A 3D epithelial–mesenchymal co-culture model of human bronchial tissue recapitulates multiple features of airway tissue remodeling by TGF-β1 treatment
Ishikawa et al. Respiratory Research
A 3D epithelial-mesenchymal co-culture model of human bronchial tissue recapitulates multiple features of airway tissue remodeling by TGF-β1 treatment
Shinkichi Ishikawa 0
Kanae Ishimori 0
Shigeaki Ito 0
0 Scientific Product Assessment Center, R&D Group, Japan Tobacco Inc. , 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512 , Japan
Background: The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However, the involvement of bronchial epithelial cells in this process should also be investigated. Methods: We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) β1 as an inducer of tissue remodeling for 21 days, and measured gel size, histological changes, and expression of factors related to extracellular matrix homeostasis. Results: TGF-β1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-β1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium, suggesting the induction of epithelial-mesenchymal transition. In addition, the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model. Conclusion: Our findings indicate that TGF-β1 can affect both epithelial and mesenchymal cells, and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells, fibroblasts, and their interactions in the airway remodeling process.
Bronchi; Transforming growth factor β1; Co-culture; Remodeling; Gel contraction
Airway remodeling is an important aspect in the
pathogenesis of various lung diseases [
], and involves
persistent changes in the normal architecture of airway walls.
These changes include mesenchymal alterations such as
increased numbers of smooth-muscle cells,
myofibroblast accumulation, and increased matrix protein
]. Fibroblasts embedded in 3D collagen
matrices are a well-known in vitro mesenchymal model.
Various studies employing this model have shown that
transforming growth factor beta (TGF-β) enhances
contraction of collagen gels and is involved in the
remodeling process [
]. These findings suggest the
usefulness of the 3D collagen-matrices model with lung
fibroblasts in investigating the airway remodeling
process in connective tissue. However, this model lacks
the bronchial epithelial layer over the mesenchymal layer
of airway walls.
Bronchial epithelium plays an important role in the
defense against inhaled allergens, viral infections, and
airborne pollutants [
]. Exposure to these materials
induces injury and repair or inflammatory responses in
bronchial epithelium, and constant stimulation may
result in imbalances in these responses [
involvement of several mediators, including TGF-β, has been
reported in these abnormal responses . Thus both
mesenchymal cells and bronchial epithelial cells
participate in the airway remodeling process. Several studies
have indicated the importance of the
epithelialmesenchymal trophic unit (EMTU) in the tissue
remodeling process [
]. EMTU consists of epithelial cells,
mesenchymal cells, and their extracellular matrix
(ECM), and local exchange of information between
EMTU components is important in the response to
In this study, we investigated the effect of TGF-β1 on
human bronchial tissue remodeling using our original
3D co-culture model. We applied human bronchial
epithelial cells (HBECs) onto the 3D collagen matrices,
and cultured the cells in an air-liquid interface (ALI),
which enables differentiation of pseudostratified
bronchial epithelium with goblet and ciliated cells [
previous report shows that this model has a mucociliary
differentiated bronchial epithelial layer on a
fibroblastembedded mesenchymal collagen layer [
]. Various in
vitro studies have reported interactions between
epithelial cells and fibroblasts. However, these in vitro
studies were performed with conditioned medium or
indirect co-culture (e.g. floating co-culture,
transmembrane co-culture) [
]. Different from these
methods, our in vitro EMTU model reproduces direct
interactions between epithelial cells, fibroblasts, and
We analyzed the effects of TGF-β1 on collagen gel
contraction and epithelial and mesenchymal cell layers
in the 3D co-culture model of human bronchial tissue.
Other endpoints related to ECM homeostasis were
investigated, including expression of matrix
metalloproteinases (MMPs), tissue inhibitor of metalloproteinase
(TIMP), and ECM proteins.
Human fetal lung fibroblasts (IMR-90) were obtained
from the American Type Culture Collection (Manassas,
VA, USA) and grown in minimum essential medium
(MEM) (Life Technologies, Carlsbad, CA, USA) with
10% fetal bovine serum (FBS; MP Biomedicals, Santa
Ana, CA, USA). Normal HBECs (Lonza, Basel,
Switzerland) were grown in Airway Epithelial Cell
Growth Medium with SupplementPack (PromoCell,
The methodological details for the 3D co-culture of
IMR-90 cells and HBECs were described in our previous
]. Cellmatrix type I-A (Nitta Gelatin, Osaka,
Japan), 10 × MEM and reconstitution buffer (Nitta
Gelatin) were mixed with 8:1:1 by volume ratios and
applied to cell culture insert (10.5 mm diameter, 1.0-μm
pore size, BD Biosciences, Franklin Lakes, NJ, USA) in
100 μL aliquots to prepare base layer. The base layer
was gelled by placing in an incubator at 37 °C with a 5%
CO2 atmosphere for more than an hour. IMR-90 cells
(approximately 2.5 × 106 cells/mL in FBS), Cellmatrix
type I-A, Cellmatrix type I-P (Nitta Gelatin), 10 × MEM,
and reconstitution buffer were mixed with 1:4:4:1:1 by
volume ratios and poured onto the base layer in 250 μL
aliquots to prepare the collagen-embedded fibroblast
layer. The fibroblast layer was gelled by placing in an
incubator at 37 °C with a 5% CO2 atmosphere for more
than an hour. After 2 days of cultivation with MEM
containing 10% FBS, HBECs suspended in Airway Epithelial
Cell Growth Medium (approximately 3.0 × 105 cells/mL)
were seeded onto the collagen layer to prepare the
coculture model, and cultured under submerged
conditions until reaching a semi-confluent state. The
fibroblast mono-culture model was prepared without
seeding of HBECs. The ALI culture was then initiated to
induce mucociliary differentiation. The mono-culture
model was also cultivated under ALI conditions.
PneumaCult-ALI medium (Stemcell Technologies,
Vancouver, BC, Canada) was supplemented with heparin
(Stemcell Technologies) and hydrocortisone (Stemcell
Technologies) according to the manufacturer’s
instructions. GM6001 (30 nM, Sigma-Aldrich, St. Louis, MO,
USA) and 1% FBS were also added to prepare the ALI
culture medium. The apical and basolateral media were
removed, and 600 μL of ALI culture medium were
added to the bottom well. Stimulation of TGF-β
signaling with TGF-β1 (R&D Systems, Minneapolis, MN,
USA) and inhibition of TGF-β signaling with a TGF-β
receptor type I blocker (SB525334; Wako Pure
Chemical Industries, Osaka, Japan) commenced on the first
day of ALI culturing. The ALI culture was maintained
for 21 days. Images of each collagen gel were obtained,
and gel contraction was analyzed with ImageJ software
(National Institutes of Health, Bethesda, MD, USA). Data
are expressed as the percentage to the initial gel area.
After fixation in 4% paraformaldehyde at 4 °C on ALI
culture day 21, bronchial tissue samples were embedded
in paraffin and 5-μm sections were prepared using a
microtome. Sections were deparaffinized and subjected
to hematoxylin and eosin staining or immunostaining.
Immunostaining was performed with Polink-2 Plus (GBI
Labs, Bothell, WA, USA) using the following antibodies:
Anti-vimentin antibody (1:250; ab92547; Abcam,
Cambridge, UK), anti-alpha-smooth muscle actin
(α-SMA) antibody (1:1000; ab5694; Abcam),
anti-Ecadherin antibody (1:250; ab40772; Abcam),
antiacetylated α-tubulin antibody (1:250; ab24610, Abcam),
anti-MUC5AC antibody (1:250; ab3649; Abcam),
anticytokeratin (CK) 5 antibody (1:100; ab52635; Abcam),
anti-fibronectin antibody (1:250; ab2413; Abcam), and
anti-tenascin-C antibody (1:250; ab108930; Abcam).
Sections were subjected to heat-induced antigen retrieval
in a 10-mM sodium citrate buffer (pH 6.0) for α-SMA,
E-cadherin, acetylated α-tubulin, MUC5AC, CK5 and
fibronectin staining at approximately 95 °C for 30 min.
EDTA (1 mM, pH 8.0) was used for vimentin and
tenascin-C staining. Image analysis of immunostained
sections was conducted with ImageJ software. Three
sections were prepared in each experimental condition,
and the area of the epithelial layer and mesenchymal
collagen layer was measured. Colour Deconvolution
] was used for diaminobenzidine (DAB) and
hematoxylin stain separation, and the DAB-positive
areas in the epithelial and mesenchymal layers were
measured. The results are expressed as the percentage of
DAB-positive area in each layer.
Culture medium collected on ALI culture day 21 was
subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis in 7.5% acrylamide gels containing
0.9 mg/mL gelatin. After electrophoresis, gels were
washed twice (30 min and 45 min) in wash buffer (0.5%
Triton X-100, 2.5 mM Tris-HCl, 150 mM NaCl) and
incubated for 19 h in incubation buffer (2.5 mM
Tris-HCl, 20 mM NaCl, 10 mM CaCl2), then stained
with 0.1% Coomassie blue. Images were obtained with
ImageQuant LAS 4000 (GE Healthcare, Little Chalfont,
UK), and signal densities were quantified using
ImageQuant TL (GE Healthcare). Data are expressed as
the fold change to the band density in the control.
Measurement of TIMP by multi-plex assay
The concentrations of TIMPs secreted into the culture
medium were analyzed with a Bio-Plex Pro Human TIMP
Panel (Bio-Rad, Hercules, CA, USA) using the Bio-Plex
system (Bio-Rad) according to the manufacturer’s
instructions. Culture medium collected on ALI culture day 21
was diluted at 1:10,000 for TIMP-1 analysis or 1:100 for
TIMP-2 analysis. The concentrations of TIMP-3 and
TIMP-4 were lower than detection limits, although the
culture medium was analyzed without dilution.
Total RNA was isolated from tissues using RNeasy
(Qiagen, Hilden, Germany), and RNA quality was
analyzed with an Agilent 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA, USA). The RNA
integrity number of the samples was ≥7.6. cDNA was
synthesized with a High-Capacity cDNA Reverse Transcription
Kit (Applied Biosystems, Waltham, MA, USA). The gene
expression profile was analyzed with the Human
Extracellular Matrix and Adhesion Molecules RT 2
Profiler PCR Array (PAHS-013, SABiosciences,
Frederick, MD, USA) on an ABI 7900 PCR system
With the exception of the PCR array results, data are
presented as the means and standard deviations of
triplicate inserts. Multiple-comparisons tests were used in the
analysis of the results obtained with co-culture model.
Bartlett’s test was used to confirm homogeneity of
variances from multiple groups. A parametric one-way
analysis of variance followed by Dunnett’s test was
performed to detect statistically significant differences.
Results were considered significant at p < 0.05. For the
analysis of the fibroblast mono-culture model, student’s
t-test was used and the results were considered to be
significant at p < 0.05. All statistical analyses except for
those of PCR array data were performed using
EkuseruToukei (SSRI Co., Ltd., Tokyo, Japan). The PCR array
data were analyzed using RT 2 Profiler PCR Array Data
Analysis version 3.5, provided by SABiosciences. The
results are presented as the means and 95% confidence
intervals of the triplicate inserts. Student’s t-test was
used; the results were considered significant at p < 0.05.
Collagen gel contraction following TGF-β1 stimulation
The co-culture human bronchial tissue model with an
epithelial cell layer and collagen-embedded fibroblast
layer was stimulated with TGF-β1 for 21 days under ALI
culture conditions (Fig. 1a). Collagen gel contraction
was observed in the untreated control on ALI culture
days 7, 14, and 21 (Fig. 1b). Gel contraction was
significantly enhanced by stimulation with 4 or 10 ng/mL
TGF-β1, compared with contraction in the the untreated
control at each time point (p < 0.05). The collagen gel
was clearly detached from the wall of the cell culture
insert on ALI culture day 21 following stimulation with 4
or 10 ng/mL TGF-β1 (Fig. 1a, arrowheads). When the
co-culture model was stimulated with 10 ng/mL TGF-β1
with simultaneous inhibition of TGF-β receptor type I
by 5 μM SB525334, gel contraction was not detectable
Histological changes induced by TGF-β1 stimulation
To further understand the effects of TGF-β1 on our
coculture model, we performed hematoxylin and eosin
staining on histological sections of tissues collected on
ALI culture day 21. We found effects of TGF-β1 on the
cellular morphology and increased numbers of elongated
HBECs (Fig. 2a). In accordance with this increase, a
concentration-dependent decrease in the thickness of
the epithelial layer was observed following stimulation
with TGF-β1 (Fig. 2a). This decrease was prevented by
the addition of 5 μM SB525334. In the mesenchymal
collagen layer, we found an increase in heavily stained
fibroblasts (arrowheads in Fig. 2a). Next, we performed
immunostaining to characterize the changes observed in
the sections stained with hematoxylin and eosin. We
stained for vimentin as a mesenchymal marker, and
found a significant increase in vimentin-positive
fibroblasts in the mesenchymal layer following
stimulation with 4 or 10 ng/mL TGF-β1 (p < 0.05) (Fig. 2a and
b). Vimentin expression was also confirmed by TGF-β1
stimulation in the basal cells of the epithelial layer (Fig. 2a,
arrows). The percentage of the vimentin-positive area in
the epithelial layer increased significantly following
stimulation with 4 or 10 ng/mL TGF-β1 (p < 0.05) (Fig. 2c).
Immunostaining was also performed with an anti-α-SMA
antibody to confirm the presence of myofibroblasts.
Intracellular expression of α-SMA in fibroblasts increased
significantly following TGF-β1 stimulation (0.4, 4, or
10 ng/mL; p < 0.05) (Fig. 2a and d). This increase was
suppressed by blocking TGF-β1 signaling with 5 μM
SB525334. We also performed immunostaining with an
anti-E-cadherin antibody to confirm the epithelial status.
E-cadherin is adhesion molecule that plays a major role in
the maintenance of epithelial intercellular junctions. In
contrast with the increased vimentin expression (Fig. 2a
and c), we observed a decrease of E-cadherin in the
epithelium following stimulation with TGF-β1 (Fig. 2a and e).
We also analyzed the effect of TGF-β1 on the
differentiation of bronchial epithelial cells. We used acetylated
α-tubulin as a ciliated cell marker, MUC5AC as a goblet
cell marker, and CK5 as a basal cell marker. These
differentiation markers were found to be expressed under
control conditions (Additional file 1: Figure S1),
following stimulation with TGF-β1, and even when blocking
TGF-β1 signaling by 5 μM SB525334 (Additional file 1:
Effect of TGF-β1 on expression of MMPs and TIMPs
In addition to collagen gel contraction, altered ECM
homeostasis is a factor in the airway remodeling process.
We therefore analyzed the expression levels of
proteolytic MMP-2 and MMP-9 in the culture medium of
coculture model collected on ALI culture day 21 by gelatin
zymography. Pro-MMP-9 (92 kDa), pro-MMP-2
(72 kDa), and active MMP-2 (62 kDa) were detected
(see co-culture data in Additional file 2: Figure S2);
expression levels of each MMP were quantified as the fold
change to the control (Fig. 3a–c). Expression of
proMMP-9 in the co-culture model did not change after
stimulation with TGF-β1 (Fig. 3a). However, pro-MMP-9
expression was suppressed significantly when TGF-β1
signaling was blocked with 5 μM SB525334 (p < 0.05).
Expression of pro-MMP-2 and active MMP-2 in the
coculture model increased in a concentration-dependent
manner following stimulation with TGF-β1, and
significant increases were detected following stimulation with
10 ng/mL TGF-β1 (p < 0.05) (Fig. 3b and c). This
increase in expression was blocked by the addition of
5 μM SB525334.
We also analyzed the concentrations of natural MMP
inhibitor TIMPs in the co-culture medium on ALI
culture day 21. Levels of TIMP-1 increased significantly
following stimulation with 4 or 10 ng/mL TGF-β1 (p < 0.05)
(Fig. 3d). Secretion of TIMP-1 following stimulation with
10 ng/mL TGF-β1 was suppressed to control levels by the
addition of 5 μM SB525334. Secretion of TIMP-2 showed a
similar trend, and increased significantly in response to
TGF-β1, compared with levels in the control (p < 0.05)
To confirm the role of epithelial-mesenchymal
interaction in ECM homeostasis in the co-culture model, a
mono-culture model with lung fibroblast cells was
prepared. The fibroblast mono-culture model was stimulated
with 10 ng/mL of TGF-β1, MMP secretion was analyzed
with gelatin zymography. Unlike findings in the co-culture
model, pro-MMP-9 expression was very low in the
monoculture even with TGF-β1 stimulation (see fibroblast
mono-culture data in Additional file 2: Figure S2).
Similarly to secretion in the co-culture model,
pro-MMP2 secretion in the mono-culture model increased
significantly following stimulation with TGF-β1 (p < 0.05)
(Fig. 4a). However, levels of active MMP-2 did not
increase with TGF-β1 stimulation (Fig. 4b). Secretion of
TIMPs in the mono-culture model stimulated with
TGFβ1 was also analyzed; a significant increase in TIMP-1 and
TIMP-2 levels was observed (p < 0.05) (Fig. 4c and d).
Effect of TGF-β1 on ECM-related genes
We analyzed the expression of ECM-related genes in the
co-culture model on ALI culture day 21. Among the 84
genes analyzed, the expression of 33 genes increased
significantly following stimulation with TGF-β1 (p < 0.05)
(Fig. 5). These genes were categorized as follows: TGF-β
and integrins (Fig. 5a), TIMPs and MMPs (Fig. 5b),
adhesion molecules (Fig. 5c), collagens (Fig. 5d), and ECM
glycoproteins and proteoglycans (Fig. 5e). The
expression of most of these genes was upregulated in a
concentration-dependent manner by TGF-β1
stimulation. These increases were suppressed to control levels
or lower by the addition of 5 μM SB525334 (p < 0.05)
(Fig. 5a–e). The top five genes upregulated following
treatment with 10 ng/mL TGF-β1 in the co-culture
model were FN1 (29.3-fold change; Fig. 5e), TNC
(10.5-fold change; Fig. 5e), VCAN (7.7-fold change;
Fig. 5e), MMP9 (7.38-fold change; Fig. 5b), and COL1A1
(6.8-fold change; Fig. 5d).
We also found that 14 of the 33 genes were
upregulated in the fibroblast mono-culture model following
stimulation with 10 ng/mL TGF-β1 (Fig. 6). However,
the level of fold increase of several genes was lower
in the mono-culture model compared with the
coculture model. For example, FN1 expression showed a
29.3-fold change in the co-culture model after
stimulation with 10 ng/mL TGF-β1 (Fig. 5e), compared
with only a 2.2-fold change in the mono-culture
model (Fig. 6).
Histological analysis of the ECM protein expression pattern
As FN1 and TNC expression was markedly upregulated
(Fig. 5e), we analyzed the expression pattern of proteins
encoded by these genes in the co-culture. The results
revealed fibronectin expression mainly across the
mesenchymal layer (Fig. 7). However, we also found
fibronectinpositive basal cells in the epithelial layer following TGF-β1
stimulation (Fig. 7, arrowheads). Tenascin-C was also
expressed across the mesenchymal layer, and the staining
appeared to become denser with higher TGF-β1
concentrations. Strong expression of tenascin-C was observed in the
sub-epithelial basement membrane of the co-culture model
(Fig. 7, arrows). Expression decreased as TGF-β signaling
was suppressed by the addition of 5 μM SB525334.
The development of an in vitro lung model is essential
to design new therapies for various lung diseases. Recent
advances in bioengineering technology have enabled the
generation of lung organoid models with matrigels and
biocompatible alginate beads [
]. One of the
novelties of these models is the co-culture of multiple types of
cells (such as fibroblasts, epithelial cells, and endothelial
cells). Here, we report another type of co-culture model
involving the ALI culture of HBECs on collagen gel
matrices with fibroblasts.
The collagen gel contraction assay is well-established,
and multiple studies have shown that TGF-β signaling
can enhance the contractility of gels in submerged
]. As our co-culture model has a
collagenembedded fibroblast layer, we examined whether
stimulation by TGF-β1 could promote gel contraction in an
ALI culture. The cells were cultured for 21 days under
ALI conditions to induce the mucociliary differentiation
of HBECs [
]; TGF-β1 stimulation was conducted
during the ALI culture period. Collagen gel contraction was
detected in the untreated (control) culture, and TGF-β1
treatment enhanced contraction at each time point
during the ALI culture period. We also found that gel
contraction was suppressed by the TGF-β type I receptor
blocker SB525334, which inhibits activin receptor-like
kinase (ALK) 5-mediated Smad2/3 phosphorylation [
]. This finding suggests that the effect of TGF-β1 on
gel contraction in our experiment was mediated through
the TGF-β1/ALK5 pathway, and is supported by a report
that Smad3-mediated TGF-β1 signaling is necessary for
collagen gel contraction .
To elucidate the mechanisms behind the gel
contraction, we analyzed histological changes in our in vitro
EMTU. We found increased numbers of elongated HBECs
by hematoxylin and eosin staining. Induction of
epithelialmesenchymal transition (EMT) is one of the most
prominent effects of TGF-β in various tissues [
HBECs cultivated under ALI conditions have been
reported to exhibit an elongated shape with EMT
characteristics following stimulation with TGF-β . Epithelial
cells show decreased expression of the epithelial marker
E-cadherin when undergoing EMT [
]. In our co-culture
model, we confirmed a decrease in E-cadherin expression
in the epithelial layer following TGF-β1 stimulation. We
also found an increase in basal cells positive for the
mesenchymal marker vimentin in the epithelial layer and in
vimentin-positive fibroblasts in the mesenchymal layer.
These results suggest that TGF-β1 promotes the
proliferation of fibroblasts in the mesenchymal layer, and that basal
cells in the epithelial layer undergoing EMT could be a
source of fibroblasts. In addition, we found that the
fibroblasts were α-SMA-positive myofibroblasts. Induction of
contractile myofibroblasts is considered important in
collagen gel contraction [
]. Thus, a
concentrationdependent induction of myofibroblasts in the
mesenchymal layer may have promoted the gel contraction we
observed. Histological analysis indicated that TGF-β1
induced EMT in the epithelial layer and myofibroblasts in
the mesenchymal layer. This suggests that our culture
model successfully reproduced multiple events in the
The effect of TGF-β1 on the epithelial layer was also
analyzed using antibodies against markers of ciliated cells,
goblet cells, and basal cells, which are all present in a
differentiated bronchial epithelium. We found the existence
of all three cell types with or without TGF-β1 stimulation.
This suggests that the effect of TGF-β1 on bronchial
epithelial differentiation is limited. Moreover, we even found
that the differentiation markers were expressed when
TGF-β1 signaling was blocked by SB525334. This is
consistent with a previous study in which mice with a deletion
of ALK5 in the lung showed a differentiation of ciliated
cells similar to control mice [
Apart from myofibroblast induction, an increase in
ECM protein deposition is an important event in the
airway remodeling process induced by TGF-β1 [
both MMPs and TIMPs are involved. MMPs digest
ECM components and are considered important
mediators of tissue remodeling [
]. MMPs are regulated
by their natural inhibitors, TIMPs . The MMP/TIMP
balance is a critical factor in controlling the overall
proteolytic activity in various tissues [
measured the expression levels of MMP-9 and MMP-2 in
the co-culture model on culture day 21 by gelatin
zymography, as well as the levels of TIMP-1 (main inhibitor
of MMP-9) and TIMP-2 (main inhibitor of MMP-2).
TGF-β1 stimulation promoted secretion of pro-MMP-2
and active MMP-2 in a concentration-dependent
manner (Fig. 3b and c). Levels of pro-MMP-9 secreted into
the medium did not change with TGF-β1 stimulation
(Fig. 3a). However, the involvement of TGF-β signaling
in pro-MMP-9 secretion was seen when TGF-β signaling
was blocked with SB525334. In line with the increases
observed in MMP expression, secretion of TIMP-1 and
TIMP-2 also increased (Fig. 3d and e). As both MMPs
and TIMPs were upregulated, the MMP/TIMP balance
could be maintained in the co-culture model even
following stimulation with TGF-β1. In a study with a
fibroblast mono-culture model, we found that pro-MMP-9
and active MMP-2 were not induced by TGF-β1 (Fig. 4b
and Additional file 2: Figure S2), while TIMP-1 and
TIMP-2 were (Fig. 4c and d). These findings suggest that
epithelial cells were necessary for secretion of
proMMP-9 and active MMP-2 in the co-culture, and
proteolytic activity of these MMPs was controlled by the
TIMPs secreted from mesenchymal fibroblasts. Thus the
concept that epithelial-mesenchymal interaction is
important in controlling ECM protein degradation is
To further characterize the effects of TGF-β1 on ECM
deposition in the co-culture model, we examined
expression changes in genes related to ECM production.
Thirty-three genes were significantly upregulated. We
surmise that these genes are regulated by the TGF-β1/
ALK5 pathway, as the TGF-β1 blocker SB525334
suppressed increases in their expression. Integrins are
transmembrane receptors that exert various effects on
tissue remodeling and contribute to EMTU homeostasis
]. The integrin families upregulated in our assays
have been shown to affect tissue remodeling by
mechanisms such as myofibroblast induction and activation of
TGF-β . Consistent with the findings for TIMPs and
MMPs, gene expression of MMP2, MMP9, and TIMP1
was regulated by TGF-β1 signaling. In addition, we
found increases in the expression of MMP14, MMP10,
MMP12, MMP7, and MMP13. Higher levels of the
MMPs encoded by those genes have been reported in
idiopathic pulmonary fibrosis patients and in a murine
model of lung disease, and these increases may play
various roles in airway tissue remodeling [
]. We also
found increased expression of genes encoding various
collagens and ECM glycoproteins and proteoglycans,
which could be substrates for the proteolytic activity of
MMPs. Expression of these genes suggests the
promotion of ECM production.
We obtained gene expression data from both the
coculture model and the fibroblast mono-culture model
(Figs. 5 and 6). We found that 14 of the 33 genes whose
expression was increased following stimulation with
TGF-β1 were also up-regulated in the fibroblast
monoculture model after TGF-β1 simulation. The reduced
number of up-regulated genes in the fibroblast
monoculture model suggests that co-culture conditions are
necessary for the induction of several genes. This
suggests an involvement of HBECs in the remodeling
process induced by TGF-β1.
We found that FN1 and TNC expression increased
notably following stimulation by 10 ng/mL TGF-β1 in
the co-culture model. The expression pattern in the lung
of proteins encoded by these genes, fibronectin and
tenascin-C, was reported to be related to lung disease
]. Histological analysis determined that these
proteins were expressed across the mesenchymal layer.
However, we found an increase in fibronectin-positive
basal cells following TGF-β1 stimulation. In addition to
vimentin expression, fibronectin expression in basal cells
is considered one aspect of EMT . We also found
strong expression of tenascin-C in the basement
membrane of the co-culture model, in a pattern consistent
with in vivo expression [
]. The expression of both
fibronectin in basal cells and tenascin-C in the
subepithelial region decreased following treatment with the
TGF-β1 blocker SB525334, indicating that the TGF-β1/
ALK5 pathway is involved in ECM deposition in
bronchial tissues. Reproduction of the sub-epithelial region
in vitro is possible when epithelial cells are directly
cocultured with collagen-embedded mesenchymal cells,
and fibrotic changes in this region (i.e. sub-epithelial
fibrosis) are important steps in the pathogenesis of
]. Hence, our in vitro EMTU model could
be useful in elucidating how changes in the
subepithelial region are induced by the interaction between
epithelial cells and mesenchymal layer.
One of the mechanisms in the epithelial–mesenchymal
interaction is paracrine signaling involving growth
factors and cytokines [
]. The interaction is also
mediated by integrins, which could be used as therapeutic
targets in airway hyperresponsiveness and remodeling
]. Various studies suggest that the ECM composition
affects cell signaling, cell proliferation, apoptosis, and
EMT in the epithelial cells through adhesion through
]. Our co-culture model demonstrated
changes in ECM protein and integrin gene expression
following TGF-β1 stimulation. Thus, our model has the
potential to recapitulate cell–cell and cell–ECM
interactions mediated through adhesion by integrins, which is
possible when epithelial cells are directly co-cultured
with a mesenchymal layer.
We developed a co-culture model of human bronchial
tissue that enables direct interactions to occur between
epithelial cells, mesenchymal cells, and their ECM
components. This model successfully reproduced
multiple events including EMT in the epithelial layer,
myofibroblast accumulation in the mesenchymal layer, and
ECM deposition in the airway remodeling process
induced by TGF-β1. These events were blocked with
SB525334. Thus, our model could be useful in the
study of airway remodeling in vitro, as well as drug
testing. Interestingly, the effect of TGF-β1 was different
in the co-culture model compared with the fibroblast
mono-culture model. In the future, we plan to
introduce a mono-culture model of HBECs to our
experimental design, which will enable us to investigate
differences in the response to TGF-β1 stimulation
among these three culture models to clarify the effect
of co-culture in more detail. Moreover, treatment with
target-specific small interfering RNAs or antibodies
should help determine the mechanism underlying
epithelial–mesenchymal cross-talk in EMTU components.
The application of cells from patients will also aid an
investigation of epithelial–mesenchymal cross-talk in
Additional file 1: Figure S1. Histological analysis of differentiation
markers of bronchial epithelium on culture day 21. Tissue sections were
immunostained with an anti-acetylated α-tubulin antibody (ciliated cell
marker), anti-MUC5AC antibody (goblet cell marker), and anti-CK5 antibody
(basal cell marker). Scale bar: 50 μm. (JPEG 2811 kb)
Additional file 2: Figure S2. Gelatin zymography of culture medium
from the co-culture model and fibroblast mono-culture model collected
on culture day 21. (JPEG 669 kb)
ALI: Air-liquid interface; ALK: Activin receptor-like kinase; CK: Cytokeratin;
DAB: Diaminobenzidine; ECM: Extracellular matrix; EMT:
Epithelialmesenchymal transition; EMTU: Epithelial-mesenchymal trophic unit;
HBEC: Human bronchial epithelial cell; MMP: Matrix metalloproteinase;
TGF: Transforming growth factor; TIMP: Tissue inhibitor of metalloproteinase
We are grateful to Dr. Tomoki Nishino and Dr. Yuichiro Takanami for their
support and advice for the project. We also thank Dean Meyer, PhD, ELS,
from Edanz Group (www.edanzediting.com/ac) for editing a draft of this
All authors are employees of Japan Tobacco Inc. Japan Tobacco Inc. is the
sole source of funding of this project.
Availability of data and materials
S. Ishikawa and S. Ito developed the concept and design of the research. S.
Ishikawa and K. Ishimori performed the experiments. S. Ishikawa and S. Ito
interpreted the results of the experiments. All authors read and approved
the final version of the manuscript.
Ethics approval and consent to participate
Consent for publication
The authors are employees of Japan Tobacco Inc. and report that they have
no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Jeffery PK . Remodeling in asthma and chronic obstructive lung disease . Am J Respir Crit Care Med . 2001 ; 164 : S28 - 38 .
2. Carroll N , Elliot J , Morton A , James A . The structure of large and small airways in nonfatal and fatal asthma . Am Rev Respir Dis . 1993 ; 147 : 405 - 10 .
3. Kuhn C , McDonald JA . The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis . Am J Pathol . 1991 ; 138 : 1257 - 65 .
4. Montesano R , Orci L. Transforming growth factor beta stimulates collagenmatrix contraction by fibroblasts: implications for wound healing . Proc Natl Acad Sci U S A . 1988 ; 85 : 4894 - 7 .
5. Finesmith TH , Broadley KN , Davidson JM . Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors . J Cell Physiol . 1990 ; 144 : 99 - 107 .
6. Kurosaka H , Kurosaka D , Kato K , Mashima Y , Tanaka Y. Transforming growth factor-beta 1 promotes contraction of collagen gel by bovine corneal fibroblasts through differentiation of myofibroblasts . Invest Ophthalmol Vis Sci . 1998 ; 39 : 699 - 704 .
7. Berube K , Prytherch Z , Job C , Hughes T. Human primary bronchial lung cell constructs: the new respiratory models . Toxicology . 2010 ; 278 : 311 - 8 .
8. Kolb M , Margetts PJ , Anthony DC , Pitossi F , Gauldie J . Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis . J Clin Invest . 2001 ; 107 : 1529 - 36 .
9. Temelkovski J , Hogan SP , Shepherd DP , Foster PS , Kumar RK . An improved murine model of asthma: selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen . Thorax . 1998 ; 53 : 849 - 56 .
10. Doherty T , Broide D . Cytokines and growth factors in airway remodeling in asthma . Curr Opin Immunol . 2007 ; 19 : 676 - 80 .
11. Evans MJ , Van Winkle LS , Fanucchi MV , Plopper CG . The attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit . Am J Respir Cell Mol Biol . 1999 ; 21 : 655 - 7 .
12. Holgate ST , Davies DE , Lackie PM , Wilson SJ , Puddicombe SM , Lordan JL . Epithelial-mesenchymal interactions in the pathogenesis of asthma . J Allergy Clin Immunol . 2000 ; 105 : 193 - 204 .
13. Emura M , Aufderheide M , Mohr U . Target cell types with stem/progenitor function to isolate for in vitro reconstruction of human bronchiolar epithelia . Exp Toxicol Pathol . 2015 ; 67 : 81 - 8 .
14. Ishikawa S , Ito S. Repeated whole cigarette smoke exposure alters cell differentiation and augments secretion of inflammatory mediators in airliquid interface three-dimensional co-culture model of human bronchial tissue . Toxicol in Vitro. 2017 ; 38 : 170 - 8 .
15. Hostettler KE , Roth M , Burgess JK , Gencay MM , Gambazzi F , Black JL , Tamm M , Borger P . Airway epithelium-derived transforming growth factor-beta is a regulator of fibroblast proliferation in both fibrotic and normal subjects . Clin Exp Allergy . 2008 ; 38 : 1309 - 17 .
16. Mio T , Liu XD , Adachi Y , Striz I , Skold CM , Romberger DJ , Spurzem JR , Illig MG , Ertl R , Rennard SI . Human bronchial epithelial cells modulate collagen gel contraction by fibroblasts . Am J Phys . 1998 ; 274 : L119 - 26 .
17. Hill AR , Donaldson JE , Blume C , Smithers N , Tezera L , Tariq K , Dennison P , Rupani H , Edwards MJ , Howarth PH , et al. IL -1alpha mediates cellular crosstalk in the airway epithelial mesenchymal trophic unit . Tissue Barriers . 2016 ; 4 : e1206378 .
18. Ruifrok AC , Johnston DA . Quantification of histochemical staining by color deconvolution . Anal Quant Cytol Histol . 2001 ; 23 : 291 - 9 .
19. Tan Q , Choi KM , Sicard D , Tschumperlin DJ . Human airway organoid engineering as a step toward lung regeneration and disease modeling . Biomaterials . 2017 ; 113 : 118 - 32 .
20. Wilkinson DC , Alva-Ornelas JA , Sucre JM , Vijayaraj P , Durra A , Richardson W , Jonas SJ , Paul MK , Karumbayaram S , Dunn B , Gomperts BN . Development of a three-dimensional bioengineering technology to generate lung tissue for personalized disease modeling . Stem Cells Transl Med . 2017 ; 6 : 622 - 33 .
21. Ross AJ , Dailey LA , Brighton LE , Devlin RB . Transcriptional profiling of mucociliary differentiation in human airway epithelial cells . Am J Respir Cell Mol Biol . 2007 ; 37 : 169 - 85 .
22. Higashiyama H , Yoshimoto D , Kaise T , Matsubara S , Fujiwara M , Kikkawa H , Asano S , Kinoshita M. Inhibition of activin receptor-like kinase 5 attenuates bleomycin-induced pulmonary fibrosis . Exp Mol Pathol . 2007 ; 83 : 39 - 46 .
23. Thomas M , Docx C , Holmes AM , Beach S , Duggan N , England K , Leblanc C , Lebret C , Schindler F , Raza F , et al. Activin-like kinase 5 (ALK5) mediates abnormal proliferation of vascular smooth muscle cells from patients with familial pulmonary arterial hypertension and is involved in the progression of experimental pulmonary arterial hypertension induced by monocrotaline . Am J Pathol . 2009 ; 174 : 380 - 9 .
24. Kobayashi T , Liu X , Wen FQ , Kohyama T , Shen L , Wang XQ , Hashimoto M , Mao L , Togo S , Kawasaki S , et al. Smad3 mediates TGF-beta1-induced collagen gel contraction by human lung fibroblasts . Biochem Biophys Res Commun . 2006 ; 339 : 290 - 5 .
25. Zavadil J , Bottinger EP . TGF-beta and epithelial-to-mesenchymal transitions . Oncogene . 2005 ; 24 : 5764 - 74 .
26. Katsuno Y , Lamouille S , Derynck R . TGF-beta signaling and epithelialmesenchymal transition in cancer progression . Curr Opin Oncol . 2013 ; 25 : 76 - 84 .
27. Hackett TL , Warner SM , Stefanowicz D , Shaheen F , Pechkovsky DV , Murray LA , Argentieri R , Kicic A , Stick SM , Bai TR , Knight DA . Induction of epithelialmesenchymal transition in primary airway epithelial cells from patients with asthma by transforming growth factor-beta1 . Am J Respir Crit Care Med . 2009 ; 180 : 122 - 33 .
28. Kalluri R , Weinberg RA . The basics of epithelial-mesenchymal transition . J Clin Invest . 2009 ; 119 : 1420 .
29. Desmouliere A , Chaponnier C , Gabbiani G. Tissue repair, contraction, and the myofibroblast . Wound Repair Regen . 2005 ; 13 : 7 - 12 .
30. Xing Y , Li C , Li A , Sridurongrit S , Tiozzo C , Bellusci S , Borok Z , Kaartinen V , Minoo P . Signaling via Alk5 controls the ontogeny of lung Clara cells . Development . 2010 ; 137 : 825 - 33 .
31. Halwani R , Al-Muhsen S , Al-Jahdali H , Hamid Q . Role of transforming growth factor-beta in airway remodeling in asthma . Am J Respir Cell Mol Biol . 2011 ; 44 : 127 - 33 .
32. Lagente V , Manoury B , Nenan S , Le Quement C , Martin-Chouly C , Boichot E. Role of matrix metalloproteinases in the development of airway inflammation and remodeling . Braz J Med Biol Res . 2005 ; 38 : 1521 - 30 .
33. Nagase H , Woessner JF Jr. Matrix metalloproteinases . J Biol Chem . 1999 ; 274 : 21491 - 4 .
34. Visse R , Nagase H . Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry . Circ Res . 2003 ; 92 : 827 - 39 .
35. Mercer PF , Shute JK , Bhowmik A , Donaldson GC , Wedzicha JA , Warner JA . MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation . Respir Res . 2005 ; 6 : 151 .
36. Perez P , Kwon YJ , Alliende C , Leyton L , Aguilera S , Molina C , Labra C , Julio M , Leyton C , Gonzalez MJ . Increased acinar damage of salivary glands of patients with Sjogren's syndrome is paralleled by simultaneous imbalance of matrix metalloproteinase 3/tissue inhibitor of metalloproteinases 1 and matrix metalloproteinase 9/tissue inhibitor of metalloproteinases 1 ratios . Arthritis Rheum . 2005 ; 52 : 2751 - 60 .
37. Watanabe N , Ikeda U . Matrix metalloproteinases and atherosclerosis . Curr Atheroscler Rep . 2004 ; 6 : 112 - 20 .
38. Cox D , Brennan M , Moran N. Integrins as therapeutic targets: lessons and opportunities . Nat Rev Drug Discov . 2010 ; 9 : 804 - 20 .
39. Araya J , Cambier S , Morris A , Finkbeiner W , Nishimura SL . Integrin-mediated transforming growth factor-beta activation regulates homeostasis of the pulmonary epithelial-mesenchymal trophic unit . Am J Pathol . 2006 ; 169 : 405 - 15 .
40. Margadant C , Sonnenberg A. Integrin-TGF-beta crosstalk in fibrosis, cancer and wound healing . EMBO Rep . 2010 ; 11 : 97 - 105 .
41. Pardo A , Cabrera S , Maldonado M , Selman M. Role of matrix metalloproteinases in the pathogenesis of idiopathic pulmonary fibrosis . Respir Res . 2016 ; 17 : 23 .
42. Annoni R , Lancas T , Yukimatsu Tanigawa R , de Medeiros MM , de Morais FS , Bruno A , Fernando Ferraz da Silva L , Roughley PJ , Battaglia S , Dolhnikoff M , et al. Extracellular matrix composition in COPD . Eur Respir J . 2012 ; 40 : 1362 - 73 .
43. Estany S , Vicens-Zygmunt V , Llatjos R , Montes A , Penin R , Escobar I , Xaubet A , Santos S , Manresa F , Dorca J , Molina-Molina M . Lung fibrotic tenascin-C upregulation is associated with other extracellular matrix proteins and induced by TGFbeta1 . BMC Pulm Med . 2014 ; 14 : 120 .
44. Gohy ST , Hupin C , Fregimilicka C , Detry BR , Bouzin C , Gaide Chevronay H , Lecocq M , Weynand B , Ladjemi MZ , Pierreux CE , et al. Imprinting of the COPD airway epithelium for dedifferentiation and mesenchymal transition . Eur Respir J . 2015 ; 45 : 1258 - 72 .
45. Laitinen A , Altraja A , Kampe M , Linden M , Virtanen I , Laitinen LA . Tenascin is increased in airway basement membrane of asthmatics and decreased by an inhaled steroid . Am J Respir Crit Care Med . 1997 ; 156 : 951 - 8 .
46. Homer RJ , Elias JA . Airway remodeling in asthma: therapeutic implications of mechanisms . Physiology (Bethesda) . 2005 ; 20 : 28 - 35 .
47. Roche WR , Beasley R , Williams JH , Holgate ST . Subepithelial fibrosis in the bronchi of asthmatics . Lancet . 1989 ; 1 : 520 - 4 .
48. Wright DB , Meurs H , Dekkers BG . Integrins: therapeutic targets in airway hyperresponsiveness and remodelling? Trends Pharmacol Sci . 2014 ; 35 : 567 - 74 .
49. Han SW , Roman J . Fibronectin induces cell proliferation and inhibits apoptosis in human bronchial epithelial cells: pro-oncogenic effects mediated by PI3-kinase and NF-kappa B. Oncogene . 2006 ; 25 : 4341 - 9 .
50. Aoshiba K , Rennard SI , Spurzem JR . Cell-matrix and cell-cell interactions modulate apoptosis of bronchial epithelial cells . Am J Phys . 1997 ; 272 : L28 - 37 .
51. Mamuya FA , Duncan MK . aV integrins and TGF-beta-induced EMT: a circle of regulation . J Cell Mol Med . 2012 ; 16 : 445 - 55 .