Cell Transplantation, Vol. 22, pp. 461–468, 2013
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DOI: http://dx.doi.org/10.3727/096368912X656063
E-ISSN 1555-3892
www.cognizantcommunication.com
Influenza Virus Infects Bone Marrow Mesenchymal Stromal Cells In Vitro:
Implications for Bone Marrow Transplantation
Mahesh Khatri and Yehia M. Saif
Food Animal Health Research Program, Ohio Agricultural Research and Development Center,
The Ohio State University, Wooster, OH, USA
Mesenchymal stromal cells (MSCs) have differentiation, immunomodulatory, and self-renewal properties
and are, therefore, an attractive tool for regenerative medicine and autoimmune diseases. MSCs may be of
great value to treat graft-versus-host disease. Influenza virus causes highly contagious seasonal infection and
occasional pandemics. The infection is severe in children, elderly, and immunocompromised hosts including
hematopoietic stem cell transplant patients. The objective of this study was to determine if MSCs are permissive to influenza virus replication. We isolated MSCs from the bone marrow of 4- to 6-week-old germ-free
pigs. Swine and human influenza virus strains were used to infect MSCs in vitro. MSCs expressed known
influenza virus a-2,3 and a-2,6 sialic acid receptors and supported replication of swine and human influenza
viruses. Viral infection of MSCs resulted in cell lysis and proinflammatory cytokine production. These findings demonstrate that bone marrow-derived MSCs are susceptible to influenza virus. The data also suggest that
transplantation of bone marrow MSCs from influenza virus-infected donors may transmit infection to recipients. Also, MSCs may get infected if infused into a patient with an ongoing influenza virus infection.
Key words: Mesenchymal stromal cells (MSCs); Influenza virus; Cell therapy
INTRODUCTION
Mesenchymal stromal cells (MSCs) were first identi
fied by Friendstein in bone marrow as precursors for
fibroblasts or stromal cells. MSCs have the potential to
differentiate into cells of mesenchymal, mesodermal,
neuroectodermal, or endodermal lineage (16).
MSCs constitute a small population (approximately
0.001–0.01% nucleated cells) of adult human bone marrow cells. MSCs support and regulate hematopoiesis via
the secretion of cytokines and growth factors (14,29).
Several studies using a variety of animal models have
shown the beneficial effects of MSCs in conditions such
as infarcted myocardium, lung injury, damaged bone,
tendon, and cartilage (1,31,33,36).
Bone marrow-derived and tissue resident MSCs isolated
from humans as well as from other mammalian species
are strongly immunosuppressive and inhibit inflammation
and immunological responses both in vitro and in vivo
(15). The mechanisms of MSC-mediated immunosuppression are not completely understood; however, MSCs
have been shown to secrete several immunosuppressive
mediators including interleukin-10 (IL-10), transforming growth factor-b (TGF-b), prostaglandin E2, nitric
oxide (NO), indoleamine 2,3-dioxygenase (IDO), and
hepatocyte growth factor (HGF) and promote generation
of regulatory T-cells (37). MSCs improve the outcome
of allogeneic transplantation by promoting hematopoietic
engraftment (22) and also reduce graft-versus-host disease (25,26) autoimmunity and other immune disorders
(37). Due to their differentiation and immunoregulatory
properties, pluripotent stem cells are an attractive tool for
tissue engineering and regenerative medicine.
Influenza viruses cause a highly contagious respiratory infection in humans and animals. These viruses cause
seasonal epidemics and infrequent pandemics in humans
with young children, elderly, and immunocompromised
individuals at high risk. In hematopoietic stem cell transplantation recipients, influenza virus caused persistent
infection and severe respiratory failure (2,5,9,11). During
the last century, influenza viruses caused three human
pandemics. The 2009 human pandemic was caused by a
novel H1N1 virus of swine origin (13). Influenza virus
mainly replicates in the respiratory tract; however, virus
replication has been detected in nonpulmonary tissues
including the bone marrow (43,49). Moreover, highly
pathogenic avian influenza (HPAI) H5N1 virus, which has
Received October 17, 2011; final acceptance April 7, 2012. Online prepub date: September 21, 2012.
Address correspondence to Dr. Mahesh Khatri, Food Animal Health Research Program, Ohio Agricultural Research and Development Center,
The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, USA. Tel: +1-330-263-3751; Fax: +1-330-263-3677; E-mail: [email protected]
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crossed the species barrier and can infect humans without prior adaptation, replicates extensively in the lungs
and extra-pulmonary tissues leading to acute respiratory
distress syndrome, multiple-organ dysfunction, lympho
penia, and hemophagocytosis (6,40,47). In the bone marrow, influenza virus caused the apoptosis of primitive
B-cells by inducing the production of proinflammatory
cytokines, mainly tumor necrosis factor-a (TNF-a) (39).
However, the phenotype of cell(s) permissive to influenza virus and the cellular source of cytotoxic cytokines
in virus-infected bone marrow are not known.
Swine is an excellent animal model to study the pathogenesis of influenza virus because the clinical manifestations and pathogenesis of influenza in pigs closely
resemble that observed in humans (23). In the present
study, we investigated whether MSCs are susceptible to
influenza virus infection. Here we show, for the first time,
that swine bone marrow MSCs are permissive to replication of swine and human influenza viruses. Transplanta
tion of virus-infected MSCs may transmit infection to the
host and viral infection of MSCs may affect the outcome
of the transplantation. Virus-infected MSCs produce pro
inflammatory cytokines and, therefore, may play an impor
tant role in the pathogenesis of influenza infection.
MATERIALS AND METHODS
Chemicals are from Sigma, USA, unless otherwise
stated.
Isolation and Culture of MSCs
Near term pregnant Landrace-Yorkshire white-Duroc
crossbred sows [Swine herd, The Ohio State University
(OSU), Columbus, OH, USA] were delivered by hysterectomy and piglets were maintained in sterile gnotobiotic
isolator units as described previously (46). The piglets of
either sex were maintained and euthanized in accordance
with the standards of the Institutional Laboratory Animal
Care and Use Committee, The Ohio State University.
Femur bones were obtained from 4- to-6-week-old germfree pigs (n = 4). MSCs were isolated by previously
described methods (19,35). Briefly, the tip of each bone
was removed and the marrow was harvested by inserting a syringe needle into one end of the bone and flushing with Dulbecco’s modified Eagle’s medium (DMEM;
Gibco, USA). The bone marrow cells were filtered
through a 70-μm nylon mesh filter (BD Falcon, USA),
and mononuclear cells were obtained by density gradient centrifugation over Ficoll-Hypaque (GE Healthcare,
USA). Cells (1–5 ´ 105/cm2) were plated in 25-cm2 cell
culture flasks in DMEM containing 10% fetal bovine
serum (FBS, Gibco), 2 mm l-glutamine (Gibco), 1% antibiotic solution (Gibco). Cultures were incubated at 37°C
in a humidified atmosphere containing 95% air and 5%
CO2. The nonadherent cells were removed after 72 h of
Khatri and Saif
culture. When primary cultures became nearly confluent,
the cells were detached with 0.025% trypsin containing
0.02% EDTA (Life Technologies, Invitrogen, USA) and
used for future experiments.
Expression of Stem Cell Markers
and Phenotyping of MSCs
Porcine MSCs cultured on coverslips were fixed in
methanol/acetone (1:1) for 3 min and then blocked with
3% bovine serum albumin (BSA) for 30 min. Cells were
incubated at 4°C with the following primary antibodies: rabbit anti-human octamer binding transcription
factor (Oct3/4) (Santa Cruz Biotechnology, USA) and
mouse anti-human stage-specific embryonic antigen-1
(SSEA-1) (Millipore, USA). After overnight incubation,
cells were washed and incubated for 1 h at room temperature with the following respective fluorescein isothiocyanate (FITC)-labeled secondary antibodies: goat
anti-rabbit IgG and goat anti-mouse IgG. Cell nuclei were
then counterstained with 4¢,6-diamidino-2-phenylindole
(DAPI; Life Technologies).
Phenotypic analysis of MSCs was performed by flow
cytometry analysis (17). MSCs were detached by treatment with 0.25% trypsin-EDTA, and single cell suspension was stained with following primary antibodies:
mouse anti-pig CD29, mouse anti-human CD90 (BD
Biosciences, Pharmingen, USA), mouse anti-pig CD44
and CD45 (VMRD, USA) for 20 min at 4°C in the dark.
After washing, cells were incubated with secondary antibodies conjugated with allophycocyanin (APC) or spectral red (BD Biosciences, Pharmingen, USA) for 30 min.
Relevant isotype and secondary antibodies were used as
controls for nonspecific binding. The cells were acquired
by C6 flow cytometer (BD Accuri Cytometers, USA) and
analyzed using CFlow® plus Software (Accuri).
Anti-human Oct3/4, SSEA-1, and CD90 antibodies
are cross-reactive with pig Oct3/4, SSEA-1 and CD90
antigens, respectively (3,21).
MSC Differentiation
MSCs were examined for differentiation into adipocytes and osteocytes (19,35). Differentiation of MSCs
into adipocytes was induced by culturing confluent MSC
cultures in DMEM supplemented with 10% FBS, 1 μM
dexamethasone, 10 μg/ml insulin, and 0.2 mM indomethacin for 21 days. The medium was replaced every
3–4 days. Adipogenesis was assessed by Oil Red O staining. Osteogenic differentiation was induced by culturing
the cells in DMEM supplemented with 10% FBS, 100 nM
dexamethasone, 10 mM b-glycerophosphate, 0.05 mM
l-ascorbic acid-2-phosphate. The medium was refreshed
every 3–4 days, and cells were maintained in these conditions for 21 days. Calcium deposition indicative of osteogenesis was detected by Von Kossa staining.
INFLUENZA VIRUS INFECTION OF MESENCHYMAL STROMAL CELLS
Detection of Influenza Virus Receptors on MSCs
Influenza viruses infect cells by binding to sialic acid
receptors. Sialic acid receptors on MSCs were detected
by immunocytochemical staining (48). MSCs were grown
on coverslips to confluency and were fixed with 3.7%
formaldehyde in PBS for 30 min. After blocking the cells
with 2% BSA in PBS for 30 min, the cells were incubated
with FITC-labeled Maackia amurensis lectin II (MAA)
or Sambucus niagra agglutinin (SNA) (EY Laborato
ries, USA) for 30 min in dark. Cell nuclei were stained
with DAPI.
Infection of MSCs With Influenza Virus
Swine (Sw/OH/24366/07; H1N1) and human (Hu/
OH/K1130/06; H1N1) influenza viruses (in house); referred
to as SwH1N1 and HuH1N1, (respectively, in the text)
were propagated in 10-day-old embryonated chicken
eggs (Charles River Laboratories, USA)(18). MSCs were
infected with SwH1N1 and HuH1N1 at a multiplicity of
infection (MOI) of 1. Following washing with PBS, the
cells were incubated with viruses diluted in DMEM supplemented with 0.02 μg/ml TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin and 0.2% BSA.
After adsorption for 1 h, the cells were washed and fresh
medium was added to the cells. At indicated intervals, the
463
supernatants containing viruses were titrated in Madin
Darby Canine Kidney (MDCK) cells (in house).
Influenza Virus-Infected MSCs Produce TNF-a and IL-6
The production of TNF-a and IL-6 in cultures of mock-,
SwH1N1-, and HuH1N1-infected MSCs was quantified at
24 and 48 h after infection by ELISA (Thermoscientific,
USA, and R&D Systems, USA, respectively) as described
earlier (17).
RESULTS
Characteristics of Porcine Bone Marrow MSCs
MSCs isolated from pig bone marrow showed a fibroblast-like morphology (Fig. 1A). MSCs expressed stem
cell markers: Oct4 and SSEA-1 (Fig. 1B). Oct4 has been
shown to regulate the pluripotency and self-renewal of
embryonic and adult stem cells of human and animal
origin (24). Phenotypically, these cells expressed CD29,
CD44, and CD90 (Thy-1) but not CD45, indicating that
these cells were of the mesenchymal lineage (Fig. 1C).
Upon culturing in appropriate induction media, swine
bone marrow-derived MSCs readily differentiated into
adipocytes and osteocytes. MSCs incubated for 21 days
in adipogenic medium showed differentiation into adipocytes. In adipogenic cultures, intracellular accumulation
Figure 1. Characteristics of porcine bone marrow-derived MSCs. Bone marrow mesenchymal stromal cells (MSCs) were isolated
from the femur bones of germ-free pigs. (A) Morphology of MSCs. (B) MSCs express stem cell markers octamer binding transcription
factor 4 (Oct4) and stage-specific embryonic antigen-1 (SSEA-1). (C) Phenotype of porcine MSCs. Phenotype of MSCs was analyzed
by flow cytometry. MSCs were positive for mesenchymal markers CD29, CD44, and CD90 and negative for hematopoietic marker
CD45. Black line, isotype control; red line, specific antibody. (D) Oil Red O staining for lipid-filled vesicles (red) in cultures of MSCs
in adipogenic differentiation media. (E) von Kossa staining for calcified mineral nodules (black) in cultures of MSCs in osteogenic
differentiation media.
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Khatri and Saif
of lipid droplets were stained with Oil Red O, indicating
adipogenesis (Fig. 1D). In osteogenic cultures mineralized nodule-like structures showing calcium deposition
were observed, which stained black with Von Kossa
staining (Fig. 1E).
with SwH1N1 or HuH1N1. Cells were incubated with
viruses at a MOI of 1 for 1 h. As shown in Figure 2, both
SwH1N1 and HuH1N1 replicated in MSCs and produced
cytopathic changes (Fig. 2B); however, SwH1N1 replicated to higher titers as compared to HuH1N1 (Fig. 2C).
MSCs Express Receptors for Influenza Virus
Influenza virus infects cells through binding to cell
surface sialic acid receptors. To examine the susceptibility of MSCs to influenza virus, we first evaluated by
immunocytochemistry these cells for the presence of
sialic acid receptors. The a-2,3- and a-2,6-linked sialic
acid receptors were detected on the surface of MSCs by
MAA and SNA staining, respectively. Expression of both
a-2,3- and a-2,6-linked sialic acid receptors was detected
on a majority of the MSCs, suggesting that viruses of
avian and mammalian lineages may be able to replicate
in these cells (Fig. 2A).
Influenza Virus Induces the Production of TNF-a
and IL-6 in MSCs
Influenza virus infection in humans and animal species is associated with the production of proinflammatory
cytokines (4,41,42). To determine whether influenza
virus-infected MSCs produce proinflammatory cyto
kines, MSCs were infected with SwH1N1 or HuH1N1
influenza viruses. After 24 and 48 h, culture super
natants were harvested and examined for the production
of TNF-a and IL-6 by ELISA. The levels of TNF-a and
IL-6 were substantially greater in virus-infected MSCs
than in uninfected control at both time points tested
(Fig. 3). The levels of TNF-a were higher at 24 h whereas
IL-6 production was higher at 48 h after infection. There
was no significant difference in TNF-a and IL-6 production between influenza viral strains at both time points
(Fig. 3).
Influenza Virus Replicates in MSCs
After confirming the stemness and expression of influenza virus receptors in MSCs, we assessed the susceptibility of MSCs to influenza virus. MSCs were infected
Figure 2. Replication of influenza virus in swine bone marrow-derived MSCs. (A) MSCs express a-2,3- and a-2,6-linked sialic acid
receptors. MSCs were stained with fluorescein isothiocyanate (FITC)-labeled Maackia amurensis lectin II (MAA) specific for a-2,3linked sialic acid receptors (avian influenza virus) and Sambucus niagra agglutinin (SNA), specific for a-2,6-linked sialic acid receptors (mammalian influenza virus). (B) MSCs were infected with swine (SwH1N1; SwIV) or human (HuH1N1; HuIV) influenza virus
at a multiplicity of infection (MOI) of 1. Virus-induced cytopathic changes in MSCs at 24 h after infection. (C) Virus production in
infected culture supernatants as measured by titration in Madin Darby Canine Kidney (MDCK) cells. TCID50 = median tissue culture
infective dose.
INFLUENZA VIRUS INFECTION OF MESENCHYMAL STROMAL CELLS
Figure 3. Influenza virus infection induces inflammatory cyto
kines in MSCs. MSCs were infected with SwH1N1 or HuH1N1.
After indicated times, supernatants from MSC cultures were
examined for (A) tumor necrosis factor (TNF)-a and (B) interleukin (IL)-6 by ELISA. The data are expressed as mean ± SEM
of two separate experiments.
DISCUSSION
In this study, for the first time, we have shown that
influenza virus productively infects the primary swine
bone marrow MSCs in vitro. Swine MSCs expressed
receptors for influenza viruses and virus-infected MSCs
produced proinflammatory cytokines: TNF-a and IL-6.
In this study, we first examined MSCs for the expression of receptors for influenza virus. Immunofluorescence
staining showed that MSCs express a-2,3 and a-2,6
sialic acid receptors utilized by avian and mammalian
influenza viruses, respectively. Based on our results on
the presence of virus receptors in MSCs, these cells were
expected to support influenza virus replication. Indeed,
we were able to detect productive infection of MSCs by
swine and human influenza viruses. Previously, we and
others have demonstrated the susceptibility of bone marrow derived MSCs to a variety of viruses (20,32,38). The
susceptibility of MSCs to influenza virus expands the
population of host target cells the virus can use for replication. Virus-infected MSCs could potentially contribute
to the virus induced pathology by directly or indirectly
affecting other known targets of the virus, including progenitor epithelial and other immune cells. In bone marrow compartments, virus-infected MSCs may transmit
465
virus to other stem and precursor cells in the bone marrow. As reported in this study, virus infection may cause
the lysis of bone marrow MSCs, thus significantly reducing their potential for renewal and ability to differentiate
into specialized cells and tissues.
MSCs are known to regulate hematopoiesis via secretion of growth factors. We detected the production of
IL-6 and TNF-a in influenza virus-infected MSCs. Virus
infection of MSCs and factors secreted by infected cells
could affect hematopoiesis and may alter the characteristics of immune progenitor cells. Production of IL-6 and
TNF-a was also detected in human MSCs stimulated
with polyinosinic–polycytidylic acid (poly (I:C)) (27,44).
IL-6 regulates inflammatory responses and hematopoiesis
(34), and its overproduction interferes with differentiation of monocytes into dendritic cells, thereby decreasing
their stimulation ability on T-cells (8). IL-6 also enhances
the pathology of autoimmune diseases and tissue remodeling. TNF-a is present in the inflammatory milieu (12).
The production of IL-6 and TNF-a thus suggests that
infection of MSCs with influenza virus might affect their
differentiation potential and potentiate the virus-induced
inflammatory response as observed in humans and animal species following infection with influenza virus
(4,41,42). MSCs have been shown to possess immunoregulatory properties and inhibit both innate and adaptive
immune cells. Infection and lysis of MSCs by influenza
virus as observed in this study may result in immune dysregulation allowing hyperactivation and proliferation of
inflammatory cells and excessive production of proinflammatory cytokines. Studies are underway to examine
the effect of virus infection of MSCs on their differentiation potential and their ability to support hematopoiesis.
A previous report (39) showed that influenza virus
caused depletion of pre-B and immature B-cells in
bone marrow of infected mice which was mediated by
release of TNF-a produced locally in the bone marrow.
However, the precise identity of the cellular source of
TNF-a was not determined. Although we have not examined the direct effect of MSCs and secreted TNF-a on
bone marrow immune cells, our results provide evidence
that TNF-a produced by virus-infected bone marrow
MSCs may mediate the apoptosis of pre-B and immature
B-cells as previously observed (39). The depletion of
B-cells may suppress humoral immune response against
influenza virus and/or other coinfecting pathogens.
In light of our data demonstrating that MSCs are susceptible to influenza virus infection in vitro, it is likely
that they may be viral transmitters. MSCs derived from a
donor with ongoing influenza virus infection could therefore carry virus to the recipient. Based on MSCs transplantation studies in animal models, MSCs are known to
first home in the lungs after transplantation (10). Similar
to humans, the respiratory tract is the primary site of
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Khatri and Saif
influenza virus replication in swine also. Therefore,
transplantation of influenza virus-infected MSCs could
infect susceptible cell populations in the lung and induce
fatal interstitial pneumonitis after infusion. Infusion of
cytomegalovirus (CMV)-infected MSCs caused widespread CMV infection in different tissues and organs in
recipients (7). Taken together, these data indicate that
bone marrow cells need to be tested for influenza virus
before transplantation to prevent virus transmission to
recipients.
MSCs have been shown to possess potent immunosuppressive properties and are being evaluated in clinical
settings to reduce graft-versus-host disease and transplantation rejection. In immunocompromised hosts such
as hematopoietic stem cell transplant (HSCT) patients,
MSC-induced immunosuppression may suppress the
immune responses to infections. Several studies have
reported the higher incidence and prolonged persistence
of respiratory viral agents in HSCT patients (28,30,45).
Our results clearly demonstrate that MSCs are susceptible to infection with influenza virus; therefore, ongoing
virus infection in the recipient may infect transplanted
MSCs and alter their immunomodulatory and differentiation abilities. Further studies are needed to confirm the
infection of infused MSCs in influenza virus-infected
pigs/animal models.
In summary, we provide evidence that the bone
marrow-derived MSCs are permissive to productive influ
enza virus infection. Future study of influenza virus
infection of MSCs in vivo and effects of virus infection
on MSCs self-renewal, differentiation, and immunoregulatory functions will help to understand the critical role of
MSCs in the pathogenesis of influenza virus.
ACKNOWLEDGMENTS: We acknowledge the salaries and
research support provided by state and federal funds appropriated to the Ohio Agriculture Research and Development Center,
The Ohio State University. We are indebted to Dr. Linda Saif’s
laboratory for providing the femur bones from germ-free pigs.
We are grateful to Dr. Kuldeep S. Chattha for valuable assistance with the flow cytometry and cytokines ELISA. We also
thank Kelly Scheuer for critically reviewing the manuscript. The
authors have no financial conflict of interest.
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