A sensory neuronal ion channel essential for airway
inflammation and hyperreactivity in asthma
Ana I. Caceresa, Marian Brackmanna, Maxwell D. Eliaa, Bret F. Bessaca, Donato del Caminob, Marc D’Amoursb,
JoAnn S. Witekb, Chistopher M. Fangerb, Jayhong A. Chongb, Neil J. Haywardb, Robert J. Homerc, Lauren Cohnd,
Xiaozhu Huange, Magdalene M. Moranb,1, and Sven-Eric Jordta,1
aDepartment of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520;bHydra Biosciences, Inc., 790 Memorial Drive,
Cambridge, MA 02139;cDepartment of Pathology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520;dSection of Pulmonary and
Critical Care Medicine, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520; andeLung Biology Center, University of California, Box
2922, San Francisco, CA 94143-2922
Edited by Lily Y. Jan, University of California, San Francisco, CA, and approved April 22, 2009 (received for review January 21, 2009)
allergens and chemical irritants. Studies focusing on immune,
smooth muscle, and airway epithelial function revealed many
aspects of the disease mechanism of asthma. However, the limited
efficacies of immune-directed therapies suggest the involvement
of additional mechanisms in asthmatic airway inflammation.
TRPA1 is an irritant-sensing ion channel expressed in airway
chemosensory nerves. TRPA1-activating stimuli such as cigarette
smoke, chlorine, aldehydes, and scents are among the most prev-
alent triggers of asthma. Endogenous TRPA1 agonists, including
drivers of allergen-induced airway inflammation in asthma. Here,
we examined the role of TRPA1 in allergic asthma in the murine
ovalbumin model. Strikingly, genetic ablation of TRPA1 inhibited
allergen-induced leukocyte infiltration in the airways, reduced
cytokine and mucus production, and almost completely abolished
airway hyperreactivity to contractile stimuli. This phenotype is
recapitulated by treatment of wild-type mice with HC-030031, a
TRPA1 antagonist. HC-030031, when administered during airway
allergen challenge, inhibited eosinophil infiltration and prevented
the development of airway hyperreactivity. Trpa1?/?mice dis-
played deficiencies in chemically and allergen-induced neuropep-
tide release in the airways, providing a potential explanation for
the impaired inflammatory response. Our data suggest that TRPA1
is a key integrator of interactions between the immune and
nervous systems in the airways, driving asthmatic airway inflam-
mation following inhaled allergen challenge. TRPA1 may represent
a promising pharmacological target for the treatment of asthma
and other allergic inflammatory conditions.
airway hyperreactivity ? TRP channel ? TRPA1
United States and world-wide (1, 2). The inflammatory response
in asthma is orchestrated by CD4 Th2 cells inducing eosinophil
infiltration and mast cell activation, followed by tissue remod-
eling, mucus hypersecretion, and airway hyperresponsiveness
(3). While it is clear that immune mechanisms play a significant
role in the development and maintenance of asthma, the limited
efficacy of immune therapies suggests the involvement of addi-
tional mechanisms and physiological systems in the disease
The airways are densely innervated by peripheral sensory
neurons expressing specific receptors activated by noxious chem-
icals contained in the inhaled air (5). Over the last decades,
evidence has mounted for bi-directional feedback between im-
munogenic and neurogenic mechanisms in airway inflammation
(6, 7). Neuronal activation causes pain and irritation, neurogenic
inflammation, mucus secretion, and reflex responses, such as
cough, sneezing, and bronchoconstriction (8, 9). Members of the
transient receptor potential (TRP) superfamily of ion channels
he dramatic increase in the number of asthma cases over the
last decades is of great concern for public health in the
play a key role in the response of sensory neurons to inflam-
matory mediators (10–12). The 2 major pro-inflammatory TRP
ion channels in sensory neurons are TRPV1, the capsaicin
receptor, and TRPA1, activated by mustard oil (13–16).
Agonists of TRPV1 and TRPA1, such as capsaicin, acrolein,
or chlorine, are potent tussive agents and have been associated
with allergic and occupational asthma and reactive airway
dysfunction syndrome (RADS) (12, 17–23). Potential endoge-
nous TRPA1 agonists include reactive oxygen species, hypochlo-
rite, and lipid peroxidation products (18, 24–26). Similar to
TRPV1, TRPA1 is activated or sensitized downstream of in-
flammatory PLC-coupled receptor pathways and mediates in-
flammatory pain sensitization (12–14, 27). In animal models,
TRPA1 antagonists block chemically induced inflammatory ther-
mal and mechanical hyperalgesia, neuropathic pain, and diminish
acute airway responses to chemical exposures (17, 19, 28).
The roles of TRPV1 and TRPA1 in asthmatic airway inflam-
mation remain unknown. Using a murine model of acute asthma,
we identify a critical role of TRPA1 in this disease. We show that
genetic deletion of TRPA1 or pharmacological channel inhibi-
tion diminishes allergen-induced inflammatory leukocyte infil-
tration, mucus production, cytokine and chemokine levels, and
airway hyperreactivity. Trpa1?/?mice also show impaired acute
and inflammatory neuropeptide release in the airways. In con-
trast, all aspects of asthmatic airway inflammation were normal
in Trpv1?/?mice. These results suggest that TRPA1 is a major
neuronal mediator of allergic airway inflammation and may
represent a promising target for suppression of inflammation
and airway hyperreactivity in asthma.
Diminished Leukocyte Airway Infiltration and Airway Hyperreactivity
in OVA-Challenged Trpa1?/?Mice. We used the ovalbumin (OVA)
mouse model of asthma to induce a Th2-directed allergic re-
sponse, comparing leukocyte levels in the bronchoalveolar la-
vage fluid (BALF) of OVA-challenged wild-type, Trpa1?/?, and
Trpv1?/?mice (Fig. 1A and B). Leukocyte numbers were greatly
elevated in BALF of OVA-challenged wild-type C57BL/6 mice
Author contributions: N.J.H., L.C., X.H., M.M.M., and S.-E.J. designed research; A.I.C., M.B.,
M.D.E., B.F.B., M.D., J.S.W., J.A.C., R.J.H., and X.H. performed research; D.d.C. and L.C.
contributed new reagents/analytic tools; A.I.C., M.B., M.D.E., B.F.B., J.A.C., R.J.H., X.H., and
S.-E.J. analyzed data; and A.I.C., C.M.F., L.C., M.M.M., and S.-E.J. wrote the paper.
Conflict of interest statement: S.-E.J. is serving on the scientific advisory board of Hydra
Biosciences, Cambridge, MA. Hydra Biosciences developed the TRPA1-antagonist, HC-
employees of Hydra Biosciences, and receive options.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/cgi/content/full/
June 2, 2009 ?
vol. 106 ?
no. 22 ?
(Fig. 1A). Eosinophils represented the majority of leukocytes
(Fig. 1A). In OVA-challenged TRPA1-deficient mice, we ob-
served a remarkable reduction in BALF leukocyte numbers
(?50%), with eosinophilia reduced by ?80% (Fig. 1A). In
contrast, OVA-challenged Trpv1?/?mice showed robust leuko-
cyte infiltration, with BALF cell counts indistinguishable from
those of wild-type mice (Fig. 1B).
Airway hyperreactivity (AHR) is another important hallmark
of asthma. Airway resistance was measured by forced oscillation
in response to i.v. administration of increasing concentrations of
acetylcholine (Fig. 1C). OVA-challenged wild-type C57BL/6
mice developed robust AHR (Fig. 1C). In OVA-challenged
Trpa1?/?mice, AHR was very mild, only differing from control
animals at the highest doses of acetylcholine. Basal reactivity of
vehicle-treated wild-type and Trpa1?/?mice was identical. We
conclude that TRPA1 plays an essential role in asthma-related
Airway eosinophilia and hyperreactivity are consequences of
an allergen-induced Th2-lymphocyte response leading to the
production of allergen-specific IgE antibodies. We measured
OVA-reactive IgE in serum by EIA to verify whether Trpa1?/?
mice produce a normal Th2-response following immunization
and airway challenge with OVA (Fig. 1D). OVA-reactive IgE
serum levels in Trpa1?/?mice were indistinguishable from those
in wild-type C57BL/6 mice, suggesting a normal Th2-dependent
systemic immune response to OVA (Fig. 1D). These data
indicate that TRPA1 has a crucial role in later events leading to
airway inflammation following allergen challenge.
Quantitative comparison of inflammatory cell densities near
airways in lung sections of OVA-challenged mice confirmed
diminished eosinophilia in Trpa1?/?mice and reduced hyper-
plasia of mucus-producing goblet cells (Fig. 1E and supporting
information (SI) Fig. S1A).
Reduced Mucus Production and Th2 Cytokine Levels in Airways of
OVA-Challenged Trpa1?/?Mice. Using quantitative Taqman PCR,
we compared the transcriptional levels of the muc5ac mucin
genes in whole lung cDNA (Fig. 2A). Mucins are mucus proteins
highly expressed in asthmatic airways. OVA-challenged wild-
type mice displayed robust induction of muc5ac transcription (Fig.
2A). In contrast, muc5ac levels were reduced by 50% in lungs of
normal in OVA-challenged Trpv1?/?mice (Fig. S1B).
Th2 leukocytes orchestrate the allergic inflammatory response
in the airways through the release of cytokines, such as inter-
leukin 5 (IL-5). We examined transcriptional activity of the IL-5
gene by Taqman PCR of whole lung cDNA from wild-type,
Trpa1?/?, and Trpv1?/?mice as a measure for Th2 leukocyte
infiltration and activity (Fig. 2B). Strikingly, while OVA-
challenged wild-type mice showed a robust increase in IL-5
transcriptional activity, IL-5 levels in OVA-challenged Trpa1?/?
mice were indistinguishable from those in vehicle-treated mice
(Fig. 2B). Trpv1?/?mice showed normal induction of IL-5
expression (Fig. S1C).
A systematic comparison of peptide concentrations of cyto-
kines and chemokines was performed using Luminex multiplex
protein analysis of BAL fluid (Fig. 2C). As predicted from our
qPCR analysis, IL-5 protein levels in BALF of OVA-challenged
Trpa1?/?mice were much lower (?20%) than in wild-type mice
(Fig. 2C, Inset). Trpa1?/?mice also showed significantly dimin-
ished levels of IL-13, IL-17, eotaxin, MCP-1, RANTES, and
TNF?, suggesting a profound defect in the Th2-directed local
inflammatory response in the airways (Fig. 2C). Levels of IFN
?, an indicator for a Th1 leukocyte activity, were below the
detection limit in all mouse groups, showing that the observed
reduction in airway eosinophilia was not due to a shift toward a
Th1-directed immune response in Trpa1?/?mice. Cytokine
R (cm H O/ml/s)
OVA IgE (serum)
Trpa1 -/- veh
BAL cells x 104
BAL cells x 104
Density of Inflammation
Trpa1 -/- OVA
mice. Cell differentials are shown for total cells, eosinophils, macrophages, lymphocytes, and neutrophils in BALF collected from vehicle (veh, PBS)- and
OVA-challenged Trpa1?/?and Trpa1?/?mice. Animal groups: Trpa1?/?OVA: n ? 8, Trpa1?/?OVA: n ? 8, Trpa1?/?veh: n ? 7, Trpa1?/?veh: n ? 6.*, P ? 0.05;
**, P ? 0.01;***, P ? 0.001 . (B) Normal inflammatory leukocyte infiltration in OVA-challenged Trpv1?/?mice. BALF leukocyte cell differentials are shown for
vehicle (veh, PBS)- and OVA-challenged Trpv1?/?and Trpv1?/?mice. Animal groups: Trpv1?/?OVA: n ? 6, Trpv1?/?OVA: n ? 4, Trpv1?/?veh: n ? 6, Trpv1?/?
veh: n ? 4. (C) Comparison of airway resistance (R) in OVA-challenged Trpa1?/?(blue) and Trpa1?/?mice (red), as well as vehicle (veh)-treated Trpa1?/?(green)
and Trpa1?/?(purple) mice, measured by forced oscillation in response to increasing dosages of acetylcholine. Animal groups: Trpa1?/?OVA: n ? 4, Trpa1?/?
OVA: n ? 4, Trpa1?/?veh: n ? 6, Trpa1?/?veh: n ? 6. (*, ? ? 0.05;**, ? ? 0.01;***, ? ? 0.001). (D) Induction of OVA-reactive Ig E in OVA-challenged wild-type
Trpa1?/?and Trpa1?/?mice, scored by counting of inflammatory cells near bronchial bundles (n ? 4 mice per group).
Decreased inflammatory response to inhaled OVA in TRPA1-deficient mice. (A) Reduced leukocyte infiltration in airways of OVA-challenged Trpa1?/?
www.pnas.org?cgi?doi?10.1073?pnas.0900591106 Caceres et al.
levels were normally elevated in OVA-challenged Trpv1?/?mice
A TRPA1 Antagonist Reduces Airway Inflammation and Hyperreactiv-
ity when Administered During OVA Airway Challenge. TRPA1-
antagonists showed efficacy in animal models of acute and
agonists known to cause asthma-related conditions (17, 19, 20,
28, 29). We asked whether a TRPA1 antagonist would prevent
or diminish airway inflammation when administered to OVA-
sensitized Balb/C mice during the airway challenge phase of the
OVA protocol. HC-030031, the most thoroughly studied TRPA1
antagonist, was injected i.p. into OVA-sensitized animals on the
day before (200 mg/kg) and twice daily (100 mg/kg) during the
MC)-treated mice showed robust OVA-induced increases in
BALF leukocyte numbers, cell numbers were diminished in
OVA-challenged HC-030031-treated mice (Fig. 3A). Moreover,
treatment with HC-030031 led to a nearly complete suppression
of airway hyperreactivity in OVA-challenged Balb/C mice (Fig.
3B). Mucin5ac transcription was strongly suppressed by HC-
030031 treatment, indicating diminished production of airway
mucus (Fig. 3C). Antagonist-injected mice also showed dimin-
ished levels of Th2 cytokines IL-5 and IL-13 in BALF (Fig. 3D).
Histological sections of airways from antagonist-treated mice
showed much lower densities of inflammatory cells (Figs. 3E and
S1E). Similar to Trpa1?/?mice, treatment with HC-030031 did
not affect serum levels of OVA-specific IgE in OVA-challenged
wild-type BALB/C mice, indicating a normal Th2-directed sys-
temic inflammatory response (Fig. S1F). These data suggest that
TRPA1 plays a crucial role in the development of asthma during
airway allergen challenge, enabling inflammatory leukocyte
infiltration, airway hyperreactivity, and mucus production.
tide Release in the Airways. It is unclear whether the pro-
inflammatory action of TRPA1 in asthma can be explained
through purely neurogenic effects. TRPA1 may play an as yet
undetected role in cells of the immune system or in airway tissue.
To assess this possibility, we used Taqman quantitative PCR to
compare TRPA1 transcript levels in cDNA derived from spleen
harboring a large variety of leukocyte precursors, Th2 lympho-
cytes, whole mouse lung and BALF leukocytes of OVA-
challenged mice, and DRG. Relative transcript quantities in
spleen, Th2 cells, whole lung, and leukocytes were minimal, with
DRG expression several 100-fold higher (Fig. S1G). Additional
qPCR experiments using cDNA prepared from primary leuko-
cytes and leukocyte cell lines failed to detect the presence of
TRPA1 cDNA. These results point to a key role for sensory
neuronal TRPA1 channels in allergic airway inflammation.
TRPA1 may be a critical trigger for neuropeptide release
crucial for leukocyte infiltration and inflammatory progression
in asthmatic airways. To investigate this possibility, we compared
neuropeptide release in airways of wild-type and and Trpa1?/?
mice in response to 2-chloroacetophenone (CN), a potent in-
flammatory TRPA1 agonist (20). We performed a 30-s BAL in
mice with CN (4 mM) contained in the BAL buffer (PBS) and
measured the resultant release of CGRP, substance P (SP) and
neurokinin A (NKA) using EIA (Fig. 4 A–C). CN induced strong
increases in the levels of all 3 neuropeptides in BALF of
wild-type C57BL/6 mice (Fig. 4 A–C). In Trpa1?/?mice CN-
induced peptide release was clearly diminished (?50% of wild-
type levels), supporting a specific and essential role for TRPA1
in chemically stimulated neurogenic peptide release in the
airways (Fig. 4 A–C). Acute CN-induced neuropeptide release
was suppressed by prior treatment with HC-030031 in Balb/C
mice (Fig. 4D).
Exogenous TRPA1 agonists such as CN are likely to mimic the
actions of endogenous reactive products and inflammatory
signaling pathways activating TRPA1 (16). Since Trpa1?/?mice
showed clear deficiencies in acute neurogenic peptide release in
the airways, we asked whether neuropeptide levels would also be
reduced during airway allergen challenge. We compared neu-
ropeptide levels in BALF of OVA-challenged wild-type and
Trpa1?/?-deficient C57/BL6 mice, and in antagonist treated
Balb/C mice, focusing on NK-A, the most abundant neuropep-
tide in airway lining fluid (30) (Fig. 4 E and F). Indeed, we
observed that the OVA-induced increase in BALF NK-A levels
was clearly diminished in Trpa1?/?mice and in antagonist-
treated Balb/C mice (Fig. 4 E and F).
Our results reveal a crucial role for the sensory neuronal ion
channel TRPA1 in experimental asthma. TRPA1-deficient mice
showed profound deficits in airway infiltration by inflammatory
leukocytes in the OVA model of allergic airway inflammation,
accompanied by a reduction in inflammatory Th2 cytokines,
such as IL-5 and IL-13, and pro-inflammatory chemokines, such
as TNF? and the eosinophil attractant, eotaxin. As a conse-
quence, OVA-induced mucus production is impaired in
Trpa1?/?mice. Airway hyperreactivity, another important hall-
mark of asthma, was strongly reduced. Pharmacological inhibi-
tion of TRPA1 during the airway challenge phase of the OVA
Mucin 5AC (RQ)
Trpa1 -/- veh
Trpa1 -/- OVA
challenged airways of Trpa1-deficient mice. (A) Relative quantities (RQ) of
mucin5ac gene transcript, determined by Taqman qPCR of whole mouse lung
cDNA. Mucin5ac induction is diminished in Trpa1?/?OVA mice. GAPDH tran-
script levels were used for normalization as endogenous control. Animal
transcript, as determined by Taqman real-time quantitative PCR of whole
mouse lung cDNA. OVA-challenged Trpa1?/?mice show no significant
changes in IL-5 transcription compared with vehicle-treated mice. GAPDH
transcript levels were used for normalization as endogenous control. Animal
OVA: n ? 6.***, P ? 0.001. (C) Comparison of cytokine and chemokine levels
in BALF of OVA-challenged Trpa1?/?(white) and Trpa1?/?(black) mice, as
measured by Luminex peptide analysis. Groups: Trpa1?/?n ? 8–10, Trpa1?/?
n ? 8–10 for each analyte.**, P ? 0.01;***, P ? 0.001
Impaired induction of mucin, cytokines, and chemokines in OVA-
Caceres et al.PNAS ?
June 2, 2009 ?
vol. 106 ?
no. 22 ?
protocol confirmed the essential function of this receptor,
blocking leukocyte infiltration, cytokine and neuropeptide re-
lease, mucus production, and abolishing airway hyperreactivity.
Trpa1?/?mice are deficient in the neuronal detection of
multiple pro-inflammatory exogenous and endogenous agents.
These include asthma-inducing agents such as chlorine, unsat-
urated aldehydes in smoke and smog, chloramines, tear gas
agents, and industrial isocyanates, as well as endogenous reactive
oxidative species and lipid mediators (12, 17–20, 24, 25). Some
of these endogenous mediators are produced by infiltrating
leukocytes or inflamed airway tissue and can reach concentra-
tions high enough to chronically activate TRPA1 in airway nerve
a lack of neuronal excitation and Ca2?influx activated by these
reactive compounds during inflammatory progression in asthma,
resulting in reduced reflex hyperreactivity and neuropeptide re-
lease. Indeed, we find that sensory neuropeptide release, a prereq-
the airways of Trpa1?/?mice. This defect applies to both acute
neuropeptide release, induced by a TRPA1 agonist, and inflam-
matory peptide release following OVA challenge in the airways.
The ability of a TRPA1 antagonist to recapitulate the knock-
out phenotype suggests that TRPA1 fulfills an acute role in
promoting local inflammation in the airways, rather than causing
a developmental defect in immune system function. Adminis-
tration of HC-030031 during the OVA airway challenge phase
was sufficient to potently suppress airway leukocyte infiltration,
mucus production and hyperreactivity. The role of TRPA1 in
local inflammatory responses to allergen challenge is also sup-
ported by the observation that genetic deletion of TRPA1, or
treatment with the TRPA1 antagonist HC-030031, did not seem
to affect the systemic Th2-mediated response to allergen immu-
nization, as evidenced by normal serum levels of OVA-reactive
IgE. Future experiments are needed to address the detailed
mechanistic role of TRPA1 in neurogenic responses affecting
Th2 lymphocyte migration into the airways, cytokine production,
leukocyte recruitment, as well as in airway hyperreactivity.
Our experiments clearly show that TRPV1, the capsaicin
receptor, is not required for allergic airway inflammation in the
OVA mouse model of asthma. A TRPA1-specific stimulus,
possibly a reactive TRPA1-specific mediator, appears to be
required to drive the pro-inflammatory activity of airway C-
fibers in asthma. Nevertheless, TRPV1 is clearly involved in the
symptomatic consequences of airway inflammation, as evi-
denced by a recent report showing anti-tussive activity of a
TRPV1 antagonist in a model of chronic cough (32).
The data in our present study support the idea that TRPA1
may function as an integrator of chemical and immunological
stimuli modulating inflammation in the airways. This integrative
activity can explain the pro-inflammatory effects of chemical
exposures in asthma patients (16). By activating TRPA1, chem-
ical irritants may trigger the release of neuropeptides and
chemokines in the airways, thereby exacerbating the cellular and
tissue inflammatory response observed in our present study. Our
as a potential target for anti-asthmatic drugs. Future studies will
Density of Inflammation
BAL cells x 104
R (cm H O/ml/s)
Mucin 5AC (RQ)
IL-4 IL-5 IL-13 Eotaxin
differentials for total leukocytes, eosinophils, macrophages, lymphocytes, and neutrophils in BALF collected from vehicle (veh, PBS)- or OVA-challenged mice
injected i.p. with HC-030031 or with methyl cellulose (MC) during the airway challenge phase. Animal groups: MC veh: n ? 8, HC-030031 veh: n ? 9, MC OVA,
n ? 10, HC-030031 OVA: n ? 8.*, P ? 0.05. (B) Comparison of airway hyperresponsiveness to i.v. acetylcholine in vehicle (veh, PBS) - or OVA-challenged mice
injected i.p. with HC-030031 or with just methyl cellulose (MC) during OVA airway challenge. Animal groups: MC veh: n ? 7, HC-030031 veh: n ? 7, MC OVA,
n ? 7, HC-030031 OVA: n ? 6 (*, ? ? 0.05;**, ? ? 0.01;***, ? ? 0.001). (C) Decreased lung mucin5ac transcription in OVA-challenged mice treated with TRPA1
or with just methyl cellulose (MC) during OVA airway challenge. GAPDH transcript levels were used for normalization as endogenous control. Animal groups:
MC veh: n ? 4, HC-030031 veh: n ? 4, MC OVA, n ? 8, HC-030031 OVA: n ? 8.*, P ? 0.05. (D) Cytokine and eotaxin levels in bronchoalveolar lavage fluid (BALF)
of OVA-challenged Balb/C mice treated with TRPA1 antagonist HC-030031 (black) or carrier methyl cellulose (white). (n ? 4 mice/group)*, P ? 0.05;**, P ? 0.01.
(E) Density of inflammation in H&E-stained airway sections from OVA-challenged HC-030031-treated and ?untreated (MC) Balb/C mice, scored by counting of
inflammatory cells near bronchial bundles (n ? 4 mice per group).
Decreased inflammatory response in Balb/C mice treated with the TRPA1 antagonist HC-030031 during the OVA airway challenge phase. (A) Cell
www.pnas.org?cgi?doi?10.1073?pnas.0900591106Caceres et al.
address the action of TRPA1 antagonists in additional animal
models of asthma and in other allergic inflammatory conditions.
Materials and Methods
Animals. Experimental procedures were approved by the Institutional Animal
Care and Use Committees of Yale University, the University of California, San
Francisco, and Hydra Biosciences. Mice were housed at facilities accredited
by the Association for Assessment and Accreditation of Laboratory Animal
Care in standard environmental conditions (12-h light-dark cycle and
23 °C). Food and water were provided ad libitum. Trpa1?/?mice were a gift
from David Julius (University of California, San Francisco). The Trpa1-
knockout allele was backcrossed into the C57BL/6 background (?99.5%) by
marker assisted accelerated backcrossing (Charles River Laboratories).
Trpv1?/?mice were purchased from Jackson Laboratories and C57/Bl6 and
BALB/c mice from Charles River Laboratories. For experiments on C57BL/6,
Trpv1?/?, and Trpa1?/?mice, animals were matched for age (12–22 weeks)
and gender. Six- to 8-week-old BALB/c mice were used for OVA sensitiza-
tion and antagonist studies.
Sensitization and Airway Challenge Procedure. Mice were sensitized on days 0,
2 mg alum gel (Sigma-Aldrich) in a total volume of 200 ?L PBS. Control animals
received alum only. Subsequently, lightly anesthetized mice (isoflurane) were
days 21, 22, and 23. For therapeutic intervention with TRP channel antagonist
once on day 20 and 100 mg/kg twice a day on days 21, 22, and 23. On the day of
morning and subject to the end point of the experiment. All measurements and
sample collection were performed 24 h after the final intranasal challenge.
samples of dose solutions were analyzed by LC/MS/MS using a standard protein
precipitation extraction with the addition of an internal standard.
Measurement of Airway Reactivity. Twenty-four hours following the last OVA
challenge, mice were anesthetized with pentobarbital (60 mg/kg of body
weight) and urethane (1 g/kg). A tracheostomy was performed, and the
trachea was cannulated. Mice were attached to a Flexivent pulmonary me-
chanics analyzer (SCIREQ) and ventilated at a tidal volume of 9 mL/kg, at a
frequency of 150 bpm. Positive end-expiratory pressure was set at 2 cm H2O.
Mice were paralyzed with pancuronium (0.1 mg/kg i.p.). A 27-gauge needle
was used to administer acetylcholine (0.03, 0.1, 0.3, 1.0, and 3.0 mg/mL)
through the subclavial/tail vein to generate a concentration-response curve.
Measurements of airway mechanics were made continuously applying the
Quantitative Analysis of Cytokines, Chemokines, and Neuropetides in BAL Fluid.
MAP Mouse Cytokine/Chemokine Kit (Millipore) on Luminex 200 analyzer
(Luminex), following the manufacturer’s recommendations. Neuropeptide
levels in BAL were measured by EIA (CGRP: Cayman Chemical; Substance P:
Phoenix Pharmaceuticals; Neurokinin A: Bachem). To measure acute peptide
were inflated with CN-BAL buffer for 30 sec. HC-030031 was injected i.p. in
Balb/C mice at 200 mg/kg 24 h, 100 mg/kg 6 h, and 100 mg/kg 30 min before
CN challenge (200 ?M).
Asthma Foundation (Grant 07–0212 to S.J.), the National Institute of Environ-
mental Health Sciences (ES015056 and ES017218 to S.J.), a Spanish Ministry of
fellowship from the German Research Foundation to M. B. (BR 3773/1–1.) and
National Institutes of Health (NIH) training grant 5T32GM07205 to M. D. E. Its
contents are solely the responsibility of the authors and do not necessarily
represent the official views of the NIH or federal government. S. J. serves on
the Scientific Advisory Board of Hydra Biosciences. All members of Hydra
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Substance P (ng/ml)
SP release / HC
Trpa1 -/- veh
Trpa1 -/- OVA
OVA NK-A release
OVA NK-A release / HC
Substance P (pg/ml)
release in the airways, measured by EIA. (A) Diminished chemically induced
release of CGRP in Trpa1?/?mice following lung exposure to CN (2-
chloroacetophenone) during BAL. Averaged CGRP levels in BAL fluid of
induced release of Substance P (SP) in Trpa1?/?mice following lung exposure
to CN (2-chloroacetophenone) during BAL. Treatments and mouse groups as
in Fig. 4A. (C) Diminished chemically induced release of neurokinin A (NK-A)
in Trpa1?/?mice following lung exposure to CN (2-chloroacetophenone)
during BAL. Treatments and mouse groups as in Fig. 4A. (D) Diminished
CN-induced release of SP in mice injected i.p. with TRPA1 antagonist HC-
030031. Averaged SP concentrations are shown in BAL fluid of Balb/C mice
treated with PBS (n ? 4), 200 ?M CN and methylcellulose vehicle (MC CN, n ?
4), or with 200 ?M CN and HC-030031 (HC CN, n ? 4) are shown.*, P ? 0.05.
(E) Reduced level of neurokinin A in BAL fluid of OVA-challenged Trpa1?/?
mice. NK-A levels were compared by EIA in BAL fluid of vehicle-treated
wild-type mice (Trpa1?/?veh, n ? 11), vehicle-treated Trpa1?/?mice (n ? 7),
OVA-challenged wild-type mice (Trpa1?/?OVA, n ? 12), and OVA-challenged
Trpa1?/?mice (n ? 12).*, ??0.05. (F) Reduction of NK-A in BAL fluid of OVA-
challenged Balb/C mice due to injection of TRPA1-antagonist HC-030031. NK-A
mice (MC veh, n ? 4), HC-030031- and vehicle-treated (HC veh, n ? 4), methyl
cellulose treated and OVA-challenged (MC OVA, n ? 7), and HC-030031-treated
and OVA-challenged mice (HC OVA, n ? 8).*, ??0.05
Caceres et al.PNAS ?
June 2, 2009 ?
vol. 106 ?
no. 22 ?
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