus – Asp) using our in vitro model. These, both
Control cells introduced at each experiment corresponded to cells cultured in
known to be frequently simultaneously detected in domestic envi-
submerged conditions without any exposure. Each experiment was realized four
times in triplicates with an interval between two tests of one week. 2. Methods
Membrane integrity was determined by measuring the release of intracellu-
lar lactate dehydrogenase (LDH) into the supernatant medium. LDH was measured
using an in vitro assay kit in which LDH oxidizes the substrate l-lactate buffered at
penicillin–streptomycin and trypsin were purchased from Gibco via Invitro-
a pH of 9.4 in the presence of NAD+ to yield pyruvate and NADH,H+ which absorbs
gen (WI, USA). Formaldehyde, TNF␣, Tween 80 and saponin were obtained from
light at 340 nm. Concentrations were initially expressed as unity of consumed sub-
Sigma–Aldrich (MO, USA). The LDH assay kit (K2054) was from Siemens and used
strate per liter (U/L) and cellular viability, expressed as a %, was calculated with
with an automaton Dimension Vista 1500 (Siemens Healthcare Laboratories). The
sample concentration and maximal LDH activity (positive control obtained by cell
DuoSet kits for ELISA assays (DY208 and DY271) were produced by R&D Systems
lysis after 2 h Triton X-100 1% treatment), each normalized on control cells concen-
(MN, USA). Inserts and ﬂasks for cell culture were provided from Greiner Bio-One
tration Control cell LDH concentration was 25 ± 4 U/L (n = 4)
and maximal activity was 1652 ± 237 U/L (n = 4).
2.2. Atmosphere generation and cell exposure device
Local cellular inﬂammation was assessed by quantiﬁcation of Interleukin-8
The Vitrocell® device used for exposure experiments has been previously
(IL-8) and Monocyte Chemoattractant Protein 1 (MCP-1) concentrations in the
described by this device consists of three exposure
apical culture supernatants of A549 cells using ELISA assay kits following the
C. Persoz et al. / Toxicology Letters 207 (2011) 53–59
Cell viabilityCytokines producƟon and expression
Exposure 2 Nomenclature for condiƟons in protocol B Exposure 1 Fig. 1. Exposure protocol.
manufacturer’s instructions as stated previously. Results were expressed as a ratio
were conﬁrmed using nonparametric Mann–Whitney U test. Difference was signif-
of cytokine production (± standard error) compared to the one of the control cells. 3. Results
mRNA extraction and puriﬁcation were performed with Qiagen RNeasy® plus
mini kit (Qiagen, Germany) according to the manufacturer’s protocol for culturedcells. Samples were examined for concentration with the NanoDrop® ND-1000 Spec-
trophotometer (NanoDrop Technologies, USA) and for integrity with the RNA 6000Nano LabChip® kit and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). All
To apply our in vitro model to sequential exposure to a chemical
the RNA Integrity Numbers of the samples were >7. RNA was then stored at −80 ◦C
pollutant and molds, two limiting factors concerning post-exposure
cDNA synthesis was performed using the cDNA verso® kit from Thermo Scien-
incubation time had to be taken into account: limiting the cell line
tiﬁc according to the manufacturer’s speciﬁcations. Reverse transcription products
growth to 24 h, and avoiding the germination of conidia.
were ampliﬁed using a Thermal Cycler 2720 (Applied Biosystems). Primers for
In a preliminary work, conidia germination was monitored at
cytokines (IL-8: Hs00174103 m1, MCP-1: Hs00234140 m1) and reference genes
different times after Asp exposure, and showed that an incubation
(GAPDH: Hs99999905 m1, B2M: Hs00984230 m1, HPRT1: Hs01003267 m1) were
time of 10 h resulted in germination and limited hyphal growth
obtained from Applied Biosystems. The real-time PCR run on the LightCycler® 480Real-Time PCR System (Roche Applied Sciences, Basel, Switzerland). Each gene was
whereas a longer incubation time resulted in a deleterious fun-
tested in duplicate within the same PCR run on an additional 96-well plate. After
gal invasion of the culture (Supplementary data, We
being diluted by ten, cDNA (5 L) from each reaction for all genes was added to a
therefore considered 10 h as the maximal incubation time after Asp
PCR reaction mix containing 1X TaqMan® Universal Master Mix II without Uracil-N-
spore exposure and scheduled this exposure after FA exposure.
Glycosylase and 1 L Endogenous Control assay or Gene expression Assay in a 20 Lreaction volume. Standard cycling conditions were used [95 ◦C for 10 min, (95 ◦C for
For the repeated exposure protocol, given cell conﬂuence was
15 s, 60 ◦C for 60 s) ×40 cycles].
reached after 24 h and maximum 10 h post-incubation after Asp
The expression levels obtained were normalized against the reference gene,
exposure, post-incubation after chemical pollutant exposure, being
and changes in expression levels were given relative to the untreated controls.
the ﬁrst exposure, had to be 14 h. We compared the results after
Data quantiﬁcation normalization of expression of marker genes was achieved by
the ﬁrst exposure with our previous results, where experimen-
normalization against a subset of three reference genes on the array that did notshow signiﬁcant changes in expression under our experimental conditions (B2M
tal conditions were designed with 24 h of post-incubation after
(␤2-microglobulin), HPRT (hypoxanthine phosphoribosyltransferase) and GAPDH
air–liquid exposure After 14 h post-incubation
(glyceraldehyde 3-phosphate dehydrogenase)).
air-exposure induced an increase of 42.6 ± 6.9% of IL-8 production
Stability of the 3 reference genes was assessed with the geNorm applet
compared to the control cells (p < 0.01), and MCP-1 production was
(). The Cq values were transformed toquantities using gene speciﬁc efﬁciencies, according to the geNorm manual. Gene
not modiﬁed, similar to exposure followed by 24 h post-incubation
expression stability (M) was calculated and the genes were ranked from best to
(In the same way, FA did not signiﬁcantly mod-
worst, based on the M value. The gene with the highest M, i.e. the least stable
ify IL-8 (ratio to control cells = 1.2 ± 0.3 vs. 1.4 ± 0.1 for air) and
gene, was then excluded in a stepwise fashion until the most stable genes were
MCP-1 (ratio = 0.6 ± 0.1 vs. 0.8 ± 0.1 for air) production by A549 cells
after 14 h incubation. Although experimental post-incubation time
Cq, the quantiﬁcation cycle number at which the amount of ampliﬁed target
reaches a ﬁxed threshold was determined (default threshold
was reduced, the cellular response was similar to that previously
settings were used in all instances). The cycle number above which the Cq was
considered as a false positive (cycle cutoff point) was set up at 35. 2−
Results were expressed as arithmetic mean of 2−
three independent experiments after normalization on the most stable gene in ourexperiments, HPRT.
3.2.1. Sequential exposure to air (Air–Air)
To validate sequential exposure, we ﬁrst studied the cellu-
lar effects of repeated exposure to the same gas, air. The doubleexposure to air (protocol B: Air–Air) did not impact cell
Statistical analyses were performed using R software version 2.12.2
project.org). The difference between two conditions was considered using Student’s
viability and only slightly modiﬁed the inﬂammation parame-
t-test for cellular viability, cytokines production and cytokines expression. Results
ters. Indeed that cell viability (2A) and IL-8 hourly
C. Persoz et al. / Toxicology Letters 207 (2011) 53–59
Fig. 2. Cell viability (A) and IL-8 production per hour (B) of A549 cells after a single or repeated exposure to air (n = 4).
production (2B) of A549 cells after one or two exposures to air
on the inﬂammatory response caused by FA. This sequential expo-
were not modiﬁed by the number of exposures to air. Similarly
sure to FA and Asp (FA–Asp) did not alter cell viability (96.5 ± 3.2%)
IL-8 expression, evaluated by RT-qPCR, was not modiﬁed between
when compared to FA–Air exposure (96.6 ± 2.0%).
Cq = 2.41 ± 0.92 vs. 2.47 ± 0.76, respec-
The reaction of cells to sequential exposure to FA and Asp was
tively). Considering MCP-1, the production of this cytokine was not
moderate. Indeed FA–Asp induced an increase, although this did not
modiﬁed by sequential air-exposure, but its expression was sig-
reach signiﬁcance, of IL-8 production (p = 0.10) when we compared
Cq = 1.05 ± 0.13 vs. 1.65 ± 0.17, p < 0.05,
these results with those obtained with FA–Air A slight
respectively after one and two exposures).
trend, but not signiﬁcant, was also observed for the expression
These results indicate that cells reacted to one or two air expo-
of this cytokine Finally, production of MCP-1 tended to
decrease whilst its expression tended to increase (p = 0.16) (and D).
3.2.2. Sequential exposure to air and Aspergillus fumigatus(Air–Asp, grey)
In order to represent a complex environmental exposure, we
also exposed cells to a biological contaminant, spores of fungal
In industrialized countries, about 90% of our time is spent
species, Asp. Because of the limiting factors related to the use of
indoors where there are numerous pollutants, including chemical
such a pollutant and the duration of total incubation, cells were
and biological contaminants, to which we are chronically exposed
exposed to Asp as the second exposure pollutant, after an initial
The difﬁculties in representing environmental
exposure to air. In our experimental conditions, cell viability at
exposure in vitro are: (1) establishing the duration and repetition
the end of experiment was not altered (96.2 ± 3.7% vs. 92.4 ± 1.9%
of exposure, (2) assessing the variety of real exposure, associat-
for Air–Air). IL-8 production did not change (and a slight
ing chemical, biological and physical compounds
but not signiﬁcant trend was observed for IL-8 expression (p = 0.21)
and (3) selecting the chosen levels for tested pollutants.
compared to Air–Air exposure. As regards MCP-1 the pro-
In the present study, FA was chosen to typify chemical pol-
duction in supernatant was unchanged in comparison to Air–Air
lution: it is a major indoor air pollutant and targets mainly the
exposure and MCP-1 expression was not modiﬁed after
of FA used in our experiments (50 g/m3) was chosen according tolevels typically observed in the domestic environment (
3.2.3. Sequential exposure to FA and Air (FA–Air, grey)
A549 cells were next exposed, ﬁrst to FA at 50 g/m3 and
tamination, on the other hand, was represented using the species
then to Air. At the end of experiment, no impact on viability
Aspergillus fumigatus. This fungi is frequently found in the indoor
was observed when we compared Air–Air and FA–Air (92.4 ± 1.9%
and 96.6 ± 2.0%, respectively). Results concerning the inﬂamma-
known for its numerous respiratory health effects (
tory response showed a decrease of production of IL-8 (and
The concentration chosen for our study is typical of that found at the
MCP-1 (but both were non-signiﬁcant (p = 0.17; p = 0.30).
high end of concentrations in contaminated indoor environments
The expression of these two cytokines was not modiﬁed
The use of these pollutants has been previously reported,
Taking these results together, that is sequential exposure to air
although more often in submerged conditions (
with or without another pollutant (Air–Air, Air–Asp and FA–Air),
we conﬁrmed that a second exposure did not induce deleterious or
pared to airborne levels. Some authors have reported FA or Asp
speciﬁc effects on A549 cell behavior.
atmosphere generation for in vivo studies, but levels were oftenhigh (Some others
3.2.4. Sequential exposure to FA and Asp: (FA–Asp,
Finally, we tested the combination of exposures to FA and Asp, in
order to study the synergic effects of a microbiological contaminant
and 250 g/m3 respectively) that were relevant compared to
C. Persoz et al. / Toxicology Letters 207 (2011) 53–59
Fig. 3. Production (A) and expression (B) of IL-8 and MCP-1 (C and D) by A549 cells for each condition of the exposure experimental protocol. Productions are presented as a ratio of unexposed cells and expressions are normalized on HPRT gene (n = 4).
occupational exposures, but far above the concentrations found in
post-incubation, as previously described (In
domestic environment (mainly <100 g/m3)
addition, we reﬁned the study of inﬂammatory response to expo-
In this context, we paid very careful attention to the gener-
sures by assessing the expression of these cytokines at the end of
ation of realistic pollutant levels and to the checking of these levels
the exposure. Testing release as well as de novo synthesis helped
to describe the early inﬂammatory response of the cells more com-
Since environmental exposure is rarely a one-off event, we
repeatedly exposed cells to FA and Asp in our cellular model. To the
The ﬁrst issue in our experimental protocol was to make sure
best of our knowledge, this is the ﬁrst time that such an air–liquid
that the shortening of the ﬁrst post-incubation time, from 24 h
sequential exposure has been performed. In addition, we made
to 14 h, did not profoundly modify cellular response to gas expo-
sure that our protocol overcame certain problems inherent in the
sure. For viability as well as cytokine production, we obtained an
design. We wanted to sequentially expose A549 cells to the two
analogous response, and once again observed the increase of IL-8
pollutants during a 24 h cycle after the ﬁrst exposure, in order to
production after air exposure and equivalent levels between air and
maintain suitable conditions as previously deﬁned (seeding and
cellular monolayer growth) (The choice of expo-
The second issue concerned the feasibility of repetitions, since
sure sequence and duration of exposure to Asp was determined
cells exposed at the air–liquid interface undergo a certain degree
by kinetics of germination and hyphal development (
of stress due to the air ﬂow of exposure itself, this during even
a single exposure (Previous research into cell
tact between epithelial cells and spores until the early stages of
viability has shown that one exposure of 60 min induced cellular
germination and hyphal growth. Maintaining cell culture over 10 h
damages (contrarily to two exposures of 30 min
after spore exposure would however lead to uncontrolled fungal
with an in-between period of 14 h. Considering cytokines, a second
growth that could induce cellular damage and markedly bias the
exposure to air did not induce a supplementary increase of IL-8 pro-
interpretation of the observed results. Considering this constraint
duction or expression whereas MCP-1 production and expression,
FA exposure was performed before Asp.
not modiﬁed after the ﬁrst air exposure, were slightly increased
Our purpose was to set up an in vitro model to assess the
impact of repeated exposures to airborne pollutants at environ-
Results from the other exposure combination data led to the
mental levels on cell inﬂammation, and we therefore had to avoid
same conclusion: no cytotoxicity and no major stress. Indeed, when
any cytotoxic condition: whatever the conditions, no cytotoxic
repeated exposures were performed with other compounds in
effect was observed. The biomarkers selected to study the inﬂam-
addition to air, either FA or Asp, none induced cell toxicity. Although
mation response were two chemokines that play a major role in
cellular response was always modest and indeed should be con-
the inﬂammatory process of the respiratory tract, IL-8 and MCP-1,
ﬁrmed by further experiments, repeated exposure was feasible in
respectively neutrophil and monocyte chemoattractants
our experimental conditions, with cells keeping their metabolic
We assessed their production in apical supernatants during
C. Persoz et al. / Toxicology Letters 207 (2011) 53–59
The third and last issue dealt with the feasibility of cell exposure
Appendix A. Supplementary data
to fungal spores. To the best of our knowledge this is the ﬁrst timethat fungal contaminants have been used in air–liquid exposure
Supplementary data associated with this article can be found, in
experiments. Fungal conidia are usually in contact with respiratory
cells by suspension Interestingly, Asp spore exposure coupled with prior air exposurewas not cytotoxic, even after 10 h post-incubation and early hyphal
With regard to inﬂammation biomarkers, repeated exposure
Anderson, S.E., Jackson, L.G., Franko, J., Wells, J.R., 2010. Evaluation of dicarbonyls
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observed. It could be due to an overtaking in biomarkers’ time of
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expression. Our results differ from those reporting a major increase
Bakand, S., Winder, C., Khalil, C., Hayes, A., 2006. An experimental in vitro model for
dynamic direct exposure of human cells to airborne contaminants. Toxicol. Lett.
of IL-8 expression 8 h after exposure of A549 cells to Aspergillus
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This difference could be due to the experimental conditions, espe-
Bellanger, A.P., Million, L., Khoufache, K., Rivollet, D., Bièche, I., Laurendeau, I., Vidaud,
cially airborne vs. submerged exposure, as well as to the exposure
M., Botterel, F., Bretagne, S., 2009. Aspergillus fumigatus germ tube growth and
not conidia ingestion induces expression of inﬂammatory mediator genes in the
We have previously shown that FA had no effect on A549
human lung epithelial cell line A549. J. Med. Microbiol. 58, 174–179.
Berkova, N., Lair-Fulleringer, S., Féménia, F., Huet, D., Wagner, M.C., Gorna, K.,
cells with TNF␣ post-sensitization whereas with pre-sensitization
Tournier, F., Ibrahim-Granet, O., Guillot, J., Chermette, R., Boireau, P., Latgé,
of this cytokine, FA induced increased IL-8 production
J.P., 2006. Aspergillus fumigatus conidia inhibit tumour necrosis factor- or
The design we used in this study does in fact ﬁt in
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Bernstein, J.A., Alexis, N., Bacchus, H., Bernstein, I.L., Fritz, P., Horner, E., Li, N., Mason,
with post-sensitization with Asp in the role of activator. Taken
S., Nel, A., Oullette, J., Reijula, K., Reponen, T., Seltzer, J., Smith, A., Tarlo, S.M.,
together, these results indicate that the time sequence of sen-
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sitization is an important parameter for A549 response to FA
exposure. Finally, these results show that sequential exposure
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to different types of environmental pollutants can be modeled
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in vitro with concomitant assessment of deleterious effect on pul-
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monary cell viability and measurement of inﬂammatory cytokine
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of inhalation toxicity and disease. The report of a FRAME workshop. Altern. Lab.
Whilst our in vitro model can be used for preliminary assessment
of cellular activity modiﬁcation induced by these airborne contam-
Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R.,
Nolan, T., Pfafﬂ, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The
inants, it does however suffer from the classic limits of in vitro
MIQE guidelines: minimum information for publication of quantitative real-
studies. With regard to the A549 cell line we used to model res-
time PCR experiments. Clin. Chem. 55, 611–622.
piratory epithelial cells, the next step will be to assess the effects
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