(2022) 23:165
Le et al. Respiratory Research
https://doi.org/10.1186/s12931-022-02078-7
Open Access
RESEARCH
Deficiency of leukocyte‑specific protein
1 (LSP1) alleviates asthmatic inflammation
in a mouse model
Nguyen Phuong Khanh Le1,2, Amanda Florentina do Nascimento1, David Schneberger4, Chi Cuong Quach1,
Xiaobei Zhang4, Gurpreet K. Aulakh1,3, Wojciech Dawicki4, Lixin Liu5, John R. Gordon4 and Baljit Singh1,6*
Abstract
Background: Asthma is a major cause of morbidity and mortality in humans. The mechanisms of asthma are still not
fully understood. Leukocyte-specific protein-1 (LSP-1) regulates neutrophil migration during acute lung inflammation.
However, its role in asthma remains unknown.
Methods: An OVA-induced mouse asthma model in LSP1-deficient (Lsp1−/−) and wild-type (WT) 129/SvJ mice
were used to test the hypothesis that the absence of LSP1 would inhibit airway hyperresponsiveness and lung
inflammation.
Results: Light and electron microscopic immunocytochemistry and Western blotting showed that, compared with
normal healthy lungs, the levels of LSP1 were increased in lungs of OVA-asthmatic mice. Compared to L sp1−/− OVA
mice, WT OVA mice had higher levels of leukocytes in broncho-alveolar lavage fluid and in the lung tissues (P < 0.05).
The levels of OVA-specific IgE but not IgA and IgG1 in the serum of WT OVA mice was higher than that of Lsp1−/− OVA
mice (P < 0.05). Deficiency of LSP1 significantly reduced the levels of IL-4, IL-5, IL-6, IL-13, and CXCL1 (P < 0.05) but not
total proteins in broncho-alveolar lavage fluid in asthmatic mice. The airway hyper-responsiveness to methacholine in
Lsp1−/− OVA mice was improved compared to WT OVA mice (P < 0.05). Histology revealed more inflammation (inflammatory cells, and airway and blood vessel wall thickening) in the lungs of WT OVA mice than in those of L sp1−/− OVA
mice. Finally, immunohistology showed localization of LSP1 protein in normal and asthmatic human lungs especially
associated with the vascular endothelium and neutrophils.
Conclusion: These data show that LSP1 deficiency reduces airway hyper-responsiveness and lung inflammation,
including leukocyte recruitment and cytokine expression, in a mouse model of asthma.
Keywords: Macrophages, Leukocytes, LSP1, Asthma model
Background
As one of the most prevalent chronic respiratory diseases, asthma is responsible for huge economic losses
and high mortality [1]. The pathogenesis of this disease
*Correspondence: baljit.singh@usask.ca
6
Western College of Veterinary Medicine, University of Saskatchewan,
Saskatoon S7N5B4, Canada
Full list of author information is available at the end of the article
is complicated because of the combination of genetic
and environmental factors [2, 3]. Consensus statements
regarding the various phenotypes and endotypes of
asthma have been developed by ATS/ERS [4]. Asthmatic
patients have symptoms such as chest tightness, shortness of breath, wheezing, and coughing, especially early
in the morning or during the night. The clinical signs
during an asthma attack are an outcome of increased
amounts of mucous in the airways, narrowing of the
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Le et al. Respiratory Research
(2022) 23:165
airway lumen, contraction of hypertrophied smooth
muscles and inflammation. The most commonly used
mouse model of ovalbumin (OVA)-induced asthma mimics acute asthma [5].
Several changes are observed in the airways of asthmatic lungs. Firstly, there is exuberant migration of
inflammatory cells including T lymphocytes, eosinophils, macrophages, and neutrophils into the lung. Secondly, airway albumin levels are significantly increased
across the spectrum of asthma severity and are correlated
with tryptase levels [6–9]. Thirdly, the airway epithelium suffers pathologic changes characterized by shedding of ciliated columnar cells and goblet and squamous
cell metaplasia. The sub-epithelial basement membrane
thickens due to fibroblast activation and deposition of
extracellular matrix (e.g., collagen) [10]. Fourthly, mucus
plugging is a common feature in the case of acute asthma
[11]. The main reason for high mortality in asthmatic
patients is airway obstruction due to airway hyperresponsiveness (AHR) and mucus hypersecretion by goblet
cells, causing asphyxia [12, 13].
There is a clinical and pathologic correlation between
the eosinophilic and neutrophilic inflammation and
the severity of asthma [11]. The tracheal mucus aspirated from acute severe asthmatic humans has more
aggregated neutrophils than eosinophils, consistent
with increased levels of the neutrophil chemoattractant
CXCL8/IL-8 [14]. In vitro data suggests that human neutrophil elastase enhances eosinophil degranulation and
eosinophil cationic protein production [15]. In chronic
asthma, sputum eosinophil percentages are strongly
associated with reduced forced expiratory volume (FEV1)
values. Similarly, sputum neutrophil percentages are positively correlated with older age and lower levels of the
pre-bronchodilator FEV1 [16, 17]. Thus, neutrophilic airway inflammation is thought to play a major role in the
progression of persistent airflow limitation in asthma
[16]. It is interesting that most asthma treatments neither control neutrophil migration in severe cases [17] nor
hasten clearance of neutrophils [18]. The lack of effective
treatment for the 5–10% of cases that comprise severe
asthma account for bulk of the asthma-related healthcare
costs [19]. Inflammatory mediators such as IL-4, IL-13
and CXCL1 have important regulatory roles in asthmatic cell recruitment and activation [20, 21]. It appears
that excessive migration of neutrophils and eosinophils
may underlie the inflammation-associated structural and
physiologic changes in the asthmatic lung. Therefore,
a better understanding of molecular regulation of their
migration may provide better ways of managing asthma.
Leukocyte-specific protein 1 (LSP1), discovered in
1988 in lymphocytes [22, 23] and initially named lymphocyte-specific protein 1, is now found in monocytes,
Page 2 of 16
macrophages, neutrophils, and endothelium [22–27].
The varied functions of LSP1, in various organs and in
distinct contexts, are still complicated and poorly understood. This protein plays an important role in leukocyte
chemotaxis in inflamed organs [28]. We reported that
the absence of LSP1 moderated endotoxin-induced acute
lung inflammation in a mouse model and reduced migration of neutrophils into the lungs [29]. Although there
were no differences in MAPK phosphorylation between
endotoxin challenged Lsp1-/- and WT mice, our data
pointed towards a direct role of phosphorylated LSP-1
in modulating neutrophil cytoskeleton [29]. Increased
expression of LSP1 has been implicated in Neutrophil
Actin Dysfunction disorder, which is a rare immunologic condition [30], but LSP1 has also been implicated
in T-cell migration in rheumatoid arthritis [31]. Because
we still don’t fully understand the mechanisms that regulate migration of neutrophils and eosinophils in asthma,
we tested a hypothesis that, in a mouse model of OVAinduced asthma, deficiency of LSP1 will suppress airway
inflammation by inhibiting inflammatory cell recruitment into the lungs. We found that Lsp1−/− asthmatic
mice showed significantly decreased inflammatory cell
emigration into the lungs and lower levels of related
cytokines in broncho-alveolar lavage (BAL) fluids, as well
as serum IgE, airway hyperresponsiveness (AHR), and
histopathology.
Methods
Murine asthma model and airway hyperresponsiveness
(AHR) measurement
LSP1-deficient (Lsp1−/−) mice were generated by Dr.
Jenny Jongstra-Bilen and colleagues on the background
of 129/SvJ mice at the University of Toronto [32, 33].
Both WT and the LSP knockout strains were transferred to and bred in the Laboratory Animal Services
Unit at the University of Saskatchewan. The mice used
in this study were produced just after the backcrossing
and genotyping of L
sp1−/−. Sixteen-week-old male wildtype (WT) 129/SvJ and Lsp1−/− mice were used (n = 6
mice per treatment group). All the animal experiments
were approved by the University of Saskatchewan’s Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines. All mice were
housed in a 12-h dark/light cycle, were fed a standard
laboratory diet in the Laboratory Animal Services Unit
at the University of Saskatchewan and allowed to acclimatize for one week before treatment. The OVA-induced
asthma mouse model was designed as described previously [34, 35]. In general, mice were injected intraperitoneally (i.p.) with 2 µg OVA/2 mg alum twice, two weeks
apart; two weeks they were given three aerosol challenges with 1% OVA in saline for 20 min per day, 2 days
Le et al. Respiratory Research
(2022) 23:165
apart. Two weeks after the final aerosol challenge, AHR
to methacholine (MCh) was determined using a headout plethysmograph and a small animal ventilator (Kent
Scientific, Litchfield, CT) and changes in the airflow were
monitored with a flow sensor (TRS3300; Kent Scientific)
linked via a preamplifier and A/D board (Kent Scientific)
to a computer-driven real-time data acquisition/analysis
system (DasyLab 5.5; DasyTec USA, Amherst, NH). [35,
36]. AHR data reflects 50% point in the expiratory cycle
(Flow@50%TVe1) responding to the aerosols of saline
0.9%, then doubling doses of MCh (1.5–25 mg/mL) which
enhance airway contraction [37, 38]. The next day all
mice were challenged with 1% OVA in saline aerosols at
a delivery rate of 0.5 L/min in an enclosed flow-through
chamber for 20 min using an ultrasonic nebulizer (UltraNeb 99 by Devilbiss, Somerset, PA). After 24 h, the mice
were euthanized with 200 mg/kg ketamine hydrochloride
(Vetalar® injection U.S.P, Bioniche, Belleville, ON, Canada) and 10 mg/kg xylazine (Rompun®, Bayer, Toronto,
ON, Canada) followed by collection of blood, BAL fluids
and lung tissues.
Blood and broncho‑alveolar lavage cell counts
BAL fluid was collected as described previously [29].
Briefly, the trachea was exposed and the airways were
lavaged with 1.5 mL of cold sterile 0.1 M PBS supplemented with 0.01% bovine serum albumin. The BAL
fluid was centrifuged at 1500g for 10 min at 4 °C and the
supernatants collected and stored at − 80 °C for protein,
chemokine, and cytokine detection. The BAL fluid total
leukocytes were counted and the cells resuspended at
106 cells/ml in 0.1 M PBS. One hundred µL of each sample was cytospun onto a microscope slide, and the cells
were stained with Hemacolor stain kit (EMD Chemicals,
Gibbstown, NJ, USA) for differential leukocyte counts (4
random fields at 400 × magnification).
Peripheral blood was collected into heparinized tubes
by cardiac puncture. The total number of leukocytes/ml
of blood was assessed after erythrocyte hemolysis with
2% acetic acid [39]. Simultaneously, a blood smear was
stained with Hemacolor stain kit for differential leukocyte count in 10 fields at 400 × magnification.
Cytokine and chemokine analyses in BAL fluid
BAL fluid levels of interleukin 4 (IL-4), IL-5, IL-6, IL-13,
IL-17, interferon-γ (IFN-γ), CCL11 (eotaxin-1) and
CXCL1 (keratinocyte-derived chemokine) were quantified using Bio-Plex Pro assays kit (Bio-Rad, Mississauga,
ON, Canada), following the manufacturer’s instructions.
Briefly, 96-well plates were washed with Bio-Plex assay
buffer before multiplex bead working solution was added.
Then beads were washed, after which diluted standards and samples were added to the wells. Detection
Page 3 of 16
antibodies were next added, followed by 1 × streptavidinPE. Washing unbound proteins with Bio-Plex wash buffer
was done in between each step. The plate was read on the
Bio-Plex system (Bioplex 200 Luminex machine with Bioplex manager 6.1 software).
Enzyme‑linked immunosorbent assay (ELISA) measuring
OVA‑specific IgA, IgG1, IgE
ELISA was used to detect OVA-specific antibody IgA,
IgG1 [35] and IgE in heparin anticoagulated plasma.
Briefly, ELISA plates were coated with 100 μL of OVA
(10 μg/mL) in coating buffer overnight at 4 °C. Nonspecific binding was blocked by incubating plates with 200
μL of 10% fetal calf serum in 0.1 M PBS for two hours at
room temperature. Then 100 μL of mouse serum samples diluted in blocking buffer were added and incubated
overnight at 4 °C. Following that 100 μL of biotinylated
anti-IgA/IgG1/IgE detecting antibody (0.5 − 2.5 μg/mL
in PBST) was added to the plate and incubated at room
temperature for 90 min. Wells were then incubated with
streptavidin-HRP conjugated following ABTS substrate
to develop color. Plates were washed five times with
PBST in between each step. A stop solution was added to
reduce variability when reading the plate at OD 405 nm.
Histopathological and pulmonary vascular permeability
analysis
Mouse lungs were processed as described [29]. The right
bronchus was ligated with a thread before the intratracheal instillation of 1 mL of cold 4% paraformaldehyde
into left lung in situ. After the left lung was inflated, the
right lung was cut and stored at − 80 °C for further analysis. The left lung was immersion fixed in 4% paraformaldehyde, processed, and embedded in paraffin in three
pieces. The sections taken from all three pieces (5 µm
thickness) were placed on poly-l-lysine coated glass slides
and stained with hematoxylin and eosin for histopathological examination. Histopathology scoring was adopted
from the previous description [40]. Briefly, the thickness
of the bronchiolar and blood vessel walls, which we used
to represent the thickness of the smooth muscle layer,
was determined as the average distance between the
inner edge to the outer edge of the wall at four different
places on each sample. The inflammatory cells infiltrating out of the blood vessels and goblet cells along the
bronchiolar epithelium were quantified. All lung sections
were evaluated at 1000 × magnification and scored using
a 4-point scale, as follows: 0, normal lung architecture; 1,
minimal, a diffuse reaction in alveolar walls, congestion,
1 − 10 immune-cells/field in peribronchiolar vascular
space; 2, mild, 11 − 20 immune-cells/field, congestion,
slightly thickened bronchiolar and blood vessel walls,
some goblet cells along the bronchiolar epithelium with
Le et al. Respiratory Research
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their mucus product; 3, moderate, 21 − 30 immune-cells
/field, congestion, thickened bronchiolar and blood vessel
walls, light epithelial damage, moderate goblet cell hyperplasia; and 4, severe, ≥ 31 immune-cells /field, congestion, very thickened bronchiolar and blood vessel walls,
severe goblet cell hyperplasia with a lot of their mucus
product, more than 10% of lung consolidated, epithelial
damage.
To evaluate vascular permeability, we performed protein analysis on BAL fluids by Bradford protein assay [41]
using a protein assay kit (Bio-Rad, Hercule, CA.) following the manufacturer’s instructions.
Immuno‑gold electron microscopy for LSP1
After in situ intra-tracheal fixation, a piece of left mouse
lung (1 × 2 mm2) was cut and fixed in 2% paraformaldehyde with 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight at 4 °C. Next, the samples were
rinsed in three changes of 0.1 M sodium cacodylate buffer
at 4 °C. After being dehydrated in ethanol, the tissues
were infiltrated in fresh white resin three times before
being placed next to a Sylvania Blacklight Blue A4485151T8/BLB in a cryostat at − 4 °C for polymerization.
The tissues were then sectioned 100 nm thickness on
nickel grids. Immuno-gold staining procedure with LSP1
antibody followed a protocol from our previous paper
[29]. The tissues were imaged using a transmission electron microscope (Hitachi HT7700—XFlash 6T160, Germany) operated at 80 kV.
Lung Myeloperoxidase (MPO) and Eosinophil Peroxidase
Assay (EPO) quantification
MPO and EPO assay protocols were adapted from a previous protocol [29, 42]. Briefly, mouse lung samples were
homogenized in 500 µl of 50 mM HEPES (Invitrogen,
Burlington, ON, Canada) and then re-homogenized in
500 µl of 0.5% cetyltrimethyl ammonium chloride solution. Diluted MPO standards from human leukocytes
(Sigma-Aldrich, St. Louis, MO, USA) and mouse lung
samples were added to 96-well plate. The MPO substrate (3, 3’, 5, 5’-tetramethylbenzidine) was then added,
followed by use of 1 M H2SO4 to terminate the reaction. The plate was read at 450 nm OD using NOVOstar
software (Bio-Rad). Total protein concentrations in each
sample was quantified using a protein assay kit (Bio-Rad).
The data are expressed as units of MPO per mg of lung
protein.
To assess EPO levels, samples and EPO standards
were added to 96-well plates. Stop solution was added
after two-minute incubation with eosinophil peroxidase
assay substrate solution (3 mM O-phenylenediamine).
EPO levels were read at 490 nm OD using NOVOstar
software (Bio-Rad). Total protein concentration in each
Page 4 of 16
sample was quantified using a protein assay kit (Bio-Rad).
Data were expressed as the units of EPO per mg of lung
protein.
LSP1, Gr1 and MPO immunohistochemical
and immunofluorescent staining
The immunohistochemical staining protocol for LSP1
and the immunofluorescent protocols for staining LSP1
and Gr1, or LSP1 and MPO were modified from our previous report [29]. Briefly, sections were de-paraffinized,
and treated to quench endogenous peroxidase activity
and then for antigen retrieval. The non-specific binding
in the lung sections was blocked with 1% BSA, followed
by incubation with primary and appropriate secondary
antibodies. Tissues were stained with 20 µg/ml rabbit
anti-mouse LSP1 polyclonal antibody (Novus Biological, Oakville, ON, Canada) followed by secondary polyclonal goat anti-rabbit immunoglobulins/horse radish
peroxidase (HRP). In immunofluorescent staining for
LSP1 and MPO, tissues were stained with 20 µg/ml rabbit
anti-mouse LSP1 polyclonal antibody (Novus Biological,
Oakville, ON, Canada) and 20 µg/ml purified polyclonal
goat anti-human/mouse myeloperoxidase antibody (R&D
Systems, Minneapolis, MN, USA.) followed by 1:100
polyclonal goat anti-rabbit immunoglobulins IgG /conjugated Cy5 (Abcam, Toronto, ON, Canada) and 1:200
polyclonal donkey anti-goat immunoglobulins/conjugated AF488 (Life technology, Waltham, MA., USA.),
respectively. In LSP1 and Gr1 immunofluorescent staining, tissues were stained with 20 µg/ml rabbit anti-mouse
LSP1 polyclonal antibody, reactive in mouse and human
(Novus Biological, Oakville, ON, Canada), and purified
rat anti-mouse Gr1 antibody (Ly-6G and Ly-6C) (BD
Biosciences Pharminogen™, Mississauga, ON, Canada),
followed by goat anti-rabbit IgG secondary antibody conjugated Alexa fluor 488 (Life technology, Waltham, MA.,
USA.), and chicken anti-rat IgG secondary antibody conjugated Alexa fluor 647 (Life technology, Waltham, MA.,
USA.), respectively.
We have used LSP-1 antibody in our previous studies and have even performed pre-absorption controls
[29]. The negative controls included staining with isotype antibody matching control rabbit IgG (Novus Biological, Oakville, ON, Canada), rat IgG2bκ and goat
IgG isotype control antibodies (Santa Cruz Biotechnology, Mississauga, ON, Canada) instead of primary antibody for LSP1, Gr1 and MPO, respectively. Another
negative control was the omission of primary antibody.
Also, Lsp1−/− murine lungs, stained with LSP1 antibody, acted as an important negative control for the
non-specific staining by the antibody (Additional file 1:
Fig. S1). Tissues were incubated for 5 min in 0.33 µg/ml
DAPI in immunofluorescent staining or methyl green in
Le et al. Respiratory Research
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immunohistochemical staining for staining the DNA of
nuclei. For immunohistochemical staining, the color was
developed by Vector® VIP peroxidase substrate kit for
peroxidase (Vector Laboratories, Burlingame, CA, USA).
For immunofluorescent staining, samples were imaged
using a confocal scanning laser microscope (Leica TCS
SP5 LSCM, Ontario, Canada) with a 63 × oil immersion
objective lens.
LSP1, Gr1 and MPO immunohistochemical
and immunofluorescent staining on human lungs
Normal and asthmatic human lungs in paraffin were
obtained from the Department of Pathology in the College of Medicine at the University of Saskatchewan (n = 3
each group). Human lung sections were stained with
rabbit anti-mouse LSP1 antibody (Novus Biological,
Oakville, ON, Canada) followed by polyclonal goat antirabbit immunoglobulins IgG conjugated Cy5 secondary
antibody (1:100, Abcam, Toronto, ON, Canada). Human
lung sections stained with bovine serum albumin or IgG
rabbit isotype control instead of LSP1 primary antibody
served as negative controls. Samples were imaged using
Olympus IX83 inverted microscope with a total internal
reflection fluorescence (TIRF) system under a 10 × , and
60 × oil immersion objective lens. The controls are shown
in Additional file 1: Fig. S2.
Western blot analyses for LSP1
Frozen mouse lungs were homogenized in T-PER Tissue
Protein Extraction Reagent (Thermo Scientific, Rockford, IL., USA) with protease and phosphatase inhibitor
cocktails as described [43]. Total protein concentration in
each sample was quantified using a protein assay kit (BioRad). The Western blot procedure has been reported
previously [29, 44]. Densitometry quantification was
performed using ImageJ software (from the National
Institutes of Health and the Laboratory for Optical and
Computational Instrumentation, University of Wisconsin) to evaluate relative density of total LSP1 expression
levels adjusted to beta-actin. Full blots are included in
Additional file 1: Fig. S2.
Statistical analysis
Statistical analysis was performed using GraphPad Prism
software version 5.04 (San Diego, CA, USA). Quantitative results were expressed as mean ± SEM. The normal
distribution of residuals was tested by histogram and
Shapiro–Wilk test. Data were analyzed by ANOVA, followed by Bonferroni multiple comparison test. Student
t-test or Wilcoxon Signed Rank Test was used to compare
two groups. The critical value of α was set to 0.05 as a significant difference (two-tailed).
Page 5 of 16
Results
LSP1 expression was increased in OVA‑induced murine
asthma
Immunohistochemistry confirmed the expression of
LSP1 in the macrophages, bronchiolar epithelium, airway
epithelium, and vascular endothelium in normal healthy
mouse lungs of WT mice (Fig. 1A). The LSP1 staining was more intense in these cells in the lungs of OVA
challenged mice (Fig. 1B). The controls for the immunohistochemistry are included in Additional file 1: Fig. S1.
Dual immunostaining for LSP-1 and MPO, as a marker
for neutrophils [45] (although it may also be expressed in
some monocytes) showed strong LSP1 expression in the
plasma membrane and cytoplasm with weaker staining
in the nuclei of neutrophils (Fig. 1C). Western blot data
demonstrated higher levels of total LSP1 in the lungs of
OVA-challenged WT mice compared to control mice
(P < 0.05, Fig. 1D, E Additional file 1: Fig. S2).
Dual immune-fluorescence revealed LSP1 expression in
all Gr1-positive granulocytes, which would include neutrophils and eosinophils (Fig. 2). Interestingly, the LSP1
fluorescence intensity was stronger in Gr1 cells adhering
to endothelium or alveoli than those cells in blood vessels
(Fig. 2C, D). We also observed LPS1 staining in the macrophages and lymphocytes in OVA-challenged mouse
lungs (Fig. 2E). The negative immunohistochemical
controls showed no staining (Additional file 1: Fig. S3).
Immuno-gold staining with LSP1 antibody further confirmed the expression of LSP1 on the plasma membrane,
cytoplasm, and nucleus of pulmonary intravascular macrophages (Fig. 3A) and alveolar macrophages (Fig. 3B).
LSP1 deficiency reduced the histopathologic signs of lung
inflammation and AHR in mouse
The data showed that control WT and L
sp1−/− mice
responded similarly when exposed to increasing doses
of methacholine. However, when compared to WT OVA
mice, Lsp1−/− OVA mice showed significantly more
decline in AHR when exposed to 25 mg/mL methacholine aerosols. The statistical linear regression further
showed significant differences in the slopes of WT OVA
mice (Y = -1.958*X—19.48) and
Lsp1−/− OVA mice
(Y = − 1.206*X—22.79) (Fig. 4).
The normal lung sections from WT (Fig. 5A) and
Lsp1−/− mice (Fig. 5B) showed no inflammation and
normal histology with thin alveolar septa, clear alveolar spaces, and occasional alveolar macrophages. Lungs
from OVA-challenged WT mice (Fig. 5C) showed more
lung inflammation compared to
Lsp1−/− OVA mouse
lungs (Fig. 5D). The histological scoring as described in
the methods revealed significantly more lung pathology
in WT OVA mice compared to all other groups (Fig. 5E).
Le et al. Respiratory Research
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Page 6 of 16
Fig. 1 The increased expression of LSP1 in asthma mouse lungs. LSP-1 staining is observed in lung sections from control mouse A and
OVA-challenged mouse lungs (B). The staining is observed in bronchiolar epithelium, and endothelium (arrows). High magnification insets in A and
B show staining in alveolar macrophages (Magnification: 400 × and 1000 ×). C The representative confocal images of OVA mouse lung sections
show staining for LSP-1 (green) and myeloperoxidase (MPO; red). The merged image show LSP-1 to be predominantly in neutrophils. Western blots
D and the densitometry E for LSP1 (at about 52 kD) and β-actin (47 kD) showed that OVA mouse lungs have higher LSP1 levels than control mouse
lungs. Data were expressed as mean ± SEM. Asterisk (*) indicates significant difference from wildtype control (P < 0.05, n = 3 each group). As alveolar
space, Bv blood vessel, Br bronchiole, N Neutrophil, WT wildtype
The BAL fluid of both genotypes of mice challenged with
OVA had a significantly higher level of protein concentration compared to respective normal controls. There was
no difference between OVA-treated WT and
Lsp1−/−
mice in protein concentration in BALF (Fig. 5F).
LSP1 deficiency dramatically down‑regulated
inflammatory cells recruitment into inflamed lungs
Compared to OVA-challenged
Lsp1−/− mice, WT
OVA mice had significantly more leukocytes, including
eosinophils, neutrophils, macrophages, and lymphocytes
in BAL fluid (P < 0.05, n = 6 each group) (Fig. 6A − 6E;
Additional file 1: Fig. S3). There were however no differences in peripheral blood leukocyte numbers between
control and OVA-challenged as well as between WT
and Lsp1−/− mice (Fig. 6F). The data also showed that
OVA-challenged WT mice had higher levels of MPO
and EPO compared to
Lsp1−/− OVA mice (P < 0.05,
n = 6 each group) (Fig. 7A, B). The Gr1 antibody staining showed significantly higher numbers of neutrophils
Le et al. Respiratory Research
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Page 7 of 16
Fig. 2 Immunofluorescent staining LSP1 and granulocytes in mouse lungs. Mouse lung sections display a lack of LSP-1 staining (green) and
granulocytes (red) in LSP-1−/− control (KO) and OVA-challenged mice (KO) compared to the wild-type control (WC) and wild-type OVA (WO) mice.
As alveolar space, Bv blood vessel, Br bronchiole, E endothelium, G granulocytes, L lymphocytes, M macrophages, N neutrophils. n = 3 each group
Le et al. Respiratory Research
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Page 8 of 16
Fig. 3 The immuno-gold electron micrographs for the expression of LSP1 in the mouse lung. The transmission electron micrograph of an
OVA-induced asthmatic WT mouse lung showed LSP1 staining in the plasma membrane, nucleus (N) and cytoplasm of an intravascular
macrophage A and alveolar macrophage (red arrows, B), an endothelial cell (E, yellow arrows, A), and type I pneumocytes (p1, blue arrows, A).
Original magnification 20,000 ×
Le et al. Respiratory Research
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Page 9 of 16
numbers of inflammatory cells in alveolar and perivascular spaces. These inflammatory cells including macrophages and neutrophils were intensely positive for
LSP1 in their cytoplasm and the plasma membrane
(Fig. 10C–E).
Fig. 4 Knocking-out LSP1 ameliorated AHR in the OVA-induced
asthma mouse model. Statistical linear regression shows that the
slopes of the AHR curves for the WT OVA mice (Y = − 1.958*X–19.48)
and Lsp1−/− OVA mice (Y = − 1.206*X–22.79) are significant different
(P < 0.05, n = 6 each group). It suggested that the AHR of Lsp1−/−
OVA mice was improved compared with that of WT OVA mice. Data
expressed as mean ± SEM at each methacholine dose
and eosinophils in the lungs of WT OVA mice compared
to Lsp1−/− OVA mouse lungs (P < 0.05, n = 3 each group)
(Fig. 2, and Fig. 7D). Also, Lsp1−/− OVA mice had a fewer
MPO-labeled cells, likely neutrophils, recruited to the
perivascular space than WT OVA mice (P < 0.05, n = 3
each group) (Fig. 7C).
The absence of LSP1 gene significantly attenuated
the levels of IL‑4, IL‑5, IL‑6, IL‑13, and CXCL1 in the BAL
fluid as well as ovalbumin‑specific IgE in the serum
of OVA‑challenged mice
Bioplex assays to measure the concentrations of IL-4,
IL-5, IL-6, IL-13, CXCL1, IL-17, CCL11, and IFN-γ in
BAL showed no difference in IL-17, CCL11, and IFN-γ
between WT or L
sp1−/− mice treated with OVA. However, the concentrations of IL-4, IL-5, IL-6, IL-13, and
CXCL1 were increased in the BAL of WT OVA mice
compared to WT control mice as well as L
sp1−/− OVA
mice (P < 0.05, n = 6 each group, Fig. 8).
ELISA data showed higher concentrations of ovalbumin-specific IgE and IgG1 in serum of both WT and
Lsp1−/− OVA-challenged mice compared to control
mice (P < 0.05, n = 6 each group) (Fig. 9). The ovalbumin-specific IgE but not IgA concentration in serum
of OVA-challenged WT mice was higher than that of
Lsp1−/− OVA mice (P < 0.05, n = 6 each group; Fig. 9).
Finally, normal human lungs were reactive for LSP1
in the endothelium, macrophages and the alveolar septa
( Fig. 10A, B). Asthmatic lung tissues showed large
Discussion
We provide new data on the role of LSP-1 in regulation
of AHR and lung inflammation in a mouse model of
asthma. The data show deficiency of LSP-1 reduces AHR
and lung inflammation. In addition, we also reported
increases in the expression of LSP1 in various resident
and recruited cells in asthmatic lungs from the mice and
humans. These data build on our previous report [29]
and further establishes the role of LPS1 as an important
regulator of inflammation in the lungs.
The OVA-induced murine model of asthma is an
important tool in elucidating the mechanisms of acute
asthma in humans such as recruitment of inflammatory
cells and AHR [46–48]. The recruitment of inflammatory cells such as eosinophils, neutrophils and lymphocytes into the lungs is an important feature of asthma
[49] and we observed the same in our model based on
BAL analyses, immunohistology and MPO assays. The
deficiency of LSP1 led to significant reduction in the
recruitment of neutrophils, eosinophils, lymphocytes
and macrophages into the airways of OVA-treated mice
compared to their WT counterparts. These data align
with our previous findings of LSP1 deficiency being associated with decreased inflammatory cell recruitment
in an endotoxin-induced lung inflammation study [29].
LSP1 has been shown to regulate T-lymphocyte migration in rheumatoid arthritis [31]. Our finding of significant reduction in lymphocyte numbers in lungs of LSP1
deficient OVA-challenged mice may be of significance
considering there are earlier studies linking the alveolar
migration of T-lymphocytes in asthma [50, 51]. It has
been previously reported that the increasing numbers
of eosinophils, mast cells and neutrophils along with
their enzymic products cause damage to lung tissues in
and determine severity of asthma [52–54]. The recurring episodes of eosinophilia and pulmonary migration
of eosinophils in asthmatics lead to thickening of the
sub-epithelial basement membrane, bronchial hyperresponsiveness, and epithelial damage [53, 54]. Neutrophil
production of mediators such as elastase, or neutrophil
interactions with goblet cells leading to mucus accumulation can narrow the airway. Degranulation of goblet
cells depends on interactions with migrated neutrophils,
and specifically their elastase activity and the expression
of the adhesive molecules such as intercellular adhesion
molecule-1 (ICAM-1), CD18, and CD11b in vivo [55].
Therefore, reduced recruitment of inflammatory cells
Le et al. Respiratory Research
(2022) 23:165
Page 10 of 16
Fig. 5 The histopathological examination of mouse lungs with hematoxylin and eosin staining. Control wildtype A and L sp1−/− mice B display
normal appearing alveolar septa and alveoli. In comparison to Lsp1−/− OVA lungs (D), WT OVA mouse lungs C showed more inflammatory cell
infiltration in alveolar septa, alveoli, and peri-bronchial and peri-vascular spaces. E Semi-quantitative scoring showed more severe pathology in WT
OVA and Lsp1−/− OVA mice than in their respective control controls, and more in WT OVA mice, than in Lsp1−/− OVA mice. F The analysis of protein
concentration in BAL fluid showed that both types of mice challenged with OVA had significantly higher protein concentration than control but
there was no significant difference between WT and Lsp1−/− mice. As Alveolar space, Bv Blood vessel, Br Bronchiole, PVS Peribronchiolar vascular
space, WT wildtype. Magnification: 400 × . Asterisk (*) indicates a significant difference (P < 0.05, n = 6 each group)
observed in L
sp1−/− may lead to better physiological outcomes in asthma. Previous data has indicated that lack
of LSP1 may not affect MAPK cell signaling, but phosphorylation of LPS1 itself may lead to modulation of the
actin cytoskeleton of neutrophils, facilitating their migration [29, 56, 57].
The inflammatory mediators in asthma have been
extensively studied for their roles in cell recruitment and
Le et al. Respiratory Research
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Page 11 of 16
Fig. 6 Total and differential leukocyte counts in bronchoalveolar lavage (BAL) fluid and peripheral blood. LSP1-knockout attenuates
immunoinflammatory cell recruitment into the alveolar space in the OVA-induced asthma mouse model (A–E). There were not any statistically
significant differences between any groups in terms of peripheral blood leukocyte numbers (F). Data were expressed as mean ± SEM. Asterisk (*)
indicates a significant difference (P < 0.05, n = 6 each group)
cell activation [20, 21, 58]. We also quantified a number
of cytokines in the BAL from the mice and found that
WT OVA mice had significantly higher concentrations
of IL-4, IL-5, IL-6, IL-13, and CXCL1 compared to the
Lsp1−/− mice. There could be multiple reasons for this
observation. The reduced migration of inflammatory
cells, which are major sources of cytokines, in OVAchallenged Lsp1−/− mice would have contributed to the
lower levels of selected cytokines. The absence of LSP1
expression, which can act as a substrate for MAPK, [56]
in lymphocytes may attenuate cytokine production by T
lymphocytes (IL-4, IL-5, IL-13), leading to reduced eosinophil recruitment and IgE production by B plasma cell. It
is well-known that IL-4, IL-5, and IL-13 cytokines produced by C
D4+ natural killer T cells and C
D4+ T MHCIIrestricted cells enhance eosinophilia and increase the
severity of asthma [59, 60]. IL-13 stimulates the epithelium in the airways [61]. The airway epithelium also produces IL-5, IL-2, TGF-β, IL-6 and IL-10, which promote
B cell differentiation into plasma cells to produce IgA [62,
63]. These cytokines also cause the metaplasia of goblet
cells and alterations in epithelial-mesenchymal signaling resulting in sub-epithelial fibrosis or smooth muscle
hyperplasia [64]. Eosinophils, which were reduced in
numbers in Lsp1−/− asthmatic mice, produce IL-16 to
attract CD4+ T cell in asthma [65]. Previous studies have
shown that binding of IgA, IgG, and IgE to their receptors on eosinophils activates and degranulates them [62,
63]. We found higher levels of OVA-specific IgG but not
IgA in the serum of WT asthmatic mice compared to
Lsp1−/− OVA mice. Taken together, we believe that LSP1
deficiency disrupts recruitment of inflammatory cells and
production of cytokines and thereby alleviates physiological and inflammatory outcomes in our murine model of
asthma.
The AHR to methacholine is a reliable method to evaluate airway function and has been shown to correlate
well with the invasive methods that are considered gold
standard for measuring lung mechanics [66] [67]. Along
with diminished recruitment of inflammatory cells and
expression of certain cytokines, there was reduction in
AHR, one of our major physiological measurements, in
Lsp1−/− asthmatic mice. Our experiments don’t directly
address the underlying mechanisms through which LSP-1
influences AHR, but the association between AHR and
inflammation has also not yet been fully resolved [68].
There however are data to show that the asthma-related
AHR is an outcome of excessive broncho-constriction
due to hypertrophy or hyperplasia of bronchiolar smooth
muscles with repeated episodes of eosinophil-related
Le et al. Respiratory Research
(2022) 23:165
Page 12 of 16
Fig. 7 The quantification of neutrophils and eosinophils left in mouse lungs after bronchoalveolar lavage. LSP1 deficiency reduced
asthma-associated neutrophil and eosinophil migration into the lungs. A, B Mouse lung lysates were analyzed using MPO and EPO assays as
surrogate measures for the numbers of neutrophils and eosinophils, respectively, remaining in the lung after bronchoalveolar lavage (n = 6 each
group). C, D We counted the number of neutrophils and granulocytes in MPO and Gr1 immunofluorescent-stained mouse lungs, respectively (n = 3
each group). Data were expressed as mean ± SEM. Asterisk (*) indicates a significant difference (P < 0.05)
airway inflammation [68, 69] [70] [71]. We believe that
reduced lung inflammation in L
sp1−/− mice may have
led to improvement in airflow into the lungs by reducing
AHR.
Lastly, to set the stage for the next set of experiments
focused on human cells and tissues, we evaluated expression of LSP-1 in normal and asthmatic human lungs.
The increase in LSP1 expression on various resident and
recruited cells in the asthmatic lungs alludes to a potential for this protein in the pathogenesis of asthma. These
data are similar to the increase in LSP1 staining in lung
samples from sepsis patients [29]. The next set of studies
will focus on the role of LSP1 in regulating the function
of human immune cells.
Conclusions
The study provides new data that deficiency of LSP-1
reduces lung inflammation as well as AHR in a murine
model of OVA-induced asthma. LSP-1 deficiency likely
disrupts the fundamental inflammatory process of
recruitment of neutrophils and eosinophils and the associated network of cytokines to reduce inflammation and
the physiological outcome of increased AHR. These data
are in line with the role of LSP-1 in inflammatory cell
recruitment in endotoxin-induced lung inflammation.
Le et al. Respiratory Research
(2022) 23:165
Page 13 of 16
Fig. 8 The quantification of cytokines and chemokines in BAL fluid using Bioplex assay. L SP1−/− OVA mice had significantly lower BAL
concentrations of IL-4, IL-5, IL-6, IL-13, and CXCL1, but not of IL-17, CCL11, or IFN-γ compared to wildtype OVA mice. Data were expressed as
mean ± SEM. Asterisk (*) indicates a significant difference (P < 0.05, n = 6 each group). IL interleukin, CXCL1 keratinocyte-derived chemokine, IFN-γ
interferon γ
Fig. 9 The impact of LSP1 expression on OVA-specific IgE, IgG1, and IgA levels in serum of asthmatic mice. Wildtype OVA mice had statistically
significant increases inOVA-specific IgE, IgG1, IgA levels compared with WT control mice. The levels of OVA-specific IgE found in WT OVA mice was
significantly higher than that in L sp1−/− OVA mice. Data were expressed as mean ± SEM. Asterisk (*) indicates significant difference (P < 0.05, n = 6
each group)
Le et al. Respiratory Research
(2022) 23:165
Page 14 of 16
Fig. 10 LSP1 expression in normal and asthmatic human lungs. In the normal human lungs (A, B), LSP1 (red) is seen in the alveolar septa and
blood vessels (BV) and in macrophages (arrowheads). The expression is more robust in the asthmatic lung C, D and E in blood vessels (BV) and
bronchioles (Br), which also show many more inflammatory cells. Alveolar macrophages (arrowheads) and granulocytes (arrows) show staining for
LSP1. (n = 3 each group)
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12931-022-02078-7.
Additional file 1: Figure S1. LSP1 immunohistochemical staining
controls for Fig. 2A, B. Figure S2. LSP1, MPO, and Gr1 immunofluorescent
staining controls for Fig. 2C and Fig. 3. Figure S3. Cytospun BAL cells were
stained with Hemacolor stain kit
Acknowledgements
We thank Ms. LaRhonda Sobchishin for the technical support provided for the
electron microscope technique and Ms. Eiko Kawamura for technical support
in using a confocal microscope.
Author contributions
NPKL, LL, JG and BS the conception and design of research and experiments;
NPKL, AN, DS, CCQ, XZ, DW and GKA performed experiments; NPKL and BS
analyzed data. NPKL and B.S. interpreted the results of experiments; NPKL and
BS prepared graphs and figures; NPKL and B.S. drafted the manuscript; NPKL,
DS, LL, JRG and BS edited and revised manuscript; NPKL, AN, DS, CCQ, XZ, DW,
GKA, LL, JRG, and BS approved the final version of the manuscript. All authors
read and approved the final manuscript.
Funding
For supporting this research, we thank the Natural Science and Engineering
Research Council (NSERC) to Dr. Baljit Singh, the Canadian Institutes for Health
Research (MOP53167) to Dr. John Gordon, the Graduate Student Scholarship
program from the Integrated Training Program in Infectious Disease, Food
Safety and Public Policy (ITraP), the Devolved Graduate Scholarship program
from the Department of Veterinary Biomedical Sciences, and the Graduate
Student Scholarship program from the Western College of Veterinary Medicine, the University of Saskatchewan, Canada. The funding bodies had no role
in study design, analyses, data interpretation and writing of this manuscript.
Availability of data and materials
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request.
Le et al. Respiratory Research
(2022) 23:165
Declarations
Ethics approval and consent to participate
The studies contained in this manuscript were approved by the University of
Saskatchewan’s Committee on Animal Care in accordance with the guidelines
of Canadian Council on Animal Care.
Consent for publication
Not applicable.
Competing interests
The authors declare they have no competing interests.
Author details
1
Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon,
Canada. 2 Faculty of Animal Science and Veterinary Medicine, Nong Lam
University, Ho Chi Minh City, Vietnam. 3 Small Animal Clinical Sciences,
University of Saskatchewan, Saskatoon, Canada. 4 Department of Medicine,
University of Saskatchewan, Saskatoon, Canada. 5 Department of Anatomy,
Physiology and Pharmacology, University of Saskatchewan, Saskatoon,
Canada. 6 Western College of Veterinary Medicine, University of Saskatchewan,
Saskatoon S7N5B4, Canada.
Received: 23 June 2021 Accepted: 7 June 2022
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