Human iPSC-MSCs prevent steroid-resistant neutrophilic airway inflammation via modulating Th17 phenotypes
Fang et al. Stem Cell Research & Therapy
Human iPSC-MSCs prevent steroid-resistant neutrophilic airway inflammation via modulating Th17 phenotypes
Shu-Bin Fang 1
Hong-Yu Zhang 1
Ai-Yun Jiang 1
Xing-Liang Fan 0 1
Yong-Dong Lin 1
Cheng-Lin Li 0 1
Cong Wang 1
Xiang-Ci Meng 1
Qing-Ling Fu 0 1
0 Centre for Stem Cell Clinical Research and Application, The First Affiliated Hospital of Sun Yat-sen University , Guangzhou 510080 , China
1 Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University , 58 Zhongshan Road II, Guangzhou 510080, Guangdong , China
Background: Human induced pluripotent stem cells-derived mesenchymal stem cells (iPSC-MSCs) have been shown to be effective in Type 2 helper T cells (Th2)-dominant eosinophilic allergic airway inflammation. However, the role of iPSC-MSCs in Type 17 helper T cells (Th17)-dominant neutrophilic airway inflammation remains poorly studied. Therefore, this study was to explore the effects of iPSC-MSCs on an experimental mouse model of steroidresistant neutrophilic airway inflammation and further determine the underlying mechanisms. Methods: A mouse model of neutrophilic airway inflammation was established using ovalbumin (OVA) and lipopolysaccharide (LPS). Human iPSC-MSCs were systemically administered, and the lungs or bronchoalveolar lavage fluids (BALF) were collected at 4 h and 48 h post-challenge. The pathology and inflammatory cell infiltration, the T helper cells, T helper cells-associated cytokines, nuclear transcription factors and possible signaling pathways were evaluated. Human CD4+ T cells were polarized to T helper cells and the effects of iPSC-MSCs on the differentiation of T helper cells were determined. Results: We successfully induced the mouse model of Th17 dominant neutrophilic airway inflammation. Human iPSC-MSCs but not dexamethasone significantly prevented the neutrophilic airway inflammation and decreased the levels of Th17 cells, IL-17A and p-STAT3. The mRNA levels of Gata3 and RORγt were also decreased with the treatment of iPSC-MSCs. We further confirmed the suppressive effects of iPSC-MSCs on the differentiation of human T helper cells. Conclusions: iPSC-MSCs showed therapeutic potentials in neutrophilic airway inflammation through the regulation on Th17 cells, suggesting that the iPSC-MSCs could be applied in the therapy for the asthma patients with steroidresistant neutrophilic airway inflammation.
Dexamethasone; Immunoregulation; iPSC-MSCs; Neutrophilic airway inflammation; Type 17 helper T cells
Asthma is characterized by heterogeneous upper airway
inflammation in which different inflammatory cells are
]. Based on the inflammatory cell profiles in
induced sputum, neutrophilic asthma has been defined
as a distinct phenotype from Type 2 helper T cells
(Th2)-dominant eosinophilic asthma [
]. It has been
reported that almost 50% asthma patients are attributable
to this subgroup, in which a substantial presence of
neutrophils is found in the airway [
]. Type 17 helper T cells
(Th17) have been implicated in the pathogenesis of
neutrophil-predominant asthma and the insensitivity to
glucocorticoid in severe asthma [
]. After being
stimulated by Th17-derived cytokines, the airway epithelial
cells further release neutrophil-attracting cytokines or
chemokines for the recruitment of the neutrophils .
Previous studies have shown that the neutrophils present
in the airway were highly associated with the severity of
airway inflammation [
] and insensitivity to
corticosteroid treatment in asthma patients [
The steroid therapy is an important treatment for
asthma patients in clinical practice. However, the
patients with neutrophil-predominant asthma sometimes
respond poorly to the steroid treatment even with
high dosages, making it increasingly a great concern
in the asthma therapies [
]. Although some advances
have been made in the development of novel
monoclonal antibodies for severe asthma, none of these
biologics produced positive effects on asthma patients
with severe neutrophilic airway inflammation [
Recently, some novel antagonists and inhibitors have
been reported to reduce neutrophilic airway
inflammation in experimental animal models of asthma
]. However, chemical therapies are often
associated with adverse side effects, and further studies on
the safety and efficacy are required before being
applied to humans. Therefore, it is evident that no
effective therapies are currently available for the
treatment of steroid-resistant neutrophilic airway
inflammation, and the need for novel therapies has
become extremely urgent.
We have successfully developed mesenchymal stem
cells (MSCs) from human induced pluripotent stem cells
], and identified that human iPSC-MSCs have
the potentials to modulate T cell phenotypes in human
allergic rhinitis [
] and ameliorate
Th2/eosinophil-dominant allergic airway inflammation in mice [
In addition, previous studies have shown that MSCs
had exerted promising immunosuppressive effects on
Th17 cells in some other immunoinflammatory
]. Thus, we hypothesized that
iPSC-MSCs could exhibit therapeutic effects in
steroid-resistant neutrophilic airway inflammation via
the Th17 signaling pathway. It has been reported that
murine bone marrow-derived MSCs (BM-MSCs) [
or human umbilical cord blood-derived MSCs
] suppressed neutrophilic airway
inflammation. In their reports, they induced mouse
models of neutrophilic airway inflammation using the
fungal or viral infections as adjuvants. However, they
did not report whether the models were
steroid-resistant inflammation or not. Actually,
exposure to environmental bacterial endotoxin has been
considered a great risk factor for neutrophilic airway
] and thus the steroid-resistant mouse
model of neutrophilic airway inflammation triggered
by allergen with an environment-relevant dose of
lipopolysaccharide (LPS) would more closely mimic
the pathogenesis of neutrophilic asthma in human
]. We have previously reported that, compared to
BM-MSCs and fetus-derived MSCs, iPSC-MSCs have
a stronger immune privilege after transplantation [
It may attribute to a better therapeutic efficacy in an
allogeneic transplantation. Currently, the effects of
iPSC-MSCs on steroid-resistant neutrophilic airway
inflammation and the underlying mechanisms remain
to be further understood.
In the present study, we aimed to explore the effects
of iPSC-MSCs on steroid-resistant neutrophilic airway
inflammation triggered by allergen plus an
environment-relevant dose of LPS, and evaluate the
immunoregulatory function of iPSC-MSCs on T helper
cells, especially the Th17 cells.
Female C57BL/6 mice (for neutrophil-dominant model)
and Balb/c mice (for eosinophil-dominant model) (aged
6–8 weeks) were purchased from the Guangdong
Medical Laboratory Animal Center (Guangzhou, China). All
the animals were maintained in the specific
pathogen-free environment. All the procedures
performed in this study were approved by the Ethics
Committee of The First Affiliated Hospital, Sun Yat-sen
Preparation and identification of human iPSC-MSCs
The human iPSC-MSCs used in this study were
prepared and identified as reported in our previous study
]. Briefly, iPSCs reprogrammed from human
urine-derived cells were further induced into
iPSC-MSCs, which were characterized by the similar
expression of general surface markers to BM-MSCs and
potentials of osteogenic, chondrogenic, and adipogenic
Mouse model of neutrophilic airway inflammation
The neutrophilic airway inflammation mouse model was
developed as previously reported with minor
]. As shown in Fig. 1a, the mice were
sensitized with 100 μg low-endotoxin Ovalbumin (OVA,
Grade V, Sigma-Aldrich, St. Louis, MO, USA) and 0.1
μg LPS (Escherichia coli O111:B4, Sigma-Aldrich, St.
Louis, MO, USA) in 40 μL sterile phosphate-buffered
saline (PBS) on day 1 and 7 and then challenged daily with
5% aerosolized OVA for 40 min on day 14 through an
air-compressing nebulizer (0.2 mL/min, Yueyue, Jiangsu,
China). The negative control mice were administered
with 40 μL sterile PBS and then challenged daily with
PBS for 40 min on day 14. The mice were sacrificed at
4 h, 24 h, 48 h or 72 h after the challenge. Where
indicated, the OVA-sensitized mice were administered with
1 × 106 iPSC-MSCs (OVA/OVA/iPSC-MSC, n = 6 for 4 h
and 48 h) intravenously or 1 mg/kg/mice dexamethasone
(DEX) intraperitoneally (OVA/OVA/DEX, n = 6 for 4 h,
n = 5 for 48 h) in 200 μL PBS on day 13 and both the
negative (PBS/PBS/PBS, n = 5 for 4 h and 48 h) and
positive control mice (OVA/OVA/PBS, n = 5 for 4 h and
48 h) were administered intravenously with only 200 μL
PBS. The frequencies of nasal rubbing and sneezing were
evaluated within 10 min after the challenge. The Th2/
eosinophil-dominant airway inflammation mouse model
was developed as our previous report [
the mice (n = 5) were sensitized with 40 μg of OVA and
4 mg of aluminum hydroxide (Thermo Fisher Scientific,
Waltham, MA, USA) on days 1, 7, 14. After the
administration of 200 μL PBS on day 20, the mice were further
challenged with 5% OVA on days 21–25 and sacrificed
at 4 h after the last challenge. After the mice were
sacrificed, the bronchoalveolar lavage fluids (BALF) was
collected and lung perfusion was performed to remove the
remaining blood. Then the lung tissues were collected
for further analysis (Left: Histopathologic analysis; Right
middle lobe: PCR and western blot analysis; others:
Collection of bronchoalveolar lavage fluids (BALF)
The BALF was collected as previously reported
]. Briefly, about 0.8 mL BALF was obtained by
performing the lung lavage with 1 mL cold PBS for
three times. The total cell numbers were counted
with a hemocytometer and the BALF was further
centrifuged at 400 g for 5 min. After the
centrifugation, the supernatants were collected for the
evaluation of Th1- (IFN-γ), Th2- (IL-4/13) or
Th17(IL-17A) derived cytokines (R&D Systems,
Minneapolis, MN, USA). The pellets were smeared onto glass
slides and stained with Diff-Quick (Baso Diagnostics
Inc., Zhuhai, Guangdong, China) for differential cell
counts, including neutrophils, eosinophils,
lymphocytes and macrophages.
Histopathologic evaluation of lung tissues
Lung sections were fixed with 4% paraformaldehyde for
hematoxylin and eosin (H&E) staining and inflammation
scores were evaluated in a blind fashion by two
independent investigators based on the scoring standard as
shown in Additional file 1: Table S1. Where indicated,
the lung sections were also stained with Periodic acid–
Schiff (PAS) for the evaluation of Goblet cell counts in
Quantitative real-time PCR
Real-time PCR was performed to detect the expression
of T-bet, Gata-3 and RORγt in the lung tissues. All the
primers for PCR were mouse specific. A brief description
is presented in Additional file 1.
Western blot analysis was performed to analyze the
expression of p-STAT1, p-STAT3 and p-STAT6 in the lung
tissues at 4 h after challenge. The detailed information is
presented in Additional file 1.
Flow cytometry analysis of T helper cells in lung tissues
Flow cytometry analyses were performed to examine the
T helper cells in lung tissues of the mouse. The detailed
information is presented in Additional file 1.
Induction of human T helper cells and co-culture with iPSC-MSCs
To investigate the effects of iPSC-MSCs on the
differentiation of T helper cells, human peripheral blood
mononuclear cells (PBMCs) were isolated and co-cultured
with iPSC-MSCs in the presence of cytokines or
antibodies for T helper cells polarization. The detailed
information is presented in the Additional file 1.
All the data were analyzed using GraphPad 6.0 (San
Diego, CA, USA) and all the results were expressed as
Mean ± SEM. Statistical analyses were performed using
Mann-Whitney test or t test as indicated. A P value less
than 0.05 were considered statistically significant.
The neutrophilic airway inflammation elicited different responses in a time-dependent manner
To establish the mouse model of neutrophilic airway
inflammation, we first explored the responses at multiple
sampling time points in the development of neutrophilic
airway inflammation (n = 3 per group). The H&E
staining of the lung tissues showed that the airway
inflammation in OVA-sensitized mice was observed at 4 h
post-challenge. The inflammatory status continued
exacerbating at 24 h, but attenuated slowly at 48 h and
72 h (Fig. 1b). However, almost no PAS-positive cells
were observed in the mice with a single challenge as
shown by the PAS staining of the lung tissues (Fig. 1c),
suggesting that goblet cell hyperplasia in the model of
neutrophilic airway inflammation was not as robust as
that in the model of eosinophilic airway inflammation.
Diff-Quik staining for the inflammatory cells showed
that neutrophils but not eosinophils were the dominant
infiltrated inflammatory cells in the airway at different
sampling time points and the levels of macrophages and
lymphocytes were also much lower than the neutrophils
(Fig. 1d). We found many eosinophils for the Diff-Quik
staining in BALF in the eosinophilic airway
inflammation (Fig. 1d). Unlike the scores of airway inflammation,
the levels of IL-17A in mice peaked at 4 h
post-challenge and then declined sharply at 24 h, 48 h
and 72 h (Fig. 1e). Therefore, it suggests that we should
examine the effects of iPSC-MSCs on the airway
inflammation or Th17 levels at different time points
Human iPSC-MSCs ameliorated inflammatory cell infiltration in murine neutrophilic airway inflammation
Human iPSC-MSCs were administered one day before
the challenge and we evaluated the effects of
iPSC-MSCs (n = 6) and DEX (n = 5) on murine
histopathology for lung tissues, and the profiles of
inflammatory cells in BALF at 48 h post-challenge. Obvious
peribronchial inflammation was observed in the OVA/
OVA/PBS mice (Fig. 2a and c, P < 0.01, n = 5). The
treatment with DEX did not exhibit therapeutic effects
on the airway inflammation. However, the airway
inflammation was significantly attenuated by iPSC-MSCs
(Fig. 2a and c, P < 0.05). Additionally, we investigated
the effects of iPSC-MSCs on the profiles of
inflammatory cells in BALF, in which substantial infiltration of
neutrophils was found (Fig. 2b). We observed significant
decreases in the numbers of total cells (P < 0.05) and
neutrophils (P < 0.01) in the iPSC-MSC group, which
were still poorly controlled by DEX (Fig. 2d). Also, the
levels of total protein in BALF were increased in the
OVA/OVA/PBS mice, and reduced by iPSC-MSCs but
not DEX (Fig. 2e). All the pathogenic improvements in
neutrophilic airway inflammation were consistent with
the functional recovery of the frequencies of nasal
rubbing (Fig. 2f ) and sneezing (Fig. 2g) post-challenge with
the treatment of iPSC-MSCs. These data suggest that
the neutrophilic airway inflammation in the settings of
the established murine model was resistant to DEX, but
could be ameliorated by iPSC-MSCs.
We also examined the effects of DEX or iPSC-MSCs
on airway inflammation at 4 h after the challenge.
Obvious infiltration of inflammatory cells in peribronchial
interstitial tissues and more total inflammatory cells,
neutrophils in BALF were observed in the mice that
were sacrificed at 4 h post-challenge (P < 0.05). However,
the airway inflammation at 4 h post-challenge was
not significantly decreased by DEX or iPSC-MSCs
(Additional file 1: Figure S1).
Human iPSC-MSCs inhibited Th17 levels in murine neutrophilic airway inflammation
Th17 was reported to be involved in the neutrophilic
airway inflammation [
], and our above data showed that
the level of IL-17A peaked at 4 h post-challenge in our
model mice. Next, we investigated the
immunomodulation of iPSC-MSCs on Th cells in this neutrophilic
airway inflammation model at 4 h post-challenge. The
single lung cells present in lung tissues were obtained
for flow cytometry analysis for Th1 (CD4+ IFN-γ+ T
cells), Th2 (CD4+ IL-4+ T cells) and Th17 (CD4+
IL-17A+ T cells) (Fig. 3a and b). We observed higher
Th1 (P < 0.05), much higher Th17 (P < 0.01) but not Th2
percentages in OVA/OVA/PBS mice (n = 5) compared to
control mice (n = 5) (Fig. 3c), suggesting that Th17 was
the prime T helper cells in this neutrophilic airway
inflammation. Both Th2 and Th17 levels were decreased after
the administration of iPSC-MSCs (P < 0.01, n = 6), while
Th1 were oppositely increased (Fig. 3c, P < 0.01). DEX (n
= 6) had no effects on Th1 and Th17 levels but slightly
decreased Th2 level (Fig. 3c). Furthermore, similar
tendencies to the levels of T helper cells were found for the Th1
(IFN-γ)- and Th17 (IL-17A)-derived cytokines in BALF, in
which higher IL-17A was significantly decreased by
iPSC-MSCs (P < 0.01) but not DEX while IFN-γ was
significantly increased (Fig. 3d and e, P < 0.05). Additionally,
the Th2 (IL-4/13)-derived cytokines were undetectable in
all of the groups (data not shown). Both the DEX and
iPSC-MSCs had no effects on the total levels of protein in
BALF at 4 h post-challenge (Fig. 3f ).
We further confirmed the above results by analyzing
the expressions of related nuclear transcription factors
and cytokines. Accordingly, the quantification for the
mRNA levels of Th1 (T-bet)-, Th2 (Gata3)- and Th17
(RORγt)-associated transcription factors in lung tissues
at 4 h post-challenge showed that RORγt were
significantly decreased after the treatment with iPSC-MSCs
(Fig. 3g, P < 0.05). The administration of iPSC-MSCs
also decreased mRNA level of Gata3 (Fig. 3h) but had
no effects on T-bet (Fig. 3i). DEX administration had no
effects on the mRNA levels of T-bet, Gata3 and RORγt
We also investigated the effects of iPSC-MSCs or DEX
on T helper cells at 48 h post-challenge. No significant
changes were observed for any of the subsets of T helper
cells at 48 h post-challenge, and the treatments of both
iPSC-MSCs and DEX had no effects on the T helper
cells (Additional file 1: Figure S2).
These findings showed that iPSC-MSCs exhibited the
immunomodulation on Th cells especially Th17 level
mainly at the early stage of 4 h post-challenge. However,
DEX exhibited no effects on Th1/17 cells and all the T
cells-associated genes and cytokines, which further
confirmed that neutrophilic airway inflammation was
steroid-resistant. Taken together, these results revealed
that iPSC-MSCs had the potential to inhibit the
development and activity of Th17 cells in steroid-resistant
The effects of iPSC-MSCs on p-STAT3 signaling pathway in the neutrophilic airway inflammation
It was reported that STAT3 promotes the differentiation
of Th17 cells [
]. To further explore the underlying
mechanisms involved in the effects of iPSC-MSCs on
neutrophilic airway inflammation and Th17, we
determined the protein level of p-STAT3 in the mouse lungs
at 4 h after the challenge. The western blot of the lung
tissues showed that p-STAT3 was significantly increased
after the induction of neutrophilic airway inflammation
(Fig. 4a). We further identified that the level of p-STAT3
was significantly decreased after the administration of
iPSC-MSCs (n = 6) but not DEX (n = 6) (Fig. 4b),
suggesting that iPSC-MSCs may inhibit the differentiation
of Th17 cells in this model of neutrophilic airway
inflammation via downregulating p-STAT3 level.
Additionally, we found that there was no expression of p-STAT1
and p-STAT6, which were involved in Th1, Th2 after the
induction of neutrophilic airway inflammation (Fig. 4a).
Human iPSC-MSCs inhibited the differentiation of Th cells in vitro
We next investigated the effects of iPSC-MSCs on the
differentiation of human Th cells in vitro (n = 5).
Purified human CD4+ T cells were polarized to Th1, Th2
and Th17 in different conditions respectively (Additional
file 1: Table S3), activated with anti-CD3/CD28, and
treated with or without human iPSC-MSCs for 5 days.
Then the CD4+ T cells were harvested for flow
cytometry analysis (Fig. 5a). We observed significant increases
in the proportions for Th1 (Fig. 5b and e, P < 0.01), Th2
(Fig. 5c and f, P < 0.01) and Th17 (Fig. 5d and g, P <
0.01) under their polarizing conditions. The treatment
with iPSC-MSCs markedly reversed the levels of all the
three subsets of Th cells (Fig. 5e-g, P < 0.01), suggesting
(See figure on previous page.)
Fig. 3 Human iPSC-MSCs inhibited Th17 level at 4 h post-challenge in a mouse model of steroid-resistant airway inflammation. a Representative
gating strategies of flow cytometry analysis for T helper cells in mouse lung tissues. b Representative dot plots showing the percentages of Th1/
Th2/Th17 cells in CD4+ T cells in different groups. c Statistical analysis of T helper cell percentages in lung CD4+ T cells. The percentage of Th17
but not Th1 and Th2 was significantly increased in neutrophilic airway inflammation. Both Th1 and Th17 were resistant to DEX and only Th2 was
sensitive to DEX. iPSC-MSCs decreased both Th2 and Th17 cell levels while increased Th1 cell level in the model mouse. d-e Statistical analysis of
IFN-γ and IL-17A levels in BALF. f The levels of total protein in BALF at 4 h post-challenge. g-i Statistical analysis of Gata-3, RORγt and T-bet levels
in the lung tissues. *P < 0.05, **P < 0.01 by the Mann-Whitney U test. Abbreviations: BALF bronchoalveolar lavage fluids, DEX dexamethasone,
iPSCMSCs induced pluripotent stem cell-derived mesenchymal stem cells, ns not significant, PBS, phosphate-buffered saline, OVA ovalbumin. n = 5 for
PBS/PBS/PBS and OVA/OVA/PBS, n = 6 for OVA/OVA/DEX and OVA/OVA/MSC
that iPSC-MSCs significantly suppressed the
differentiation of all the three subsets of human Th cells in vitro.
In this study, for the first time, we demonstrated that
human iPSC-MSCs inhibited the Th17 level, and further
ameliorated airway inflammation in a mouse model of the
steroid-resistant neutrophilic asthma. Moreover, we found
that STAT3 signaling was the potential pathway involved
in the immunoregulatory functions of iPSC-MSCs on
neutrophilic airway inflammation. Additionally, we
identified that iPSC-MSCs were capable of inhibiting the
differentiation of human T helper cells in vitro.
Although the effects of the murine BM-MSCs or
human UBC-MSCs in neutrophilic airway inflammation
has been reported, the animal model established in this
study was quite different from the two previous reports
]. In our study, LPS was used as the adjuvant to
develop not only the neutrophilic but also
steroid-resistant airway inflammation model. It is a good
model to study the candidates for the treatment of
steroid-resistant neutrophilic airway inflammation. We
have previously reported that human iPSC-MSCs were
effective in Th2/eosinophil-dominant asthma , thus
we further evaluated the effects of iPSC-MSCs on
steroid-resistant neutrophilic airway inflammation in our
current report. MSCs derived from iPSCs, which were
reprogrammed from human urine cells, were utilized in
this study. For the urine cells are exfoliated renal system
epithelial cells that are able to be collected under most
of the circumstances except for renal failure, making it
the practical and non-invasive way to collect unlimited
source of human cells for reprogramming. In addition,
we previously reported that the U-iPSC-MSCs exhibited
obviously higher growth ability than BM-MSCs and
almost no senescent cells were found even at Passage 50
]. These advantages enable us to provide plentiful of
iPSC-MSCs in the future clinical application. We found
that the administration of iPSC-MSCs prior to the
challenge prevented the development of steroid-resistant
neutrophilic airway inflammation, and decreased the
frequencies of nasal rubbing and sneezing at 48 h
post-challenge in mice. Clinically, it has been reported
that the care for patients with severe asthma account for
60% of the cost of asthma even though it only makes up
3–10% of the population [
]. Among the severe
asthmatics, the neutrophilic asthma is the most troublesome
phenotype that is more frequently accompanied by
severe symptoms and poor quality of life [
]. To our
knowledge, the corticosteroid is currently the mainstay
treatment for the asthmatics but fails to achieve good
responses in some asthma patients with neutrophilic
airway inflammation and no other effective therapies are
currently available for this subpopulation [
the development of some chemicals in experimental
models provided possible approaches to the therapies
for this type of asthma [
15, 17, 18
], the general side
effects of chemical treatments such as severe headache
and gastrointestinal reactions should be highly
concerning and could be the major obstacle in clinical
application. Our findings provided us with the strong evidence
that iPSC-MSCs were clinically promising in the
application for the treatment of asthmatics, especially for the
patients that are insensitive to steroid therapy.
As far as we know, our study was the first time to explore
the effects of human iPSC-MSCs on steroid-resistant
neutrophilic airway inflammation in a mouse model triggered
with allergen plus an environment-relevant dose of LPS
]. Previous studies have reported that mouse BM-MSCs
or human UCB-MSCs were effective in the mouse models
of neutrophilic airway inflammation, but the effects of
glucocorticoid were not evaluated in these studies [
Additionally, the models were induced with adjuvants that
simulated the fungal and viral infections, which were
different risk factors for neutrophilic asthma. Thus, these animal
models could not possibly mimic the important feature of
steroid resistance in some neutrophilic asthma patients as
our model did. Also, human iPSC-MSCs have been shown
to have the higher regenerative capacity and lower
immunogenicity compared with BM-MSCs , suggesting
that our iPSC-MSCs were more promising for the therapy
of steroid-resistant asthma patients.
It has been demonstrated that Th17 is the major
player in the pathogenesis of murine and human
steroid-resistant neutrophilic asthma, in which IL-17A
derived from Th17 cells further promotes the
recruitment of neutrophils mainly by stimulating the
production of neutrophil-attracting cytokines or chemokines
from airway epithelial cells [
]. Whitehead et al. [
previously reported that after being sensitized with
allergen and increasing doses of environmentally relevant
LPS, the mice that initially displayed Th2 responses
gradually exhibited Th17-associated neutrophilia and
they subsequently reported that 100 ng LPS was able to
induce Th17-associated neutrophilia in mice [
Similarly, the mice were sensitized with allergen plus 0.1 μg
LPS in our model and we found that the Th17 cells
primed in the neutrophilic airway inflammation and was
resistant to DEX treatment, suggesting that the Th17
cells should be responsible for the steroid-insensitivity of
neutrophilic airway inflammation as previously reported
]. Intriguingly, we found that the levels of Th17 cells,
as well as the Th17-associated cytokine (IL-17A) and
nuclear transcription factor (RORγt), were all significantly
decreased by iPSC-MSCs at 4 h post-challenge, further
suggesting that iPSC-MSCs exhibited the therapeutic
effects on neutrophilic airway inflammation by the
regulation of Th17 cells.
Interestingly, we demonstrated that human
iPSC-MSCs regulated the Th17-mediated neutrophilic
airway inflammation in a time-dependent manner, in
which the Th17 level was decreased at 4 h
post-challenge and the airway inflammation was further
ameliorated at 48 h post-challenge. It suggests that the
different parameters exhibited their good responses to
the induction of airway inflammation and the treatment
of iPSC-MSCs in different time points. We identified
that iPSC-MSCs decreased the Th17 level at the early
phase, and further decreased the airway inflammation at
the later phase. The decrease of the high level of Th17
may be helpful to the reduction of inflammation
infiltration in the later phase.
It has been demonstrated that STAT-1, STAT-6 and
STAT-3 are involved in the differentiation of Th1, Th2
and Th17 respectively [
]. We observed that only
p-STAT-3 but not p-STAT-1 and p-STAT-6 were
expressed with high levels after the induction of
steroid-resistant neutrophilic airway inflammation.
Moreover, the level of p-STAT3 was significantly
decreased after the administration of iPSC-MSCs. All these
findings were consistent to the effects of iPSC-MSCs on
the neutrophilic airway inflammation and Th17 level,
which collectively suggested that iPSC-MSCs were
effective in the steroid-resistant neutrophilic airway
inflammation and p-STAT3 was the underlying pathway
We also investigated the effects of iPSC-MSCs on the
polarization of human Th cells in vitro. We found that
the differentiation of Th1, Th2 and Th17 were all
significantly inhibited by iPSC-MSCs. It is important that these
results further confirmed the effects of iPSC-MSCs on
Th17 cells. However, these findings were not totally
consistent with the above in vivo experiments in which the
levels of Th2 and Th17 were decreased while Th1 were
reciprocally increased with the administration of
iPSC-MSCs. The inconsistency could possibly be
elucidated by the different activation statuses of the T cells
and the different microenvironments that the
iPSC-MSCs encountered between in vitro and in vivo
There are some limitations in our study. First, we only
focused on the effects of iPSC-MSCs on Th17 cells in
our current report. It has been acknowledged that many
other immune cells such as the pulmonary macrophages
] also contribute to the steroid-insensitivity of
neutrophilic airway inflammation. Therefore, further studies
are required to fully explain the underlying mechanisms.
Second, we only reported the prevention effects of
iPSC-MSCs in our model; iPSC-MSCs were only
confirmed to be effective when administered prior to OVA
challenge, which somehow limits the therapeutic
applicability of iPSC-MSCs. The effects of iPSC-MSCs
administrated after the challenge on neutrophilic airway
inflammation should be further studied in the future.
Third, we used lung tissues instead of purified T cells for
the qPCR and WB in our study, and we also used the
PBMCs from healthy donors but not steroid-insensitive
asthmatics for in vitro experiment, these could possibly
lead to some unexpected results.
In summary, our study showed that iPSC-MSCs were
effective in steroid-insensitive neutrophilic airway
inflammation. These findings emphasized that iPSC-MSCs
were promising and significant alternative therapy for
asthma, especially steroid-insensitive asthma.
Additional file 1: Figure S1. Human iPSC-MSCs showed no effects on
murine steroid-resistant airway inflammation at 4 h post-challenge. (A)
Representative H&E staining of lung tissues with different treatment (×
200). (B) Representative Diff-Quik staining for the inflammatory cells
present in BALF with different treatment (× 200). (C) Statistical analysis of
inflammatory scores for the mice that were sacrificed. No significant
decreases could be observed in the mice that were treated with DEX or
iPSC-MSCs. (D) Statistical analysis of cell counts for the infiltrated
inflammatory cells in BALF. Neither DEX nor iPSC-MSCs could reduce the
infiltration of inflammatory cells in BALF. *P < 0.05 by the Mann-Whitney
U test. Abbreviations: BALF bronchoalveolar lavage fluids, DEX
dexamethasone, iPSC-MSCs induced pluripotent stem cell-derived
mesenchymal stem cells, ns not significant, PBS phosphate-buffered
saline, OVA ovalbumin. n = 5 for PBS/PBS/PBS and OVA/OVA/PBS, n = 6 for
OVA/OVA/DEX and OVA/OVA/MSC. Figure S2. Human iPSC-MSCs had no
effects on the Th17 level at 48 h post-challenge in a mouse model of
steroid-resistant airway inflammation. (A) Representative dot plots
showing the percentages of Th1/Th2/Th17 cells in CD4+ T cells at 48 h
post-challenge in murine lung tissues. (B) Statistical analysis of T helper
cell percentages in lung CD4+ T cells at 48 h post-challenge. No
significant changes of the T helper cells could be observed at 48 h
post-challenge. Abbreviations: DEX dexamethasone, iPSC-MSCs induced
pluripotent stem cell-derived mesenchymal stem cells, ns not significant,
PBS phosphate-buffered solution, OVA ovalbumin. n = 6 for OVA/OVA/
MSC, n = 5 for the other groups. (DOCX 972 kb)
BALF: Bronchoalveolar lavage fluids; BM-MSC: Bone marrow-derived
mesenchymal stem cell; DEX: Dexamethasone; H&E: Hematoxylin and eosin;
iPSCMSC: Induced pluripotent stem cell-derived mesenchymal stem cell;
LPS: Lipopolysaccharide; OVA: Ovalbumin; PAS: Periodic acid–Schiff;
PBMCs: Peripheral blood monocytes; PBS: Phosphate-buffered saline;
Th1: Type 1 helper T cells; Th17: Type 17 helper T cells; Th2: Type 2 helper T
cells; UCB-MSC: Umbilical cord blood-derived MSC
The authors thank Guangzhou Blood Center for providing the buffy coats of
the healthy volunteers in our study.
This study was supported by grants from NSFC for Excellent Young Scholars
(81322012 to Prof. QL Fu), NSFC (81373174, 81471832, 81671882 and
81770984), the key grant from the Science and Technology Foundation of
Guangdong Province of China (2015B020225001) and the Natural Science
Foundation of Guangdong Province (2014A030313051, 2016A030308017,
Availability of data and materials
All data generated or analyzed for this study are included in this published
article and the Additional files.
SBF and HYZ contributed to collection and/or assembly of data, initial
manuscript writing and primary data analysis, data collection and analysis.
XLF contributed to manuscript writing and data analysis. AYJ, YDL, CLL, CW
and XCM contributed to collection and/or assembly of data. QLF contributed
to concept and design, data analysis, manuscript writing and final approval
of the manuscript. All authors read and approved the manuscript.
Ethics approval and consent to participate
The protocol of this study was reviewed and approved by the Ethics
Committee of The First Affiliated Hospital, Sun Yat-sen University.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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