Smooth Muscle Hypocontractility and Airway Normoresponsiveness in a Mouse Model of Pulmonary Allergic Inflammation

Abstract

The contractility of airway smooth muscle (ASM) is labile. Although this feature can greatly modulate the degree of airway responsiveness in vivo, the extent by which ASM’s contractility is affected by pulmonary allergic inflammation has never been compared between strains of mice exhibiting a different susceptibility to develop airway hyperresponsiveness (AHR). Herein, female C57BL/6 and BALB/c mice were treated intranasally with either saline or house dust mite (HDM) once daily for 10 consecutive days to induce pulmonary allergic inflammation. The doses of HDM were twice greater in the less susceptible C57BL/6 strain. All outcomes, including ASM contractility, were measured 24 h after the last HDM exposure. As expected, while BALB/c mice exposed to HDM became hyperresponsive to a nebulized challenge with methacholine in vivo, C57BL/6 mice remained normoresponsive. The lack of AHR in C57BL/6 mice occurred despite exhibiting more than twice as much inflammation than BALB/c mice in bronchoalveolar lavages, as well as similar degrees of inflammatory cell infiltrates within the lung tissue, goblet cell hyperplasia and thickening of the epithelium. There was no enlargement of ASM caused by HDM exposure in either strain. Unexpectedly, however, excised tracheas derived from C57BL/6 mice exposed to HDM demonstrated a decreased contractility in response to both methacholine and potassium chloride, while tracheas from BALB/c mice remained normocontractile following HDM exposure. These results suggest that the lack of AHR in C57BL/6 mice, at least in an acute model of HDM-induced pulmonary allergic inflammation, is due to an acquired ASM hypocontractility.

Full text

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ORIGINAL RESEARCH
published: 29 June 2021
doi: 10.3389/fphys.2021.698019
Edited by:
Andrew John Halayko,
University of Manitoba, Canada
Reviewed by:
Jill Johnson,
Aston University, United Kingdom
Maggie Lam,
Monash University, Australia
*Correspondence:
Ynuk Bossé
ynuk.bosse@criucpq.ulaval.ca
Specialty section:
This article was submitted to
Respiratory Physiology,
a section of the journal
Frontiers in Physiology
Received: 20 April 2021
Accepted: 04 June 2021
Published: 29 June 2021
Citation:
Boucher M, Henry C,
Dufour-Mailhot A, Khadangi F and
Bossé Y (2021) Smooth Muscle
Hypocontractility and Airway
Normoresponsiveness in a Mouse
Model of Pulmonary Allergic
Inflammation.
Front. Physiol. 12:698019.
doi: 10.3389/fphys.2021.698019
Smooth Muscle Hypocontractility
and Airway Normoresponsiveness in
a Mouse Model of Pulmonary Allergic
Inflammation
Magali Boucher, Cyndi Henry, Alexis Dufour-Mailhot, Fatemeh Khadangi and
Ynuk Bossé*
Institut Universitaire de Cardiologie et de Pneumologie de Québec – Université Laval, Québec, QC, Canada
The contractility of airway smooth muscle (ASM) is labile. Although this feature can
greatly modulate the degree of airway responsiveness in vivo, the extent by which
ASM’s contractility is affected by pulmonary allergic inflammation has never been
compared between strains of mice exhibiting a different susceptibility to develop
airway hyperresponsiveness (AHR). Herein, female C57BL/6 and BALB/c mice were
treated intranasally with either saline or house dust mite (HDM) once daily for 10
consecutive days to induce pulmonary allergic inflammation. The doses of HDM were
twice greater in the less susceptible C57BL/6 strain. All outcomes, including ASM
contractility, were measured 24 h after the last HDM exposure. As expected, while
BALB/c mice exposed to HDM became hyperresponsive to a nebulized challenge with
methacholine in vivo, C57BL/6 mice remained normoresponsive. The lack of AHR in
C57BL/6 mice occurred despite exhibiting more than twice as much inflammation than
BALB/c mice in bronchoalveolar lavages, as well as similar degrees of inflammatory
cell infiltrates within the lung tissue, goblet cell hyperplasia and thickening of the
epithelium. There was no enlargement of ASM caused by HDM exposure in either
strain. Unexpectedly, however, excised tracheas derived from C57BL/6 mice exposed
to HDM demonstrated a decreased contractility in response to both methacholine
and potassium chloride, while tracheas from BALB/c mice remained normocontractile
following HDM exposure. These results suggest that the lack of AHR in C57BL/6 mice,
at least in an acute model of HDM-induced pulmonary allergic inflammation, is due to
an acquired ASM hypocontractility.
Keywords: respiratory mechanics, airway responsiveness, mouse models, asthma, resistance
INTRODUCTION
The degree of airway responsiveness to a direct challenge, such as methacholine, is highly variable
between individuals (Sparrow et al., 1987;Woolcock et al., 1987;Rijcken et al., 1988;Kennedy et al.,
1990;Bakke et al., 1991;Paoletti et al., 1995;Ulrik, 1996). To tease out the underlying mechanisms
liable for such disparity, different strains of mice exhibiting variable degrees of responsiveness have
been widely utilized. While some strains are innately more responsive (Table 1), others rather
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Boucher et al. Acquired Smooth Muscle Hypocontractility
display a different susceptibility to develop airway
hyperresponsiveness (AHR) after exposure to offending
triggers (Table 2). These murine models have been instrumental
to deepen our understanding of the numerous elements
contributing to AHR in diseases.
One element that is mandatory for airway responsiveness
is the contraction of airway smooth muscle (ASM). On
the one hand, the contractility of ASM has been compared
between strains of mice exhibiting different innate degrees of
responsiveness. A/J mice, for example, have been shown to
be hyperresponsive because of enhanced muscarinic signaling
(Gavett and Wills-Karp, 1993) and excessive ASM shortening
(Duguet et al., 2000;Wagers et al., 2007). On the other hand, the
contractility of ASM has never been compared between mouse
strains in studies showing a different susceptibility to develop
AHR after exposure to offending triggers (Table 2). This is rather
strange. We think it is due to the false perception that assessing
airway responsiveness in vivo is a good surrogate for measuring
ASM contractility. In fact, the level of ASM contractility rarely
matches the degree of in vivo responsiveness in both mice
(Weinmann et al., 1990) and humans (Armour et al., 1984a,b;
Taylor et al., 1985;Cerrina et al., 1986;Roberts et al., 1987;
Thomson, 1987;de Jongste et al., 1988;Whicker et al., 1988).
ASM contractility should thus be regarded as one of many
elements affecting the degree of in vivo airway responsiveness
(Bosse et al., 2010).
It is also important to understand that the contractility of ASM
is labile (Auger et al., 2016). The concept of lability stipulates that
the contractility of ASM is not fixed but can rather change in
response to different interventions. This lability is independent
from a change in muscle size. It truly refers to an ASM of a
TABLE 1 | Innate degree of airway responsiveness.
References Mouse strains Readouts
Levitt and Mitzner, 1988 A/J and C3H/HeJ APTI
Levitt and Mitzner, 1989 A/J and C3H/HeJ APTI
Gavett and Wills-Karp,
1993
A/J and C3H/HeJ APTI
De Sanctis et al., 1995 A/J and C57BL/6 RL
Ewart et al., 1995 A/J and C3H/HeJ Rrs, Ers, and APTI
Longphre and Kleeberger,
1995
AKR/J and C3H/HeJ APTI
Ewart et al., 1996 A/J and C3H/HeJ Rrs and APTI
De Sanctis et al., 1997 C57BL/6 and A/J RL
Nicolaides et al., 1997 C57BL/6 and DBA/2 APTI
De Sanctis et al., 1999 A/J and C3H/HeJ RL
Duguet et al., 2000 C57BL/6, BALB/c, A/J and
C3H/HeJ
Rrs and Ers
Ackerman et al., 2005 C57BL/6 and A/J Penh
Leme et al., 2010 36 strains, including
C57BL/6 and BALB/c
RN
Berndt et al., 2011 29 strains, including
C57BL/6 and BALB/c
Penh, RN, and H
APTI, airway pressure time index; Ers, respiratory system elastance; H, tissue
elastance; Penh, enhanced pause measured by whole-body plethysmography; RL,
lung resistance; RN, Newtonian resistance; Rrs, respiratory system resistance.
given size being able to generate a force of different magnitudes
in response to a given contractile stimulus. Typical examples of
lability include the increased contractility caused by numerous
inflammatory mediators (Auger et al., 2016).
The contractile lability of ASM may obviously contribute
to the development of AHR observed in murine models of
pulmonary allergic inflammation. The extent by which it occurs
may also contribute to the different inter-strain susceptibility
to develop AHR in a context of inflammation. Herein, we
hypothesized that while ASM from BALB/c mice, a strain
generally considered vulnerable for the development of AHR
TABLE 2 | Changes in airway responsiveness induced by offending triggers.
References Mouse strains Offending
triggers
Readouts
Longphre and
Kleeberger, 1995
AKR/J and C3H/HeJ PAF APTI
Zhang et al., 1997 C57BL/6 and BALB/c OVA GLand CL
Miyabara et al., 1998 BALB/c and C3H/HeJ DEP Rrs and Crs
Brewer et al., 1999 12 strains, including
C57BL/6 and BALB/c
OVA GL
Zhang et al., 1999 BALB/c and BP2 OVA Penh
Ewart et al., 2000 C57BL/6, BALB/c, A/J,
AKR/J, and C3H/HeJ
OVA APTI
McIntire et al., 2001 BALB/c and DBA/2 OVA Penh
Takeda et al., 2001 C57BL/6 and BALB/c OVA RLand CL
Kenyon et al., 2003 C57BL/6 and BALB/c OVA Penh
Shinagawa and Kojima,
2003
C57BL/6, BALB/c, A/J,
and C3H/HeJ
OVA Penh
Whitehead et al., 2003 9 strains, including
C57BL/6 and BALB/c
OVA Penh
Adler et al., 2004 C57BL/6 and BALB/c OVA Penh, RL,
and CL
Koya et al., 2006 C57BL/6 and BALB/c OVA RLand CL
Gueders et al., 2009 C57BL/6 and BALB/c OVA Penh and RN
(or Rrs – not
clear)
Hirota et al., 2009 C57BL/6 and BALB/c OVA Rrs
Kearley et al., 2009 C57BL/6 and BALB/c OVA RL
Van Hove et al., 2009 C57BL/6 and BALB/c OVA Penh
Zhu and Gilmour, 2009 C57BL/6, BALB/c, and
FVB/NJ
OVA Penh
De Vooght et al., 2010 7 strains, including
C57BL/6 and BALB/c
TDI Rrs
Kodama et al., 2010 C57BL/6 and BALB/c OVA Rrs
Sahu et al., 2010 C57BL/6 and BALB/c HDM Rrs
Kelada et al., 2011 C57BL/6 and BALB/c Der p 1 Rrs
Chang et al., 2013 C57BL/6 and BALB/c Der f 2 Penh
Evans et al., 2015 C57BL/6 and BALB/c OVA and
Aspergillus
Rrs, RN, G
and H
Li et al., 2017 C57BL/6 and BALB/c FA and OVA Riand Re
APTI, airway pressure time index; CL, lung compliance; Crs, respiratory system
compliance; DEP, diesel exhaust particles; G, tissue damping; Ers, respiratory
system elastance; FA, formaldehyde; GL, lung conductance; H, tissue elastance;
OVA, ovalbumin; PAF, platelet-activating factor; Penh, enhanced pause measured
by whole-body plethysmography; Re, expiratory resistance; Ri, inspiratory
resistance; RL, lung resistance; RN, Newtonian resistance; Rrs, respiratory system
resistance; TDI, toluene-2,4-diisocyanate.
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(Table 2), acquires hypercontractility in response to pulmonary
allergic inflammation, the ASM from C57BL/6 mice, a strain
generally considered less susceptible for the development of AHR
(Table 2), remains normocontractile.
MATERIALS AND METHODS
Mice
Sixty pathogen-free female C57BL/6 (Jackson, Bar Harbor, MA,
United States) and 60 female BALB/c (Charles River, Saint-
Constant, PQ, Canada) mice were purchased at 6- or 7-week-old.
We chose females because they are more susceptible than males
to the development of pulmonary allergic inflammation (Melgert
et al., 2005;Blacquiere et al., 2010). They were provided food and
water ad libitum and were housed until they reached 8 weeks of
age before starting the protocol. All procedures were approved by
the Committee of Animal Care of Université Laval in accordance
with the guidelines of the Canadian Council on Animal Care
(protocol 2018-046-2).
Experimental Protocol
Mice from each strain were divided into three groups of 20
mice (Figure 1): one control group exposed to saline and two
experimental groups exposed to one of two doses of house dust
mite (HDM) extract (Dermatophagoides pteronyssinus; Greer,
Lenoir, NC, United States). The endotoxin concentration was
47.3 EU per mg of HDM extract. They were exposed by intranasal
instillation once daily for 10 consecutive days under isoflurane
anesthesia. While C57BL/6 mice received 25 µL of 0, 4, or 6 mg
of HDM extract per mL, BALB/c mice received 25 µL of 0, 2, or
3 mg of HDM extract per mL. HDM concentrations were twice
higher in C57BL/6 mice in an attempt to aggravate inflammation
and to promote AHR in this less susceptible strain (Table 2). All
outcomes were measured 24 h after the last exposure. Half of
the mice in each group (n= 10) was used to assess respiratory
mechanics and to collect bronchoalveolar lavages (BAL). The
other half was used to assess tracheal contractility and to collect
the lung for histology (Figure 1).
Respiratory Mechanics
Mice were anesthetized with ketamine (100 mg/Kg) and xylazine
(10 mg/Kg). They were then tracheotomized and connected to the
flexiVent (SCIREQ, Montreal, PQ, Canada) through an 18-gauge
cannula in a supine position. To avoid leakage, a surgical thread
was passed around the trachea and tightened to securely seal the
tracheal wall against the cannula. The mice were also paralyzed
by injecting pancuronium bromide (0.1 mg/Kg) intramuscularly.
This was to avoid spontaneous breathing during the procedure.
They were ventilated mechanically at a tidal volume of 10 mL/Kg,
at a breathing frequency of 150 breaths/min and at a positive end
expiratory pressure of 3 cmH2O.
Respiratory mechanics was assessed using the SnapShot-150
and the Quick Prime-3, two perturbation maneuvers inflicted
by the flexiVent. The volume perturbation imparted by the
SnapShot-150 is a single sine wave oscillation that allows one
to infer values for resistance (Rrs) and elastance (Ers) of the
respiratory system based on the linear single-compartment model
(Bates, 2009). The volume perturbation imparted by the Quick
Prime-3 is a composite signal, made of 13 sine waves of mutually
prime frequencies and of different amplitudes and phases, that
allows one to infer values for Newtonian resistance (RN), tissue
damping (G), and tissue elastance (H) based on the constant
phase model (Hantos et al., 1992).
The degree of airway responsiveness was assessed by
nebulizing incremental concentrations of methacholine over
25 s at 5 min intervals. The concentrations used were 0, 3,
10, 30, and 100 mg/mL for C57BL/6 mice and 0, 1, 3, 10,
and 30 mg/mL for BALB/c mice. These concentrations were
tailored for each mouse strain according to their respective
FIGURE 1 | Experimental protocol to induce pulmonary allergic inflammation. While 60 C57BL/6 mice were exposed to saline (n= 20) or either 4 (n= 20) or
6 mg/mL (n= 20) of house dust mite (HDM), 60 BALB/c mice were exposed to saline (n= 20) or either 2 (n= 20) or 3 mg/mL (n= 20) of HDM once daily for 10
consecutive days. At day 11, the degree of in vivo airway responsiveness to methacholine was measured in half of the mice within each group (n= 10) using the
flexiVent. The same mice were used to collect the bronchoalveolar lavages in order to measure cellular inflammation. The trachea was collected in the other half of
mice within each group (n= 10), also at day 11, in order to measure the contractile capacity of airway smooth muscle in response to incremental concentrations of
methacholine and potassium chloride. The left lung of the same mice was collected and processed for histology to quantify the infiltration of inflammatory cells within
the tissue, the content of airway smooth muscle, the number of goblet cells and the thickness of the epithelium.
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degree of airway responsiveness and to avoid doses that may be
causing death (Mailhot-Larouche et al., 2018). Again, respiratory
mechanics was assessed with the SnapShot-150 and the Quick
Prime-3. Each of these volume-perturbation maneuvers was
actuated 10 times in an alternating fashion after each dose,
starting 10 s after dose delivery. Eight seconds of tidal breathing
was intercalated between each volume-perturbation maneuver
to avoid desaturation. A deep inflation was also imposed after
the last volume-perturbation maneuver, ∼2 min before the
subsequent dose. The peak value for each parameter (Rrs, Ers,
RN, G, and H) after each dose were used to assess the response.
Bronchoalveolar Lavage (BAL)
One mL of phosphate-buffered saline (PBS) was injected in the
lung through the trachea and aspirated to recover the BAL. This
was repeated three times and the recovered BAL was pooled
together. The total volume was recorded and centrifuged at
500 ×gfor 5 min. The supernatant was discarded and the
pellet was resuspended in 100 µL of PBS for control groups
and 500 µL for HDM groups. Total cells in BAL were stained
with crystal violet and counted using a hemacytometer. Seventy
five thousand cells were also cytospin and stained with modified
May-Grünwald Giemsa to count the number of macrophages,
lymphocytes, neutrophils, and eosinophils.
Tracheal Contractility
The whole trachea was collected and immersed in Krebs solution
(pH 7.4, 111.9 mM NaCl, 5.0 mM KCl, 1.0 mm KH2PO4, 2.1 mM
MgSO4, 29.8 mM NaHCO3, 11.5 mM glucose, and 2.9 mM
CaCl2). It was then mounted horizontally in a 10-mL organ bath
containing Krebs solution maintained at 37◦C and coupled to a
force transducer (Harvard Apparatus, St-Laurent, PQ, Canada).
The latter measured the isometric force generated by the trachea
in response to contractile activation. A resting distending force
of 5 mN was applied. Before the contractile assays, the trachea
was subjected to a period of conditioning, during which time
it was stimulated to contract repeatedly for 5 min at 10-min
intervals with 10−5M of methacholine until a reproducible
force was recorded.
Cumulative concentration-response curves were generated
with methacholine and potassium chloride (KCl). Methacholine
was added in log increments at 5-min intervals from 10−7to 10−4
M. The concentration of KCl was doubled at 5-min intervals from
20 to 160 mM. The peak force obtained at each concentration was
used to generate the concentration-response curves. The trachea
was left untreated and washed repeatedly with fresh Krebs for at
least 30 min between methacholine and KCl.
Lung Histology
The left lung was excised and immersed in 4%
paraformaldhehyde (PFA) during 24 h for fixation. The PFA was
replaced by progressively upraising the ethanol concentration
to dehydrate the tissue. The lung was then embedded in
paraffin and cut transversally in 5 µm-thick sections. The
sections were deposited on microscopic slides and stained with
hematoxylin and eosin (H&E), Masson trichrome or Periodic
acid-Schiff (PAS) with alcian blue. They were then scanned
with a NanoZoomer Digital scanner (Hamamastu photonics,
Bridgewater, NJ, United States) at 40X.
H&E stain was performed to evaluate the infiltration
of inflammatory cells within the lung tissue. Fifteen non-
overlapping photomicrographs (1440 ×904 pixels) from three
non-contiguous lung sections were blindly scored from zero
(no inflammation) to five (very severe inflammation) by one
observer. The scores from each of the 15 photomicrographs
were averaged to obtain one value per mouse and the values
from each mouse within one group were compiled to obtain
a mean per group.
Masson trichrome was used to quantify the content of smooth
muscle within the airway wall. All bronchi cut transversally in
three non-contiguous lung sections were analyzed, representing
1–6 bronchi per mouse (3.6 ±1.5 and 3.3 ±1.2 for C57BL/6
and BALB/c mice, respectively). The content of ASM in each
bronchus was calculated by the area occupied by ASM divided
by the square of the basement membrane perimeter. A mean was
calculated for each mouse and the values from each mouse within
one group were compiled to obtain a mean per group.
Periodic acid-Schiff with alcian blue was used to assess
the number of goblet cells and the epithelium thickness.
All bronchi cut transversally in three non-contiguous lung
sections were analyzed, representing 1–7 bronchi per mouse
(4.0 ±1.9 and 2.6 ±1.5 bronchi for C57BL/6 and BALB/c
mice, respectively). The number of goblet cells within each
bronchus was divided by its basement membrane perimeter.
The epithelium thickness was analyzed by measuring the area
occupied by the epithelium divided by the basement membrane
perimeter. For both outcomes, a mean was calculated for each
mouse and the values from each mouse within one group were
compiled to obtain a mean per group.
Data Analysis
Unless otherwise indicated, data are presented as
means ±standard deviations. The parameters of respiratory
mechanics and the readouts used for the contractile assays with
excised tracheas were analyzed by repeated measures two-way
ANOVA followed by Sidak’s multiple comparison tests. One-way
ANOVA were used to compare inflammatory cells in BAL, the
infiltration of inflammatory cells within the tissue, the content
of ASM, counts of goblet cells and the epithelium thickness
between groups. All statistical analyses were performed using
Prism 9 (version 9.0.0, GraphPad, San Diego, CA, United States).
P≤0.05 was considered sufficient to reject the null hypothesis.
RESULTS
The degree of airway responsiveness to methacholine in C57BL/6
and BALB/c mice exposed to either saline or one of two
doses of HDM is depicted in Figure 2. While BALB/c mice
exposed to HDM developed AHR, C57BL/6 mice remained
normoresponsive. This was true for all parameters used to assess
respiratory mechanics, including Rrs, Ers, RN, G, and H. There
was no significant difference in the degree of AHR between doses
of 2 vs. 3 mg/mL of HDM in BALB/c mice, except for H, which
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FIGURE 2 | The degree of in vivo airway responsiveness in C57BL/6 (left panels) and BALB/c (right panels) mice exposed to either saline or one of two doses of
HDM. Phosphate-buffered saline and incremental doses of methacholine were delivered by nebulization and the changes in several parameters were used to
evaluate the degree of airway responsiveness, including: (A) respiratory system resistance (Rrs); (B) respiratory system elastance (Ers); (C) Newtonian resistance
(RN); (D) tissue damping (G); and (E) tissue elastance (H). *, #, and $ designate significant differences in BALB/c mice for saline vs. HDM 3 mg/mL, for saline vs.
HDM 2 mg/mL, and for HDM 3 vs. 2 mg/mL, respectively (P<0.05). Data are shown as means ±SD (some error bars seem absent because their length is smaller
than the symbol). n= 10 mice per group.
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FIGURE 3 | The contractile capacity of excised tracheas derived from C57BL/6 (left panels) and BALB/c (right panels) mice exposed to either saline or one of two
doses of HDM. The graphs in (A,B) show the isometric force generated by the tracheas in response to increasing concentrations of methacholine and potassium
chloride, respectively. *, #, and $ designate significant differences in C57BL/6 for saline vs. HDM 6 mg/mL, for saline vs. HDM 4 mg/mL, and for HDM 6 vs.
4 mg/mL, respectively (P<0.05). Data are shown as means ±SD. n= 10 mice per group.
reached a significantly higher value after the highest dose of
methacholine for the 3 vs. 2 mg/mL of HDM.
The isometric force generated by excised tracheas derived
from C57BL/6 and BALB/c mice exposed to either saline or one
of two doses of HDM in response to incremental concentrations
of methacholine and KCl is depicted in Figure 3. While
the repeated measures two-way ANOVA indicates that HDM
exposure significantly decreased tracheal contraction in C57BL/6
mice (p<0.0001), it did not influence contraction in BALB/c
mice. This was true for both methacholine and KCl. Post hoc
analyses demonstrate that in vivo exposure to 4 mg/mL of
HDM significantly decreased the ex vivo contraction of tracheas
in response to 10−6M of methacholine and in response to
40 mM of KCl. The same tests also demonstrate that in vivo
exposure to 6 mg/mL of HDM significantly decreased the ex vivo
contraction of tracheas in response to 10−6, 10−5, and 10−4M of
methacholine and in response to 80 and 160 mM of KCl. There
was no significant difference between doses of 4 vs. 6 mg/mL
of HDM for the contraction of tracheas derived from C57BL/6
mice, except for the highest concentration of methacholine (10−4
M), which was significantly lower for tracheas derived from mice
exposed to 6 vs. 4 mg/mL of HDM.
The number of total cells per mL of BAL in C57BL/6 and
BALB/c mice exposed to either saline or one of two doses of
HDM is depicted in Figure 4A. In both C57BL/6 and BALB/c
mice, the number of total cells increased significantly in response
to both doses of HDM. For either strain, the increases were not
significantly different between the two doses of HDM. However,
the increases caused by HDM were approximately twice greater
in C57BL/6 than BALB/c mice. The differential cell counts
in all groups are depicted in Figure 4B. In C57BL/6 mice,
macrophages, lymphocytes and eosinophils were significantly
increased by 3.1, 52.0, and 371.5-fold, respectively, in response to
4 mg/mL of HDM, and by 5.3, 104.8, and 402.8-fold, respectively,
in response to 6 mg/mL of HDM. In BALB/c mice, only
macrophages and eosinophils were significantly increased by 1.1
and 11.2-fold, respectively, in response to 2 mg/mL of HDM,
and by 1.4 and 14.5-fold, respectively, in response to 3 mg/mL
of HDM. For each cell type in either strain, the increases were not
significantly different between the two doses of HDM. Notably,
the number of eosinophils were approximately fourfold greater
in C57BL/6 than BALB/c mice after HDM exposure.
The infiltration of the lung tissue with inflammatory cells in
C57BL/6 and BALB/c mice exposed to either saline or one of
two doses of HDM is depicted in Figure 5. In both C57BL/6
and BALB/c mice, cellular infiltration increased significantly
in response to both doses of HDM. For either strain, the
increases were not significantly different between the two doses
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FIGURE 4 | Inflammatory cells in bronchoalveolar lavages of C57BL/6 (left panels) and BALB/c (right panels) mice exposed to either saline or one of two doses of
HDM. The scatter plots in (A,B) show the number of total cells/mL and the differential cell counts in percentages, respectively. Note that the scale on the y-axes in
(A) is twice greater for C57BL/6 than BALB/c mice. *Designates significantly different from saline-treated mice within the same mouse strain (P<0.05). Data are
shown as means ±SD. n= 10 mice per group. Macro, macrophages; Lympho, lymphocytes; Neutro, neutrophils; and Eosino, eosinophils.
of HDM. BALB/c mice demonstrated a greater infiltration of
inflammatory cells compared to C57BL/6 mice after exposure to
saline. However, the degree of infiltration after HDM exposure
was similar between the two mouse strains.
The ASM content within the airway wall of C57BL/6 and
BALB/c mice exposed to either saline or one of two doses of HDM
is depicted in Figure 6. The content of ASM was neither affected
by HDM nor different between the two mouse strains.
The goblet cell counts and the epithelium thickness in
C57BL/6 and BALB/c mice exposed to either saline or one of
two doses of HDM are depicted in Figure 7. In both C57BL/6
and BALB/c mice, the goblet cell counts (Figure 7B) and
the epithelium thickness (Figure 7C) increased significantly in
response to both doses of HDM. For either strain, the increases
were not significantly different between the two doses of HDM.
The goblet cell counts and the epithelium thickness were also not
significantly different between the two mouse strains.
DISCUSSION
The present study investigated the underlying mechanisms
accounting for the different susceptibility to develop AHR upon
pulmonary allergic inflammation between C57BL/6 and BALB/c
mice. Although previous studies comparing these two strains in
models of pulmonary allergic inflammation abound (Table 2),
we are aware of only one study showing such comparisons
in the model used herein (Sahu et al., 2010); i.e., intranasal
exposure to HDM once daily for 10 consecutive days without
prior intraperitoneal sensitization. This model is considered
more genuine to human pathology (Doras et al., 2018), mainly
because: (1) its route of sensitization mimics natural exposure
to airborne allergens through the nasal mucosa; and (2) HDM
is an allergen for which a large proportion of humans develops
atopy. Another unique feature of our study is that we doubled
allergen doses in the less susceptible strain in an attempt to
promote AHR by furthering inflammation. Despite increasing
BAL inflammation by more than twice and achieving same levels
of tissue inflammation, goblet cell hyperplasia and epithelium
thickness compared to BALB/c mice, C57BL/6 mice remained
normoresponsive. In contradistinction with our hypothesis, the
development of AHR in BALB/c mice and the lack thereof in
C57BL/6 mice was not due to an increased contractility of ASM in
the former and the lack thereof in the latter. It was rather caused
by an acquired hypocontractility in C57BL/6 mice.
Innate airway responsiveness (i.e., in the absence of induced
inflammation) is generally greater in BALB/c vs. C57BL/6 mice
(Levitt and Mitzner, 1989;Duguet et al., 2000;Leme et al., 2010;
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FIGURE 5 | Inflammatory cell infiltrates within the lung tissue. The images in
(A) show representative lung sections from C57BL/6 (upper panels) and
BALB/c (lower panels) mice exposed to incremental doses of house dust mite
(HDM) (from left to right). The zone enclosed by the gray square on images of
the third row is zoomed on the next image on the right. The scale bar is
250 µm for the six first images from the left and 25 µm for the last two images
on the right. An inflammatory score was assigned to each of these images
and average results for each mouse in all groups are presented in the scatter
plot shown in (B). * and # designate significantly different from saline-treated
mice within the same mouse strain and from the other mouse strain exposed
to the same treatment, respectively (P<0.05). Data are shown as medians
with 95% confidence intervals. n= 10 mice per group.
Berndt et al., 2011). Yet, the magnitude and the direction of these
different responses depend on the readout used to assess airway
responsiveness. For example, when RN(Newtonian resistance,
which is an indicator of resistance to airflow within conducting
airways) is used to assess airway responsiveness, BALB/c mice
are more responsive than C57BL/6 mice (Berndt et al., 2011).
However, when H (i.e., tissue elastance) is used to assess airway
responsiveness, BALB/c mice are sometimes more responsive
than C57BL/6 mice (Berndt et al., 2011). To add to the confusion,
when Penh (i.e., enhanced pause) is used to assess airway
responsiveness, C57BL/6 mice are sometimes equally (Whitehead
et al., 2003) or more responsive (Zhu and Gilmour, 2009;Berndt
et al., 2011;Kelada et al., 2011) than BALB/c mice. It is worth
FIGURE 6 | The content of smooth muscle within the airway wall. The images
in (A) show representative lung sections from C57BL/6 (upper panels) and
BALB/c (lower panels) mice exposed to incremental doses of house dust mite
(HDM) (from left to right). The scale bar is 100 µm. For each bronchus
analyzed, the area occupied by the airway smooth muscle (ASM) was divided
by the square of the basement membrane (BM) perimeter and average results
for each mouse in all groups are presented in the scatter plot shown in (B).
Data are shown as means ±SD. n= 10 mice per group.
mentioning though that Penh is no longer considered suitable
to assess airway responsiveness (Mitzner and Tankersley, 2003;
Adler et al., 2004;Bates et al., 2004).
In the case of acquired AHR (i.e., induced by inflammation),
the general consensus is again that BALB/c mice are more
prone than C57BL/6 mice (Table 2). A few exceptions were
reported, however, showing no significant differences between
strains (Brewer et al., 1999;Kearley et al., 2009;Kodama et al.,
2010) or even the opposite (Kodama et al., 2010;Chang et al.,
2013). In the latter cases, where it was shown that the magnitude
of the acquired AHR was greater in C57BL/6 than BALB/c
mice, it was attributed to a different route of sensitization
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FIGURE 7 | Goblet cell counts and epithelium thickness. The images in (A) show representative lung sections from C57BL/6 (upper panels) and BALB/c (lower
panels) mice exposed to incremental doses of house dust mite (HDM) (from left to right). The scale bar is 25 µm. For each of these images, the number of goblet
cells/basement membrane perimeter and the area occupied by the epithelium/basement membrane perimeter were calculated and average results for each mouse
in all groups are presented in scatter plots shown in (B,C), respectively. *Designates significantly different from saline-treated mice within the same mouse strain
(P<0.05). Data are shown as means ±SD. n= 10 mice per group.
(Kodama et al., 2010) (epicutaneous instead of peritoneal, nasal
or tracheal) or the use of an atypical adjuvant (Chang et al., 2013)
(Freund’s adjuvant with pertussis toxin instead of aluminum
hydroxide or none). The time at which the degree of airway
responsiveness is assessed after the last allergenic exposure should
also be taken into consideration, as the kinetics was reported to
differ between the two mouse strains (Whitehead et al., 2003;
Kearley et al., 2009). In particular, AHR seemed to recede more
quickly in C57BL/6 mice (Kearley et al., 2009).
In the study that has used an identical model of pulmonary
allergic inflammation as ours (Sahu et al., 2010), the degree
of airway responsiveness was greater in BALB/c than C57BL/6
mice after exposure to HDM. Unfortunately, however, the degree
of airway responsiveness was not measured in naïve (i.e., not
exposed to HDM) mice, making it impossible to compare the
magnitude of the acquired AHR between strains (Sahu et al.,
2010). Our study demonstrated that BALB/c mice, but not
C57BL/6 mice, acquire AHR in this model.
In other models of pulmonary allergic inflammation, the
increased susceptibility of BALB/c mice to develop AHR
compared to C57BL/6 mice was sometimes ascribed to an
increased propensity to accumulate inflammatory cells within the
lung tissue, either T cells (Parkinson et al., 2021), eosinophils
(Takeda et al., 2001), or mast cells (Gueders et al., 2009). In
the present study, we doubled the doses of HDM in C57BL/6
mice, which has led to a twofold and a fourfold greater increases
in BAL total inflammatory cells and eosinophils, respectively,
compared to BALB/c mice. These increased BAL cell counts in
C57BL/6 mice (Figure 4A) resulted in a degree of inflammatory
cell infiltration within the lung tissue that was equivalent to
the one observed in BALB/c mice (Figure 5). Yet, C57BL/6
remained normoresponsive, suggesting that a different degree of
infiltration of inflammatory cells within the tissue may not be
the factor accounting for the different susceptibility to develop
AHR upon pulmonary allergic inflammation between C57BL/6
and BALB/c mice.
Strangely, among all studies comparing the degree of
airway responsiveness between C57BL/6 and BALB/c mice after
exposure to offending triggers, none of them measured (and
then compared between strains) the change in ASM contractility
caused by inflammation (Table 2). A few other studies merit to be
discussed though.
One study compared the contractility of excised tracheal
segments between C57BL/6 and BALB/c mice that had been
either exposed or not to pulmonary allergic inflammation (Herz
et al., 1998). This later study did not measure the degree of in vivo
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airway responsiveness. The contractility of ASM was assessed
by measuring the response to electrical field stimulation (EFS).
More specifically, the readout to assess ASM contractility was
the frequency causing 50% (ES50) of the maximal response.
There was no difference between strains in mice not exposed
to pulmonary allergic inflammation. However, in both strains,
the ES50 decreased with pulmonary allergic inflammation,
decreasing more in BALB/c than C57BL/6 mice (Herz et al.,
1998). This would suggest an acquired ASM hypercontractility
in both strains, being more pronounced in BALB/c mice.
However, a previous study had reported similar findings in
BALB/c mice but also showed that, in contrast to ES50, the
response to methacholine was not affected by pulmonary allergic
inflammation (Larsen et al., 1992), which is consistent with
our finding. In combination, these studies suggested that the
decrease in ES50 in tracheas derived from mice exposed to
pulmonary allergic inflammation (Herz et al., 1998) is more
relevant to the neural control of their airways than a true change
in ASM contractility.
Previous comparisons of responsiveness between C57BL/6
and BALB/c mice were also performed in lung isolated perfusion
system (Held and Uhlig, 2000;Landgraf and Jancar, 2008). While
the lung from BALB/c mice was more responsive than the lung
from C57BL/6 mice in one study (Held and Uhlig, 2000), the
other study showed the opposite (Landgraf and Jancar, 2008).
The increased responsiveness in the former was not restricted
to methacholine, as the lung of BALB/c mice was also more
responsive to endothelin-1 and the thromboxane A2analog U-
46619 (Held and Uhlig, 2000). Because of these discrepancies, we
do not think, at least at this point, that these previous experiments
can help us interpreting our findings.
It is also worthy to mention that comparisons in tracheal
ASM contractility between C57BL/6 and BALB/c mice were
previously performed before and after in vitro exposures to single
inflammatory stimuli, including tumor necrosis factor α(TNFα),
lipopolysaccharide (LPS) and poly-inosinic:polycytidylic acid
(poly I:C) (Safholm et al., 2011). Consistent with our findings,
this study demonstrated no difference in ASM contractility
between C57BL/6 and BALB/c mice in untreated preparations.
The response to a muscarinic agonist (carbachol) was also not
affected by in vitro exposure to any of the tested inflammatory
stimulus in both strains. However, hypercontractility to serotonin
and bradykinin was acquired in both strains after exposure to
inflammatory stimuli, and the magnitudes of these responses
were greater in ASM from BALB/c than C57BL/6 mice. The
authors concluded that these different inter-strain responses
may contribute to the increased propensity of BALB/c mice to
develop AHR in a context of inflammation (Safholm et al., 2011).
Although this type of study is useful to assess the direct influence
of single inflammatory mediators on ASM contractility, it is
important to understand that exposure to a single inflammatory
stimulus does not recapitulate the whole spectrum of cellular
and molecular events occurring in vivo upon pulmonary allergic
inflammation. There are plenty of inflammatory mediators that
are dysregulated in a context of pulmonary allergic inflammation
that can either increase or decrease the contractility of ASM
(Auger et al., 2016;Gazzola et al., 2020). Therefore, although
Safholm et al. (2011) study clearly demonstrated, once again, that
the contractility of ASM is labile, the relevance of their findings
to the inter-strain difference obtained in the present study in
response to HDM is uncertain.
Another previous study reported different changes in ex vivo
ASM contractility induced by in vivo exposure to an allergen,
although this was not in C57BL/6 and BALB/c mice but in
ASW/SnJ and SJL/J mice (Fan et al., 1997). The latter study
did not measure the degree of in vivo airway responsiveness. It
was thus not possible to determine whether the acquired ASM
hypercontractility caused by pulmonary allergic inflammation
in SJL/J mice, and the lack thereof in ASW/SnJ, translated
into a different change in the degree of in vivo airway
responsiveness between mouse strains. Yet, this study confirmed
that ex vivo ASM contractility is modulated by in vivo pulmonary
allergic inflammation (viz. ASM contractility is labile) and this
degree of modulation is strain specific. In our study, we show
that BALB/c mice became hyperresponsive while their ASM
remained normocontractile and that C57BL/6 mice remained
normoresponsive while their ASM became hypocontractile.
It represents the second study to suggest that the different
susceptibility of C57BL/6 and BALB/c mice to develop AHR
upon pulmonary allergic inflammation is ascribed to a different
modulation of ASM contractility. The first study was the one
from Kelada et al. (2011). These authors have shown that
while BALB/c mice developed AHR in response to pulmonary
allergic inflammation, C57BL/6 mice inversely developed airway
hyporesponsiveness. They used the purified single protein Der
p 1, the immunodominant allergen from the Dermatophagoides
pteronyssinus species of HDM. The protocol consisted of
sensitizing the mice with two intraperitoneal injections without
adjuvant followed by a single orotracheal challenge. The mice
were then studied 72 h after the challenge. Their protocol
was therefore different from ours. Yet, the conclusions drawn
were very much the same. Although they did not measure
ASM contractility directly, they demonstrated that several
genes, including many G protein-coupled receptors involved in
ASM contraction, were downregulated by pulmonary allergic
inflammation in C57BL/6 but not BALB/c mice. Of particular
interest was the downregulation of the M2 muscarinic receptor.
The authors concluded that the hyporesponsiveness acquired by
C57BL/6 mice in response to pulmonary allergic inflammation
was probably due to a decreased contractility of ASM. We extend
these findings by actually showing that ASM derived from HDM-
exposed C57BL/6 mice generates less force in response to both
methacholine and KCl. Taken together, these results strongly
suggest that ASM from C57BL/6 mice becomes hypocontractile
in response to pulmonary allergic inflammation.
How normocontractile ASM can lead to AHR in BALB/c
mice and how hypocontractile ASM can lead to a normal degree
of airway responsiveness in C57BL/6 mice can be baffling for
some. However, these notions are very well understood (Bosse
et al., 2010). Hypercontractility of ASM is not required to
cause AHR (Bosse, 2021). The combined effects of pulmonary
inflammation and ASM contraction are not only additive but
usually synergistic in the manifestation of AHR (Bosse et al.,
2010). It is thus expected that in the presence of inflammation,
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a normal ASM contraction should lead to AHR, as we observed
in BALB/c mice. In fact, this was previously suggested in BALB/c
mice (Wagers et al., 2004;Wagers et al., 2007). Based on
computational modeling, Wagers et al. (2004, 2007) convincingly
demonstrated that the development of AHR upon pulmonary
allergic inflammation in BALB/c mice can be entirely explained
by airway wall thickening and small airway closure. It is
thus not totally surprising that the inter-strain difference in
the susceptibility to develop AHR upon pulmonary allergic
inflammation was not due to an acquired hypercontractility
in BALB/c mice. The lack of AHR in C57BL/6 mice was
more puzzling in that matter, since this strain clearly develops
inflammation that is often worse than the one seen in BALB/c
mice (Zhang et al., 1997;Morokata et al., 1999;Takeda et al.,
2001;Whitehead et al., 2003;Gueders et al., 2009;Van Hove et al.,
2009;Kelada et al., 2011;Evans et al., 2015). We should have
anticipated a counterbalancing phenomenon, such as an acquired
ASM hypocontractility, to explain their normoresponsiveness.
The present study has some limitations. First, we have used
the trachea to assess the contractility of ASM. The validity of
our findings thus rests on the assumption that the ASM from
the trachea is appropriate to assess the overall contractility and
that the ASM from other airways are similarly affected by HDM.
Previous studies comparing ASM derived from the trachea versus
lower airways have generally found no differences in contractility
(Gunst and Stropp, 1988;Jiang and Stephens, 1990;Ijpma et al.,
2015). However, ASM from different locations within the airway
tree are sometimes, but not always (Ijpma et al., 2015), differently
affected in asthma (Ijpma et al., 2020), heaves (Matusovsky et al.,
2016), and murine model of asthma (Donovan et al., 2013). More
precisely, while asthma (or asthma-like conditions) is sometimes
associated with increased contractility of the peripheral airways
but not with changes in tracheal contractility (Matusovsky et al.,
2016;Ijpma et al., 2020), it is sometimes associated with increased
contractility of the trachea and a decreased contractility of
peripheral airways (Donovan et al., 2013). A second limitation of
our study is that the underlying molecular mechanisms were not
investigated. For example, previous studies have shown that IL-4
and IL-13 signaling are required for the manifestation of AHR in
BALB/c mice exposed to HDM (Johnson et al., 2007;McKnight
et al., 2020). Further studies will be required to determine
whether these types of signaling pathways are ultimately shaping
the different susceptibility of C57BL/6 and BALB/c mice to
develop AHR by differently modulating ASM contractility.
CONCLUSION
Our results suggest that the lack of AHR in the presence of
pulmonary allergic inflammation may sometimes be ascribed
to a decrease in the contractility of ASM. This is relevant
to humans because not everyone with atopy or pulmonary
inflammation exhibits AHR. A failure to downregulate ASM
contractility under pulmonary inflammation may allow ASM
to remain normocontractile and, thereby, cause AHR upon
activation by synergizing with inflammation. We propose that
mechanisms downregulating ASM contractility deserve further
investigations as they may provide alternative and important
clues for treatments.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The animal study was reviewed and approved by the Committee
of Animal Care of Université Laval in accordance with
the guidelines of the Canadian Council on Animal Care
(protocol 2018-046-2).
AUTHOR CONTRIBUTIONS
All the authors edited the manuscript, and read and approved
the final manuscript. MB developed the experimental design,
performed the laboratory experiments pertaining to respiratory
mechanics, histology and the contractile assays with excised
tracheas, analyzed the data, and wrote the manuscript. CH
developed the experimental design, performed some of the
laboratory experiments pertaining to BAL and histology, and
analyzed the data. AD-M assisted in laboratory experiments
pertaining to contractile assays with excised tracheas. FK
performed the laboratory experiments pertaining to saline or
HDM exposure. YB secured funding, developed the experimental
design, analyzed the data, and wrote the manuscript.
FUNDING
This work was supported by the Natural Sciences and
Engineering Research Council of Canada (NSERC; RGPIN-2020-
06355), the Canadian Institutes of Health Research (CIHR: PJT-
387910), and the Fondation de l’IUCPQ (Institut Universitaire de
Cardiologie et de Pneumologie de Québec). FK was supported by
the Fondation de l’IUCPQ. YB was supported by FRQS (Fonds de
Recherche du Québec – Santé).
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