Roles of Bronchopulmonary C-fibers in airway Hyperresponsiveness and airway remodeling induced by house dust mite
Yang et al. Respiratory Research
Roles of Bronchopulmonary C-fibers in airway Hyperresponsiveness and airway remodeling induced by house dust mite
Zhimei Yang 1 2 3 4 5
Jianguo Zhuang 3
Lei Zhao 3
Xiuping Gao 3
Zhengxiu Luo 1 2 4 5
Enmei Liu 1 2 4 5
Fadi Xu 0 3
Zhou Fu 0 1 2 4 5
0 Equal contributors
1 Department of Respiratory Medicine, Children's Hospital of Chongqing Medical University , Chongqing , China
2 Pediatrics Research Institute, Ministry of Education Key Laboratory of Child Development and Disorders, Children's Hospital of Chongqing Medical University , No.136, Zhong Shan 2nd Road, Yuzhong District, Chongqing 400014 , China
3 Pathophysiology Program, Lovelace Respiratory Research Institute , Albuquerque, NM , USA
4 Department of Respiratory Medicine, Children's Hospital of Chongqing Medical University , Chongqing , China
5 Pediatrics Research Institute, Ministry of Education Key Laboratory of Child Development and Disorders, Children's Hospital of Chongqing Medical University , No.136, Zhong Shan 2nd Road, Yuzhong District, Chongqing 400014 , China
Background: Asthma is characterized by chronic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling. While exposure of house dust mites (HDM) is a common cause of asthma, the pathogenesis of the HDMinduced asthma is not fully understood. Bronchopulmonary C-fibers (PCFs) contribute to the neurogenic inflammation, viral infection induced-persistent AHR, and ovalbumin induced collagen deposition largely via releasing neuropeptides, such as substance P (SP). However, PCF roles in the pathogenesis of the HDM-induced asthma remain unexplored. The goal of this study was to determine what role PCFs played in generating these characteristics. Methods: We compared the following variables among the PCF-intact and -degenerated BALB/c mice with and without chronic HDM exposure (four groups): 1) AHR and pulmonary SP; 2) airway smooth muscle (ASM) mass; 3) pulmonary inflammatory cells; and 4) epithelium thickening and mucus secretion. Results: We found that HDM evoked AHR associated with upregulation of pulmonary SP and inflammation, ASM mass increase, epithelium thickenings, and mucus hypersecretion. PCF degeneration decreased the HDM-induced changes in AHR, pulmonary SP and inflammation, and ASM mass, but failed to significantly affect the epithelium thickening and mucus hypersecretion. Conclusion: Our data suggest an involvement of PCFs in the mechanisms by which HDM induces allergic asthma via airway inflammation, AHR, and airway remodeling.
Substance P; Airway smooth muscle; Airway inflammation; Airway epithelial cells
Asthma is an airway chronic inflammatory disease that is
mainly characterized by airway inflammation, airway
hyperresponsiveness (AHR), and airway remodeling [
AHR could result from excessive contraction of airway
smooth muscle (ASM), thickening of the airway wall, and
over-sensitization of sensory nerves [
], while airway
remodeling stems from an enhanced ASM mass and
thickened abnormal epithelium with mucus gland
]. Most asthma begins in childhood with
sensitized airway responses to common aeroallergens, such as
house dust mite (HDM), cockroaches, animal dander,
fungi and pollens [
]. Animal model of the
HDMinduced asthma has been well established with the key
features including AHR, airway remodeling (ASM
thickening and mucus hypersecretion), and airway
inflammation (abnormal airway eosinophilia) [
]. However, it is
unclear how HDM affects epithelial cells, such as Clara
cells and ciliated cells, and how HDM induces the airway
inflammation, AHR, and airway remodeling.
Bronchopulmonary C-fibers (PCFs) represent ∼80% of
the vagal bronchopulmonary afferents innervating the
airways and lungs. PCFs have been reported to participate in
the allergic airway inflammation, the ovalbumin-induced
pulmonary remodeling, and the RSV-induced AHR.
Previous studies showed that PCF degeneration by neonatal
capsaicin (CAP) pretreatment decreased the allergic
airway inflammation [
] and the pulmonary remodeling
characterized by collagen and elastic fiber deposition in
] in ovalbumin-induced asthma model. PCF
degeneration also eliminated the AHR induced by
respiratory syncytial viral infection [
]. These PCF effects are
thought to be achieved by the PCF-released
neuropeptides, especially the substance P (SP) that is responsible
for neurogenic inflammation, potentiation of airway
constriction, epithelial cells proliferation, and mucus
hypersecretion in asthma [
]. However, their roles in the
pathogenesis of the HDM-induced asthma remain
unexplored. While the HDM-induced asthma possesses the
asthmatic characteristics (AHR, airway inflammation and
remodeling) similar to those induced by OVA, ozone, and
virus; however, the pathological processing to cause these
characteristics is likely not the same. For example, ozone
and respiratory syncytial virus either fails or only affects
some types of inflammatory cells [
], but HDM
increases all or more types of inflammatory cells in our
pilot and previously reported studies [
9, 17, 18
HDM induces chronic airway inflammation and
remodeling with a multifaceted immune responses initiated in the
lungs, but the classical OVA model is more related to
acute airway inflammation with the Th2 skewing adjuvant
aluminum hydroxide and sensitization originated in the
]. Most importantly, HDM has shown to be
able to uniquely and directly stimulate the cell bodies of
PCFs resident in the nodose ganglion [
]. These lines of
information encourage us to test the PCF involvement in in
the HDM-induced AHR, airway remodeling, and airway
inflammation. Revealing a causal impact of PCFs on airway
remodeling including ASM, epithelial cells (Clara cells and
ciliated cells), and mucus is novel as this PCF impact was
not investigated in previous animal models. To examine our
hypothesis, we evaluated the roles of PCFs in developing
the chronic HDM exposure-induced AHR, airway
remodeling (airway smooth muscle, airway epithelial cells, mucus
secretion), and airway inflammation (BALF cells) in mice.
All animals were managed in accordance with the Guide
for the Care and Use of Laboratory Animals and approved
by the Institutional Animal Care and Use Committee
(IACUC), which is accredited by the Association for
Assessment and Accreditation of Laboratory Animal Care
International, USA (protocol# FY15-024).
Adult female and male BALB/c mice were purchased
from Charles River Laboratories, Inc. (Wilmington,
MA), housed and mated in ABSL2 animal facility at
Lovelace Respiratory Research Institute. Mouse pups
born by spontaneous vaginal delivery were housed with
their mother and siblings (24-25 °C, and 12:12 h light/
dark cycle). In all experiments, no more than three
female pups from each litter with similar overall litter size
were used in each study series to minimize the possible
effect of genetic difference between litters on the results.
Degeneration of c-fibers
Capsaicin (CAP, 50 mg/kg diluted in the solution
containing 18 μl saline, 1 μl ethanol, and 1 μl Tween 80) or
its vehicle was injected subcutaneously one or 2 days
after birth [
]. The females were exposed to HDM or
saline at 6-8 weeks old and correspondingly grouped as
CtrlIntact, HDMIntact, CtrlPCFX, and HDMPCFX. To ensure
the viability of the C-fiber degeneration after CAP
pretreatment, one drop (10 μl) of 0.01% capsaicin solution
was applied onto the cornea at 6-8 weeks old. All vehicle
pretreated mice displayed rigorous eye-wiping (> 30
wipes in 30 s), while those pretreated with CAP were
virtually unresponsive or had fewer than 5 wipes with
Female BALB/c mice pretreated with vehicle or CAP at
6-8 weeks old were exposed to HDM whole body
extracts (Greer Laboratories, Lenoir, NC, USA.
XPB82D3A25) intranasally (25 μg HDM protein diluted
in 10 μl saline, HDM group) or saline (Ctrl group) every
day for five consecutive days followed by 2 days rest and
this treatment was repeated for five consecutive weeks
]. Pups were randomly assigned to five Study Series as
Study Series I was designed to study the effects of PCF
degeneration on the HDM-induced AHR. CtrlIntact,
HDMIntact, CtrlPCFX, and HDMPCFX mice (n = 12, 12, 9,
and 9) were placed in the chamber. After stabilization,
the sRaw responses to aerosolized saline and
methacholine solutions were recorded.
Study Series II aimed to clarify the HDM effect on
pulmonary SP and its dependency on PCFs. We detected
the SP level in lung homogenates by ELISA in CtrlIntact,
HDMIntact, CtrlPCFX, and HDMPCFX mice (n = 7, 7, 5, 5).
The upper lobe of the left lung was collected and
homogenized; then supernatant was harvested.
Series III was conducted to study the effect of PCF
degeneration on the HDM-induced airway smooth muscle
remodeling. We compared the expression of airway
smooth muscle by α-SMA immunofluorescence that was
quantified by morphological analysis and western blot
among the four groups of mice (n = 5/group,
respectively). To functionally confirm the effect of PCFs on the
contractile function of airway smooth muscle, we
compared the tracheal contraction evoked by high potassium
(67 mM) and methacholine in vitro among the four
groups (n = 6/group).
Study Series IV was carried out to study the effect of
PCF degeneration on the HDM-induced airway
inflammation. Total cells and percentage of differential cell in BALF
were counted (n = 7/group) and compared among the four
groups of mice, while the morphology changes of airway
and lung were detected by H&E stain (n = 3/group).
Study Series V was applied to study the influence of
PCF degeneration on the HDM-induced changes in
airway epithelial cells and airway mucus secretion. In four
groups of mice, Clara cells (CCSP-positive) and ciliated
cells (tubulin-positive) were labeled by
immunofluorescence and quantified by morphological analysis.
Moreover, airway mucus secretion was detected by AB-PAS
satin (n = 6/group).
Measurement of airway Hyperresponsiveness (AHR)
During the final 3 days of exposure, the mice were
individually placed in a double-chamber whole body
plethysmograph (Buxco Electronics, Inc., Wilmington,
USA) for 30 min × 2 times daily for environmental
conditioning. After the final exposure, AHR was measured
in conscious mice by means of specific airway resistance
(sRaw). In brief, the mice were individually placed in the
double-chamber to assess sRaw [
]. The mice were
exposed to aerosolized saline (0 mg/ml) and methacholine
solutions in a dose-increasing manner (3.125, 6.25, 12.5,
25, and 50 mg/ml; Sigma). Each exposure was last for
1 min and apart from a 4 min-interval between the
neighboring two exposures.
Measurement of tracheal constriction in vitro
Tracheal segments were collected 48-72 h after the final
HDM or saline exposure. Following removal of the
surrounding tissues, the trachea (approximately 5 mm long)
was cut below the larynx and above the bronchial
bifurcation and gently mounted on two L-shaped metal
prongs. The lower support was fixed and the upper
support was connected to a force transducer (model FT03;
Grass Instruments, Quincy, MA, USA) linked to a
PowerLab data acquisition system (AD Instruments Ltd)
via a preamplifier. The trachea was then immersed in a
10 ml tissue bath (model 47,264, World Precision
Instruments) filled with normal physiological salt solution
(PSS) (in mM: 119 NaCl, 4.7 KCl, 1.18 KH2PO4,1.17
MgSO4, 18 NaHCO3, 0.026 EDTA, 2.0 CaCl2, 11
Glucose and 12.5 Sucrose) that was maintained at 37 °C and
constantly bubbled with 95%/5% of O2/CO2 mixture.
The resting tension of 0.5 g was applied and maintained
during a 60 min equilibration period with changes of
fresh PSS solution every 15-20 min. Following
equilibration, the trachea was challenged with a high potassium
PSS solution (67 mM KCl, in mM: 56.7 NaCl and 67
KCl, others same as above PSS). When the contraction
reached a steady state for 5-10 min, the tissue bath was
rinsed with normal PSS to completely relax the trachea.
After another 20 min equilibration at 0.5 g resting tension,
the trachea was challenged with cumulative
concentrations of methacholine (10−9-10−4 M). Concentration of
methacholine solution was increased every 10-15 min or
until the contraction approached a plateau [
]. Data are
normalized to baseline level.
Bronchoalveolar lavage fluid (BALF) and cell count
The mice were euthanized with 1:9 diluted Euthasol
(150-200 mg/kg, intraperitoneally) 48-72 h after the final
HDM or saline exposure. The trachea was cannulated
with a polyethylene tube (Becton Dickinson, Sparks,
MD). Prior to lavage, the left bronchus was tied and the
right lung was lavaged three times with 0.5 ml cold
saline for collecting BALF. Approximately 1.3 ml of the
instilled fluid was consistently recovered. The BALF
samples were centrifuged for 5 min at 1500 rpm at 4 °C.
Supernatants were decanted and immediately frozen at
−80°. Cell pellets were resuspended in saline. The total
cell numbers were counted using a hematocytometer.
BALF cell smears (100 μL) were prepared using cytospin
apparatus and stained with Diff-Quik solution (Life
Technologies, Auckland, New Zealand) to determine the
differential cells counts in accordance with conventional
morphological criteria. At least 300 cells per slide were
evaluated in order to obtain the differential cell counts.
Histology and Immunofluorescence
The left lobes were inflated with 4% paraformaldehyde
at 25 cm H2O pressure. Paraffin-embedded tissue
sections (5 μm) were then prepared and the sections were
evenly divided into three groups. Sections were stained
with hematoxylin and eosin (H&E) or stained with
Alcian blue and PAS (AB-PAS).
For immunofluorescence staining, paraffin-embedded
sections were dewaxed through xylene (2 changes) and
rehydrated through descending alcohol
(100%-95%-80%70%) to deionized H2O. Antigen recovery was performed
by using pre-heated Na-Citrate buffer (10 mM, pH 6.0)
for 10 min in the microwave. Sections were blocked in
blocking buffer (3% BSA, 0.3% Triton X-100 in PBS) at
room temperature for 1 h. Sections were incubated with
primary antibody at 4 °C overnight, incubated with
secondary antibody at room temperature for 1 h. Cover
slips were mounted on stained sections with anti-fade
reagent containing DAPI (Invitrogen, USA). The primary
antibody used was Anti-Clara cell secretory protein (CCSP
also known as CC10 OR CC16) (1:2000 dilution, 07-623,
EMD Millipore), monoclonal anti-acetylated tubulin
(1:10,000 dilution, T7451, Sigma), and monoclonal anti-α
smooth muscle actin (1:400 dilution, A 2547, Sigma). The
secondary antibody used was Alexa Fluor labeled goat
anti-rabbit antibody, goat anti-mouse antibody (1:200
dilution, A-11008, A-11005, Thermo Fisher, USA).
Digital images of the immunohistochemistry were
obtained with the use of a light microscope (ZEISS)
equipped with a digital camera (ZEISS) linked to a
computer, and then analyzed with the use of ImageJ software
(NIH, Bethesda, MD, USA). This was used because
histologic analysis reduces three-dimensional structures to two
dimensions in which volumes become areas and surfaces
become lines. The ratio of tissue area to the length of the
basement membrane was used to express the ratio of the
volume to the surface area (V:SA) or the thickness of the
airway wall and its compartments [
Airway smooth muscle remodeling was evaluated by
measuring the thickness of airway smooth muscle,
defined by the ratio of the α-SMA positive area to the
length of the subepithelial basement membrane. The
expression of epithelial cells (Clara cells and ciliated cells)
was evaluated by measuring the ratio of the volume to
the surface area (V:SA), which is defined by the ratio of
the epithelial cell positive area to the length of the
subepithelial basement membrane. Five [
] airways per
mouse were quantified and averaged.
The SP levels in lung homogenates were measured by
commercial ELISA kit (Cayman). The protocols were
followed according to the manufacturer’s instructions.
The upper lobe of the left lung was collected, ground
and homogenized with M2 lysis buffer (v:v 1:9)
containing protease inhibitor PMSF, followed by centrifugation
at 13000 rpm for 5 min at 4 °C. The supernatant was
harvested and stored at −80 °C.
The trachea and bronchi tissues were homogenized with
M2 buffer (20 mM Tris-HCl pH 7.6, 0.5% NP40,
250 mM NaCl, 3 mM EDTA, 2 mM DTT, 0.5 mM
phenylmethylsulfonylfluoride, 20 mM β-glycerophosphate,
1 mM sodium vanadate, and 1 μg/ml leupeptin). Protein
concentrations were detected by using the BCA assay
reagent (Bioteke). Equal quantities of protein homogenates
were run in a 12% SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis) and then transferred
to PVDF membranes (Millipore, Billerica, MA). The
membranes were probed with primary antibody
overnight at 4 °C and followed by a second antibody for 1 h
at room temperature. Signals were detected by enhanced
chemiluminescence according to the manuals (Millipore,
Billerica, MA). The band density was quantified using
ImageJ software (NIH, Bethesda, MD, USA) and
normalized relative to GAPDH. The primary antibody used was
monoclonal anti-α smooth muscle actin (1:2000 dilution,
mouse, A 2547, Sigma), GAPDH (1:2000 dilution, mouse;
sc-32,233, Santa Cruz, CA, USA). The secondary antibody
used was alkaline phosphatase-conjugated goat
antimouse antibody (1:2000 dilution, Santa Cruz, CA, USA).
Data were analyzed using GraphPad Prism 6.0 software
(GraphPad, San Diego, CA) and presented as mean ±
SEM. Statistical significance was assessed by two-way
analysis of variance (ANOVA) to assess significant
differences of the tested variables among the four groups.
Tukey’s test was utilized for specific comparisons
between individual groups. Any P values < 0.05 were
considered statistically significant.
PCF degeneration decreased the HDM-induced AHR.
To investigate whether PCF degeneration affects the
HDM-induced AHR, we compared the responses of
airway resistance to increasing doses of methacholine
challenge among CtrlIntact, HDMIntact, CtrlPCFX, and
HDMPCFX mice. sRaw responses to methacholine
concentrations at 6.25, 12.5, 25, and 50 mg/ml were
significantly higher in HDM than Ctrl mice in the intact group
(Fig. 1). Interestingly, this HDM-evoked AHR was
almost eliminated by PCF degeneration. In contrast,
PCF degeneration did not markedly alter the sRaw
response in Ctrl mice. It is worthy to note that no
significant difference of body weights (19.7 ± 0.9 g vs. 19.8
± 0.7 g) and baseline ventilation (65.3 ± 6.4 ml/min vs.
63.6 ± 8.8 ml/min) was observed between CtrlPCFX and
CtrlIntact groups, pointing to the lack of PCF
degeneration impact on normal physiological function.
PCF degeneration reduced pulmonary substance P levels.
To understand HDM effect on the PCF-originated
pulmonary SP, we tested the SP levels in the lungs using
ELISA in the four groups of mice. As shown in Fig. 2,
HDM profoundly elevated pulmonary SP in the intact
mice, while this HDM-evoked response disappeared
after PCF degeneration (no significant difference of SP
between HDMPCFX and CtrlPCFX mice). Furthermore,
the SP level in Ctrl mice was significantly lowered after
PCF degeneration. These data suggest a PCF-origination
of the pulmonary SP, especially SP response to HDM.
PCF degeneration attenuated the HDM-induced airway smooth muscle remodeling.
Airway smooth muscle plays an important role in AHR
of asthma. In order to investigate the effect of PCF
degeneration on expression of airway smooth muscle, we
used α-SMA to label airway smooth muscle by
immunofluorescence. We found the highest density and
continuousness of airway α-SMA expression in HDMIntact mice
among the four groups (Fig. 3a). In other words, α-SMA
expression in the airway was increased by HDM and the
response was diminished by PCF degeneration.
Statistically, the thickness of airway smooth muscle was
significantly higher in HDMIntact than CtrlIntact mice and the
thickened airway smooth muscle by HDM disappeared
after PCF degeneration (i.e., no difference between
CtrlIntact and HDMPCFX; Fig. 3b). The similar results
were also observed in expression of airway smooth
muscle quantified by western blot (Fig. 3c).
PCF degeneration failed to affect tracheal contraction evoked by high potassium and methacholine in vitro.
In this study, we asked if HDM-exposure would increase
the contractility of airway smooth muscle in response to
high potassium (67 mM) and methacholine in vitro and
what the role of PCFs was in the contractility.
Unexpectedly, we observed that the tracheal contraction evoked
by high potassium (67 mM) was similar in the four
groups (Fig. 4a). Moreover, the contraction of the
tracheal ring was gradually increased during exposure to
methacholine concentrations from 10−9 M to 10−4 M as
exhibited in Fig. 4b and c. The contraction increased
obviously at methacholine 10−7 M, and approached plateau
at 10−5 M, but there was no significant difference
between the responses among the four groups.
PCF degeneration altered the HDM-induced airway inflammation.
Airway chronic inflammation is an evident character
of asthma. Figure 5a showed that HDM exposure
significantly doubled total BALF cells and this response
was markedly reduced by PCF degeneration. In
contrast, PCF degeneration did not change the total
BALF cell count in Ctrl mice. With respect to the
cell differentiations, HDM exposure strikingly
elevated the percentages of lymphocytes, eosinophils
and neutrophils in total BALF cells, especially
eosinophils, and these changes were unaffected by PCF
degeneration (Fig. 5b). We also examined the
absolute cell number in each cell type and found that
PCF degeneration significantly reduced the absolute
cell number of neutrophils in BALF with little
changes in other cell types (Fig. 5c).
Morphologically, cell infiltration in the airways and lungs induced
by HDM exposure were not profoundly altered by
PCF degeneration (Fig. 5d).
PCF degeneration did not significantly affect the HDMinduced changes in epithelial cells and mucus secretion.
To quantify the change of epithelial cells induced by
HDM and the role of PCFs in these changes,
CCSPlabeled Clara cells and tubulin-labeled ciliated cells in
the airways were stained by immunofluorescence (Fig. 6a
and b). As presented in Fig. 6a and c, the layer of
CCSPpositive Clara cells was significantly thickened and
highly hypertrophied with stratified-like aspect by HDM
exposure with limited effect produced by PCF
degeneration. Different from CCSP-labeled Clara cells, neither
HDM exposure nor PCF degeneration affected the
expression of tubulin-positive ciliated cells (Fig. 6b and d).
Fig. 4 Tracheal contraction evoked by high potassium (67 mM) and methacholine in vitro. a High potassium (67 mM) contractile response of
tracheal ring. b Tracheal ring contractile response curves to 10−9-10−4 M methacholine. c The methacholine-induced contractile response normalized
to the high potassium (67 mM) response. Data are presented as mean ± SEM. Numbers of mice were six per group
Parallel to the data of CCSP-positive Clara cells, HDM
exposure induced a remarkable increase in mucus
production independent of PCFs (Fig. 6e).
PCF degeneration has shown an abolishment of airway
resistance response to allergic stimulations by ovalbumin
] and ozone [
]. Our finding that PCF degeneration
eliminated the HDM-induced AHR convincingly
demonstrates a critical involvement of PCFs in the AHR
response to HDM exposure, which is novel. To probe the
mechanisms underlying the PCFs’ contribution to the
HDM-induced AHR, we detected the level of pulmonary
SP. The latter is primarily synthesized in the cell bodies
of PCFs [
] and released locally from peripheral sensory
terminals upon stimulation [
]. It is responsible for
airway/lung neurogenic inflammation, promotes ASM
contraction, AHR, mucus hypersecretion and inflammation
in asthma [
13, 14, 16
]. In fact, blockade of vesicle
release/recycling machinery of lung sensory neurons to
minimize neuropeptides’ release (including SP) inhibits
the allergic AHR induced by ovalbumin [
]. In the
present study, we found a PCF-dependent increase of
pulmonary SP in HDM-exposed mice, supporting a
stimulatory impact of HDM in the release of SP from
PCFs. It has been unclear how PCFs participate in the
AHR to HDM-exposure; however, two factors are likely
involved. First, the cell bodies of PCFs residing in the
nodose ganglion could be stimulated by HDM acting via
PAR2 receptors in mice [
]. Interestingly, PAR2 is
expressed in PCFs and its activation synergizes the
transient receptor potential vanilloid 1 (TRPV1)-mediated
]. Second, vagal TRPV1 may be responsible
for the HDM-induced AHR because selective ablation of
TRPV1-neurons in the vagal ganglion completely
prevented AHR induced by ovalbumin [
]. Recent reports
have revealed an interaction of SP receptor (neurokinin
1 receptor, NK1R) and TRPV1. The majority of TRPV1
positive neurons co-express NK1R, and NK1R can
potentiate TRPV1 activity [
]. Therefore, we reason
that the critical role PCFs play in the HDM-induced
AHR is, at least in part, realized by the direct HDM
stimulation of PCFs via acting on PAR2 and PCFs’
released SP acting on NK1R to synergize the TRPV1.
We next tested the role of PCF degeneration on ASM
change induced by HDM. Similar to a previous report in
], we found that HDM increased AMS thickening
and more importantly that PCF degeneration almost
eliminated the HDM-induced ASM thickening,
indicating PCF involvement in the mechanism of ASM
remodeling. The assumption of the PCF involvement in the
development of ASM thickening by releasing SP is
supported by following findings. First, SP can induce ASM
cell proliferation in vitro [
]. Second, SP induces
ASM contraction  to produce mechanical force on
airway structures that may, in turn, evoke airway
remodeling, such as elevated ASM thickening [
]. To reveal
the effect of HDM on the contractile function of ASM,
we examined tracheal contraction in vitro. Unexpectedly,
HDM exposure did not increase tracheal contraction
response to cholinergic agonist (methacholine). This
finding is consistent with a report showing no difference
between tracheal response to methacholine in ovalbumin
immunized and nonimmune mice, although
acetylcholine (ACh) release was significantly increased in the
]. In fact, the dominant role of PCFs in the
HDM-induced AHR as noted in this study may account
for the lack of difference in tracheal contraction
response to methacholine in vitro. In other words, PCF
tonic over-excitation required for the HDM-induced
AHR is absent in vitro. Additionally, bronchial
constriction is known to be the major contributor to the AHR
], but its contribution was not examined in vitro in
the present study. Recent studies reveal a crucial role of
the abnormality of Abelson tyrosine kinase [
glycogen synthase kinase 3 beta [
] in generating
ASM hyperplasia (airway remodeling) and airway
inflammation in asthmatic mice models. Further
investigation is needed to verify whether this abnormality is
involved in HDM-induced airway inflammation and ASM
hyperplasia and whether PCF degeneration is able to
prevent or diminish this abnormality.
Airway remodeling also includes thickened abnormal
airway epithelium with mucus gland hypertrophy that is
characterized by hypersecretion in goblet cells in
patients with asthma. In mice, Clara cells are the
predominant secretory cell type of airway epithelial cells
]. It is known that ovalbumin-exposure can induce
mucous metaplasia of airways in Clara cells and the
latter produces mucin in mice [
]. However, it is
unknown whether and how HDM-exposure affects Clara
cells although HDM-induced mucus hypersecretion has
been identified in mice. Thus, we inspected the
morphological changes of epithelial cells (Clara cells and ciliated
cells) and mucus secretion in HDM-exposed airways.
Interestingly, HDM exposure induced Clara cell layer
thickening and mucus hypersecretion without a
significant change in the thickness of ciliated cells layer. The
pathophysiological functions of these changes evoked by
HDM exposure are unclear but may be related to
strengthening airway epithelium defensive barrier.
Previous studies have shown anti-inflammatory functions of
CCSP in asthma, instead of labeling Clara cells , and
the protective influence of Clara cells on airway
epithelium via secreting surfactant proteins and
], along with trapping inhaled pathogens
by mucus. Interestingly, we found that PCF degeneration
did not reverse the changed Clara cells and mucus
hypersecretion induced by HDM, suggesting that airway
epithelium may not be the main target of PCFs in the
HDM-induced asthma model.
Airway inflammation is a prominent feature of
asthma, involving multiple inflammatory cells and
cellular mediators. These inflammatory cells in asthmatic
airways include eosinophils, T lymphocytes,
macrophages, and neutrophils [
]. We found that HDM
exposure increased inflammatory cells including
elevation of total BALF cells and absolute numbers of
eosinophils, lymphocytes, neutrophils and macrophages
in BALF, which is similar to a previous report [
The new finding here is that PCF degeneration
significantly reduced the total BALF cells and the number of
neutrophils. Our finding demonstrates a modulatory effect
of PCFs on airway inflammatory cell response to HDM,
confirming a neuro-immune interaction in asthmatic
development. Neuropeptides are suggested as the key
mediators of a neuro-immune pathway . It is well
documented that SP mainly released from PCFs mediates the
neurogenic inflammation in the airways [
contributes to the neuro-immune crosstalk in asthma [
Therefore, the PCF degeneration-induced changes in
inflammatory cell responses to HDM exposure may be
related to SP. We conclude that PCFs modulate
neurogenic inflammation by releasing neuropeptides, such as
SP, and participate in the mechanism of allergic airway
inflammation induced by HDM.
In summary, our major findings in this study are that PCFs
degeneration: 1) decreases the HDM-induced AHR with
the pulmonary SP response to HDM diminished; 2) reduces
HDM-induced ASM mass in airway remodeling; 3) lowers
BALF cells, especially neutrophils, in response to HDM
without effect on the percentage of different cells; and 4)
fails to significantly affect the epithelium thickening and
mucus hypersecretion. Our results have translational
significance. In the clinical setting, inhaled corticosteroids are
currently the most effective anti-inflammatory medication;
however, it has limited effect on protecting against airway
]. Our data show the key role PCFs play
in the genesis of allergic asthma, which highlights a new
potential way to therapeutically intervene ASM remodeling
by inhibition of PCFs.
AHR: Airway hyperresponsiveness; ASM: Airway smooth muscle;
BALF: Bronchoalveolar lavage fluid; CAP: Capsaicin; CCSP: Clara cell secretory
protein; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HDM: House
dust mite; NK1R: Neurokinin receptor 1; PAR2: Protease-activated receptor 2;
PCFs: Bronchopulmonary C-fibers; PSS: Physiological salt solution;
SP: Substance P; sRaw: Specific airway resistance; TRPV1: Transient receptor
potential vanilloid 1
We thank Dr. Yong Lin and Wenshu Chen for project coordination and
technical assistance throughout the course of this work. The authors are
grateful to Ellen Blake for editing.
This work was supported by National Institutes of Health grants (HL-119683)
and Respiratory Group funding in Children’s Hospital of Chongqing Medical
Availability of data and materials
Please contact author for data requests.
ZY had full access to all of the data in the study and takes responsibility for
the integrity of the data and the accuracy of the data analysis, including and
especially any adverse effects. JZ contributed to the study design, data
analysis and interpretation, and the writing of the manuscript. LZ, XG
contributed to study design and data collection. ZL and EL contributed to
drafting the manuscript. FX and ZF have seen the original study data,
reviewed the analysis of the data, approved the final manuscript, and are the
authors responsible for archiving the study files. All authors read and
approved the final manuscript.
All animals were managed in accordance with the Guide for the Care and
Use of Laboratory Animals and approved by the Institutional Animal Care
and Use Committee (IACUC), which is accredited by the Association for
Assessment and Accreditation of Laboratory Animal Care International, USA
Consent for publication
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