Bone Marrow-Derived Mesenchymal Stromal Cells Inhibit Th2-Mediated Allergic Airways Inflammation in...

TRANSLATIONAL AND CLINICAL RESEARCH

Bone Marrow-Derived Mesenchymal Stromal Cells Inhibit

Th2-Mediated Allergic Airways Inflammation in Mice

MEAGAN GOODWIN,aVIRANUJ SUEBLINVONG,

aPHILIP EISENHAUER,

aNICHOLAS P. ZIATS,

bLAURIE LECLAIR,

a

MATTHEW E. POYNTER,a CHAD STEELE,c MERCEDES RINCON,a DANIEL J. WEISSa

aDepartment of Medicine, University of Vermont College of Medicine, Burlington, Vermont, USA; bDepartment

of Pathology, Case Western Reserve University, Cleveland, Ohio, USA; cDepartment of Medicine, University of

Alabama at Birmingham, Birmingham, Alabama, USA

Key Words. Mesenchymal stromal cell • Allergic airways disease • CD4 lymphocyte

ABSTRACT

Bone marrow-derived mesenchymal stromal cells (BMSCs)mitigate inflammation in mouse models of acute lunginjury. However, specific mechanisms of BMSC actions on

CD4 T lymphocyte-mediated inflammation in vivo remainpoorly understood. Limited data suggests promotion of Th2phenotype in models of Th1-mediated diseases. However,

whether this might alleviate or worsen Th2-mediated dis-eases such as allergic asthma is unknown. To ascertain theeffects of systemic administration of BMSCs in a mouse

model of Th2-mediated allergic airways inflammation, oval-bumin (OVA)-induced allergic airways inflammation was

induced in wild-type C57BL/6 and BALB/c mice as well asin interferon-c (IFNc) receptor null mice. Effects of sys-temic administration during antigen sensitization of either

syngeneic or allogeneic BMSC on airways hyperreactivity,lung inflammation, antigen-specific CD4 T lymphocytes,

and serum immunoglobulins were assessed. Both syngeneic

and allogeneic BMSCs inhibited airways hyperreactivityand lung inflammation through a mechanism partly de-pendent on IFNc. However, contrary to existing data,

BMSCs did not affect antigen-specific CD4 T lymphocyteproliferation but rather promoted Th1 phenotype in vivo asassessed by both OVA-specific CD4 T lymphocyte cytokine

production and OVA-specific circulating immunoglobulins.BMSCs treated to prevent release of soluble mediators anda control cell population of primary dermal skin fibroblasts

only partly mimicked the BMSC effects and in some casesworsened inflammation. In conclusion, BMSCs inhibit Th2-

mediated allergic airways inflammation by influencing anti-gen-specific CD4 T lymphocyte differentiation. Promotionof a Th1 phenotype in antigen-specific CD4 T lymphocytes

by BMSCs is sufficient to inhibit Th2-mediated allergic air-ways inflammation through an IFNc-dependent process.

STEM CELLS 2011;29:1137–1148

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Mesenchymal stromal cells (MSCs) are adult, multipotent pro-genitor cells that were first identified in the bone marrow andhave the ability to differentiate into bone, fat, and cartilage[1]. MSCs have subsequently been isolated from virtually allpostnatal tissues as well as umbilical cord blood, placenta,and amniotic fluid and can be induced in vitro to differentiateinto a variety of cell types [2]. While MSCs isolated from dif-ferent sources share key identifying characteristics, differen-ces in gene expression and their secretome have beenobserved. Bone marrow-derived MSCs (BMSCs) have beenbest characterized and have been found to have significantimmunomodulatory and nonimmunogenic properties, allowingadministration of allogeneic BMSCs without eliciting an im-munogenic response within the host [3–5]. BMSCs inhibit theproliferation and function of a broad range of immune cells

in vitro, including T-cells, B-cells, natural killer cells, anddendritic cells [6–19]. Notably, BMSCs can suppress CD4 Tlymphocyte responses in vitro by inhibiting T-cell prolifera-tion induced by mitogens or specific antigens [20–31]. Theseeffects likely occur through a paracrine effect by the releaseof soluble mediators by the BMSCs, although cell-cell contactmay also be involved [22, 23, 26–28, 30–33]. However,whether the mechanisms by which BMSCs suppress immunecells in vitro are similar to those in vivo remains unclear.

Published reports evaluating BMSC effects on CD4 Tlymphocyte differentiation in in vitro model systems generallydemonstrate that MSCs promote a Th2 phenotype in CD4lymphocytes. In vivo, systemic administration of humanBMSCs (hBMSCs) decreased numbers of Th1 and Th17 CD4T lymphocytes, with corresponding decreases in interferon-c(IFNc) and interleukin (IL)-17 production, and increased num-bers of IL-4 producing Th2 lymphocytes, when administeredin a mouse model of experimental autoimmune encephalitis

Author contributions: M.G.: concept and design, collection and/or assembly of data, data interpretation, manuscript writing, finalapproval of manuscript; V.S., P.E., N.Z., and C.S.: data analysis and interpretation; L.L., M.P., and M.R.: concept and design; D.W.:concept and design, manuscript writing, final approval of manuscript.

Correspondence: Daniel J. Weiss, M.D., Ph.D., 226 Health Sciences Research Facility, University of Vermont College of Medicine,Burlington, Vermont 05405, USA. Telephone: 802-656-8925; Fax: 802-656-8926; e-mail: [email protected] Received October 27,2010; accepted for publication March 28, 2011; first published online in STEM CELLS EXPRESS May 4, 2011. VC AlphaMed Press1066-5099/2009/$30.00/0 doi: 10.1002/stem.656

STEM CELLS 2011;29:1137–1148 www.StemCells.com

[34]. These findings suggest that administration of BMSCswould be either ineffective or could potentially exacerbate theimmune response in diseases characterized by Th2-mediatedinflammation, such as allergic asthma. As little is known aboutthe in vivo effects of BMSC administration in Th2 models ofinflammation, it was of particular interest to investigate theeffects of BMSCs on the generation of antigen-specific CD4 T-cells in allergic airways inflammation, a mouse model of aller-gic asthma. Sensitization to ovalbumin (OVA) with the Th2-promoting adjuvant aluminum hydroxide, followed by chal-lenge with aerosolized OVA is a well-established model ofinducing Th2-mediated eosinophilic allergic airways inflamma-tion in mice [35]. Initial clonal expansion and differentiationof antigen-specific CD4 T-cells occurs during the sensitizationphase of the OVA model. Given this, we investigated whetheradministration of either syngeneic or allogeneic BMSCs duringantigen sensitization would effect the generation of allergic air-ways inflammation, including clonal proliferation and differen-tiation of antigen-specific CD4 Th2 lymphocytes.

MATERIALS AND METHODS

Mice

Female C57BL/6, BALB/c, and IFNcRKO (IFNc�/�) mice (6–8weeks) from the Jackson Laboratory (Bar Harbor, ME, www.jax.-org) were maintained in accordance with institutional and Ameri-can Association for Accreditation of Laboratory Animal Carestandards, with studies subject to Institutional Animal Care and UseCommittee review at the University of Vermont (Burlington, VT).

Bone Marrow-derived Mesenchymal Stem Cells

MSCs derived from bone marrow of adult male C57BL/6 micewere obtained from the Tulane University Center for Gene Ther-apy. BMSCs were cultured in Iscoves Modified Dulbecco Me-dium with 2 mM L-glutamine, 100 U/ml penicillin, 100 lg/mlstreptomycin, 10% fetal bovine serum, and 10% horse serum(GIBCO, Invitrogen, Carlsbad, CA, www.invitrogen.com/site/us/en/home.html). Cells were used at passage six or lower and main-tained in culture at confluency no greater than 70%. Purity wasdetermined by expression of Sca-1, CD106, CD29, absent expres-sion of CD11b, CD11c, CD34, CD45, and the ability to differen-tiate into chondrocytes and adipocytes in vitro.

OVA Sensitization

Mice were immunized by i.p. injection with 20 lg of OVA(grade V; Sigma Aldrich, St. Louis, MO, www.sigmaaldrich.com/united-states.html) or phosphate-buffered saline (PBS) with 2.25mg aluminum hydroxide (Alum; ImjectAlum; Pierce, Rockford,IL, www.piercenet.com) or complete (day 0) or incomplete (day7) Freud’s adjuvant on days 0 and 7. Immediately before eachimmunization, experimental mice received tail vein injection of 2� 106 MSCs, whereas control mice received the same number ofMSCs treated with the cross-linker 1-ethyl-3-[3-dimethylamino-propyl] carbodiimide hydrochloride (EDCI) [36], primary dermalC57BL/6 fibroblasts, viable MSCs with PBS/alum-immunization,or PBS control. Mice were challenged with aerosolized 1% OVAin saline for 30 minutes on days 14, 15, and 16.

Respiratory Mechanics

Pulmonary function was analyzed on day 18 using the forced os-cillation technique (flexiVent; SCIREQ Scientific RespiratoryEquipment, Chandler, AZ, www.scireq.com) as previouslydescribed [37]. The peak responses for airway resistance (RN),tissue resistance (G), or tissue elastance (H) were determined inresponse to sequential inhalation of nebulized saline, 3.125 and12.5 mg/ml of methacholine.

BAL, Total and Differential Cell counts, and Cyto-kine Assessment

Bronchoalveolar lavage (BAL) fluid was collected by instillingand recovering 1 ml of saline into the airways. Cells werecounted using an Advia cell counter (Bayer, Leverkusen, Ger-many, www.bayer.com), cytospun onto glass slides, and stainedwith Hema-3 (Fisher Scientific, Pittsburgh, PA, www.fishersci.-com). A total of 200 cells per slide were scored by characteristicmorphology and staining. BAL cytokine levels were measuredusing a mouse 23-plex kit on the Bio-Plex suspension array sys-tem (Bio-rad, Hercules, CA, www.bio-rad.com).

Lung Histology and Inflammation Scoring

Lungs were inflated, gravity fixed (20 cm) with 4% paraformalde-hyde, paraffin embedded, cut into 5-lm sections, and stained withhematoxylin and eosin. Inflammation was scored on a scale from 0to 3, in blinded fashion by a collaborating veterinary pathologist(Dr. Ziats) based on the presence and intensity of peribronchial cellinfiltrates when compared with known positive and negative con-trols using an established scoring system. Four airways per animalwere analyzed, and six to eight animals were analyzed per condition.

Serum Collection and Immunoglobulin Analyses

Serum samples were diluted 1:50 for IgG1, 1:20 for IgG2a, and1:15 for IgE and analyzed as described previously [38].

CD4 T Lymphocyte Isolation, Proliferation, andCytokine Production

Splenic CD4 T lymphocytes were positively selected (MACS CD4T lymphocyte separation system, Miltenyi, Auburn, CA, www.mil-tenyibiotec.com); purity was above 98% by flow cytometry withantibodies against CD4 and T-cell receptor. The irradiated (2000Rads) CD4-negative fraction of splenocytes from naı̈ve C57BL/6mice provided antigen-presenting cells (APCs). Antigen-specificCD4 T lymphocyte proliferation was assessed by 3[H]-labeled thy-midine (NEN #NET027A, Waltham, MA, www.perkinelmer.com)incorporation as described previously [39] using serial OVA dilu-tions. IFNc and IL-4 were measured by enzyme-linked immunosor-bent assay (R&D Systems; DuoSet ELISA Development Systems,Minneapolis, MN, www.rndsystems.com).

Statistical Analyses

Mean values were compared by Student’s t test or analysis ofvariance. For analysis of inflammation scores, a nonparametric,Kruskal-Wallis rank sum test was performed.

RESULTS

Systemic Administration of Either Syngeneic orAllogeneic BMSCs During Antigen SensitizationInhibits Methacholine-Induced Airways Hyperreac-tivity and Eosinophilic Lung Inflammation

To determine if systemic BMSCs administration during anti-gen sensitization inhibited the OVA-stimulated increase inairways hyperreactivity of the large conducting airways, theprimary physiologic outcome, adult mice were immunized byi.p. injection of OVA in the presence of the Th2 promotingadjuvant aluminum hydroxide (alum) on days 0 and 7 (Fig.1A). BMSCs isolated from the bone marrow of adult C57BL/6 mice (Tulane Mesenchymal Stem Cell Core Facility) or ve-hicle control (PBS) were administered by tail vein injectionimmediately before immunization on days 0 and 7 to eitheradult C57BL/6 (syngeneic) or adult BALB/c (allogeneic)mice. Following aerosol challenge with OVA on days 14–16,airways hyperreactivity was assessed on day 18. C57BL/6

1138 BMSCs Inhibit Allergic Airways Inflammation

mice receiving syngeneic BMSCs during immunization dem-onstrated significant inhibition of the OVA-stimulatedincrease in methacholine-induced airways hyperreactivity inthe large conducting airways (RN) (Fig. 1B). BMSC adminis-tration had no effects on the other physiologic parametersmeasured, lung tissue elastance (H), or tissue resistance (G)[37] (Supporting Information Fig. E1A, E1B). Adult BALB/cmice receiving allogeneic BMSCs during antigen sensitizationcomparably demonstrated a significant decrease in airwayshyperreactivity (RN) in response to the higher methacholinedose (Fig. 1C). Allogeneic BMSC administration to theBALB/c mice also decreased tissue elastance (H) but no

effects were observed on tissue resistance (G) (Supporting In-formation Fig. E1C, E1D). Administration of BMSCs to micereceiving control (PBS/alum) immunizations (i.e., ‘‘MSCs,Sham OVA’’) followed by OVA challenge did not affect air-ways hyperreactivity in either the C57BL/6 or BALB/c mice(Fig. 1A, 1B).

We next evaluated whether BMSC administration duringantigen sensitization would also inhibit the Th2-mediated eo-sinophilic lung inflammation induced by OVA/alum sensitiza-tion and subsequent challenge. In OVA-treated C57BL/6 micereceiving syngeneic BMSCs during OVA/alum sensitization,the total numbers of eosinophils in BAL fluid was

Figure 1. Systemic administration of syngeneic and allogeneic bone marrow-derived mesenchymal stromal cells (BMSCs) during ovalbumin(OVA)/alum sensitization inhibits airways hyperresponsiveness (AHR) and allergic airways inflammation. (A): Schematic of experimental studies.(B): AHR in syngeneic or (C) Allogeneic BMSC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDCI)-MSC, fibroblast, or phos-phate-buffered saline (PBS)-treated OVA immunized mice as determined by peak airways resistance responses at each methacholine dose as a func-tion of percentage change from resting baseline. (D): Bronchoalveolar lavage (BAL) fluid cell differentials in syngeneic or (E) allogeneic BMSC,EDCI-MSC, fibroblast, or PBS-treated OVA immunized mice models. Data represents percentage of total cells counted. (F): Syngeneic and (G)allogeneic BAL fluid cytokine levels as measured using luminex multibead technology (Bioplex, Biorad, Hercules, CA). Data depicts means 6 SEfrom one of three similar experiments with six to eight mice per condition for each experiment. *, p < .05 when compared with OVA. Abbrevia-tions: EDCI, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; IL, interleukin; MSC, mesenchymal stromal cells; OVA, ovalbumin.

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significantly decreased 2 days after final aerosol challengewhen compared with the characteristic increase provoked byOVA/alum sensitization and OVA challenge (Fig. 1D). WhileBMSC administration had no effects on the small increase innumbers of BAL fluid lymphocytes and neutrophils observedin OVA-treated mice, administration did result in significantincreases in BAL fluid macrophages when compared withOVA treated mice. Similar effects were observed in BALB/cmice receiving allogeneic BMSCs whereby there was a signif-icant decrease in the number of eosinophils with a concordantincrease in numbers of macrophages and no effect on lympho-cyte or neutrophil counts (Fig. 1E). BMSCs administered inboth the syngeneic and allogeneic sham OVA models resultedin similar cellular profiles as to what was observed in naı̈vemice (Fig. 1D, 1E).

The Th2 cytokines IL-4 and IL-5 are known promoters ofeosinophil recruitment, and immunoglobulin class switching,and are elevated in BAL fluid on day 18 following aerosol-ized OVA challenge in both C57BL/6 and BALB/c OVA-treated mice. Administration of BMSCs in either the synge-neic or allogeneic models resulted in a significant decrease inlevels of IL-4 and IL-5 in the BAL fluid (Fig. 1F, 1G). Levelsof another prominent Th2 cytokine, IL-13, which characteris-

tically peak earlier after OVA challenge, were not elevated inBAL fluid of C57BL/6 OVA-treated mice on day 18 andwere otherwise not affected by syngeneic BMSC administra-tion (Fig. 1F). Although IL-13 levels were still detectable onday 18 in the BALB/c mice, BMSC administration did notresult in a significant decrease when compared with OVAcontrols. (Fig. 1G). No detectable levels of IFNc or IL-17,indicative of a Th1 or Th17 response, respectively, weremeasured in BAL fluid for any of the groups (data notshown). IL-6 is rapidly produced by stimulated APCs andpromotes a Th2 phenotype in CD4 cells. Syngeneic BMSCadministration significantly decreased IL-6 levels in the BALfluid when compared with OVA treated mice (Fig. 1F). Nomeasurable levels of BAL fluid IL-6 was detected in any ofthe BALB/c treated mice (Fig. 1G).

Qualitative and quantitative assessment of histologic lunginflammation demonstrated that administration of allogeneicBMSCs to BALB/c mice decreased peribronchial inflammatorycell infiltrates characteristic of this model (Fig. 2A, 2B). Simi-larly, administration of syngeneic BMSCs to C57BL/6 micealso resulted in a significant decrease in lung inflammation asassessed by the number of inflammatory cells surrounding theairways (Supporting Information Fig. E2A, E2B).

Figure 2. Systemic administration of allogeneic bone marrow-derived mesenchymal stromal cells during ovalbumin (OVA)/alum sensitizationdecreases peribronchial inflammation. (A): Representative photomicrographs, stained with hematoxylin and eosin, depicting histologic inflamma-tion on day 18 for allogeneic models are depicted for each experimental condition. Arrows designate areas of peribronchial inflammatory cellinfiltration. Original magnification �100. (B): Airways inflammation scoring. Data depicts means 6 SE from one of three similar experimentswith six to eight mice per condition for each experiment. *, p < .05 when compared with OVA. Abbreviations: EDCI, 1-ethyl-3-[3-dimethylami-nopropyl] carbodiimide hydrochloride; MSC, mesenchymal stromal cells; OVA, ovalbumin.


To determine whether soluble mediators secreted byBMSCs were required for the observed inhibition of airwayshyperreactivity and Th2-mediated, eosinophil lung inflamma-tion, administration of BMSCs treated with EDCI to preventrelease of soluble mediators (‘‘EDCI-MSCs’’) was assessed[36]. Unlike viable MSCs, administration of the EDCI-MSCsdid not inhibit all the end points assessed. EDCI-MSCs sup-pressed airways hyperresponsiveness (AHR) only at the lower3.125 mg/ml dose in the syngeneic model as opposed to theallogeneic model whereby EDCI-MSCs were only effective atsuppressing AHR at the higher 12.5 mg/ml dose (Fig. 1A,1B). While administration of EDCI-MSCs decreased levels ofIL-4 and IL-5 in both the syngeneic and allogeneic models,they were only able to decrease BAL fluid eosinophils withinthe syngeneic model (Fig. 1B–1E). The effects of EDCI-MSCs were also variable with respect to their effects on air-ways hyperreactivity. Furthermore, administration of EDCI-MSCs did not reduce histologic inflammation in either thesyngeneic or allogeneic models as assessed by the number ofinflammatory cells found surrounding the airways (Fig. 2 andSupporting Information Fig. E2A, E2B). These results suggestthat while cell surface structures contribute to the BMSCeffects, soluble mediators are required for BMSCs to exhibittheir full suppressive capabilities in this model.

To further examine whether the effects observed werespecific to the administration of BMSCs and not a result ofintroducing a foreign cell type into circulation, we assessedthe effects of a control cell type, primary mouse dermal fibro-blasts obtained from skin biopsies of C57BL/6 mice. Notably,syngeneic administration of C57BL/6 mouse primary dermalfibroblasts significantly inhibited airways hyperreactivity (Rn)in C57BL/6 mice (Fig. 1B) and decreased levels of Th2-asso-ciated cytokines in the BAL fluid (Fig. 1F). However, admin-istration of syngeneic primary dermal fibroblasts did notdecrease the numbers of BAL fluid eosinophils and, impor-tantly, significantly increased histologic airways inflammationwhen compared with the control OVA-treated mice (Fig. 1Dand Supporting Information Fig. E2A, E2B).

The above results demonstrate that both syngeneic andallogeneic administration of BMSCs during sensitization toOVA decreases AHR and eosinophilic Th2-mediated lunginflammation following subsequent challenge with aerosolizedOVA. Furthermore, administration of the control cell types,EDCI-MSCs and primary dermal fibroblasts only partiallymimicked the suppressive effects observed when comparedwith the administration of viable BMSCs and in some casesincreased cellular infiltration.

Systemic Administration of Syngeneic or AllogeneicBMSCs During Antigen Sensitization Does Not In-hibit In Vivo Antigen-Specific CD4 T LymphocyteProliferation But Alters Antigen-Specific CD4 TLymphocyte Differentiation

As BMSCs have been demonstrated to inhibit CD4 T lympho-cyte proliferation in in vitro mixed lymphocyte reactions [20,23, 40], we reasoned that inhibition of antigen (i.e., OVA)-specific CD4 T lymphocyte generation might be a mechanismby which the BMSCs inhibit allergic airways inflammation.To assess this, C57BL/6 or BALB/c mice were immunizedwith OVA/alum on days 0 and 7 and splenic CD4 T lympho-cytes were harvested on day 14 (Fig. 4A). C57BL/6 BMSCsor PBS control was administered by tail vein injection 15minutes before each immunization. Notably, BMSC adminis-tration during OVA immunization had no effect on OVA-spe-cific CD4 T lymphocyte proliferation following either synge-neic administration to C57BL/6 mice or allogeneic

administration to BALB/c mice (Figs. 3B and Supporting In-formation Fig. E3A). While the in vitro restimulation of CD4lymphocytes occurred in the absence of BMSCs, we concludethat OVA-specific T-cell proliferation was unaffected by invivo exposure to BMSCs due to the fact that any differencesin the generation of OVA-specific T lymphocytes during clo-nal expansion would be reflected by in vitro antigen-specificstimulation of proliferation.

We next assessed whether systemic BMSC administrationduring antigen sensitization would affect antigen-specific CD4T lymphocyte differentiation. Use of the adjuvant alum duringOVA immunization results in generation of Th2 antigen-spe-cific CD4 T lymphocytes. Notably, administration of alloge-neic BMSCs during OVA sensitization had no effect on IL-4release from antigen-specific splenic CD4 T lymphocytes cul-tured ex vivo in the presence of OVA and irradiated splenicAPCs (Fig. 3C)) and had either no effect or increased levelsof IL-4 as a result of syngeneic BMSC administration (Sup-porting Information Fig. E3B). However, administration of ei-ther syngeneic or allogeneic BMSCs, resulted in significantincreases in the production of IFNc by antigen-specific splenicCD4 T lymphocytes (Fig. 3D and Supporting Information Fig.E3C). No significant change in either IL-4 or IFNc release byantigen-specific splenic CD4 T lymphocytes was observed insham-OVA treated mice that received allogeneic BMSCswhen compared with naı̈ve mice (Fig. 3C, 3D).

Systemic Administration of Sygeneic or AllogeneicMSCs During Antigen Sensitization Alters Levels ofCirculating Antigen-Specific Immunoglobulins

As BMSC administration during OVA sensitization promotedTh1 differentiation of antigen-specific CD4 T lymphocytes,we assessed whether MSC administration affected levels ofcirculating antigen-specific antibodies provoked by OVA sen-sitization and challenge. Systemic administration of allogeneicBMSCs to the BALB/c mice significantly mitigated theincreases in OVA-specific IgE and IgG1 typically provokedby OVA/alum sensitization and OVA challenge (Fig. 4A,4B). However, levels of circulating antigen-specific IgG2a, in-dicative of a Th1 phenotype, were significantly increased inmice receiving BMSCs (Fig. 4C). In contrast, administrationof syngeneic BMSCs to C57BL/6 mice during OVA sensitiza-tion had no effect on the characteristic increase in antigen-specific IgE or IgG1; however, it did resulted in a significantincrease in the Th1-associated OVA-IgG2a (Supporting Infor-mation Fig. E4A, E4B, E4C). No significant change in levelsof circulating OVA-specific IgE or IgG1 were observed insham-OVA treated mice that had received either syngeneic orallogeneic BMSCs when compared with naı̈ve mice (Fig. 4A–4C and Supporting Information Fig. E4A, E4B, E4C).Increases in OVA-specific IgG2a were observed in sham-OVA treated mice receiving allogeneic BMSCs; whereby,OVA-specific responses were generated during the challengephase of the experimental protocol.

These results demonstrate that while neither syngeneic norallogeneic BMSC administration alters genesis and prolifera-tion of antigen-specific CD4 lymphocytes in vivo, differentia-tion of these cells is skewed from a Th2 phenotype toward aTh1 as measured both by release of IL-4 and IFNc as well asby patterns of circulating antigen-specific immunoglobulins.The promotion of a Th1 phenotype in CD4 lymphocytes wasobserved in both the Th1 biased C57BL/6 mouse strain andnotably in the Th2 biased BALB/c strain. Differences betweensyngeneic and allogeneic BMSC administration on levels ofcirculating IgE and IgG1 were observed; however, it is


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unclear whether this is due to strain differences in productionor a result of the different types of transplants performed.

Systemic Administration of Syngeneic BMSCs Dur-ing Antigen Sensitization Has No Effect on Th1-Mediated Allergic Airways Inflammation

As MSC administration promotes differentiation of antigen-specific Th1 CD4 T lymphocytes in this Th2 model of aller-gic airways inflammation, we questioned what the BMSCsmight do in a model of Th1-mediated allergic airways inflam-mation in the Th1-biased C57BL/6 mice. To assess this, weused complete Freund’s adjuvant (CFA) rather than alum tocreate a model of OVA-induced Th1-mediated lung inflamma-tion (Fig. 5A). Administration of BMSCs during OVA sensiti-zation had no effect on the characteristic Th1-mediatedincrease in BAL fluid neutrophils and lymphocytes (Fig. 5B).Notably, BMSC administration did not increase but ratherslightly decreased IFNc release from antigen-specific splenicCD4 T lymphocytes (Fig. 5C). IL-4 release by antigen-spe-cific CD4 T lymphocytes was not detected under any experi-mental condition (data not shown). In parallel, BMSCs didnot alter levels of the circulating antigen-specific Th1 immu-noglobulin IgG2a (Fig. 5D). These studies demonstrate thatwhile BMSCs promote a Th1 phenotype in the setting of anin vivo antigen-stimulated Th2 response in both Th1 andTh2-biased mouse strains, MSCs do not either augment theTh1-mediated inflammation or promote a Th2 phenotype inthe setting of an antigen-stimulated allergic airways Th1response in a Th1-biased mouse.

BMSC Inhibition of Allergic Airways InflammationIs Partly Dependent on IFNc

Promotion of a Th1 phenotype and production of IFNc byantigen-specific CD4 T lymphocytes has been demonstratedto be sufficient for ameliorating Th2-mediated allergic air-ways inflammation [25, 41]. To determine if amelioration ofallergic airways inflammation by BMSCs was comparably de-pendent on IFNc, studies were repeated in IFNc receptor nullmice (IFNcR�/�), which develop OVA/alum-stimulated aller-gic airways inflammation comparable in many respects towild-type mice [42, 43]. In contrast to strain control C57BL/6mice, systemic administration of syngeneic BMSCs duringantigen sensitization of IFNcR�/� mice did not inhibit theOVA-stimulated increase in BAL fluid eosinophils or BALfluid IL-4, IL-5, or IL-13 content (Fig. 6A, 6C). Another cyto-kine, transforming growth factor b1 (TGFb1), which has beenimplicated in generation of allergic AHR in the OVA model[44], was significantly increased by OVA/alum treatment inBAL fluid of both strain control C57BL/6 and IFNcR�/�

mice when compared with respective naı̈ve controls (Fig. 6B).However, while BMSC administration during antigen sensiti-zation decreased the OVA/alum-stimulated increase in BALfluid TGFb levels in the C57BL/6 mice, BMSC administra-tion significantly increased BAL fluid TGFb in the IFNcR�/�

mice (Fig. 6B). While BMSC administration inhibited theincrease in histologic inflammation provoked by OVA/alumsensitization and OVA challenge in the strain control C57BL/6 mice, histologic inflammation was not inhibited followingBMSC administration to the IFNcR�/� mice (Fig. 6D, 6E). In

Figure 3. Systemic administration of allogeneic bone marrow-derived mesenchymal stromal cells does not affect antigen-specific CD4 T lym-phocyte proliferation but promotes CD4 Th1 differentiation. (A): Schematic of experimental protocol. (B): Allogeneic antigen-specific CD4 Tlymphocyte proliferation as assessed by 3[H]-thymidine incorporation. (C): Interleukin-4 content in conditioned media from splenic CD4 T lym-phocytes isolated from allogeneic model. (D): Interferon-c content in conditioned media from splenic CD4 T lymphocytes isolated from the allo-geneic model. Data depicts means 6 SE from one of three similar experiments with six to eight mice per condition for each experiment. *, p <.05 when compared with ovalbumin. Abbreviations: IFNc, interfereon-c; IL-4, interleukin-4; MSC, mesenchymal stromal cells; OVA, ovalbumin.


contrast to strain control C57BL/6 mice, BMSC administra-tion to the IFNcR�/� mice during OVA sensitizationincreased OVA-stimulated IL-4 release from OVA-specificCD4 T lymphocytes (Fig. 7A). However, IFNc release fromOVA-specific CD4 T lymphocytes was not significantlyincreased in the IFNcR�/� mice, when compared with theincrease observed in the C57Bl/6 control mice (Fig. 7B). Cir-culating OVA-specific IgE levels were blunted in theIFNcR�/� mice when compared with strain control mice,however, BMSCs had no effect in either cohort (Fig. 7C).

MSC administration had no effect on circulating OVA-spe-cific IgG1 in either wild-type or IFNcR�/� mice (Fig. 7D),but in contrast to strain control mice in which OVA-specificIgG2a is increased in MSC–treated mice, circulating OVA-specific IgG2a was nearly undetectable under all experimentalconditions in the IFNcR�/� mice (Fig. 7E). Therefore, themechanism by which BMSCs suppress Th2-mediated allergicairways inflammation in this model is thus partly through de-velopment of a Th1 response and the ability of the recipientto respond to IFNc.

DISCUSSION

MSCs are increasingly being found to have potent anti-inflam-matory effects in a wide range of inflammatory and immune-mediated disease models [3–5]. However, the mechanisms ofMSC actions in vivo, particularly those affecting CD4 T lym-phocyte-mediated inflammation, remain poorly understood.Although MSCs can inhibit CD4 T lymphocyte proliferationin in vitro assays, whether this occurs in vivo remainsunclear. Further, the few published reports evaluating MSCeffects on CD4 T lymphocyte differentiation generally dem-onstrate that MSCs tend to shift CD4 T lymphocytes toward aTh2 phenotype both in vitro and in vivo [34, 45]. These find-ings suggest that administration of MSCs might be either inef-fective or could potentially exacerbate immune responses in amodel of Th2 mediated inflammation, such as allergic asthma.As such, it was of particular interest to investigate the effectsof MSCs on the generation and differentiation of antigen-spe-cific CD4 T-cells in a model of Th2-mediated inflammation.Using a mouse model of OVA-induced Th2-mediated eosino-philic allergic airways inflammation, we found that systemicadministration of either syngeneic or allogeneic BMSCs dur-ing antigen sensitization inhibited both airways hyperreactiv-ity and lung inflammation through a mechanism partly de-pendent on IFNc. BMSCs promoted a Th1 phenotype in vivoas assessed by both IFNc production following antigen-spe-cific CD4 T lymphocyte stimulation and antigen-specific cir-culating immunoglobulins, in both the Th1-biased C57BL/6and Th2-biased BALB/c mouse strains. As promotion of aTh1 CD4 lymphocyte and subsequent IFNc production can in-hibit Th2-mediated allergic airways inflammation [25, 41],BMSCs, therefore, inhibit Th2-mediated allergic airwaysinflammation by influencing antigen-specific CD4 T lympho-cyte differentiation and promoting IFNc production. Further,the BMSC effects on allergic airways inflammation are de-pendent on the ability of the mouse to respond to IFNc.

To address whether soluble mediators released by theBMSCs played a role in amelioration of allergic airwaysinflammation, we used BMSCs treated with EDCI to inhibitsecretion of soluble mediators yet preserve cell surface epi-topes. Administration of either syngeneic or allogeneic EDCI-treated BMSCs did not inhibit airways hyperreactivity or sig-nificantly decrease OVA-stimulated histologic inflammation.However, BAL fluid eosinophilia and levels of Th2 cytokineswere reduced similarly to that observed in the mice receivinguntreated BMSCs. This suggests that cell surface moleculesplay a role in the BMSC actions in vivo but that solublemediators are required for MSCs to exhibit their full suppres-sive potential in this model.

MSCs were first identified as an adherent, fibroblast-likepopulation of cells in the bone marrow and initially identifiedas colony-forming fibroblasts. Several recent publicationshave argued that their immunosuppressive properties are notunique to MSCs, but rather a fundamental property shared

Figure 4. Systemic administration of allogeneic bone marrow-derived mesenchymal stromal cells increases circulating antigen-spe-cific IgG2a but has no effect on IgGE or IgG1. (A): Total IgE fromthe allogeneic model. (B): Ovalbumin (OVA)-specific IgG1 from theallogeneic model. (C): OVA-specific IgG2a from the allogeneicmodel. Data depicts means 6 SE from one of three similar experi-ments with six to eight mice per condition for each experiment. *, p< .05 when compared with OVA. Abbreviations: MSC, mesenchymalstromal cells; OD, optical density; OVA, ovalbumin.


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among stromal cells and fibroblasts [46, 47]. For example,fibroblasts derived from a variety of sources including skin,are capable of inhibiting T lymphocyte proliferation and tu-mor necrosis factor a and IFNc production in response to pol-yclonal stimulation in vitro [47]. To investigate whether fibro-blasts could mimic the inhibitory affects of BMSCs inallergic airways inflammation, we included primary dermalfibroblasts obtained from skin biopsies from C57BL/6 mice asa cell control. While systemic administration of these fibro-blasts during OVA/alum sensitization decreased AHR and lev-els of Th2 cytokines in the BAL fluid, syngeneic fibroblastadministration did not decrease the number of eosinophils inthe airways and conversely increased the number of peribron-chial inflammatory cell infiltrates when compared with OVA-treated mice. While we are uncertain as to why fibroblastadministration resulted in beneficial effects for some of theend points investigated, syngeneic fibroblast administrationdid not result in comparable overall anti-inflammatory actionsand in some cases promoted inflammation and antigen-spe-cific immune responses. These data correspond to findings inendotoxin-induced injury in mouse lungs where intratrachealadministration of 3T3 fibroblasts did not improve survival orseverity of disease as did MSC administration [48].

Precisely, how BMSCs induce a Th1 response in vivo inthis model remains unclear. Notably, induction of a Th1 CD4phenotype was observed in two strains of mice, includingBALB/c mice which are known to exhibit an inherent Th2bias in response to inflammatory injuries [49]. Administrationof either syngeneic or allogeneic BMSCs to sham (i.e., PBS)-

treated mice was well tolerated and did not affect any of themeasured endpoints of airways hyperreactivity, lung inflam-mation, antigen-specific CD4 T lymphocyte differentiation, orlevels of antigen-specific circulating immunoglobulins whencompared with naı̈ve control mice. This is an important obser-vation which highlights a specific immunomodulatory role ofBMSCs only under conditions of immune stimulation or dis-ruption. Further, BMSCs administration to mice with OVA/CFA-mediated Th1-mediated allergic airways inflammationdid not worsen the inflammation suggesting that MSCs do notfurther contribute to a comparable Th1-mediated allergic air-ways injury. These data contrast with data in other experi-mental models of Th1-mediated inflammatory or immuneinjury in which MSC administration decreases injury by pro-moting Th2 CD4 T lymphocyte differentiation and demon-strates that MSC effects are dependent on the model used.This suggests that the specific local inflammatory environ-ments both in vitro and in vivo affect MSC actions as hasbeen recently found in other model systems [50, 51].

It has recently been demonstrated by Nemeth et al. [52] thatsystemic administration of either syngeneic or allogeneicBMSCs in a ragweed induced model of allergic airways diseasealso led to a decrease in airways inflammation as assessed byBAL cytokine levels, circulating antibodies, and histology. Incontrast to our study, where MSCs were administered duringimmunization, MSCs were delivered during challenge, a timewhen antigen-specific CD4 T-cells previously generated duringsensitization are restimulated. In this model, no increase inTh1-specific immunoglobulins or levels of IFNc in BAL fluid

Figure 5. Systemic administration of syngeneic bone marrow-derived mesenchymal stromal cells does not augment Th1-mediated allergic air-ways inflammation. (A): Schematic of experimental studies. (B): Bronchoalveolar lavage fluid cell differentials. Data represent percentage of totalcells counted. (C): Antigen-specific CD4 T lymphocyte interferon-c production 72 hours after ovalbumin (OVA) stimulation. (D): CirculatingOVA-specific IgG2a levels. Data depict means 6 SE of one experiment with four mice per condition *, p < .05 when compared with OVA.Abbreviations: CFA, complete Freund’s adjuvant; IFNc, interfereon-c; MSC, mesenchymal stromal cells; OD, optical density; OVA, ovalbumin.


was observed. Rather release of TGFb from the BMSCs stimu-lated an increase in T regulatory cells in the lung with subse-quent suppression of allergic airways inflammation. Thus,BMSC administration can decrease airways inflammation,when given during either immunization or challenge; however,the mechanism by which they do so differs. This highlights agrowing appreciation of MSCs to ‘‘sense’’ their microenviron-ment and respond in different ways to suppress inflammatoryand immune responses. Further, administration of dermal fibro-blasts to the ragweed-sensitized mice also partly mimicked theBMSC responses reinforcing the growing appreciation that stro-

mal cells such as fibroblasts may share some of the immunosup-pressive effects of BMSC.

Syngeneic adipose-derived MSCs (ASCs) have also beendescribed to inhibit both OVA-induced allergic rhinitis andallergic airways inflammation when administered during orbefore antigen challenge in previously sensitized mice [53,54]. Interestingly, the mechanisms appear similar to our find-ings as the ASCs appear to promote a Th1 environment asmeasured by levels of BAL fluid IFNc levels and by polyclo-nally activated CD4 T lymphocytes recovered from BAL andspleen which stained positively for intracellular IFNc. Most

Figure 6. Administration of syngeneic bone marrow-derived mesenchymal stromal cells does not abrogate allergic airways inflammation ininterferon-cR�/� mice (A) BAL cell differentials. Data represent percentage of total cells counted. (B): BAL fluid cytokine content. (C): BALfluid TGFb levels as measured by enzyme-linked immunosorbent assay. (D): Semiquantitative scoring of histologic peribronchial inflammation.(E): Representative histologic inflammation on day 18. Arrows designate areas of peribronchial inflammatory cell infiltration. Lungs sectionswere stained with hematoxylin and eosin. Original magnification �100. Values represent means 6 SE of one of three similar separate experi-ments with five to eight mice studied for each experimental condition. *, p < .05 when compared with ovalbumin. Abbreviations: BAL, bron-choalveolar lavage; IFN, interfereon; IL, interleukin; MSC, mesenchymal stromal cells; OVA, ovalbumin; TGF, transforming growth factor.


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recently, xenogeneic administration of hBMSCs during chal-lenge in a mouse model of OVA-induced chronic airwayinflammation decreased airways inflammation [55] Althoughthe mechanisms for the hBMSC effects are not clear, hBMSCadministration increased serum IFNc when in mice challengedwith OVA but not saline, indicating that the ability of MSCsto induce IFNc release only occurred in the context of an ongoing inflammatory response.

To further assess the importance of IFNc in the ability ofBMSCs to suppress inflammation in our model, we used IFNcRdeficient mice that are known to have an impaired ability togenerate a Th1 inflammatory response. We found that BMSCsnot only induce a Th1 phenotype in CD4 cells that are capable

of secreting IFNc upon antigen stimulation but also found thatthe ability of the host to respond to IFNc is necessary toachieve the suppressive effects exerted by the BMSCs. We arecurrently working to address the mechanisms by which BMSCsare inducing Th1 phenotype in CD4 cells and which are thecritical cell types that need to be able to respond to IFNc.

CONCLUSION

In conclusion, this study demonstrates that systemic adminis-tration of syngeneic or allogeneic BMSCs during antigen

Figure 7. Bone marrow-derived mesenchymal stromal cells do not promote Th1 phenotype in interferon-c (IFNc)R�/� mice. (A): Interleukin-4and (B) IFNc content in conditioned media from splenic CD4 T lymphocytes. (C): Total IgE. (D): Ovalbumin (OVA)-specific IgG1. (E): OVA-Specific Ig2a measured by enzyme-linked immunosorbent assay from serum. Values represent means 6 SE of one of three similar separateexperiments with five to eight mice studied for each experimental condition. *, p < .05 when compared with OVA. Abbreviations: IFN, interfer-eon; IL, interleukin; MSC, mesenchymal stromal cells; OD, optical density; OVA, ovalbumin.


sensitization suppresses allergic airways inflammation inmice, in part by promotion of Th1 differentiation of antigen-specific CD4 T lymphocytes. Elucidation of the mechanismsby which MSCs induce a Th1 inflammatory response andother mechanisms by which the MSCs may be acting in thismodel is the subject of ongoing investigations. Because of thetiming of administration of the BMSCs during antigen sensiti-zation, our studies were not designed to serve as a model forBMSC treatment as a possible therapy for preestablishedasthma but rather to investigate the effects of BMSCs on CD4T lymphocyte differentiation in a model of Th2-mediatedinflammation. Collectively, our study along with previouslypublished reports, whereby BMSCs were administered eitherbefore or during antigen challenge, provide a better overallunderstanding of how MSCs can suppress the inflammatoryresponse generated in asthma, beginning with the initial de-velopment of antigen-specific T-cells to their suppressiveeffects on effector functions. Understanding the immunologiceffects of MSCs on antigen sensitization and challenge willbe necessary should MSCs be used as a therapy to treat aller-gic asthma. Allergic asthma is an immune-mediated inflam-matory disease of the lung in which Th2 CD4 T lymphocytesare central to the pathogenesis. While most patients can beeffectively managed with a combination of medications, com-monly b-agonists and inhaled corticosteroids, approximately5% of patients have severe refractory disease that is difficultto treat [53–56]. In addition to higher morbidity and mortality,these patients also account for a disproportionate use of healthcare resources with estimates of up to 50% of health care

costs associated with asthma being used by 5% of patientswith severe refractory disease. Currently, available treatmentsfor severe refractory asthma are either poorly effective orhave significant toxicities and new treatment approaches arethus urgently needed [57, 58].

ACKNOWLEDGMENTS

We thank the staff of the University of Vermont Office of Ani-mal Care Management and Dr. Jeffrey Spees and Charla Pooleof the University of Vermont Stem Cell Core for technical assis-tance, Taka Ashikaga of the University of Vermont StatisticsDepartment, Drs. Jason Bates, Lennart Lundblad, and CharlesIrvin of the Vermont Lung Center, and Dr. Alan Howe of theUniversity of Vermont College of Medicine for helpful discus-sions and advice. The work was supported by grants HL087274and HL081289 and a research grant from the Cystic FibrosisFoundation (to D.J.W.), NCRR COBRE P20 RR-155557(Charles Irvin PI), and a T32 HL76122 NIH/NHBLI Multidisci-plinary Training Grant in Lung Biology (Vermont Lung Center,Charles Irvin, PI).

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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