Mesenchymal stromal cell treatment prevents H9N2 avian influenza virus-induced acute lung injury in mice
Li et al. Stem Cell Research & Therapy
Mesenchymal stromal cell treatment prevents H9N2 avian influenza virus- induced acute lung injury in mice
Yan Li 0 1 2 4 5
Jun Xu 2 3
Weiqing Shi 2 3
Cheng Chen 1 2 5
Yan Shao 1 2 5
Limei Zhu 1 2 5
Wei Lu 1 2 5
XiaoDong Han 0 2 4
0 Medical School, Nanjing University , Nanjing, Jiangsu 210093 , People's Republic of China
1 Department of Chronic Communicable Disease, Jiangsu Provincial Center for Disease Prevention and Control , Nanjing 210009 , People's Republic of China
2 Abbreviations: AI , Avian influenza; AIV, Avian influenza virus; ALI, Acute lung injury; ARDS, Acute respiratory distress syndrome; MSC, Mesenchymal stromal cell
3 Institute of Toxicology & Functional Assessment, Jiangsu Provincial Center for Disease Prevention and Control , Nanjing 210009 , People's Republic of China
4 Medical School, Nanjing University , Nanjing, Jiangsu 210093 , People's Republic of China
5 Department of Chronic Communicable Disease, Jiangsu Provincial Center for Disease Prevention and Control , Nanjing 210009 , People's Republic of China
Background: The avian influenza virus (AIV) can cross species barriers and expand its host range from birds to mammals, even humans. Avian influenza is characterized by pronounced activation of the proinflammatory cytokine cascade, which perpetuates the inflammatory response, leading to persistent systemic inflammatory response syndrome and pulmonary infection in animals and humans. There are currently no specific treatment strategies for avian influenza. Methods: We hypothesized that mesenchymal stromal cells (MSCs) would have beneficial effects in the treatment of H9N2 AIV-induced acute lung injury in mice. Six- to 8-week-old C57BL/6 mice were infected intranasally with 1 × 104 MID50 of A/HONG KONG/2108/2003 [H9N2 (HK)] H9N2 virus to induce acute lung injury. After 30 min, syngeneic MSCs were delivered through the caudal vein. Three days after infection, we measured the survival rate, lung weight, arterial blood gas, and cytokines in both bronchoalveolar lavage fluid (BALF) and serum, and assessed pathological changes to the lungs. Results: MSC administration significantly palliated H9N2 AIV-induced pulmonary inflammation by reducing chemokines and proinflammatory cytokines levels, as well as reducing inflammatory cell recruit into the lungs. Thus, H9N2 AIV-induced lung injury was markedly alleviated in mice treated with MSCs. Lung histopathology and arterial blood gas analysis were improved in mice with H9N2 AIV-induced lung injury following MSC treatment. Conclusions: MSC treatment significantly reduces H9N2 AIV-induced acute lung injury in mice and is associated with reduced pulmonary inflammation. These results indicate a potential role for MSC therapy in the treatment of clinical avian influenza.
Mesenchymal stromal cell; H9N2 avian influenza viruses; Lung injury; Cell therapy
Infections with avian influenza virus (AIV) strains have
become highly prevalent in poultry worldwide [1–4].
Avian influenza (AI) is a leading cause of morbidity,
mortality, and economic loss in many countries. Mammals
can also be infected with several AIV subtypes, including
H5N1, H9N2, H7N7, and H7N3 [5, 6]. In 1997, 18 people
were infected with avian H5N1 influenza virus and six
died, refocusing global attention on the potential role of
AIVs as precursors of human pandemic influenza virus
strains [7, 8]. The H9N2 strain has been isolated from pigs
and humans with influenza-like illnesses in Hong Kong
and mainland China since 1998 [9, 10]. These findings
indicate that the AIV can also cross species barriers and
expand its host range from birds to mammals, thus
highlighting the pandemic potential of the H9N2 virus.
Although the H9N2 avian virus subtype is generally
not highly pathogenic for avian species, it has been
associated with severe morbidity and mortality in poultry
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following coinfection with other pathogens [11, 12].
Recent findings indicate that the H5N1 viruses responsible
for severe human disease contain genetic rearrangements
that include several genes from obtained avian H9N2
However, despite decades of research, few therapeutic
strategies for clinical AI have emerged, and current
specific treatment options are limited. Treatment of AI
currently still relies on antiviral agents. A recent
Cochrane review reported that antiviral therapies had
little benefit for severe influenza patients [14, 15].
Furthermore, AI continues to require prolonged mechanical
ventilation in the intensive care unit, and AI-associated
mortality remains high at 30–50 % despite optimal
supportive care [3, 16].
The reasons for the high mortality associated with AI
are unknown; the presence of a new viral subtype to which
the human host has no prior immunity cannot totally
explain this phenomenon. AIV is well known to mainly
cause pneumonia, severe acute lung injury (ALI), and
acute respiratory distress syndrome (ARDS) in humans
. A clinically important pronounced activation of the
proinflammatory cytokine cascade perpetuates the
inflammatory response and may contribute to further tissue
damage and persistence of the systemic inflammatory
response syndrome, leading to pulmonary infection in both
animals and humans [18, 19]. We therefore hypothesized
that improving immune regulation would benefit the
treatment of AI.
Cell therapies using bone marrow-derived mesenchymal
stromal cells (MSCs) have emerged as potential novel
therapeutic approaches for several diseases [20, 21].
Several studies have shown that both ectogenic and
endogenic MSCs can migrate into the lung, adopt the
phenotype of lung cells, and play positive roles in repairing lung
injury, including that caused by ARDS, emphysema, and
idiopathic pulmonary fibrosis [22–24]. Importantly, recent
studies have demonstrated that bone marrow-derived
MSCs can exhibit immunosuppressive properties. In
addition, MSCs have been suggested to be “immune
evasive”, and thus protected from rejection, which potentially
permits their use in allotransplantation [25, 26]. MSCs
may also engraft in the injured lung and can even
differentiate into lung epithelial cells in vivo . Therefore,
MSCs may be beneficial in the treatment of AI.
The aim of this study was to evaluate the effect of
MSC treatment on lung inflammation and injury
induced by H9N2 AIV infection in a murine model.
Isolation, culture, and characterization of MSCs
MSCs were isolated from the bone marrow of C57BL/6
mice weighing 20–30 g, obtained from the Shanghai
Laboratory Animal Research Center (Shanghai, China)
and maintained under specific pathogen-free conditions.
Bone marrow was flushed from the femurs under aseptic
conditions using DMEM. The collected cells were
washed three times with PBS, resuspended in DMEM
containing 10 % fetal bovine serum and 1 % L-glutamine,
and seeded at 1 × 106 cells/ml into culture flasks. Cells
were maintained in a humidified atmosphere of 95 % air
and 5 % CO2 at 37 °C. Nonadherent cells were discarded
after 48 h and the culture medium changed every 3–4
days thereafter. Cells were harvested when they reached
approximately 90 % confluency and were diluted 1:2 or
1:3 at each passage. MSCs used in all in vivo
experiments were between passages 3 and 10.
Fluorescence-activated cell sorting analysis
Passage 3–10 MSCs were analyzed for the following
markers: CD34, CD45, CD73, CD79, CD90, and CD105.
A total of 1 × 105 cells were incubated with each
fluorescence-conjugated primary antibody at 37 °C for 2
h in the dark. After three PBS washes, cells were
processed by flow cytometry (BD FACSCalibur) and data
were analyzed using Cell Quest software. Antibodies
used were: anti-mouse CD34, CD45, and CD79 (Santa
Cruz Biotechnology Inc.); anti-mouse CD73, CD90, and
CD105 (BioLegend, San Diego, CA), and all of the
antibodies were labeled with FITC.
Differentiation of MSCs
Passage 5 MSCs were differentiated into adipocytes,
chondrocytes, and osteocytes using the Mouse Mesenchymal
Stem Cell Functional Identification Kit (R&D, USA) to
prove their ability to differentiate into multiple
mesenchymal lineages. Methods were according to the procedures
of protocol; briefly, MSCs were plated at 1 × 104/well in
24-well plates and incubated in α-MEM containing 10 %
fetal bovine serum and 1 % L-glutamine until they reached
approximately 50–70 % confluency for differentiation.
Cells were cultured in adipogenic, osteogenic, or
chondrogenic media for 10–21 days before being prepared for
lineage-specific immunocytochemistry stains. Adipocytic
differentiation was confirmed by staining with FABP4,
differentiation to osteocytes was confirmed by staining with
osteopontin, and differentiation to chondrocytes was
confirmed by staining with collagen II as previously described.
Briefly, cells were washed with PBS and fixed in 4 %
paraformaldehyde in PBS for 20 min. The cells were
permeabilized and blocked with 0.5 ml PBS containing 0.3 %
Triton X-100 and 1 % BSA at room temperature for 30
min. After blocking, the cells were incubated with Goat
Anti-mouse FABP4, osteopontin, or Sheep Anti-mouse
Collagen II antibody working solution for 1 h at 37 °C;
they were then washed and incubated with Alexa 555
labelled Rabbit Anti-Goat secondary antibody in the dark
for 60 min at room temperature. Images of the stained
cells were obtained by using a phase fluorescence
microscope (Axio Observer, Zeiss, Germany).
Murine H9N2 infected mice of H9N2 AIV-induced acute
A/Hong Kong/2108/2003 [H9N2 (HK)] AIV was kindly
provided by Prof. Zheng Xing (Medicine School, Nanjing
University). To assess pathological changes induced by
H9N2 AIV, 6-week-old female SPF C57BL/6 mice were
housed in microisolator cages in the animal facility of
the Jiangsu Provincial Center for Disease Prevention and
Control under conditions of negative pressure and
ventilated with HEPA-filtered air.
A total of 95 C57BL/6 mice were divided into seven
groups: A: controls (n = 10); B: MSCs (n = 10); C: H9N2
infected mice + physiological saline (n = 15); D: H9N2
infected mice + McCoy (n = 15); E: H9N2 infected mice +
MSCs (n = 15); F: H9N2 infected mice + physiological saline
(n = 15); G: H9N2 infected mice + MSCs (n = 15). Groups
B–D had MSCs, McCoy, or saline administered 30 min
following viral infection induction, while groups E and F
were administered 1 day after. To induce lung injury in
H9N2-infected mice, diethyl ether was used to anesthetize
the mice and 1 × 104 MID50 (Median Infective Dose) of A/
HONG KONG/2108/2003 [H9N2 (HK)] H9N2 AIV
dissolved in 10 μl sterile physiological saline was administered
intranasally. Control mice were treated with noninfectious
allantoic fluid of the equivalent dilution. Thirty minutes
after H9N2 AIV challenge, physiological saline, McCoy, or
syngeneic MSCs (1 × 105 cells; 100 μl total volume) were
slowly infused into each mouse via the caudal vein. Naive
mice (without H9N2 AIV instillation) were injected with
saline or MSCs to serve as controls for any inflammatory
response that might result from the injected MSCs. Mice
were humanely killed by ether anesthesia followed by
percutaneous left ventricular bleeding 3 days after MSC
treatment and their tissues were harvested for analysis.
McCoy was used as a negative control.
Virus titration in lung of mice
Virus titration in lung homogenate of three mice per
group was detected to quantify the infection level. Tissues
were collected and homogenized in cold
phosphatebuffered saline on day 3 after treatment. Clarified
homogenates were titrated for viral infectivity in embryonated
chicken eggs from initial dilutions of 1:2. Viral titers were
expressed as mean log10 EID50/ml ± SD (1 MID50 is
around 1 × 103 EID50).
Survival rate and lung weight
Three days after treatment, the survival rate was observed
for each experimental group. In addition, the upper right
lung lobes were weighed before and after oven desiccation
at 70 °C to determine the lung wet:dry weight ratio. Lung
wet:dry weight ratio = weight of the wet lung/weight of
the dry lung; relative lung weight (%) = weight of the
whole wet lung/body weight × 100 %. These were used as
indicators of lung edema.
Lung tissue samples of three mice per group (one cross
section of the right lower lobe, one of the right mid lobe,
and one of the right upper lobe) were fixed in 4 %
paraformaldehyde, embedded in paraffin, and cut into 4-μm thick
sections. Sections were stained with hematoxylin and
eosin, and observed using an optical microscope. All lungs
were uniformally inflated and fixed in the same way. The
average interalveolar septum thickness was quantified in a
blinded fashion by measuring the thickness of all septae
along a crosshair placed on each image (at least 100 septae
were measured per animal). Lung injury score was
observed for quantification of the lung damage; briefly,
images were evaluated by an investigator who was blinded
to the identity of the slides (WCL) according to a
previously defined scoring system . The grading system was
as follows: 0, minimal damage; 1, mild damage; 2,
moderate damage; 3, severe damage; and 4, maximal damage.
Arterial blood gas analysis
Five mice from each group were anesthetized with
diethyl ether on day 3 postinoculation. Arterial blood
samples (100 μl) of lightly anesthetized mice spontaneously
breathing room air were withdrawn into a heparinized
syringe by percutaneous left ventricular sampling. Blood
gas analysis was immediately performed with an i-STAT
300 blood gas/electrolytes analyzer (Abbott, USA).
Concentrations of the chemokines granulocyte-macrophage
colony-stimulating factor (GM-CSF), monocyte
chemoattractant protein (MCP-1), keratinocyte chemoattractant
(KC), macrophage inflammatory protein-1α (MIP-1α), and
monokine induced by IFN-γ (MIG) and the inflammatory
cytokines interleukin (IL)-1α, IL-6, IL-10, tumor necrosis
factor alpha (TNF-α), and interferon (IFN-γ) in
bronchoalveolar lavage fluid (BALF) and serum (five mice from each
group) were measured 3 days after H9N2 infection using
the Mouse Cytokine Magnetic 20-Plex Panel (Invitrogen) by
Luminex 100 (Bio-Rad, USA).
BALF of mice was collected immediately following
sacrifice. Briefly, the lungs were lavaged three times with a
total volume of 1.0 ml physiological saline (4 °C) in the
chest cavity opened by midline incision. The rate of
recovery of BALF was not less than 90 % for all of the animals
Fig. 1 Flow cytometric analysis of MSCs. Isolated MSCs do not express CD45, CD34, or CD79 (a–c), but do express C105, CD73, and CD90 (d–f).
Grey lines represent a non-stained control, red lines represent specific antibody staining
Expression of CD14, TLR4, ERK, and JNK protein in lung
tissue was measured 3 days after H9N2 infection using
Western blotting. Lung tissue proteins were obtained
from the right lower lungs of mice in each group. Briefly,
tissues were lysed in ice-cold extraction buffer containing
protease inhibitor cocktail (Roche) and then centrifuged at
12,000 g for 30 min; the protein concentration in the
supernatant was determined using BCA assays. Proteins
were separated using 10 % SDS-polyacrylamide gel
electrophoresis and electrophoretically transferred to
polyvinylidene fluoride (PVDF) membranes using
Fig. 2 Differentiation of passage 5 MSCs into osteocytes, adipocytes, and chondrocytes. Fluorescence microscopy images of osteogenic differentiation
(osteopontin staining of undifferentiated MSCs (a) and differentiated osteocytes (b)), adipogenic differentiation (FABP4 staining of undifferentiated MSCs (c),
and differentiated adipocytes (d)), and chondrogenic differentiation (collagen II of undifferentiated MSCs (e) and differentiated chondrocytes (f))
Fig. 3 Virus titration in the lungs of mice. Mean viral titers based on
three mice per group are expressed as log10 EID50 per milliliter ± SD
standard procedures. The membranes were blocked at
37 °C for 1 h in phosphate-buffered saline (PBS)
containing 0.05 % (vol/vol) Tween 20 and 5 % (wt/vol)
non-fat milk for 1 h at RT. Membranes were then
incubated in primary antibody at a 1:500 dilution in
PBSTM (PBS containing 0.05 % (vol/vol) Tween 20
and 0.1 % (wt/vol) non-fat milk) for 3 h at RT and
then washed three times for 5 min each in PBSTM.
Membranes were then incubated with the appropriate
horseradish peroxidase (HRP)-conjugated anti-species
secondary antibodies (Boster, Wuhan, China) for 1 h
at RT and then washed in PBSTM as described above.
Immunoreactive protein bands were detected using an
Odyssey Scanning System (LI-COR, Inc., Lincoln, NE,
USA). Ratios for the protein of interest (POI) were
expressed relative to GAPDH in the same sample as
a loading control. The primary antibodies rabbit
antiCD14, TLR4, ERK, JNK, and GAPDH were obtained
from Santa Cruz (USA).
Fig. 4 The survival rate of mice in different experimental groups 3 days
post-treatment. Kaplan-Meier curves are shown for mice in different
groups. MSCs could improve survival in experimental AI mice. See text
for definition of groups A–G
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health.
The protocol was approved by the Committee on the
Ethics of Animal Experiments of the University of
Minnesota (approval no. A9089). All surgery was
performed under sodium pentobarbital anesthesia, and all
efforts were made to minimize suffering.
Data are expressed as the mean ± standard deviation (S.D.).
All calculations and statistical analyses were performed
using SPSS for windows version 13.0 (SPSS Inc., Chicago,
IL, USA). One-way ANOVA followed by Dunnett’s t test
was used to analyze differences between groups. P < 0.05
was regarded as statistically significant.
Highly pure MSCs can be isolated from murine bone
After approximately 2 weeks of culture, isolated MSCs
had expanded significantly and exhibited evident plastic
adherence. After three to four passages, MSCs exhibited a
homogeneous fibroblast-like, spindle-shaped morphology.
FACS analysis demonstrated that passage 3–10 MSCs did
not express CD34, CD45, or CD79 (Fig. 1a–c), but did
express CD73, CD90, and CD105 (Fig. 1d–f ). These data
indicate that the cultured cells were of mesenchymal origin
and of a high purity. Passage 5 MSCs readily differentiated
into adipocytes, osteocytes, or chondrocytes when
incubated in differentiation medium, confirming their
pluripotent potential (Fig. 2).
Virus titration in the lungs of mice
H9N2 viral infection resulted in high viral titers in the
lungs; the viral titers of the H9N2 infected mice +
physiological saline group, H9N2 infected mice + McCoy
group, and H9N2 infected mice + MSCs group exceeded
6.0 log10 EID50/ml (Fig. 3) on day 3 post-infection.
Survival rate and lung weight
Three days after infusion of MSCs, we observed that the
survival rate of mice in the H9N2 infected mice + MSCs
group was higher than that of the H9N2-infected mice
groups; the survival rate of the H9N2 infected mice +
MSCs group was 100 %, whereas that of the
H9N2infected mice groups were approximately 80 % (3 deaths
out of 15 mice in the H9N2 infected mice + physiological
saline group and 2 deaths out of 15 mice in the H9N2
infected mice + McCoy groups) (Fig. 4).
Mean relative lung weights and lung wet:dry weight
ratios were significantly higher (p < 0.05) in
H9N2infected lungs versus control mice and significantly
Fig. 5 Effect of MSCs on lung weights of mice in comparison to control mice. a Lung wet:dry weight ratios and b relative lung weight.
*Response that is significantly different from the control (p < 0.05).▲Response that is significantly different from the H9N2 infected mice (p < 0.05).
See text for definition of groups A–G
lower (p < 0.05) in mice treated with H9N2 + MSCs
versus those of the H9N2-infected groups (Fig. 5).
Lung sections from mice in the H9N2 infected mice +
physiological saline group and H9N2 infected mice +
McCoy group displayed similar histopathological patterns
consistent with severe diffuse pneumonia and characterized
by inflammatory cellular infiltration, numerous
polymorphonuclear leukocytes and macrophages in interstitial
spaces, interstitial and alveolar edema, and hemorrhage.
Diffuse pneumonia with severe alveolar damage was
observed throughout the entire lung (Fig. 6c and d).
Administration of MSCs (H9N2 infected mice + MSCs) reduced
airspace inflammation and improved lung histopathology.
Lung structure was less abnormal than in the H9N2
infected mice + physiological saline group, suggesting that
therapy with MSCs is protective against H9N2-induced
lung injury (Fig. 6e). The severity of lung injury was also
assessed using a semiquantitative histopathological scoring
system that evaluated lung injury in four categories: alveolar
septae, alveolar hemorrhage, intra-alveolar fibrin, and
intraalveolar infiltrates. Although treatment with MSCs tended
to reduce lung injury scores (Fig. 7), the observed
differences did not reach statistical significance.
Arterial blood gas analysis
Arterial blood gas parameters in the different
experimental groups are shown in Table 1. In the
H9N2infected mice, the partial pressure of arterial oxygen
Fig. 6 Histological evaluation of the therapeutic potential of MSCs on AIV-induced lung injury in mice. Lung pathology of mice in different experimental
groups 3 days post-treatment (hematoxylin and eosin, 100× magnification). a Control group, b MSC group (lungs of control and MSC groups showed a
normal aspect), c H9N2 infected mice + physiological saline group, d H9N2 infected mice + McCoy group (diffuse pneumonia and severe alveolar injury
were observed), e H9N2 infected mice + MSCs group (MSC transplantation reduced lung injury). f H9N2 infected mice+ Physiological saline administer 1
day following viral infection induction (Severe diffuse pneumonia and extensive alveolar damage). g H9N2 infected mice+MSCs administer 1 day following
viral infection induction (MSC transplantation reduced lung inflammation and injury)
Fig. 7 Lung injury score of mice in different experimental groups 3
days post-treatment. Data are represented as mean ± standard error
of the mean; n =6 per group. *Response that is significantly different
from the control (p < 0.05). ▲Response that is significantly different
from the H9N2 infected mice (p < 0.05). See text for definition of
(PaO2) was significantly lower, while the partial pressure
of arterial carbon dioxide (PaCO2) was significantly
higher, and the values for saturation of arterial oxygen
(SaO2) and pH were slightly lower. Compared with the
H9N2 infected mice + physiological saline and H9N2
infected mice + McCoy groups, mice in the H9N2 infected
+ MSCs group had significantly higher PaO2,
significantly lower PaCO2, and slightly higher (but still
statistically significantly different) SaO2 and pH values. These
results indicate that most of the H9N2 AIV infected
mice developed severe hypoxemia, and that MSCs
effectively protected against functional pulmonary injury.
in both BALF and serum. The concentrations of all
chemokines were increased in BALF and serum (Fig. 8) in
the H9N2 infected mice + physiological saline group. In
contrast, the concentrations of chemokines were much
lower in the H9N2 infected mice + MSCs group.
The concentrations of IL-1α, IL-6, IL-10, TNF-α, and
IFN-γ were measured in BALF and serum 3 days after
H9N2 infection. In the H9N2 infected mice +
physiological saline group, the concentrations of these
inflammatory cytokines were significantly increased in both BALF
and serum (Fig. 9). In contrast, the concentrations were
significantly lower in the H9N2 infected mice + MSCs
Three days after H9N2 infection, the expression of
CD14, TLR4, ERK and JNK protein in the lung tissue
was measured. In the H9N2-infected mice, the
expression of CD14, TLR4, ERK, and JNK protein in lung
tissue was significantly increased compared with the
control group. Mice in the H9N2 infected + MSCs group
had significantly decreased ERK and JNK expression
compared with the H9N2 infected mice + physiological
saline and H9N2 infected mice + McCoy groups. The
expression of CD14 and TLR4 in mice in the H9N2
infected + MSCs group tended to be lower, although not
significantly so (Fig. 10).
The present study demonstrated that MSCs exert a
beneficial therapeutic effect on H9N2 AIV-induced lung
injury in a murine model; treatment with MSCs results
in improvements in both pulmonary inflammation and
lung tissue organization. These findings have potentially
important implications for the treatment of AI, which is
an important clinical problem characterized by high
patient morbidity and mortality.
Table 1 Effect of H9N2 avian influenza virus (AIV) and mesenchymal stromal cells (MSCs) on arterial blood gas analysis of mice
B: MSCs (1 × 105 MSCs; n = 10)
C: H9N2-infected mice (1 × 104 MID50 H9N2AIV; n = 15)
D: H9N2-infected mice + MSCs (1 × 104 MID50 H9N2AIV + 1 × 105 McCoy; n = 15)
E: H9N2-infected mice + MSCs (1 × 104 MID50 H9N2AIV + 1 × 105 MSCs;
n = 15, 30 min following infection)
F: H9N2-infected mice (1 × 104 MID50 H9N2AIV; n = 15)
G: H9N2-infected mice + MSCs (1 × 104 MID50 H9N2AIV + 1 × 105 MSCs;
n = 15, 1 day following infection)
*Response that is significantly different from the control (p < 0.05)
▲Response that is significantly different from the H9N2-infected mice (p < 0.05)
PaCO2 partial pressure of arterial carbon dioxide, PaO2 partial pressure of arterial oxygen, SaO2 saturation of arterial oxygen
Fig. 8 Levels of chemokines in bronchoalveolar lavage fluid (BALF) and serum. Concentrations of the chemokines granulocyte-macrophage
colony-stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP-1), keratinocyte chemoattractant (KC), macrophage inflammatory
protein-1α (MIP-1α), and monokine induced by IFN-γ (MIG) in each group were measured using Luminex in bronchoalveolar lavage fluid and serum.
Groups as legend. Group comparisons were made using one-way ANOVA with Dunnett’s post hoc test. *p < 0.05; **p < 0.01; n = 6 mice per group. MSC
mesenchymal stromal cell
Fig. 9 Levels of inflammatory cytokines in bronchoalveolar lavage fluid (BALF) and serum. Concentrations of the inflammatory cytokines interleukin
(IL)-1α, IL-6, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ), and of IL-10 were measured using Luminex in bronchoalveolar lavage
fluid and serum. Groups as legend. Group comparisons were made using one-way ANOVA with Dunnett’s post hoc test. *p < 0.05; **p < 0.01; n = 6
mice per group. MSC mesenchymal stromal cell
Fig. 10 Western blotting analyzed the expression of TLR4-related protein in the lung tissue after MSC transplantation for 3 days. a Expression of
CD14, TLR4, ERK, and JNK were evaluated by immunoblotting using specific antibodies. GAPDH is used as the control. 1: controls, 2: MSCs, 3: H9N2, 4:
H9N2 + McCoy, 5: H9N2 + MSCs, 6: H9N2 + Physiological saline (1 day following) 7: H9N2 +MSCs (1 day following). b Responses were quantified by
densitometry and normalized to the expression of GAPDH. Densitometry data are shown as mean ± SD. *Response that is significantly different from
the control (p < 0.05). ▲Response that is significantly different from the H9N2 infected mice (p < 0.05)
Avian H9N2 viruses have been widespread in domestic
poultry in Asian countries since the mid-1990s, with a
mortality ranging from 5–30 % [29, 30]. Many studies
have shown that H9N2 viruses can cause upper
respiratory tract illnesses in humans. This indicates that
the virus has evolved to cross the species barrier and
is capable of infecting humans, bypassing intermediate
H9N2 viruses can activate the innate immune
response. A hallmark of pulmonary infiltration associated
with AI is the presence of infiltrating leukocytes [31, 32].
Leukocyte migration is directed largely by chemokines,
and the inter-relationship of early-response cytokines,
adhesion molecules, and chemokines orchestrates the
recruitment of neutrophils into the lungs . In most
studies, cytokines have been described as forming an
inflammatory “cascade” or “network” in patients [30, 34].
Lung injury may be a direct consequence of this
Adult stem cells or progenitor cells are being evaluated
for the treatment of a number of diseases that currently
have limited or no treatment options [35, 36].
Marrowderived stem cells are hypothesized to be the source of
lung regeneration and repair. An alternative source are
exogenous stem/progenitor cells, delivered into the lung
either intravenously via the trachea or by direct injection.
Recently, many studies have confirmed that MSCs can
engraft in the injured lung [37–39] and can even
differentiate into lung epithelial cells in vivo. MSCs may
also exhibit immunosuppressive properties, suggesting
“immune-privilege”, and may even have certain immune
regulation functions [40–42]. Therefore, MSCs may, in
their own right, have beneficial effects in ALI.
Our data showed that H9N2 viral infection dramatically
increases the expression of chemokines, including
GMCSF, MCP-1, KC, MIP-1α, and MIG, in both BALF and
serum. However, MSC treatment decreases the expression
of these chemokines. We also showed that MSC-treated
mice have significantly reduced levels of some
inflammatory cytokines (IL-1α, IL-6, TNF-α, and IFN-γ) and a
corresponding increase in anti-inflammatory cytokines
(IL-10). It is believed that IL-1α, IL-6, TNF-α, and IFN-γ
play important roles in the development of ALI .
These results are consistent with several previous studies.
For example, in Mei’s study, treatment with MSCs alone
significantly reduced LPS-induced acute pulmonary
inflammation in mice . Importantly, our in vivo
experimental results suggest that the administration of MSCs
can greatly improve the hypoxemia and histopathological
changes of lung injury induced by H9N2 AIV infection.
The data indicate that administration of MSCs results in a
marked increase in survival rates, primarily due to a
decrease in ALI. These results are not consistent with the
findings of Lam et al. , who reported that MSC
therapy fails to improve outcomes in experimental HIN1
influenza. We speculate that this might be a consequence of
different pathogenic characteristics of different influenza
viruses. It is known that avian influenza virus infection
can lead to a “cytokine storm”. In consequence, the host
cellular response may be different from that with H1N1
influenza virus infection.
We have, therefore, demonstrated that MSC-based cell
therapy can attenuate the inflammatory reaction and
injury in the lungs caused by H9N2 AIV exposure, as well
as by reducing lung histopathological changes. These
beneficial effects may be mediated by the
downregulation of chemokines such as GM-CSF, MCP-1, KC,
MIP1α, and MIG, leading to the downregulation of
inflammatory cytokines such as TNF-α, IL-1α, IL-6, and IFN-γ.
Our findings provide further support for a prominent
role of TLR4-dependent immune regulation in acute
lung injury therapy. The quantity of stem cells used in
our study was 1 × 105. This is much less than used by
Wang et al. . We did not find an obvious stem cell
niche in the lung tissue of mice. Therefore, we deduce
that the immune regulation function of stem cells is via
a paracrine mechanism.
On the other hand, it is possible that MSCs may be
involved in the early stages of carcinogenesis through
spontaneous transformation. In addition, it has been
suggested that MSCs can modulate tumor growth and
metastasis, although this remains controversial and
poorly understood. Interestingly, different studies have
reported contradicting findings, with some finding that
MSCs promote tumor growth and others that they
inhibit tumor growth. Therefore, the role of MSCs in avian
influenza infection requires ongoing surveillance.
Our results suggest that MSC-based therapy can reduce
inflammatory lung injury and pulmonary vascular
permeability. This may provide a novel strategy for the treatment
of AIV-induced lung injury through improving tissue
repair and protecting against inflammation. Further
investigations into the application of MSC-based cell therapy
may give new hope for patients with AIV-induced ALI.
The authors thank Dr. Zheng Xing for his kind help. They also thank Xueting
Wang, Yu Chen, Xuemin Gong, and Yuan Zhou for their technical assistance,
and Dr. Leonardo Martinez for his editorial assistance.
This work was supported by National Natural Science Foundation of China
(81170054;81301448), Natural Science Foundation of Jiangsu Province of
China (BK2011570,), the National Basic Research Program of China (973
program 2010CB945103), and Open Research Fund of State Key Laboratory
of Bioelectronics, Southeast University, Key Medical Talent Foundation of
Jiangsu Provincial Center for Disease Prevention and Control (JKRC20110029).
YL and JX performed and analyzed the majority of the experiments and wrote
the manuscript. YL, XH, and WL were responsible for obtaining funds and
designed the whole study concept, and drafted and revised the manuscript.
WS and JX performed animal experiments, conducted the experimental analysis
and drafted and revised the manuscript. CC and YS performed the cells
experiments, conducted the experimental analysis and drafted and revised the
manuscript. LZ participated in the writing and revision of the manuscript. All
authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was carried out in strict accordance with the recommendations in
the Guide for the Care and Use of Laboratory Animals of the National
Institutes of Health. The protocol was approved by the Committee on the
Ethics of Animal Experiments of the University of Minnesota (approval no.
A9089). All surgery was performed under sodium pentobarbital anesthesia,
and all efforts were made to minimize suffering.
1. Si Y , de Boer WF , Gong P. Different environmental drivers of highly pathogenic avian influenza H5N1 outbreaks in poultry and wild birds . PLoS One . 2013 ; 8 : e53362 .
2. Qi X , Cui L , Jiao Y , Pan Y , Li X , Zu R , Huo X , Wu B , Tang F , Song Y , Zhou M , Wang H , Cardona CJ , Xing Z. Antigenic and genetic characterization of a European avian-like H1N1 swine influenza virus from a boy in China in 2011. Arch Virol . 2013 ; 158 : 39 - 53 .
3. Metras R , Stevens KB , Abdu P , Okike I , Randolph T , Grace D , Pfeiffer DU , Costard S. Identification of potential risk factors associated with highly pathogenic avian influenza subtype H5N1 outbreak occurrence in Lagos and Kano States, Nigeria, during the 2006-2007 epidemics . Transbound Emerg Dis . 2013 ; 60 : 87 - 96 .
4. Parker CD , Reid SM , Ball A , Cox WJ , Essen SC , Hanna A , Mahmood S , Slomka MJ , Irvine RM , Brown IH. First reported detection of a low pathogenicity avian influenza virus subtype H9 infection in domestic fowl in England . Vet Rec . 2012 ; 171 : 372 .
5. Younan M , Poh MK , Elassal E , Davis T , Rivailler P , Balish AL , Simpson N , Jones J , Deyde V , Loughlin R , Perry I , Gubareva L , ElBadry MA , Truelove S , Gaynor AM , Mohareb E , Amin M , Cornelius C , Pimentel G , Earhart K , Naguib A , Abdelghani AS , Refaey S , Klimov AI , Donis RO , Kandeel A. Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans , Egypt , 2007 - 2011 . Emerg Infect Dis . 2013 ; 19 : 43 - 50 .
6. Su S , Qi W , Chen J , Zhu W , Huang Z , Xie J , Zhang G . Seroepidemiological evidence of avian influenza A virus transmission to pigs in southern China . J Clin Microbiol . 2013 ; 51 : 601 - 2 .
7. Kandun IN , Tresnaningsih E , Purba WH , Lee V , Samaan G , Harun S , Soni E , Septiawati C , Setiawati T , Sariwati E , Wandra T. Factors associated with case fatality of human H5N1 virus infections in Indonesia: a case series . Lancet . 2008 ; 372 : 744 - 9 .
8. Tam JS . Influenza A, (H5N1) in Hong Kong: an overview . Vaccine . 2002 ;20 Suppl 2: S77 - 81 .
9. Peiris M , Yuen KY , Leung CW , Chan KH , Ip PL , Lai RW , Orr WK , Shortridge KF . Human infection with influenza H9N2 . Lancet . 1999 ; 354 : 916 - 7 .
10. Butt KM , Smith GJ , Chen H , Zhang LJ , Leung YH , Xu KM , Lim W , Webster RG , Yuen KY , Peiris JS , Guan Y. Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003 . J Clin Microbiol . 2005 ; 43 : 5760 - 7 .
11. Tombari W , Paul M , Bettaieb J , Larbi I , Nsiri J , Elbehi I , Gribaa L , Ghram A. Risk factors and characteristics of low pathogenic avian influenza virus isolated from commercial poultry in Tunisia . PLoS One . 2013 ; 8 : e53524 .
12. Kim HR , Lee KK , Kwon YK , Kang MS , Moon OK , Park CK. Comparison of serum treatments to remove nonspecific inhibitors from chicken sera for the hemagglutination inhibition test with inactivated H5N1 and H9N2 avian influenza A virus subtypes . J Vet Diagn Invest . 2012 ; 24 : 954 - 8 .
13. Lin YP , Shaw M , Gregory V , Cameron K , Lim W , Klimov A , Subbarao K , Guan Y , Krauss S , Shortridge K , Webster R , Cox N , Hay A. Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates . Proc Natl Acad Sci U S A . 2000 ; 97 : 9654 - 8 .
14. Ortiz JR , Rudd KE , Clark DV , Jacob ST , West TE . Clinical research during a public health emergency: a systematic review of severe pandemic influenza management . Crit Care Med . 2013 ; 41 : 1345 - 52 .
15. Hsu J , Santesso N , Mustafa R , Brozek J , Chen YL , Hopkins JP , Cheung A , Hovhannisyan G , Ivanova L , Flottorp SA , Saeterdal I , Wong AD , Tian J , Uyeki TM , Akl EA , Alonso-Coello P , Smaill F , Schunemann HJ . Antivirals for treatment of influenza: a systematic review and meta-analysis of observational studies . Ann Intern Med . 2012 ; 156 : 512 - 24 .
16. Uno Y , Usui T , Soda K , Fujimoto Y , Takeuchi T , Ito H , Ito T , Yamaguchi T. The pathogenicity and host immune response associated with H5N1 highly pathogenic avian influenza virus in quail . J Vet Med Sci . 2013 ; 75 : 451 - 7 .
17. Ramos I , Fernandez-Sesma A. Innate immunity to H5N1 influenza viruses in humans . Viruses . 2012 ; 4 : 3363 - 88 .
18. Ling MT , Tu W , Han Y , Mao H , Chong WP , Guan J , Liu M , Lam KT , Law HK , Peiris JS , Takahashi K , Lau YL . Mannose-binding lectin contributes to deleterious inflammatory response in pandemic H1N1 and avian H9N2 infection . J Infect Dis . 2012 ; 205 : 44 - 53 .
19. Viemann D , Schmolke M , Lueken A , Boergeling Y , Friesenhagen J , Wittkowski H , Ludwig S , Roth J. H5N1 virus activates signaling pathways in human endothelial cells resulting in a specific imbalanced inflammatory response . J Immunol . 2011 ; 186 : 164 - 73 .
20. Warnke PH , Humpe A , Strunk D , Stephens S , Warnke F , Wiltfang J , Schallmoser K , Alamein M , Bourke R , Heiner P , Liu Q. A clinically-feasible protocol for using human platelet lysate and mesenchymal stem cells in regenerative therapies . J Craniomaxillofac Surg . 2013 ; 41 : 153 - 61 .
21. Schnerch A , Lee JB , Graham M , Guezguez B , Bhatia M. Human embryonic stem cell-derived hematopoietic cells maintain core epigenetic machinery of the polycomb group/trithorax group complexes distinctly from functional adult hematopoietic stem cells . Stem Cells Dev . 2013 ; 22 : 73 - 89 .
22. Toonkel RL , JM Hare, MA Matthay, MK Glassberg. Mesenchymal stem cells and idiopathic pulmonary fibrosis. Potential for clinical testing . Am J Respir Crit Care Med . 2013 ; 188 : 133 - 40 .
23. Waszak P , Alphonse R , Vadivel A , Ionescu L , Eaton F , Thebaud B. Preconditioning enhances the paracrine effect of mesenchymal stem cells in preventing oxygen-induced neonatal lung injury in rats . Stem Cells Dev . 2012 ; 21 : 2789 - 97 .
24. Wang N , Li Q , Zhang L , Lin H , Hu J , Li D , Shi S , Cui S , Zhou J , Ji J , Wan J , Cai G , Chen X. Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG-6. PLoS One . 2012 ; 7 : e43768 .
25. Maria Spaggiari G , Moretta L. Cellular and molecular interactions of mesenchymal stem cells in innate immunity . Immunol Cell Biol . 2013 ; 91 : 27 - 31 .
26. Reddy BY , Xu DS , Hantash BM . Mesenchymal stem cells as immunomodulator therapies for immune-mediated systemic dermatoses . Stem Cells Dev . 2012 ; 21 : 352 - 62 .
27. Gharaee-Kermani M , Gyetko MR , Hu B , Phan SH . New insights into the pathogenesis and treatment of idiopathic pulmonary fibrosis: a potential role for stem cells in the lung parenchyma and implications for therapy . Pharm Res . 2007 ; 24 : 819 - 41 .
28. Xia J , Zhang H , Sun B , Yang R , He H , Zhan Q. Spontaneous breathing with biphasic positive airway pressure attenuates lung injury in hydrochloric acidinduced acute respiratory distress syndrome . Anesthesiology . 2014 ; 120 : 1441 - 9 .
29. Butt AM , Siddique S , Idrees M , Tong Y. Avian influenza A (H9N2): computational molecular analysis and phylogenetic characterization of viral surface proteins isolated between 1997 and 2009 from the human population . Virol J . 2010 ; 7 : 319 .
30. Ashour MM , Khatab AM , El-Folly RF , Amer WA. Clinical features of avian influenza in Egyptian patients . J Egypt Soc Parasitol . 2012 ; 42 : 385 - 96 .
31. Li C , Bankhead 3rd A, Eisfeld AJ , Hatta Y , Jeng S , Chang JH , Aicher LD , Proll S , Ellis AL , Law GL , Waters KM , Neumann G , Katze MG , McWeeney S , Kawaoka Y. Host regulatory network response to infection with highly pathogenic H5N1 avian influenza virus . J Virol . 2011 ; 85 : 10955 - 67 .
32. Cilloniz C , Shinya K , Peng X , Korth MJ , Proll SC , Aicher LD , Carter VS , Chang JH , Kobasa D , Feldmann F , Strong JE , Feldmann H , Kawaoka Y , Katze MG . Lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes . PLoS Pathog . 2009 ; 5 : e1000604 .
33. Zeng H , Pappas C , Belser JA , Houser KV , Zhong W , Wadford DA , Stevens T , Balczon R , Katz JM , Tumpey TM . Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection . J Virol . 2012 ; 86 : 667 - 78 .
34. Ferreira HL , Pirlot JF , Reynard F , van den Berg T , Bublot M , Lambrecht B. Immune responses and protection against H5N1 highly pathogenic avian influenza virus induced by the Newcastle disease virus H5 vaccine in ducks . Avian Dis . 2012 ; 56 : 940 - 8 .
35. Filali M , Liu X , Cheng N , Abbott D , Leontiev V , Engelhardt JF . Mechanisms of submucosal gland morphogenesis in the airway . Novartis Found Symp . 2002 ; 248 : 38 - 45 . discussion - 50 , 277 - 82 .
36. Kim DW , Staples M , Shinozuka K , Pantcheva P , Kang SD , Borlongan CV . Wharton's jelly-derived mesenchymal stem cells: phenotypic characterization and optimizing their therapeutic potential for clinical applications . Int J Mol Sci . 2013 ; 14 : 11692 - 712 .
37. Xue J , Li X , Lu Y , Gan L , Zhou L , Wang Y , Lan J , Liu S , Sun L , Jia L , Mo X , Li J. Gene-modified mesenchymal stem cells protect against radiation-induced lung injury . Mol Ther . 2013 ; 21 : 456 - 65 .
38. Toonkel RL , Hare JM , Matthay MA , Glassberg MK . Mesenchymal stem cells and idiopathic pulmonary fibrosis. Potential for clinical testing . Am J Respir Crit Care Med . 2013 ; 188 : 133 - 40 .
39. Mei SH , McCarter SD , Deng Y , Parker CH , Liles WC , Stewart DJ . Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1 . PLoS Med . 2007 ; 4 : e269 .
40. Zhang S , Wang Y , Mao JH , Hsieh D , Kim IJ , Hu LM , Xu Z , Long H , Jablons DM , You L. Inhibition of CK2alpha down-regulates Hedgehog/Gli signaling leading to a reduction of a stem-like side population in human lung cancer cells . PLoS One . 2012 ; 7 : e38996 .
41. Alphonse RS , Rajabali S , Thebaud B. Lung injury in preterm neonates: the role and therapeutic potential of stem cells . Antioxid Redox Signal . 2012 ; 17 : 1013 - 40 .
42. Nemeth K , Leelahavanichkul A , Yuen PS , Mayer B , Parmelee A , Doi K , Robey PG , Leelahavanichkul K , Koller BH , Brown JM , Hu X , Jelinek I , Star RA , Mezey E. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)- dependent reprogramming of host macrophages to increase their interleukin-10 production . Nat Med . 2009 ; 15 : 42 - 9 .
43. Puneet P , Moochhala S , Bhatia M. Chemokines in acute respiratory distress syndrome . Am J Physiol Lung Cell Mol Physiol . 2005 ; 288 : L3 - 15 .
44. Lam WY , Yeung AC , Ngai KL , Li MS , To KF , Tsui SK , Chan PK. Effect of avian influenza A H5N1 infection on the expression of microRNA-141 in human respiratory epithelial cells . BMC Microbiol . 2013 ; 13 : 104 .
45. Wang Y , Sun Z , Qiu X , Li Y , Qin J , Han X. Roles of Wnt/beta-catenin signaling in epithelial differentiation of mesenchymal stem cells . Biochem Biophys Res Commun . 2009 ; 390 : 1309 - 14 .