Role of Transient Receptor Potential Ion Channels and

Evoked Levels of Neuropeptides in a FormaldehydeInduced Model of Asthma in Balb/c Mice

Yang Wu1,2, Huihui You1, Ping Ma1,3, Li Li1, Ye Yuan1, Jinquan Li1, Xin Ye1, Xudong Liu1, Hanchao Yao1,
Ruchong Chen2, Kefang Lai2*, Xu Yang1*
1 Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University,
Wuhan, China, 2 State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Disease, Guangzhou Medical University, Guangzhou, China, 3 College of
Basic Medical Sciences, Hubei University of Science and Technology, Xianning, China

Abstract
Objective: Asthma is a complex pulmonary inflammatory disease characterized by the hyper-responsiveness, remodeling
and inflammation of airways. Formaldehyde is a common indoor air pollutant that can cause asthma in people experiencing
long-term exposure. The irritant effect and adjuvant effect are the two possible pathways of formaldehyde promoted
asthma.
Methodology/Principal Findings: To explore the neural mechanisms and adjuvant effect of formaldehyde, 48 Balb/c mice
in six experimental groups were exposed to (a) vehicle control; (b) ovalbumin; (c) formaldehyde (3.0 mg/m3); (d)
ovalbumin+formaldehyde (3.0 mg/m3); (e) ovalbumin+formaldehyde (3.0 mg/m3)+HC-030031 (transient receptor potential
ankyrin 1 antagonist); (f) ovalbumin+formaldehyde (3.0 mg/m3)+ capsazepine (transient receptor potential vanilloid 1
antagonist). Experiments were conducted after 4 weeks of combined exposure and 1-week challenge with aerosolized
ovalbumin. Airway hyper-responsiveness, pulmonary tissue damage, eosinophil infiltration, and increased levels of
interleukin-4, interleukin-6, interleukin-1b, immunoglobulin E, substance P and calcitonin gene-related peptide in lung
tissues were found in the ovalbumin+formaldehyde (3.0 mg/m3) group compared with the values seen in ovalbumin -only
immunized mice. Except for interleukin-1b levels, other changes in the levels of biomarker could be inhibited by HC-030031
and capsazepine.
Conclusions/Significance: Formaldehyde might be a key risk factor for the rise in asthma cases. Transient receptor potential
ion channels and neuropeptides have important roles in formaldehyde promoted-asthma.
Citation: Wu Y, You H, Ma P, Li L, Yuan Y, et al. (2013) Role of Transient Receptor Potential Ion Channels and Evoked Levels of Neuropeptides in a FormaldehydeInduced Model of Asthma in Balb/c Mice. PLoS ONE 8(5): e62827. doi:10.1371/journal.pone.0062827
Editor: Pranela Rameshwar, University of Medicine and Dentistry of New Jersey, United States of America
Received December 21, 2012; Accepted March 26, 2013; Published May 9, 2013
Copyright: 2013 Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the National Natural Science Foundation of China (51136002 and 51076079), the Open Project of State Key Laboratory of
Respiratory Disease of China (2007DA780154F0906), and the China Key Technologies R&D Program (No. 2012BAJ02B03). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: yangxu@mail.ccnu.edu.cn (XY); klai@163.com (KL)

Immunological inflammation has a key role in the development

of asthma, but does not fully account for the complex inflammatory processes in the airways of asthmatics subjects. Authors have
stated that pro-inflammatory neuropeptides are also involved in
airway inflammation and AHR [6,7]. Pro-inflammatory neuropeptides such as tachykinin substance P (SP) and calcitonin generelated peptide (CGRP) can activate their specific receptors and
induce inflammatory cells in the airway to release inflammatory
mediators such as cytokines, oxygen radicals and histamine. These
mediators potentiate tissue injury, stimulate the further recruitment of leukocytes, produce and amplify inflammatory responses
in the airway, and participate in respiratory disease (including
chronic obstructive pulmonary disease (COPD) and asthma): this
is referred to as neurogenic inflammation [810].
The innervation of the airways is supplied by the autonomic
nervous system; the autonomic nerves contribute to the regulation

Introduction
Over recent decades, asthma has become an increasingly
prevalent disease. It now represents a serious public-health
problem worldwide, with an estimated 300 million people of all
ages affected (especially children) [1]. Asthma is a common
chronic disease of the airways characterized by enhanced airway
hyper-responsiveness (AHR), reversible airway remodeling and
chronic airway inflammation, which can lead to recurrent episodes
of wheezing, breathlessness, chest tightness and cough [2]. Asthma
is considered to be primarily an atopic disease [3]. At the cellular
level, allergens are internalized by antigen-presenting cells. CD4
TH2 cells are then activated, resulting in the release of TH2associated cytokines. This action leads to the synthesis of
immunoglobulin (Ig) E antibody. This is followed by the
degranulation of mast cells and infiltration of the airway mucosa
with eosinophils, which induces tissue remodeling and AHR [4,5].
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Adjuvant Effect on Asthma via TRP Ion Channels

airway inflammation via an irritant effect. Animal studies have

demonstrated that repeat exposure to FA can enhance the
response to antigens [4446], suggesting that FA acts as an
adjuvant to common allergens in animal experiments. However,
the mechanisms of such adjuvant effects are incompletely
understood.
Recent evidence suggests that TRPV1 and TRPA1 channels
can be activated by FA, which can lead to acute thermal
hyperalgesia and neuropathic pain [4749]. In the present study,
we used a model of asthma in mice based on FA exposure. We
evaluated hyper-responsiveness, inflammation and remodeling of
the airways using selective receptor antagonists to investigate the
role of TRP ion channels and neuropeptides in FA-promoted
asthma. We also investigated the mechanisms of adjuvant effects in
FA- promoted asthma.

of airway smooth muscle tone and the transport of fluid across the
bronchial epithelium [11].The largest portion of mammalian
airway-innervating sensory nerve fibers originates from vagal
ganglia, and a smaller number of airway sensory nerves originate
from dorsal root ganglia. The cell bodies of vagal sensory fibers are
located in the jugular and nodose ganglia with projections
peripherally to the airways and centrally to the solitary tract
nucleus in the brain stem [12].Some airway-specific neurons
within the vagal sensory ganglia have relatively larger cell body
diameters give rise to faster conducting myelinated A-fibers, while
others with small diameter cell bodies that give rise to
unmyelinated C-fibers [13]. The bronchial C-fibers are present
within the airway mucosa, and the pulmonary C-fibers are located
in the lung parenchyma. C-fibers can be activated by inflammatory mediators or exogenous chemical irritants, and releases
various neuropeptides, in particular SP and CGRP [14]. The
terminals of nerve fibers and the receptors for these neuropeptides
are localized in the vessel walls, bronchial smooth muscles, the
epithelial area and around mucus glands, so local stimulation of
sensory neurons projecting to these targets and the subsequent
neuropeptide release can lead to the features of inflammation such
as hyperemia, edema, mucus hypersecretion and contraction of
bronchial smooth muscle [15].
Neuropeptides have been described having a neuronal origin,
but there is increasing evidence that these peptides may be
synthesized and released from immune cells such as macrophages,
lymphocytes and monocytes [1619].Inflammatory cytokines may
increase the expression of neuropeptide genes in inflammatory
cells, so that inflammatory cell become a major source of the
neuropeptide at the inflammatory site [20].
Transient receptor potential (TRP) channels are a group of ion
channels located mostly on the plasma membrane of sensory nerve
cells and other cell types. They are distributed widely within the
respiratory tract. Here they act as a mechanistic link between
exposure to noxious irritants and inflammation to heightened
sensitivity to airway reflexes, pathological remodeling and airflow
limitation, as well as being associated with allergic asthma and
irritant-induced asthma [21]. TRP channels can be opened by a
wide range of exogenous chemical-irritant stimuli to elicit acute
pain and neurogenic inflammation through the peripheral release
of neuropeptides [22,23]. The two important pro-inflammatory
TRP ion channels are TRPV1 (capsaicin receptor) and TRPA1
(activated by mustard oil and garlic) [2426] because almost all
major neuronal inflammatory pathways converge on TRPV1 and
TRPA1 to increase the excitability of C-fibers during airway
inflammation [27], and they are also present on non-neuronal cells
[21,28].
Ascertaining the risk factors for the increase in the prevalence of
asthma is important. Formaldehyde (FA) is used in many products,
including resins, plywood, tobacco, particle board and tissue
fixatives [29,30]. Because of its wide use and varied sources,
individuals are exposed to gaseous FA in occupational and
domestic environments. Acute and chronic exposure to FA has
been associated with various toxic effects, including irritation,
oxidative stress, nasopharyngeal cancer and respiratory disorders
[3133]. Several epidemiological studies have demonstrated that
FA exposure can induce or exacerbate asthma [3437]. Unlike
typical allergens, FA-specific IgE is rarely detected [38,39], which
suggests that FA may be involved in asthma via a non-IgEmediated mechanism. Two possible pathways for FA-induced
asthma have been proposed: the irritant effect and the adjuvant
effect [40]. Some animal studies have shown that FA alone can up
regulate the levels of IgE and interleukin (IL)-4 and increase the
eosinophil count [4143], suggesting that FA alone may cause
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Materials and Methods

All experimental procedures were approved by the Office of
Scientific Research Management of Huazhong Normal University
(Huazhong, China) with a certification of Application for the Use
of Animals dated 8 November 2011 (approval ID: CCNU-SKY2011-008).

Reagents and Kits

The TRPA1 antagonist HC-030031 (CAS 349085-38-7, 2-(1, 3Dimethyl-2, 6-dioxo-1, 2, 3, 6-tetrahydro-7H-purin-7-yl)-N-(4isopropylphenyl) acetamide) was purchased from Toronto Research Chemicals (Toronto, Canada). The TRPV1 antagonist
capsazepine (CPZ, CAS 138977-28-3,N-[2-(4-Chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2carbothioamide) was purchased from Tocris Bioscience (Bristol,
UK). Formalin solution (10%), methacholine (MCH), gelatinous
Al (OH)3, and pentobarbital sodium were purchased from Sigma
Aldrich (St. Louis, MO, USA). Methylcellulose, ovalbumin (OVA)
and bovine serum albumin (BSA) were obtained from Merck
(Darmstadt, Germany). Mouse enzyme-linked immunosorbent
assay (ELISA) kits for total IgE and OVA-IgE were purchased
from Biolegend (San Diego, CA, USA). A mouse EILSA kit for
OVA-lgG1 was purchased from Chondrex (Redmond, WA, USA).
Mouse EILSA kits for IL-4, interferon (IFN) -c and IL-1b were
purchased from Raybiotech (Norcross, GA, USA). Mouse ELISA
kits for SP and CGRP were purchased from R&D Systems
(Minneapolis, MN, USA). Rabbit anti-SP-antibody, rabbit antiCGRP- antibody, goat-anti-rabbit lgG-antibody, a rabbit lgG
peroxidase conjugated streptavidin-biotin complex (SABC-POD)
kit and a diaminobenzidine (DAB) kit were obtained from Boster
Bio-engineering (Wuhan, China).

Animals
Male Balb/c mice (56 weeks; 2261.5 g) were purchased from
the Hubei Province Experimental Animal Center (Wuhan, China)
and housed in standard environmental conditions (12-h lightdark
cycle, 5070% humidity and 2025uC). Food and water were
provided ad libitum. Mice were quarantined for $7 days before
study initiation. Eight mice in each group were used so as to
minimize the number of experimental animals needed while
ensuring the validity of statistical power.

Preparation of Gaseous FA
Gaseous FA was prepared from 10% formalin. The solution was
administered to mice through a vapor generator and automated
mouthnose only inhalation exposure chamber HOPE-MED
8052 (Hope-Med, Tianjin, China) capable of emitting gaseous
2

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Adjuvant Effect on Asthma via TRP Ion Channels

FA at the concentrations demanded. Environmental parameters

were monitored and controlled automatically by the system. Air
temperature, relative humidity and airflow rate were maintained
at 2262uC, 5065% and 1.6560.15 m3/h, respectively. During
exposure, FA concentrations were monitored every 2 h using a
Gaseous FA Analyzer (4160-2, Interscan, Simi Valley, CA, USA).
The level of gaseous FA adopted was 3.0 mg/m3 in accordance
with the occupational threshold limit in China [50].

samples were centrifuged at 1006g for 10 min at 4uC. The cell

pellets from BALF were suspended in saline for the cell counts.
Total cells, eosinophils, lymphocytes and neutrophils in cell
suspensions were counted using a Blood Cell Analysis system
(MTN-21, Motenu, Changchun, China).

Quantitative Analyses of IgE in Serum

Twenty-four hours after the final challenge, blood was taken
from the hearts of the mice. Then, serum samples were collected
after centrifugation of the blood (30006g, 15 min, 4uC) and stored
at 70uC until analyses. The serum levels of total IgE (T-lg E),
OVA-IgE and OVA- IgG1 were measured using an ELISA kit
according to manufacturer protocols. Concentrations were determined in duplicate for each sample. The sensitivities of the ELISA
kits were 0.1 ng/mL for T-lgE, 20.7 pg/mL for OVA-lgE and
0.4 ng/mL for OVA-lgG1. The coefficient of variation (CV) of
ELISA kits was ,8% (intra-assay) and ,11% (inter-assay) for TlgE, ,4% (intra-assay) and ,5% (inter-assay) for OVA-lgE,
,10% (intra-assay) and,4% (inter-assay) for OVA-lgG1.

Experimental Protocol
Mice were divided randomly into six experimental groups of 8
animals. They were exposed to or not exposed to gaseous FA for 4
weeks (days 1 to 28) at 6 h/day and 5 days/week in the inhalation
exposure chamber. They were sensitized with OVA+Al(OH)3
(80 mg OVA and 1.3 mg gelatinous Al (OH)3 in 200 mL saline
each time or saline (200 mL) saline each time) by subcutaneous
injection on days 10, 18 and 25 followed by aerosol challenge in
1% OVA (30 min/day) or saline from days 29 to 35 using an
Ultrasonic Nebulizer (402AI, Yuyue, Beijing, China). For therapeutic intervention with a TRP channel antagonist, mice were preadministered (i.p.) with 150 mL of 8 mg/kg HC-030031 (suspended in 0.5% methylcellulose) or 1.6 mg/kg CPZ (TRPV1 dissolved
in 2.5% EtOH) or vehicle 30 min before FA exposure starting
each day from days 1 to 28.
Dedicated groups of mice (for each of the regimens described
above) were employed to prevent inaccuracies upon measurements
of lung cytokines/pulmonary architecture due to the influence of
the MCH used in AHR assessments. One set of 48 mice was
treated for 35 days according to the regimen(s) stated above and
then used for AHR tests and collection of bronchoalveolar lavage
fluid (BALF) samples. A second set of 48 mice was treated as
described above and then used directly for ELISA, histopathologic
and immunohistochemical analyses. The detailed protocol of this
study is shown in Fig. 1.

Quantitative Analysis of Cytokines and Neuropeptides in

Lung Tissue
After serum sample collection, right lungs tissue were rinsed in
ice-cold phosphate-buffered saline (PBS: pH 7.5), then dried and
weighed. Afterwards, they were homogenized using a glass
homogenizer on ice, with 10 mL/g of ice-cold PBS. Next,
homogenates was centrifuged at 23006g, for 10 min at 4uC, and
supernatants collected for detection using ELISA. The tissue levels
of IL-4, IFN-c, IL-1b, SP and CGRP were measured using ELISA
kits according to manufacturer instructions. Concentrations were
determined in duplicate for each sample. The sensitivities of
ELISA kits were 0.6 pg/mL for IL-4, 5 pg/mL for IFN-c and IL1b, 31.5 pg/mL for SP and 3.9 pg/mL for CGRP. The CV of
ELISA kits were ,10% (intra-assay) and ,12% (inter-assay) for
IL-4, IL-1b and IFN-c, ,9% (intra-assay) and,13% (inter-assay)
for SP, ,8% (intra-assay) and ,10% (inter-assay) for CGRP c.

Measurement of AHR
AHR as an indicator of lung function was tested 24 h after the
final exposure to aerosol using an AniRes 2005 Lung Function
system (Bestlab 2.0, Beijing, China) according to manufacturer
instructions. Anesthesia was induced by intraperitoneal injection of
95 mg/kg pentobarbital sodium. A connection was made by a
computer-controlled ventilator via a cannula that had been
implanted surgically in the trachea. The respiratory rate and the
time ratio of expiration/inspiration were pre-set at 90/min and
1.5:1, respectively. An injector needle was inserted into the jugular
vein through which 0.025, 0.05, 0.1 and 0.2 mg/kg body weight
MCH was injected successively at 5-min intervals. After each
injection, airway resistance and lung compliance (Cdyn) in
response to increasing concentrations of MCH were recorded by
the system. Airway resistance is defined as the pressure driving
respiration divided by flow. Lung compliance refers to the
distensibility of the lung and is defined as the alteration in volume
of the lung produced by a change in pressure across the lung [51].
Airway responsiveness was assessed by indexes of expiratory
resistance (Re), inspiratory resistance (Ri), and the peak value of
Cdyn. The relative area (R-area, RL) was defined as the area
under the peak curve of Re or Ri and beyond the baseline level.

Lung Histological Assay

Left lungs from non-lavaged mice were isolated for preparation
of histopathology slices. Lung lobes were incubated in fixative
(saturated 2, 4, 6-trinitrophenol: formalin: glacial acetic acid
[15:5:1 v/v/v]) for 24 h at room temperature, then cut into pieces.
Slides of lung tissues were stained using hematoxylin and eosin
(H&E) [52], periodic acid-Schiff (PAS) [53], and Massons
trichrome (MT) [52] methods to examine the magnitude of
airway inflammation, mucus production, and peribronchial
collagen deposition according to recommended standard protocols. Stained pieces were embedded in paraffin, sectioned into 10mm slices and observed using a DM 4000B microscope (Lecia,
Berlin, Germany). Tissue sections were examined qualitatively by
two experienced pathologists in a blinded fashion.

Immunohistochemical Assay
Sections of lung tissues were quenched of endogenous peroxides
with 3% H2O2. They were boiled in sodium citrate (0.01 mol/L,
pH 6.0) for antigen retrieval to unmask antigen epitopes,
permeabilized with 0.2% Triton X-100 for 10 min, and blocked
with 5% BSA in PBSfor 30 min at room temperature. Sections
were incubated with diluted primary antibodies (rabbit anti-SP
antibody or rabbit anti-CGRP antibody, 1:200 dilution) overnight
at 4uC. Slides were washed with PBS, incubated with secondary
antibody (goat anti-rabbit IgG; 1:200 dilutions) for 30 min at 37uC
and detected with a rabbit IgG peroxidase conjugated streptavi-

Differential Cell Counts in BALF

BALF was collected immediately after the conclusion of AHR
measurements. The lungs of each mouse were lavaged in situ with
three successive 1-mL volumes of saline instilled by syringe. After
gentle pressing of the chest several times, BALF was collected and
combined. The recovery ratios of different mice were <90%. All
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Adjuvant Effect on Asthma via TRP Ion Channels

Figure 1. Study protocol. (A) Exposure, immunization and antagonist schedule. Groups: (a) vehicle control, (b) OVA, (c) FA, (d) FA+OVA, (e)
FA+OVA+HC-030031 and (f) FA+OVA+CPZ; (B) experimental design.
doi:10.1371/journal.pone.0062827.g001

din-biotin complex (SABC-POD) kit, followed by incubation with

a diaminobenzidine (DAB) kit. Immunostained sections were
viewed under a DM 4000B Microscope (Leica). The intensity of
protein SP or CGRP staining was determined as average optical
density using Image-Pro Plus 6.0 software (Media Cybernetics,
Bethesda, MD, USA). A non-stained region was selected and set as
the background. All tissue sections were examined qualitatively by
two experienced pathologists in a blinded fashion.

Effect of TRP Channel Antagonists on Leukocyte Levels in

BALF
Levels of inflammatory cells (eosinophils, total cells, lymphocytes, neutrophils) in BALF were elevated significantly in OVAchallenged mice compared with control mice except for neutrophils (total cells: p,0.01 eosinophils: p,0.05 lymphocytes:
p,0.05) (Fig. 3). FA exposure also significantly increased the
eosinophil count and total cells compared with vehicle controls
(p,0.05). OVA immunization significantly intensified the increased eosinophil infiltration induced by FA (p,0.01). Compared
with the control group, FA+OVA+HC-030031 and FA+OVA+CPZ treated groups have higher count of total cells
(p,0.01) and lymphocytes (p,0.05), and FA+OVA+CPZ treated
groups had a higher count of neutrophils (p,0.05). Treatment
with CPZ and HC-030031 significantly reduced the number of
eosinophils compared with the FA+OVA group (p,0.01).

Statistical Analyses
All data are the mean 6 standard error of the mean (except the
data for substance P and calcitonin gene-related peptide measured
by EILSA which are the mean 6 standard deviation of the mean).
Statistical graphs were generated using Origin 8.0 Software
(OriginLab, Berkeley, CA, USA). One-way ANOVA combined
with Fishers protected t-test was used to determine the significance
of differences between groups. p,0.05 was considered significant.
Data analyses were carried out using SPSS ver13 (SPSS, Chicago,
IL, USA).

Effect of TRP Channel Antagonists on Serum Levels of IgE

Figure 4A shows that, compared with the vehicle control
group: OVA-sensitized groups enhanced T-IgE (p,0.01); FA-only
exposure promoted the level of T-IgE; and the OVA+FA group
caused a significant increase in serum T-IgE (p,0.01). Treatment
with CPZ and HC-030031 significantly reduced these levels
compared with the FA+OVA group (p,0.01).
From the results of the analyses of OVA-IgE and OVA-IgG1 in
Figs. 4B and 4C, respectively, exposure to FA only did not cause
changes in the serum levels of OVA-IgE and OVA-IgG1
(p.0.05). OVA-only groups demonstrated significantly of increased levels OVA-IgE and OVA-IgG1 in relation to vehicle
controls (p,0.01). Levels of OVA-IgE and OVA-IgG1 were
significantly inhibited by the administration of TRP channel
antagonists (p,0.01).

Results
Effect of TRP Channel Antagonists on AHR
Figure 2 shows the results of measurements of airway
responsiveness. The R-area of the respiratory resistance (RL,
including Re and Ri) and peak values of Cdyn have been adopted
as sensitive indices. Three parameters of lung function (Ri, Re and
Cdyn) were recorded after each injection of MCH (0.025, 0.05, 0.1
and 0.2 mg/kg). In all groups, the expiratory and inspiratory
resistance increased with increasing MCH levels (p,0.01),
whereas Cdyn decreased (p,0.01). OVA-sensitized groups exhibited a greater airway response to MCH compared with the vehicle
control group (p,0.01) and FA exposure significantly enhanced
the degree of airway reactivity in OVA-sensitized mice (p,0.01).
Treatment with the TRPA1 antagonist HC-030031 and TRPV1
antagonist CPZ dramatically reduced Ri, Re and restored Cdyn in
FA+OVA-treated mice in response to MCH, respectively
(p,0.01).

Effect of TRP Channel Antagonists on Cytokines Levels

The levels of the Th2 cytokine IL-4 and the Th1 cytokine IFN-c
were assessed in samples of lung tissue. The FA exposure group
and OVA-sensitized group induced a significant increase in IL-4
levels (p,0.01). Among all groups, exposure to FA+OVA group
induced the highest IL-4 levels (p,0.01). Treatment with CPZ

Figure 2. Airway hyperresponsiveness (AHR) measurements. With different doses of MCH: (A) R-area of Re. (B) R-area of Ri; (C) the peak value
of Cdyn. Animal groups (in all panels): n = 3 mice per group.*: p,0.05, **: p,0.01, compared with vehicle control; #: p,0.05, ##: p,0.01, compared
with OVA-immunized group; +: p,0.05, ++: p,0.01, compared with FA+OVA group.
doi:10.1371/journal.pone.0062827.g002

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Figure 3. Leukocyte infiltration in the airways in different experimental groups. (A) eosinophil counts in BALF. (B) Total cell counts in BALF.
(C) Lymphocyte counts in BALF. (D) Neutrophil counts in BALF. Animal groups(in all panels): veh:n = 7,OVA:n = 6,FA:n = 7,FA+OVA:n = 6,FA+OVA+HC030031:n = 6,FA+OVA+CPZ:n = 6.*: p,0.05, **: p,0.01, compared with vehicle control; ##: p,0.01, compared with OVA-immunized group. ++:
p,0.01, compared with FA+OVA group.
doi:10.1371/journal.pone.0062827.g003

Immunohistochemical analyses were conducted to detect the

expression of SP and CGRP in lung tissue. SP and CGRP were
expressed mainly in the epithelial layers around the bronchioles
(Figs. 7A and 7B). Expression of these immunoreactive proteins
was up-regulated in the OVA-treated group and FA-treated group
compared with the control groups. Exposure to FA+OVA induced
the highest levels. However, when mice were treated with HC030031 or CPZ, expression of SP and CGRP in lung tissues was
restored.
The average optical density was also measured. We noted in
Figs. 7C and 7D that the OVA group presented an increase in
the content of SP and CGRP, respectively, compared with the
control group(p,0.01; p,0.05); and FA exposure significantly
enhanced the level of SP and CGRP in lung tissue(p,0.01). FA
exposure significantly enhanced the levels of SP and CGRP in
OVA-sensitized mice (p,0.05; p,0.01), HC-030031 and CPZ
reduced such content when compared with the OVA group
(p,0.05).

and HC-030031 significantly reduced IL-4 levels compared with

FA+OVA groups (p,0.01) (Fig. 5A). However, IFN-c levels were
not significantly different among all groups (Fig. 5B).
Exposure to FA and OVA enhanced the levels of the proinflammatory cytokine IL-1b in lung tissue (p,0.01) (Fig. 5C).
Exposure to the FA+OVA group induced the highest IL-4 levels
(p,0.01). Treatment with CPZ or HC-030031 did not reduce
these levels compared with FA+OVA groups (p.0.05).

Effect of TRP Channel Antagonists on Neuropeptide

Release in the Airways
FA exposure and OVA-sensitized groups enhanced the levels of
SP and CGRP in the lungs (p,0.05) (Fig. 6). Exposure to
FA+OVA induced the highest levels of release (p,0.01).
Treatment with CPZ and HC-030031 significantly reduced the
levels of SP (HC-030031 group, F = 20.67, p,0.01; CPZ group,
F = 13.66, p,0.01) or CGRP (HC-030031 group, F = 35.45,
p,0.01; CPZ group, F = 17.11, p,0.01) compared with
FA+OVA groups.

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Adjuvant Effect on Asthma via TRP Ion Channels

Figure 4 Serum IgE level. (A) Total IgE.Animal groups: veh: n = 8,OVA:n = 7,FA:n = 7,FA+OVA:n = 7,FA+OVA+HC- 030031:n = 7,FA+OVA+CPZ:n = 7.(B)
OVA-IgE.Animal groups: veh: n = 7,OVA:n = 8,FA:n = 7,FA+OVA:n = 7,FA +OVA+HC-030031:n = 7,FA+OVA+CPZ:n = 7.(C) OVA-IgG1.Animal groups:
veh:n = 8,OVA:n = 7,FA:n = 7,FA+. OVA:n = 7.FA+OVA+HC-030031:n = 7,FA+OVA+CPZ:n = 7.**: p,0.01, compared with vehicle control; ##: p,0.01,
compared with OVA-immunized group. ++: p,0.01, compared with FA+OVA group.
doi:10.1371/journal.pone.0062827.g004

exposure in allergen-induced inflammation, pulmonary tissue

damage and AHR. We also revealed that SP and CGRP
participate in the development of FA-promoted asthma or
asthma-like symptoms.
The expression of asthma is an intricate process dependent
upon the interaction between two major factors: environmental
exposure and genetic factors [54]. The present study showed that
pre-administration of low levels of HC-030031 and CPZ during
FA exposure and an OVA-sensitized phase was sufficient to
potently suppress: AHR; leukocyte infiltration; production of Th2
cytokines and IgE; neuropeptide release; pulmonary physiological
changes. These findings suggest that TRPA1 and TRPV1 have an
important role in FA-promoted asthma.
To investigate the effects of FA exposure on the structure and
function of bronchial airways, we used a murine model of asthma
based on a well-established OVA sensitization/challenge protocol
[43,46]. In the present study, to simulate environmental exposure
in the workplace, exposure for 5 days/week and 6 h/day was
adopted, and 4 weeks of a low dose of FA was administered rather
than immediately high-dose exposures before allergen challenge.

Effect of TRP Channel Antagonists on Airway Remodeling

The increase in airway responsiveness and inflammation in
mice was associated with histopathological changes in the
bronchiolar airways. The typical pathological features of airway
inflammation and structural alterations in asthmatic were
observed: infiltration of inflammatory cells in the peribronchial
and perivascular areas (H&E staining) (Fig. 8A). Mucus overproduction and hyperplasia of goblet cells (PAS staining) (Fig. 8B)
and peribronchial deposition of collagen (MT staining) (Fig. 8C)
were observed in OVA-challenged mice. Slight changes were
observed in the FA group compared with the vehicle control
group. The most severe changes in airway structure were in the
FA+OVA group. Treatment with CPZ and HC-030031 markedly
reduced the degree of infiltration of inflammatory cells, mucus
overproduction, goblet-cell hyperplasia, peribronchial deposition
of collagen and fibrosis.

Discussion
Here we reported that the TRPA1 channel antagonist HC030031 and TRPV1 channel antagonist CPZ reduced FA
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Adjuvant Effect on Asthma via TRP Ion Channels

Figure 5. Cytokine levels in lung tissue. (A) IL-4 levels. Animal groups: veh: n = 6,OVA:n = 7,FA:n = 8, FA+OVA:n = 7, FA+OVA+HC030031:n = 6,FA+OVA+CPZ:n = 6. (B) IFN-c levels. Animal groups: veh: n = 8,OVA:n = 8,FA:n = 8; FA+OVA:n = 7, FA+OVA+HC-030031:n = 6,FA+OVA+CPZ:n = 6. (C) IL-1b levels. Animal groups:veh: n = 7,OVA: n = 7;FA:n = 8;FA+OVA:n = 8,FA+OVA+HC-030031:n = 6,FA+OVA+CPZ:n = 6. **: p,0.01,
compared with vehicle control; ##: p,0.01, compared with OVA-immunized group. ++: p,0.01, compared with FA+OVA group.
doi:10.1371/journal.pone.0062827.g005

For smaller rodent species (e.g., mouse), using nose-only modes in

inhalation studies is considered superior to whole-body exposure.
This is because the nose-only route can reduce dosing from nonrespiratory routes (including wall loss as well as losses on the skin
and fur of animals), which cause the actual concentration to be
substantially less than the nominal concentration. Most applications of selective receptor antagonists are by direct injections,
particularly peritoneal injection, which is convenient to carry out.
The injection dose was decided according to the results in our
preliminary experiments before the formal ones.
Hyper-responsiveness and remodeling of the airways are the
major pathophysiological features of asthma, which lead to airway
narrowing and airflow obstruction. The degree to which AHR can
be defined by contractile responses to challenges with MCH
correlates with asthma severity. MCH as a pharmacological agent
can cause increased Ri/Re and decreased Cdyn. The R-areas of
respiratory resistances (Ri and Re) explain the variation in the
large airways, whereas the change in Cdyn represents the state of
the small airways or the parenchyma [51]. Airway remodeling is
difficult to define because it involves several structural changes.
These include thickening of the sub-basement membrane, hyperPLOS ONE | www.plosone.org

secretion of mucus, subepithelial fibrosis, infiltration of inflammatory cells, and deposition of the extracellular matrix in the
subepithelial layer. In the present study, we used three types of
staining methods to reveal changes in pulmonary histology. H&E
staining is a classical and typical staining research method for
airway remodeling. It can be used to examine the magnitude of
airway inflammation, but staining of collagen and goblet cells of
the lung sections cannot be done based on H&E staining. Hence,
we used PAS methods to examine mucus secretion and goblet
cells, and using MT methods to examine peribronchial collagen
deposition to describe airway remodeling.
The FA exposure group showed increased AHR compared with
the control group and caused slight, but statistically significant,
histological changes in the airways of mice. However, these
changes were not obviously different compared with those seen in
the OVA group. However, upon co-exposure with OVA, AHR
and airway remodeling was exacerbated.
The main pathological feature of asthma is allergic airway
inflammation, and an imbalance of Th1/Th2 immune responses is
a pathological basis of asthma and other atopic diseases [55]. IFNc represents the Th1 immune response and is associated with
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Adjuvant Effect on Asthma via TRP Ion Channels

Figure 6. Neuropeptide levels in lung tissue. (A) Substance P. Animal groups: veh: n = 6,OVA:n = 6, FA+OVA:n = 6, FA+OVA:n = 6, FA+OVA+HC030031:n = 6,FA+OVA+CPZ:n = 6. (B) CGRP. Animal groups: veh: n = 7,OVA:n = 8, FA+OVA:n = 7, FA+OVA:n = 8, FA+OVA+HC-030031:n = 7,FA+OVA+CPZ:n = 7.*: p,0.05, **: p,0.01, compared with vehicle control; ##: p,0.01, compared with OVA-immunized group. ++: p,0.01, compared with
FA+OVA group.
doi:10.1371/journal.pone.0062827.g006

protective immunity; IL-4 represents the Th2 immune response,

drives the type-2 pathway and upregulates IgE production to fight
extracellular organisms. In the present study, IFN-c levels were
not significantly different among the experimental groups. Levels

of IL-4 and T-lgE were markedly increased in OVA-immunized

mice exposed to FA. We observed up-regulated expression of IgE
and IL-4 in FA-alone groups; this finding may reflect acceleration

Figure 7. Immunohistochemical analyses. Representative images of the expression of (A) Substance P and (B) CGRP as determined by
immunohistochemical staining (brown color stain) (red arrow). Panel: (a) negative control group, (b) OVA group, (c) FA group, (d) FA+OVA group, (e)
FA+OVA+HC-030031 group, and (f) FA+OVA+CPZ group; Magnification =620. Analyses of (C) Substance P and (D) CGRP expression levels according
to average optical density. Animal groups (in all panels): n = 4 mice per group.*: p,0.05, **: p,0.01, compared with vehicle control; #: p,0.05, ##:
p,0.01, compared with OVA-immunized group. +: p,0.05, ++: p,0.01, compared with FA+OVA group.
doi:10.1371/journal.pone.0062827.g007

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Adjuvant Effect on Asthma via TRP Ion Channels

Figure 8. Representative histological images of airway remodeling of lung tissue. (A) H&E staining: showing infiltration of inflammatory
cells (red arrow) and hypertrophy of structural cells (blue arrow). N: normal conditions; S: slight changes; M: moderate changes; V: severe changes. (B)
Periodic acid-Schiff (PAS) staining: showing mucous cells (pale pink color stain) (red arrow). (C) Massons trichrome (MT) staining showing
subepithelial collagen deposition (blue color stain) (red arrow). Panel: (a) vehicle control group, (b) OVA group, (c) FA group, (d) FA+OVA group, (e)
FA+OVA+HC-030031 group and (f) FA+OVA+CPZ group. Magnification =620.Animal groups (in all panels): n = 4 mice per group.
doi:10.1371/journal.pone.0062827.g008

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Adjuvant Effect on Asthma via TRP Ion Channels

of the imbalance of Th1/Th2 cytokines and contribute to the

cause and evolution of asthma.
Increased numbers of eosinophils in peripheral blood and in
airway secretions are a characteristic feature of asthma [56] and
correlate with a greater severity of asthma. Eosinophils then pass
from the circulation via the microvascular compartment into the
airway wall. Data from the present study showed that the number
of eosinophils was markedly increased in OVA-immunized mice
exposed to FA. Hence, the eosinophil count is a major biomarker
for FA-promoted asthma.
Pro-inflammatory cytokines activate the immune system and
participate in the inflammatory response. IL-1b is an important
pro-inflammatory cytokine released by innate immune cells during
inflammation. IL-1b is increased in the bronchial epithelium and
macrophages of patients with asthma [57], and IL-1b is required
for OVA-specific IgE production and induction of eosinophilic
airway inflammation [58]. The present study showed that the coexistence of allergen (OVA) and FA can increase the highest level
of IL-1b. Hence, IL-1b may have a key role in FA-promoted
asthma. Our data also showed IL-1b levels were not inhibited by
HC-030031 or CPZ, thereby suggesting that IL-1b expression is
not mediated by TRP channels.
We confirmed our previous findings that an adjuvant effect
contributes to the development of FA-induced asthma or asthmalike symptoms [43,46]. Additionally, a significant difference in the
levels of OVA-IgE and OVA-IgG1 was found between the
OVA+FA and OVA-alone group.
The results of ELISA and immunohistochemical analyses
suggested that the OVA-treated group modulated the release of
SP and CGRP; these results are consistent with those in other
animal studies [59,60].These data may suggest that allergen
exposure stimulates immune cells to produce neuropeptides.
Neuropeptides can activate the immune system, influence the
activity of immune cells and stimulates: degranulation of mast cells
and release histamine and 5-HT; T lymphocyte and macrophages
to release pro-inflammatory cytokine, macrophages and eosinophils to produce and secrete neuropeptides [61]. Thus, our data in
sensitized animals suggest that immune interactions could be
mediated by neuropeptides. Immunity is coordinated by neural
circuits that operate reflexively, exogenous molecular products

interact with receptors that are expressed by cells of the innate

immune system, activate innate immune responses and induce the
secretion of pro-inflammatory cytokines(e.g. IL-1b).This in turn
activates the efferent arc, which is known as the cholinergic antiinflammatory pathway [62]. Release of SP and CGRP led to
vasodilation and plasma extravasation as well as bronchoconstriction and mucus production, respectively. Hence, neuropeptide
release and neurogenic inflammation may have a role in the
development of asthmatic symptoms and amplify the immune
response during allergic inflammation [63].
The present study showed that the level of SP and CGRP was
markedly increased in OVA-immunized mice exposed to FA, and
that this pathway could be blocked by TRPA1 and TRPV1
channel antagonists, These findings suggested that FA inhalation
enhances neuropeptide levels in a TRP ion channel-dependent
way. TRPV1 is expressed not only in neurons but also in mast cells
[64,65], but further more studies needed to identify the cell
origin.Evidence shows that TRPA1 channels identified in nonneuron cells including lymphocytes [66], and genetic deletion of
TRPA1 drastically attenuates inflammation and hyper-reactivity
in a mouse model of asthma [67]. TRP channels are involved in
different immune functions, which are relevant to asthma
pathophysiology, thereby, suggesting a role for TRP proteins.
In conclusion, the present study suggested that FA enhances the
sensitization of Balb/c mice to inhaled allergens and that it might
be an underlying risk factor for an increase in asthma severity;
Further, TRPV1, TRPA1 and neuropeptides play a key part in the
adjuvant effect of FA-promoted asthma or asthma-like symptoms.

Acknowledgments
We are grateful to Professors Yinping Zhang and Jan Sundell of Tsinghua
University, China, for their excellent suggestions and comments on this
work. We gratefully acknowledge Liwen Bianji for editing the article.

Author Contributions
Conceived and designed the experiments: X. Yang KL YW. Performed the
experiments: YW H. You PM LL YY JL X. Ye XL RC. Analyzed the data:
X. Yang YW. Contributed reagents/materials/analysis tools: X. Yang H.
Yao. Wrote the paper: YW X. Yang.

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