Trichostatin A Abrogates Airway Constriction, but Not
Inflammation, in Murine and Human Asthma Models
Audreesh Banerjee1*, Chinmay M. Trivedi2,4*, Gautam Damera1, Meiqi Jiang1, William Jester1,
Toshinori Hoshi3, Jonathan A. Epstein2*, and Reynold A. Panettieri, Jr.1*
1Airways Biology Initiative, Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine,2Penn Cardiovascular Institute and
Institute for Regenerative Medicine, Department of Cell and Developmental Biology, and3Department of Physiology, University of Pennsylvania,
Philadelphia, Pennsylvania; and4Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts
Histone deacetylase (HDAC)inhibitorsmayoffernovelapproachesin
the treatment of asthma. We postulate that trichostatin A (TSA),
Aspergillus fumigatus antigen (AF) and treated with TSA, dexametha-
sone, or vehicle. Lung resistance (RL) and dynamic compliance were
numbers of leukocytes and concentrations of cytokines. Human
precision-cut lung slices (PCLS) were treated with TSA and their
agonist-induced bronchoconstriction was measured, and TSA-
treated human airway smooth muscle (ASM) cells were evaluated
for the agonist-induced activation of Rho and intracellular release of
Ca21. The activity of HDAC in murine lungs was enhanced by antigen
and abrogated by TSA. TSA also inhibited methacholine (Mch)-
control mice and in AF-sensitized and -challenged mice. Total cell
counts, concentrations of IL-4, and numbers of eosinophils in BALF
were unchanged in mice treated with TSA or vehicle, whereas dexa-
trations of IL-4. TSA inhibited the carbachol-induced contraction of
PCLS. Treatment with TSA inhibited the intracellular release of Ca21
to Mch in both naive and antigen-challenged mice. TSA inhibits the
agonist-induced contraction of PCLS and mobilization of Ca21in
distinct from that of anti-inflammatory agents such as steroids, and
represent a promising therapeutic agent for airway disease.
Keywords: HDAC; asthma; allergen; mice; trichostatin A
Asthma manifests as reversible airway obstruction, hyperrespon-
siveness, and inflammation (1). Although most patients respond
to conventional therapy, some exhibit severe or refractory asthma
associated with frequent exacerbations, irreversible airflow limi-
tation, and airway inflammation, despite maximal medical therapy
(2). Patients with severe asthma also use healthcare resources
disproportionately and experience more adverse effects from high
doses of glucocorticoids (3). The need for improved therapeutic
approaches to severe asthma continues, and the pursuit of epige-
netic modifications of gene expression in asthma offers unique
therapeutic opportunities. Accordingly, the acetylation and deace-
tylation of histone represent a potential therapeutic target (4).
Histone acetyl transferases (HATs) induce the acetylation of
histone, whereas histone deacetylases (HDACs) remove the ace-
tyl groups from histones to modulate gene transcription. Cur-
rently, 11 HDACs have been identified and grouped into three
major categories by their sequence similarity to the Saccharo-
myces cerevisiae reduced potassium dependency-3 (RPD3) or
histone-deacetylase 1 (Hda1) enzyme (5), and evidence suggests
that HDACs differentially regulate genes (6). In addition to
modulating gene activity by acetylating histones, HDACs also
modulate nonhistone targets (7) that include transcription fac-
tors, cytokine receptors, cytoskeletal proteins, and nuclear hor-
mone receptors (8). Although both HATs and HDACs may play
a role in inflammatory lung disease and modulate steroid sensi-
tivity (9), the roles of HATs and HDACs in the regulation of
inflammatory and anti-inflammatory gene expression remain
controversial. Airway cells derived from subjects with asthma
demonstrate increased HAT activity and decreased HDAC ac-
tivity (10), and the inhibition of HDAC improves airway hyper-
responsiveness (AHR) and inflammation in some animal models
of airway inflammation (11, 12).
Here, we characterize the expression of HDAC isoforms in
murine lung tissue and in human airway smooth muscle (ASM)
and epithelial cells. Further, we show that trichostatin A (TSA),
a Class I and II inhibitor of HDAC, abrogates methacholine
(Mch)–induced AHR without affecting leukocyte trafficking
and concentrations of cytokines in bronchoalveolar lavage fluid
(BALF) from antigen-challenged mice, human precision-cut
lung slices (PCLS), and ASM cells.
MATERIALS AND METHODS
FemaleC57/BL6 mice, aged8 weeks, were purchased from Charles River
laboratories (Malvern, PA). All animal protocols were approved by the
Animal Use and Care Committee at the University of Pennsylvania.
Antigen Sensitization and Challenge
As shown in Figure 1, mice were sensitized by intraperitoneal injections
of 20 mg antigen, a protein extract of the ubiquitous airborne fungus,
Aspergillus fumigatus (AF; Bayer Pharmaceuticals, Spokane, WA) in
100 ml PBS solution containing 2 mg of alum (Imject Alum; Pierce,
(Received in original form July 1, 2010 and in final form July 18, 2011)
* These four authors contributed equally to this work.
This work was supported by National Institutes of Health grants F32HL096286 and
K08HL097032 (A.B.), R01HL097796, RO1 HL081824, and 5P30ES013-508-04 (R.A.P.),
K99-R00 HL098366 (C.M.T.), R01GM057654 and R01GM078579 (T.H.), and
Correspondence and requests for reprints should be addressed to Audreesh
Banerjee, M.D., Translational Research Laboratories, Division of Pulmonary, Allergy,
and Critical Care Medicine, University of Pennsylvania Medical Center, 125 South
31st St., Translational Research Laboratories Suite 1200, Philadelphia, PA 19104-
3403. E-mail: firstname.lastname@example.org
Am J Respir Cell Mol Biol
Copyright ª 2012 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2010-0276OC on August 18, 2011
Internet address: www.atsjournals.org
Vol 46, Iss. 2, pp 132–138, Feb 2012
This research demonstrates a novel role for inhibitors of
histone deacetylase (HDAC) in abrogating airway constric-
tion in response to contractile agonists in both animal and
human models. Our findings suggest a therapeutic role for
smooth muscle contraction may be epigenetically modulated.
Rockford, IL) on Days 0 and 14, and challenged on Days 25–27 with 30
ml of AF extract in PBS (25 mg) intranasally. This is a modification of
our previously described protocol (13).
Aldrich) once daily on Days 25–27. Control animals received an equal
volume of DMSO (carrier) without TSA by intraperitoneal injection.
Invasive Lung Function Measurements of Anesthetized,
Lung resistance (RL), dynamic compliance, elastance, tissue damping,
tissue elastance, and airway resistance were recorded using the FlexiVent
system (SCIREQ Scientific Respiratory Equipment, Inc., Montreal, PQ,
Canada), as described previously (14). Briefly, mice were anesthetized by
an intraperitoneal injection of a ketamine (100 mg/kg) and xylazine
(20 mg/kg) mixture. After anesthesia, a 0.5-cm incision was performed
from the rostral to caudal direction. The flap of skin was retracted, the
connective tissue was dissected away, and the trachea was exposed. The
trachea was then cannulated between the second and third cartilage rings
with a blunt-end stub adapter and secured with suture. The mouse was
next connected to the FlexiVent system, and spontaneous respirations
were terminated with an intramuscular injection of pancuronium bro-
mide (3 mg/kg). Parameters of mechanical ventilation included a rate of
140 breaths/minute and a 0.25-ml tidal volume. The respiratory mechan-
ics were measured as previously described (14). Airway responsiveness
was measured after the inhalation of nebulized saline and increasing
concentrations of nebulized Mch (1.25, 5, 10, and 20 mg/ml).
BAL Cell Count and Differential Cell Count
Aftermeasurementsof RL,lungswerelavagedwith1-mlaliquotsof sterile
saline through the tracheal cannula. After centrifuging (500 3 g for 10
minutes at 4?C), the cell pellet was resuspended in RPMI medium.
Differential cell counts were performed from cytospin preparations, as
described elsewhere (15). Cells were identified as macrophages, eosino-
phils, neutrophils, and lymphocytes according to standard morphology,
and a minimum of 300 cells was counted using a Nikon microscopic
system (Nikon Instruments, Melville, NY) (3400 magnification). The
percentages and absolute numbers of each cell type were then calculated.
The supernatants were harvested and stored at 220?C for further analysis.
Determination of IL-4 and IL-6 in BALF
Concentrations of cytokines were determined by ELISA according to
standard protocols, as previously described (16). The limits of detection
were 5 pg/ml for IL-4 and IL-6 standards. We used the recombinant
murine IL-4 and IL-6 included with the kits (R&D Systems, Minneap-
olis, MN) as control samples.
SDS-Polyacrylamide Gel Electrophoresis
and Immunoblot Analysis
(pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton
X-100, 1 mg/ml leupeptin, 2.5 mM sodium pyrophosphate, 1 mM
Na3VO4, and 1 mM b-glycerophosphate. Phenylmethylsulfonyl fluo-
ride (1 mM) was added before use. One hundred micrograms of each
sample were separated by 4–12% SDS-PAGE (Invitrogen, Grand
Island, NY) and transferred to polyvinylidene fluoride (PVDF) mem-
branes. We used antibodies to HDAC1 (1:1,000 dilution; Cell Signal-
ing), HDAC2 (1:1,000 dilution; Invitrogen), HDAC3 (1:1,000 dilution;
Sigma Chemical Co., St. Louis, MO), HDAC4 (1:1,000 dilution; Cell
Signaling, Danvers, MA), HDAC5 (1:1,000 dilution; Cell Signaling),
HDAC6 (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz,
CA), HDAC8 (1:1,000 dilution; Santa Cruz Biotechnology), HDAC10
(1:1,000; Sigma), and anti–glyceraldehyde 3-phosphate dehydrogenase
(1:5,000 dilution; Sigma). These antibodies are specific to both human
and murine samples (17, 18). Primary antibody binding was visualized
by using the Westernbreeze Kit (Invitrogen) as described previously,
and according to the manufacturer’s instructions (19, 20).
HDAC Activity Assay
The activity of HDAC was measured using a commercial fluorometric
detection HDAC activity assay kit (Millipore/Upstate Cell Signaling,
Lake Placid, NY), as described previously (19). In brief, total extract
was prepared from freshly harvested lung tissue. Fifty micrograms of
total extract were incubated with HDAC assay substrate and incubated
at 30?C for 60 minutes. After the addition of reagent, samples were
kept at room temperature for 15 minutes, and readings were taken with
a fluorescent microplate reader at an excitation of 360 nm and emission
of 450 nm. A standard curve was performed according to the manu-
Epithelial cells were derived from protected brush specimens obtained
University of Pennsylvania Committee on Studies Involving Human
Beings. Specimens were centrifuged at 1,200 rpm for 5 minutes, and
pelleted cells were seeded onto 100-mm collagen-coated tissue culture
dishes and incubated at 37?C with 5% CO2.When cells reached 80%
confluence, monolayers were transferred onto collagen-coated Trans-
well inserts, as previously described (21, 22). After the formation of
monolayers, the apical surfaces of the Transwell inserts were raised to
air–liquid interfaces (ALI), and cultured for an additional 2 weeks.
Lysates were obtained on Days 15–16 at the ALI for immunoblot
Human ASM cells were derived from tracheas obtained from the
National Disease Research Interchange (Philadelphia, PA). Human
ASM cell culture was performed as described previously (23). Briefly,
a segment of trachea just proximal to the carina was removed under
aseptic conditions, and the tracheal muscle was isolated, centrifuged,
and resuspended in buffer containing 0.2 mM CaCl2, 640 U/ml colla-
genase, 1.0 mg/ml soybean trypsin inhibitor, and 10 U/ml elastase. The
tissue was enzymatically dissociated, filtered, and washed. Aliquots of
the cell suspension were plated on plastic plates at a density of 1.0 3
104cells/cm2. The cells were cultured in Ham’s F-12 medium supple-
mented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin,
and 2.5 mg/ml amphotericin B, and this medium was replaced every
72 hours. Human ASM cells in subculture during passages 1–5 were
used, because these cells retain the expression of native contractile
protein, as demonstrated by immunocytochemical staining for smooth
muscle actin and myosin (23).
Preparation of PCLS and Airway Function
described previously (24). Briefly, healthy whole lungs were received
from the National Disease Research Interchange and inflated with
agarose solution. Lobes were then sectioned, and cores were made in
which a small airway was visible. The cores were sliced into 250-mm-
thick PCLS, which were then placed in a 12-well plate and exposed to
increasing doses of carbachol. After each dose of carbachol, images
were collected and airway diameters were measured, as previously
Figure 1. Experimental design. Animals were sensitized with two intra-
peritoneal (IP) injections on Days 0 and 14 with 20 mg of Aspergillus
fumigatus antigen (AF). Three intranasal (IN) challenges of 25 mg AF
were performed, once a day for the 3 days before the animal was killed.
Animals were treated with an HDAC inhibitor, trichostatin A (TSA), or
DMSO (diluent) alone by IP injection once a day for the 3 days before
being killed on Day 28.
Banerjee, Trivedi, Damera, et al.: TSA Inhibits Airway Contraction in Mice and Humans133
Rho Activation Assay
Cultured human ASM cells were measured for the activation of Rho,
using a Rho Activation Assay Kit (Upstate Cell Signaling Solutions)
according to the manufacturer’s protocol, as previously described
(25). Total Rho was detected using an anti-Rho antibody (Upstate Cell
Cultured ASM cells were evaluated for the mobilization of Ca21in
response to histamine, using fura-2 acetoxymethyl ester (AM) (Invitro-
gen). Approximately 30,000 cells were plated onto 25-mm coverslips.
After 4 days, cells were serum-deprived and treated with TSA or vehi-
cle. After 2 days of treatment with TSA or diluents, cells were washed in
PBS. Measurements of fura-2 were performed as described elsewhere
(26). Briefly, after incubation with fura-2 AM (1 mM) for 45–60 minutes
at room temperature, cells were constantly perfused with a saline solu-
tion containing 130 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2,
10 mM N-2-hydroxyethylpiperazine-N9-ethane sulfonic acid, and 15 mM
glucose (pH 7.4). The emission signals in response to 340 and 380 nm
were collected, and the 340 nm/380 nm signal ratio was analyzed after
subtracting the background obtained in the presence of 10 mM MnCl2
and 5 mM ionomycin in the absence of added Ca21.
TSA Inhibits the Antigen-Induced Activity of HDAC
As shown in Figure 2A, we characterized the expression of Class
I and II HDACs in human and murine lung tissue by immuno-
blotting. HDAC isoforms are differentially expressed in human
ASM cells, epithelial cells, and murine lung tissue. HDAC4 and
HDAC6 are predominantly expressed in human ASM cells,
whereas HDAC1 and HDAC3 are expressed by human airway
epithelium. Given the spectrum of expression of HDAC
isoforms in lung tissue, we examined the effects of a broad-
spectrum Class 1 and 2 HDAC inhibitor, TSA, on antigen-
induced airway inflammation and function. We used a dose of
TSA and a route of administration (0.6 mg/kg intraperitoneal,
daily) previously shown to inhibit the activity of HDAC (27).
Control mice were treated with DMSO alone. We then mea-
sured the activity of HDAC in lung tissue from naive and TSA-
treated mice. As shown in Figure 2B, lung HDAC activity was
significantly increased by exposure to antigen, whereas TSA ef-
fectively inhibited the activity of HDAC in both naive and
antigen-exposed mice. In naive mice, the basal activity of HDAC
was measured at 141 3 1046 7.7 3 104relative light units
(RLU). These results are presented relative to basal HDAC
activity. In human ASM cells, treatment with TSA decreased
the activity of HDAC in a dose-dependent manner (Figure 2C).
The Inhibition of HDAC Modulates Mch-Induced Changes
in Lung Mechanics
The effects of TSA on lung resistance and compliance were de-
termined after exposing mice to increasing doses of Mch. As
shown in Figure 3A, Mch increased airway resistance in
a dose-dependent manner (Emax¼ 2.4 6 0.1 cm H2O/ml, P ,
0.05), whereas TSA substantially abrogated this response (Emax¼
1.0 6 0.01 cm H2O/ml, P , 0.05) in non–antigen-exposed (na-
ive) mice. Treatment with dexamethasone exerted little effect
on baseline Mch-induced RLin naive mice (Emax¼ 2.3 6 0.07
cm H2O/ml). In antigen-exposed mice, RLincreased to a greater
degree in response to Mch, compared with that in naive mice
(Emax¼ 13.9 6 0.7 cm H2O/ml), and treatment with TSA sig-
nificantly blunted this response (Emax¼ 7.8 6 0.1 cm H2O/ml,
P , 0.05) (Figure 3B). Dexamethasone also inhibited antigen-
enhanced RLto a similar degree as TSA (Emax¼ 6.7 6 0.5 cm
H2O/ml, P , 0.05). As shown in Figure 4A, dynamic compli-
ance was also decreased by Mch, with a maximum inhibition at
0.016 6 0.001 cm H2O/ml. Treatment with TSA reversed this
reduction in dynamic compliance with a maximum inhibition at
Figure 2. Expression and activity of histone deacetylase
(HDAC) in human and murine tissue. (A) Immunoblot
analyses of lysates from human airway smooth muscle
(HASM) cells, lung epithelial cells, and wild-type murine
lung tissue were performed using anti-HDAC antibodies.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
was used to confirm equal protein loading. (B) Relative
HDAC activity from lung tissue lysate was quantified. Data
are reported as the ratio of measured HDAC activity to
basal HDAC activity (Relative HDAC activity). Mice were
sensitized and challenged with antigen or solvent (naive),
and received vehicle treatment (DMSO) or TSA. Data rep-
resent means 6 SD from five samples for each condition.
*P , 0.05 (treatment versus basal concentrations). (C)
Relative HDAC activity from HASM lysate was quantified.
Data are reported as the ratio of measured HDAC activity
to basal HDAC activity (Rel. HDAC activity). Cells were
treated with TSA at concentrations of 1, 10, or 100 mM
for 48 hours before lysis and assay. Data represent
means 6 SD from five samples for each condition. *P ,
0.05 (treatment versus basal concentrations).
134 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 46 2012
0.32 6 0.001 cm H2O/ml (P , 0.05). The maximum inhibition of
dynamic compliance by Mch in dexamethasone-treated mice
was measured at 0.10 6 0.001 cm H2O/ml. The maximum inhi-
bition of dynamic compliance in antigen-exposed mice was
0.004 6 0.0004 cm H2O/ml (Figure 4B), and treatment with
dexamethasone exerted little effect on dynamic compliance.
Treatment with TSA significantly improved dynamic com-
pliance in the mice, with a maximum inhibition at 0.007 6
0.0009 cm H2O/ml (P , 0.05). Together, these data demonstrate
that treatment with TSA diminishes RLand increases dynamic
compliance in response to Mch at baseline and after exposure to
antigen. By altering the response to multiple doses of Mch, TSA
reduces AHR more effectively than dexamethasone under the
TSA Exerted Little Effect on Antigen-Inducted Increases
in BALF Cell Count
To address whether inhibiting HDAC modulated antigen-
induced airway inflammation, BALF cell counts and concentra-
tions of cytokines were determined in naive mice and in mice
exposed to antigen and treated with TSA or dexamethasone
(Figure 5A). Naive mice treated with dexamethasone, TSA,
or DMSO showed no significant difference in basal BALF cell
counts. Exposure to antigen increased cell counts in BALF,
compared with naive mice. Dexamethasone decreased the
total cell counts in BALF of antigen-exposed mice, whereas
TSA and DMSO exerted little effect. Mice exposed to antigen
also exhibited increased eosinophil counts, as shown in Figure
5B. Although treatment with dexamethasone decreased the
numbers of eosinophils in BALF, TSA and DMSO exerted little
effect. Little difference was evident in the numbers of macro-
phages, neutrophils, or lymphocytes in untreated mice, mice
exposed to antigen, or mice treated with TSA, dexamethasone,
or DMSO. Regarding percentages of cells in BALF, the BALF
from naive mice predominantly manifested macrophages, whereas
exposure to antigen decreased the percentages of macrophages
and increased the percentages of eosinophils (Figure 5C). Col-
lectively, these data show that although both dexamethasone
and TSA inhibit antigen-induced AHR, dexamethasone but
not TSA inhibits antigen-induced airway inflammation.
Dexamethasone, but Not TSA, Inhibited Antigen-Induced
Concentrations of Cytokines in BALF
IL-4 and IL-6 are inflammatory cytokines implicated in asthma.
In naive mice, the concentrations of IL-4 and IL-6 in BALF were
negligible, regardless of treatment with dexamethasone, DMSO,
or TSA, as shown in Figure 6. Mice exposed to antigen exhibited
increased concentrations of IL-4 and IL-6 in the BAL, and
Figure 3. TSA inhibits lung resistance (RL) in naive and antigen-
exposed mice. (A) RLof naive (non–antigen-exposed), vehicle, TSA,
or dexamethasone (Dex)–treated mice (basal condition). Methacholine
(Mch) increased RLto a maximum of 2.46 6 0.1 cm H2O/ml. Treat-
ment with Dex exerted little effect on basal Mch responsiveness. TSA
markedly inhibited basal responsiveness to Mch. Data represent the
mean 6 SD for each condition derived from of 12 measurements at
each dose in three mice. *P , 0.05 (naive versus TSA-treated mice). (B)
RLof mice that were antigen-challenged and received vehicle (DMSO),
TSA, or Dex. Maximum RLin antigen-challenged mice was measured at
13.9 6 0.7 cm H2O/mL, that is, much higher than in naive mice. TSA
was as effective as Dex in abrogating Mch-induced airway hyperres-
ponsiveness (AHR). Data represent the means 6 SD derived from 12
measurements at each dose in six mice for each condition. *P , 0.05
(AF 1 TSA versus AF).1P , 0.05 (AF 1 Dex versus AF).
Figure 4. TSA reversed antigen-induced effects on lung compliance.
(A) Dynamic compliance was measured in naive mice and mice treated
with vehicle (DMSO), TSA, or Dex in response to increasing doses of
Mch measured by the Scireq FlexiVent system. TSA markedly inhibited
Mch-induced decreases in dynamic compliance. Data represent the
means 6 SD derived from 12 measurements at each dose in three mice
for each condition. *P , 0.05 (naive versus TSA-treated mice). (B)
Dynamic compliance of mice challenged with antigen or solvent (na-
ive), and of mice that received treatment (DMSO), TSA, or Dex. Data
represent the means 6 SD derived from 12 measurements at each dose
in six mice for each condition. *P , 0.05 (AF 1 TSA versus AF).
Banerjee, Trivedi, Damera, et al.: TSA Inhibits Airway Contraction in Mice and Humans 135
dexamethasone attenuated the increase in IL-4 but not IL-6.
Treatment with DMSO or TSA, however, exerted little effect
on concentrations of IL-4 or IL-6. These data suggest that
treatment with dexamethasone abrogates concentrations of an-
tigen-induced cytokines, whereas TSA exerts little effect on
concentrations of cytokines.
TSA Inhibits Carbachol-Induced Contraction in PCLS
To determine whether TSA modulates human ASM function,
PCLS were incubated with increasing doses of carbachol, and lu-
minal narrowing was determined in untreated cells and com-
pared with cells treated with 20 mM or 4 mM TSA. Carbachol
abrogated airway luminal diameters, and treatment with
TSA decreased luminal narrowing, with 4 mM TSA inhibiting
the luminal narrowing of PCLS by 55% 6 3.2%, and 20 mM
TSA inhibiting the luminal narrowing of PCLS by 68% 6 6%
TSA Inhibits the Release of Ca21in Human ASM without
Affecting the Activation of Rho
To determine whether TSA modulates excitation–contraction cou-
pling in humans, we evaluated the effects of TSA treatment on
agonist-induced Ca21sensitization and mobilization in human
ASM. Bradykinin and histamine were used as contractile agonists
instead of carbachol, because muscarinic M3 receptors (M3R)
responses are attenuated in cultured human ASM cells (28). Cul-
tured human ASM cells treated with 10 mM TSA did not show
a decrease in the activation of Rho in response to bradykinin after
incubation,asshown in Figure 7B,suggesting thatCa21sensitization
is not affected by treatment with TSA. In parallel, intracellular
Ca21([Ca21]i) was measured in human ASM cells in response
to an application of histamine (100 mM), to determine the effect
of TSA treatment on the agonist-induced mobilization of Ca21.
Virtually every untreated cell exhibited a robust and consistent
increase in [Ca21]iin response to histamine, but TSA inhibited
the histamine-triggered increases in [Ca21]iin a dose-dependent
manner without markedly altering the resting [Ca21]ilevel (Fig-
ure 7C). Together, these results suggest that TSA inhibits the
contraction of ASM by disrupting the release of Ca21into
ASM cells in response to stimulation with a contractile agonist.
We have shown that TSA, an inhibitor of HDAC activity, abro-
gates Mch-induced increases in airway resistance in both naive
and antigen-exposed mice. Moreover, we show that TSA inhibits
basal and antigen-induced sensitivity to Mch without altering
numbers of leukocytes or concentrations of cytokines in BALF.
Our experiments in human PCLS show a decrease in the
carbachol-induced contraction after treatment with TSA, and a
decrease in the agonist-induced intracellular release of Ca21,
with no effect on Ca21sensitization. Collectively, these data
suggest that the inhibition of Mch responsiveness by TSA is
not attributable to the anti-inflammatory activity of TSA, but
is rather a direct effect on agonist-induced smooth muscle
contraction. Further, the effects of TSA on AHR appear dis-
tinct from those of glucocorticoids, which modulate airway
inflammation in response to antigen. Therefore, glucocorti-
coids and the inhibition of HDAC may modulate AHR by
Figure 5. Dex, but not TSA, inhibits antigen-induced
increases in BALF cell counts. (A) The total number of cells
was obtained from bronchoalveolar lavage fluid (BALF) of
mice that were sensitized and challenged with antigen or
diluent (naive) and mice that received vehicle (DMSO),
TSA, or Dex. TSA exerted little effect on numbers of in-
flammatory cells in the BAL. Data represent the mean 6
SD for each condition, derived from the BALF of three
mice. *P , 0.05 (naive versus treatment cohort). (B) The
cellular profile of BALF from mice that were sensitized and
challenged with antigen or solvent (naive) and mice that
received vehicle (DMSO), TSA, or Dex. Data represent the
mean 6 SD for each condition, derived from the BALF of
three mice. *P , 0.05 (naive versus AF).1P , 0.05 (AF 1
Dex versus AF). (C) Percentages of cell subtypes. Data
represent the mean 6 SD for each condition, derived from
the BALF of three mice. *P , 0.05 (baseline versus treat-
136AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 462012
Our results diverge from those of Choi and colleagues (12),
who reported that in antigen-exposed mice, TSA inhibited air-
way inflammation, including numbers of eosinophils and con-
centrations of IL-4 in BAL. The dose of TSA used and/or the
timing of TSA treatment may explain the differential effects of
TSA on these parameters of inflammation. Choi and colleagues
treated mice with 1 mg/kg TSA on the first day of sensitization,
and the mice received 11 total doses of TSA over 22 days,
whereas in our study, mice were treated with 0.6 mg/kg TSA
on the day of antigen challenge, 26 days after the first sensiti-
zation treatment, and they received three doses of TSA over
3 days. A higher dose of TSA and/or a longer course of treat-
ment may exert a direct effect on airway inflammation as well as
smooth muscle contraction. However, our conclusion, that TSA
can abrogate airway sensitivity to Mch in naive mice that exhibit
no associated changes in inflammation, supports the hypothesis
that TSA may directly inhibit bronchoconstriction, and our
study of ASM showing a decrease in the intracellular release
of Ca21after TSA treatment further supports this hypothesis.
Our findings are in agreement with studies indicating that the
contraction of isolated guinea pig tracheal rings in response to
histamine, carbachol, and 5-hydroxytryptamine is abrogated by
inhibitors of HDAC (11).
Contraction in ASM is regulated by multiple mechanisms, in-
cluding release of Ca21stored in the sarcoplasmic reticulum, as
well as the modulation of Ca21sensitivity by the activation of
RhoA (29). Our finding that treatment with TSA inhibits the
agonist-induced mobilization of calcium in human ASM in
a dose dependent manner is novel, and elucidates a potential
mechanism by which TSA inhibits contractile, agonist-induced
AHR. Although our results demonstrate that TSA inhibits the
activity of HDAC in the lung, the effects on chromatin structure
and gene expression remain unclear. Expression profiling stud-
ies suggest that 2–10% of genes are modulated by HDACs (30),
and that the inhibition of HDAC can both up-regulate and
down-regulate gene expression (31). Mechanisms unrelated to
chromatin remodeling and gene expression may also contribute
to the effects of TSA on agonist-induced airway contraction.
For example, the inhibition of HDAC hyperacetylates tubulin,
decreases tubulin function, and impairs lysosome exocytosis (7).
HDAC8, a Class I HDAC, associates with a-actin, and the
inhibition of HDAC8 inhibits in turn the contraction, size,
and spreading of smooth muscle (32). Further studies evaluating
the inhibition of specific HDACs or the activity of specific genes
after treatment with TSA will elucidate other mechanisms by
which TSA inhibits ASM contraction in response to contractile
agonists. We previously showed that augmenting the activity of
HDAC by stimulating ASM cells with a combination of TNF-a
and IFN-g diminishes the acetylation of NF-kB (33). Thus, TSA
may inhibit AHR in mice and human ASM by modulating gene
expression or by altering the acetylation of nonhistone targets,
as demonstrated in the case of NF-kB.
ASM has long been a target in asthma therapy (34), and
newer therapies that disrupt ASM were recently approved
Figure 6. Dex, but not TSA, inhibits antigen-induced increases of IL-4
concentrations in BALF. Concentrations of (A) IL-4 and (B) IL-6 in BALF
were measured from mice that were challenged with antigen or diluent
(naive) and mice that received vehicle alone (DMSO), TSA, or Dex. TSA
exerted little effect on the secretion of cytokines in mice sensitized and
challenged with antigen. Data represent the mean 6 SD for each con-
dition, derived from the BALF of three mice. *P , 0.05 (naive versus AF).
1P , 0.05 (AF 1 Dex versus AF).#P , 0.05 (AF 1 Dex versus AF 1 TSA).
Figure 7. TSA inhibits the agonist-induced contraction of
precision-cut lung slices (PCLS) by decreasing the agonist-
induced mobilization of calcium in airway smooth muscle
(ASM). (A) Carbachol induced contractions of PCLS after
treatment with vehicle, TSA at 4 mm, or TSA at 20 mM. (B)
Human ASM was treated with TSA (10 mM) for 12, 24, 48,
and 72 hours, and then treated with 1 mM bradykinin for
1 minute, lysed, and assayed for active and total Rho by
immunoprecipitation and immunoblot, respectively. (C)
Fura-2 signal ratios from human ASM cells treated with
TSA at 0 (untreated), 1, 5, and 10 mM for 48 hours in
response to histamine (100 mM). In each group, approxi-
mately 20 cells were measured. In each graph, thin gray
curves represent individual responses, and the thick dark
curve represents the mean response.
Banerjee, Trivedi, Damera, et al.: TSA Inhibits Airway Contraction in Mice and Humans137
(35). Patients with asthma demonstrate increased airway con- Download full-text
striction in response to challenge with methacholine, which is
commonly used to diagnose patients with suspected asthma
(36). Our finding that the administration of TSA inhibits the
activity of HDAC as well as the mobilization of calcium in
response to contractile agonists in ASM suggests that the in-
creased sensitivity to methacholine in patients with asthma may
be an epigenetically regulated phenomenon, and that further
elucidation of the mechanism by which inhibiting HDAC
decreases the agonist-induced contraction of ASM could lead
to novel asthma therapies.
In conclusion, our experiments show that in both human and
murine models, TSA effectively inhibits lung HDAC activity
and decreases agonist-induced lung resistance in both naive and
antigen-challenged mice, and in human PCLS. Moreover, inhibi-
torsof HDAC appear to modulate airway resistance by decreasing
the release of Ca21in response to contractile agonists, a mecha-
nism unrelated to any effects on inflammation. Thus, the inhibi-
tion of HDAC may offer a therapeutic approach that inhibits
bronchoconstriction, apart from effects on airway inflammation.
Author disclosures are available with the text of this article at www.atsjournals.org.
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