of April 12, 2018.This information is current as

Regulatory T Cells+by Modulating the Development of Foxp3

IL-3 Attenuates Collagen-Induced Arthritis

and Mohan R. WaniMishraBarhanpurkar, Navita Gupta, Satish T. Pote, Gyan C.

Rupesh K. Srivastava, Geetanjali B. Tomar, Amruta P.

http://www.jimmunol.org/content/186/4/2262doi: 10.4049/jimmunol.1002691January 2011;

2011; 186:2262-2272; Prepublished online 17J Immunol 

Referenceshttp://www.jimmunol.org/content/186/4/2262.full#ref-list-1

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The Journal of Immunology

IL-3 Attenuates Collagen-Induced Arthritis by Modulatingthe Development of Foxp3+ Regulatory T Cells

Rupesh K. Srivastava, Geetanjali B. Tomar, Amruta P. Barhanpurkar, Navita Gupta,

Satish T. Pote, Gyan C. Mishra,1 and Mohan R. Wani1

IL-3, a cytokine secreted by Th cells, functions as a link between the immune and the hematopoietic system. We previously dem-

onstrated the potent inhibitory role of IL-3 on osteoclastogenesis, pathological bone resorption, and inflammatory arthritis. In this

study, we investigated the novel role of IL-3 in development of regulatory T (Treg) cells. We found that IL-3 in a dose-dependent

manner increases the percentage of Foxp3+ Treg cells indirectly through secretion of IL-2 by non-Treg cells. These IL-3–expanded

Treg cells are competent in suppressing effector T cell proliferation. Interestingly, IL-3 treatment significantly reduces the severity

of arthritis and restores the loss of Foxp3+ Treg cells in thymus, lymph nodes, and spleen in collagen-induced arthritis mice. Most

significantly, we show that IL-3 decreases the production of proinflammatory cytokines IL-6, IL-17A, TNF-a, and IL-1 and

increases the production of anti-inflammatory cytokines IFN-g and IL-10 in collagen-induced arthritis mice. Thus, to our

knowledge, we provide the first evidence that IL-3 play an important role in modulation of Treg cell development in both in

vitro and in vivo conditions, and we suggest its therapeutic potential in the treatment of rheumatoid arthritis and other auto-

immune diseases. The Journal of Immunology, 2011, 186: 2262–2272.

Regulatory T (Treg) cells play a crucial role in controll-ing autoimmunity, and they prevent the development ofchronic inflammatory and autoimmune diseases by sup-

pressing autoreactive T cells (1). Treg cells generated in the thy-mus are called natural Treg cells. Treg cells could also be gen-erated outside the thymus from peripheral naive CD4+CD252

T cells under both in vitro and in vivo conditions and are referredas adaptive or induced Treg cells (2, 3). The forkhead transcriptionfactor (Foxp3) is the master regulator for the development andfunction of Treg cells (4, 5). The in vitro conversion of naiveCD4+CD252Foxp32 T cells to CD4+CD25+Foxp3+ Treg cells de-pends on the presence of TGF-b1 (2, 6, 7) and of IL-2, which isessential for TGF-b–mediated induction of Foxp3 (8, 9). BothTGF-b1 and IL-2 play an important role in the induction, main-tenance of Foxp3 expression, and function of Treg cells underin vitro and in vivo conditions (10). Treg cells in rheumatoid ar-thritis (RA) patients have a defect in their ability to suppress pro-inflammatory cytokine production by activated T cells and mono-cytes (11, 12). Also, deficiency of Foxp3 results in the paucity ofCD4+CD25+ Treg cells and leads to severe multiorgan autoim-mune diseases in both mice and humans (13, 14). Thus, the de-velopment of functional Treg cells holds promise for the treatmentof RA and other autoimmune diseases.

IL-3, a cytokine secreted by Th cells, stimulates the proliferation,differentiation, and survival of pluripotent hematopoietic stem cells(15). In previous studies, we have shown that IL-3 is a potentinhibitor of osteoclastogenesis and it inhibits both RANKL- andTNF-a–induced mouse osteoclast formation (16, 17). We haverecently shown that IL-3 inhibits RANKL-induced human osteo-clasts derived from blood monocytes and bone marrow cells ofosteoporotic individuals (18). IL-3 also inhibits TNF-a–inducedpathological bone resorption in the presence of other proin-flammatory cytokines such as IL-1a, TGF-b1, TGF-b3, IL-6, andPGE2, and pretreatment with IL-3 prevents the development ofinflammatory arthritis in mice, and protects cartilage and bonedestruction in the joint (19). These results suggest that IL-3 has ananti-inflammatory activity.In RA patients and in a collagen-induced arthritis (CIA) mouse

model, Treg cells are defective and there is increased osteoclasto-genesis (11, 20–22). Decreased circulating Treg cell numbers areassociated with increased osteoclast formation and bone resorptionin humans (23). Also, Treg cells are potent inhibitor of osteoclastdifferentiation and bone resorption, and they prevent developmentof CIA (22, 24). Therefore, we hypothesized that the anti-in-flammatory activity of IL-3 may be through regulation of Tregcell development. In this study, we investigated the role of IL-3 inregulation of Treg cell differentiation in both in vitro and in vivoconditions. We demonstrate that IL-3 increases the percentage ofFoxp3+ Treg cells indirectly through secretion of IL-2 by non-Tregcells. Importantly, IL-3 treatment decreases severity of arthritis andrestores the loss of Foxp3+ Treg cells in lymph nodes, spleen, andthymus in CIA mice. Thus, we reveal the novel role of IL-3 inmodulation of Treg cell development and suggest its therapeuticpotential in treatment of RA and other autoimmune diseases.

Materials and MethodsMice

BALB/c mice 6–8 wk old and DBA/1J mice 8–10 wk old were obtained fromthe Experimental Animal Facility of the National Centre for Cell Science(NCCS, Pune, India). Water and food were provided ad libitum. All pro-

National Centre for Cell Science, University of Pune Campus, Pune 411 007, India

1G.C.M. and M.R.W. share senior authorship.

Received for publication August 9, 2010. Accepted for publication December 14,2010.

This work was supported by the Department of Biotechnology, Government of India.R.K.S. is the recipient of a Senior Research Fellowship from the Council for Scien-tific and Industrial Research, New Delhi, India.

Address correspondence and reprint requests to Dr. Mohan R. Wani, National Centrefor Cell Science, University of Pune Campus, Pune 411 007, India. E-mail address:mohanwani@nccs.res.in

Abbreviations used in this article: CBA, cytometric bead array; CIA, collagen-induced arthritis; CII, collagen type II; m-CT, microcomputed tomography; RA,rheumatoid arthritis; rm, recombinant murine; Treg, regulatory T.

Copyright� 2011 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/11/$16.00

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cedures involving animals were conducted according to the requirements andwith the approval of the Institutional Animal Ethics Committee of NCCS.

Abs and reagents

The following reagents were purchased from BD Biosciences: purified anti-CD3ε (145-2C11,NA/LE), purifiedanti-CD28 (37.51,NA/LE),PerCP-Cy5.5-anti–CD4 (RM4-5), PE-Cy7-anti–CD25 (PC61), biotinylated anti-CD123(5B11), purified anti–IL-2 mAb (S4B6), PE-anti–pSTAT5 (pY694), stre-ptavidin-PE, biotin-rat IgG2a-k, mouse IgG1-k, GolgiStop, and recombinantmurine (rm)IL-3. Allophycocyanin-anti–Foxp3 (FJK-16s), PE-anti–IL-2(JES6-5H4), allophycocyanin-rat IgG2a-k, PE-rat IgG2b-k, Foxp3 stainingbuffer, and RBC lysis buffer were obtained from eBioscience. rmIL-2and recombinant human TGF-b1 were obtained from R&D Systems. CFSEwas obtained from Molecular Probes. PMA and ionomycin were purchasedfrom Sigma-Aldrich. Serum-free medium X-Vivo 15 was obtained fromLonza.

Isolation of T lymphocytes

For isolation of CD4+CD252 and CD4+CD25+ T cells, lymphocytes har-vested from spleens of 6- to 8-wk-old mice were incubated with CD4T cell enrichment mixture (Miltenyi Biotec), and untouched CD4+ T cellswere isolated using autoMACS (Miltenyi Biotec). The purified CD4+ cellswere further incubated with PE-anti–CD25 followed by incubation withanti-PE beads (Miltenyi Biotec) to isolate CD4+CD252 and CD4+CD25+

T cells, according to the manufacturer’s instructions. The purity of sortedCD4+CD252 and CD4+CD25+ T cells was typically .96%.

In vitro Treg cell development

For in vitro Treg cell differentiation, CD4+CD252 T cells (106 cells/well of48-well plate) from splenocytes were stimulated for 4 d with plate-boundanti-CD3ε (10 mg/ml) plus soluble anti-CD28 (2 mg/ml) mAbs in serum-free X-Vivo 15 medium. Where indicated, the medium was supplementedwith recombinant human TGF-b1 (5 ng/ml) and rmIL-2 (20 ng/ml) with orwithout different concentrations of IL-3. Cells were then harvested and thepercentage of Foxp3+ Treg cells was analyzed by FACS.

In vitro suppression assay

The induced and natural Treg cells generated as described above wereharvested and various cell ratios of CD4+CD25+ Treg cells treated with orwithout IL-3 were added to freshly isolated CFSE (2.5 mM)-labeled CD4+

CD252 T cells (2.5 3 105/well) and activated with anti-CD3ε (1 mg/ml)for 72 h in the presence of irradiated (800 rad) syngenic T cell-depletedsplenocytes (5 3 104/well) in 96-well flat-bottom plates. CFSE-labeledCD4+CD252 T cells were also cocultured with unlabeled CD4+CD252

T cells serving as a “crowding control”. Proliferation of effector T cellswas analyzed by FACS for CFSE dilution.

RNA extraction and analysis by RT-PCR

Expression of IL-3Ra and GAPDH was assessed by RT-PCR analysis. Theprimer sequences used were: IL-3Ra forward, 59-GTTTACCCTCGGAAGC-TCAG9-3 and reverse, 59-GTTCCCACACGACGATCTC-39; and GAPDHforward, 59-GGTGCTGAGTATGTCGTGGA-39 and reverse, 59-CCTTCC-ACAATGCCAAAGTT-39. RNA was isolated using TRIzol reagent (Invi-trogen). Total RNA (2 mg) was used for synthesis of cDNAs by reverse tran-scription (cDNA synthesis kit; Invitrogen). The cDNAs were amplified usingPCR for 35 cycles. Each cycle consisted of 30 s denaturation at 94˚C and30 s annealing at 55˚C and 30 s extension at 72˚C. GAPDH was used as aninternal control.

Flow cytometric analysis

T cells in single-cell suspensions isolated from thymus, spleen, and lymphnodes or in vitro-induced Treg cells were stained with PerCP-Cy5.5-anti–CD4 and PE-Cy7-anti–CD25 Abs for 30 min on ice. Cells were washedthoroughly with wash buffer and fixed-permeabilized with Foxp3 per-meabilization buffer for 30 min on ice. Intracellular Foxp3 staining wasperformed using allophycocyanin-conjugated anti-Foxp3 Ab in per-meabilization buffer for 45 min on ice. Cells were washed thoroughly withpermeabilization buffer and analyzed by flow cytometry in a FACSCanto II(BD Biosciences), and the data were analyzed using either BD FACSDivaor CellQuest Pro software (BD Biosciences).

For analysis of IL-3Ra expression on Treg cells, cells were first surfacestained with PerCP-Cy5.5-anti–CD4, PE-Cy7-anti–CD25, biotinylatedanti-CD123 (IL-3Ra), and streptavidin-PE Abs for 30 min on ice. Cellswere washed thoroughly with buffer and further fixed-permeabilized withFoxp3 fixation-permeabilization buffer for 30 min on ice. Intracellular

Foxp3 staining was performed using allophycocyanin-conjugated anti-Foxp3 Ab in Foxp3 permeabilization buffer for 45 min on ice. Cellswere washed thoroughly and analyzed by flow cytometry.

Intracellular staining of IL-2 production was performed by stimulatingcells for 5–6 h with PMA (50 ng/ml) and ionomycin (500 ng/ml). After 1 h,GolgiStop (1 mg/ml) was added to block cytokine secretion. Cells weresurface stained with PerCP-Cy5.5-anti–CD4 and PE-Cy7-anti–CD25 Absfor 30 min on ice. Cells were washed thoroughly with wash buffer, fixed-permeabilized with Foxp3 permeabilization buffer for 30 min on ice, andstained intracellularly with allophycocyanin-conjugated anti-Foxp3 andPE-conjugated anti–IL-2 Abs in permeabilization buffer for 30 min on ice.Cells were washed thoroughly and analyzed by flow cytometry.

For pSTAT5 labeling, T cells were stimulated and fixed with Lyse/Fixbuffer (BD Biosciences) for 30 min at 37˚C, washed and permeabilizedwith Perm Buffer III (BD Biosciences) for 30 min at 4˚C, washed withFACS buffer, and stained with PE-pSTAT5 for 30 min at room tempera-ture. Cells were washed and analyzed by FACS.

Cytometric bead array flex

Levels of IL-1, IL-2, IL-5, IL-6, IL-10, IL-17A, TNF-a, and IFN-g inculture supernatants were assessed by fluorescent bead-based technologyusing cytometric bead array (CBA) Flex sets according to the manu-facturer’s instruction (BD Biosciences). Fluorescent signals were read andanalyzed on a FACSCanto II flow cytometer (BD Biosciences) with thehelp of BD FCAP Array software (BD Biosciences).

Induction and assessment of CIA

Native chicken collage type II (CII; Sigma-Aldrich) was dissolved in 50mMacetic acid at 4˚C overnight and then emulsified with an equal volume ofCFA (Chondrex, containing 4 mg/ml Mycobacterium tuberculosis; strainH37Ra). Male DBA/1J mice 6–8 wk old were injected s.c. at the base of thetail with 100ml emulsion containing 200mg CII. At day 21 after the primaryimmunization, mice were boosted s.c. with 200 mg CII emulsified with anequal volume of IFA (Sigma-Aldrich). Treatment with rmIL-3 began withthe secondary immunization, and cytokine was administered i.p. daily (1.5mg/mouse/d in two divided doses) until day 35 after primary immunization.Mice were observed by two independent blinded examiners every fifth dayand monitored for signs of arthritis onset using two clinical parameters: pawswelling and arthritic score. Paw swelling was assessed by measuringthickness of the affected hind paws with 0–10 mm calipers. Clinical arthritiswas assessed using the following system: grade 0, no swelling; grade 1,slight swelling and erythema; grade 2, pronounced edema; and grade 3, jointrigidity. Each limb was graded, giving a maximum possible score of 12 peranimal. Inflammatory swellings of soft tissues were radiologically evaluatedby taking soft x-ray photographs (Siemens).

Histology

For histological analysis, micewere killed by cervical dislocation on day 36.Whole knee joints were removed and fixed for 4 d in 10% formalin. Afterdecalcification in 5% formic acid, the specimens were processed for paraffinembedding. Next, 5-mm sections were prepared and stained for H&E.

Microcomputed tomography

Microcomputed tomography (m-CT) of the tibiae was performed usinga SkyScan 1176 scanner (SkyScan). Scanning was done at 100 kV, 100 mAusing a 1-mm aluminum filter and exposure set to 590 ms. In total, 1800projections were collected at a resolution of 6.93 mm/pixel. Reconstructionof sections was achieved using a modified Feldkamp cone-beam algorithmwith beam hardening correction set to 50%. CTAnalyzer software (version1.02; SkyScan) was employed for morphometric quantification of trabec-ular bone indices such as bone volume fraction (bone volume/tissue vol-ume), trabecular number, trabecular thickness, structure model index, andconnectivity density.

Statistical analysis

The results were evaluated by using ANOVAwith subsequent comparisonsby Student t test for paired or nonpaired data, as appropriate. Statisticalsignificance was defined as p# 0.05. Values are reported as mean 6 SEM.

ResultsIL-3Ra is expressed by Treg cells

To evaluate the role of IL-3 in Treg cell development, we firstdetermined the expression of IL-3Ra on both natural and induced

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Treg cells. We isolated CD4+CD25+ natural Treg cells fromsplenocytes of 6- to 8-wk-old BALB/c mice, and induced Tregcells were generated from splenic CD4+CD252 T cells by stim-ulating with plate-bound anti-CD3ε (10 mg/ml) and soluble anti-CD28 (2 mg/ml) mAbs in the presence of TGF-b1 (5 ng/ml) andIL-2 (20 ng/ml). Cells were incubated for 4 d and the expressionof IL-3Ra (CD123) was checked at mRNA level by RT-PCR. Weobserved that both natural and induced Treg cells show strongexpression of IL-3Ra (Fig. 1A). Expression of IL-3Ra was alsoanalyzed by FACS on both natural and induced Treg cells bysurface labeling for CD4, CD25, and IL-3Ra, and cells werefurther stained intracellularly for Foxp3. Gating was done onCD4+CD25+ T cells for expression of Foxp3 and IL-3Ra. Whenthe IL-3Ra+ cell population was plotted against Foxp3, we ob-served that almost all the Foxp3+ Treg cells expressed IL-3Ra(Fig. 1B). These results demonstrate that both natural and inducedTreg cells express IL-3Ra at both the gene and protein levels.

IL-3 enhances the percentage of both induced and natural Tregcells in vitro and potently suppresses the proliferation ofeffector T cells

We next examined whether IL-3 promotes conversion of CD4+

CD252 T cells into Foxp3-expressing Treg cells. Splenic CD4+

CD252 T cells stimulated with anti-CD3ε and anti-CD28 mAbswere incubated with TGF-b1 with or without IL-2 and in theabsence or presence of different concentrations of IL-3. After 4 d,cells were analyzed for Foxp3 expression by FACS. We found thatIL-3 in a dose-dependent manner increases the TGF-b1–inducedconversion of CD4+CD252Foxp32 T cells into CD4+CD25+Foxp3+

T cells (Fig. 2A). Fig. 2B represents the average percentage ofCD4+CD25+Foxp3+ cells increased by IL-3. To further checkwhether IL-3 also enhances the percentage of natural Treg cells,splenic CD4+CD25+ T cells were incubated with TGF-b1 with orwithout IL-2 and in the absence or presence of IL-3 (50 and 100ng/ml). After 4 d, cells were analyzed for Foxp3 expression byFACS. We observed that IL-3 also increases the percentage ofFoxp3+ natural Treg cells (Fig. 2C). Fig. 2D represents the averagepercentage of CD4+CD25+Foxp3+ cells in presence of IL-3. Theseresults suggest that IL-3 enhances the expression of Foxp3 in bothnatural and induced Treg cells.To check whether IL-3–expanded induced and natural Treg

cells are functional in suppressing the proliferation of responder/effector T cells, we performed a CFSE-based suppression assay.Freshly isolated, CFSE-labeled conventional CD4+CD252 T cellswere cocultured for 72 h with non-Treg cells (unlabeled CD4+

CD252 serving as crowding control), with induced Treg cellsgenerated in the presence or absence of IL-3 (100 ng/ml), or withnatural Treg cells treated in the presence or absence of IL-3 atdifferent cell ratios. Proliferation of effector T cells was analyzedby FACS for CFSE dilution. We found that IL-3–expanded in-duced and natural Treg cells inhibit the proliferation of responderT cells in a cell density-dependent manner (Fig. 2E). These resultssuggest that IL-3 promoted Treg cells are bona fide Treg cells.

IL-3 enhances percentage of Foxp3+ Treg cells indirectlythrough secretion of IL-2

IL-2 is essential for TGF-b1–induced conversion of naive CD4+

CD252Foxp32 T cells into CD4+CD25+Foxp3+ Treg cells (8,9). To check whether IL-3 enhances TGF-b1–induced Treg celldifferentiation in the absence of exogenous IL-2, we stimulatedCD4+CD252 T cells with anti-CD3ε and anti-CD28 mAbs for4 d in the presence of TGF-b1 with or without different con-centrations of IL-3. We found that IL-3 increased TGF-b1–in-duced Treg cell differentiation even in the absence of exogenousIL-2 (Fig. 3A). Next, to determine whether the stimulatory ef-fect of IL-3 on induced Treg cells is IL-2–dependent, CD4+

CD252 T cells were stimulated with anti-CD3ε and anti-CD28mAbs for 4 d in TGF-b1 with or without IL-3 and in the absenceor presence of anti–IL-2 mAb (10 mg/ml). Anti–IL-2 mAbcompletely neutralized the stimulatory effect of IL-3 on TGF-b1–induced Treg cells (Fig. 3B). Also, IL-2 secretion induced by IL-3was significantly inhibited by anti–IL-2 mAb (Fig. 3C). Theseresults suggest that IL-3 induces development of Treg cells in-directly through secretion of IL-2 by non-Treg cells. This wasfurther confirmed by a dose-dependent effect of IL-3 on secretionof IL-2 by CBA. We observed that IL-3 induces secretion of IL-2in a dose-dependent manner. There was a drastic increase in IL-2secretion at 100 and 300 ng/ml concentrations of IL-3 (Fig. 3D).Also, the enhanced secretion of IL-2 was directly correlated witha simultaneous increase in expression of IL-2Ra (CD25) on thesecells (Fig. 3E).

FIGURE 1. IL-3Ra is expressed by both natural and induced Treg cells.

A, CD4+CD25+ natural Treg cells were isolated from splenocytes of 6- to

8-wk-old BALB/c mice. Induced Treg cells were generated from splenic

CD4+CD252 T cells stimulated with plate-bound anti-CD3ε (10 mg/ml)

and soluble anti-CD28 (2 mg/ml) mAbs and incubated for 4 d in the

presence of TGF-b1 (5 ng/ml) and IL-2 (20 ng/ml). Both cell populations

were analyzed for mRNA expression of IL-3Ra (CD123) by RT-PCR. B,

Expression of IL-3Ra was also analyzed on both natural and induced Treg

cells by FACS by surface labeling for CD4, CD25, and IL-3Ra, and cells

were further stained intracellularly for Foxp3. Gating was done on CD4+

CD25+ T cells for expression of Foxp3 and IL-3Ra. Results in A and B are

representative of three independent experiments. i, induced; n, natural.

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FIGURE 2. IL-3 enhances the percentage of both induced and natural Treg cells in vitro and suppresses the proliferation of effector T cells. A, Splenic

CD4+CD252 T cells stimulated with anti-CD3ε and anti-CD28 mAbs were incubated with TGF-b1 (5 ng/ml) with or without IL-2 (20 ng/ml) in the

absence or presence of different concentrations of IL-3. After 4 d, cells were analyzed for Foxp3 expression by FACS. B, Average percentage of CD4+

CD25+Foxp3+ cells from three independent experiments of A. Values are expressed as mean 6 SEM. *p , 0.05 versus control. C, CD4+CD25+ T cells

isolated from splenocytes were incubated with TGF-b1 (5 ng/ml) with or without IL-2 (20 ng/ml) in the absence or presence of IL-3 (50 and 100 ng/ml).

After 4 d, cells were analyzed for Foxp3 expression by FACS. D, Average percentage of CD4+CD25+Foxp3+ cells from two independent experiments of C.

Values are expressed as mean 6 SEM. *p , 0.05 versus control. E, Freshly isolated CD4+CD252 T cells (2.5 3 105/well) labeled with CFSE were seeded

in 96-well plates and cocultured for 72 h with non-Treg cells (unlabeled CD4+CD252 serving as crowding control), with induced Treg cells generated in the

presence or absence of IL-3 (100 ng/ml), or with natural Treg cells treated in the presence or absence of IL-3 at different cell ratios. Proliferation of effector

T cells was analyzed by FACS for CFSE dilution. Similar results were obtained in three independent experiments. n, natural; i, induced.

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FIGURE 3. IL-3 enhances the percentage of Foxp3+ Treg cells indirectly through secretion of IL-2 by non-Treg cells. A, Splenic CD4+CD252 T cells

stimulated with anti-CD3ε and anti-CD28 mAbs were incubated with TGF-b1 (5 ng/ml) in the absence or presence of different concentrations of IL-3.

After 4 d, cells were analyzed for Foxp3 expression by FACS. B, Splenic CD4+CD252 T cells stimulated with anti-CD3ε and anti-CD28 mAbs were

incubated with TGF-b1 with or without IL-3 (100 ng/ml) or TGF-b1, IL-3, and anti–IL-2 (10 mg/ml) mAb. After 4 d, cells were analyzed for Foxp3

expression by FACS. C, Culture supernatants collected after 4 d from experiments in B were analyzed for IL-2 secretion by CBA as per the manufacturer’s

instructions. Values are expressed as mean 6 SEM. *p , 0.05 versus TGF-b, **p , 0.001 versus TGF-b1 and IL-3. D, Dose-dependent effect of IL-3 on

secretion of IL-2 in Treg cells culture supernatants was analyzed by CBA. Values are expressed as mean 6 SEM. *p , 0.001 versus TGF-b1. E, Dose-

dependent effect of IL-3 on expression of IL-2Ra (CD25) in induced Treg cells. Values are expressed as mean 6 SEM. *p , 0.05 versus TGF-b1. Data in

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The induced Treg cell population contains a significant pro-portion of Foxp32 cells, which are likely to produce IL-2. There-fore, to further check the source of IL-2 secretion we stimulatedsplenic CD4+CD252 T cells with anti-CD3ε and anti-CD28 mAbsfor 4 d in the presence of TGF-b1 with or without different con-centrations of IL-3. After 4 d, cells were activated with PMA(50 ng/ml) and ionomycin (500 ng/ml) for 5–6 h before being ana-lyzed for the secretion of IL-2 by intracellular staining by FACS.Gating was done on two cell populations, CD4+CD25high

and CD4+CD25low, and cells were analyzed for expressionof Foxp3 and IL-2. We found that IL-3 in a dose-dependentmanner increased the percentage of IL-2–producing CD4+

CD25lowFoxp32 cells from 2.3 to 9.1% (Fig. 3F). However, no IL-2> was produced by CD4+CD25highFoxp3+ cells in the absence orpresence of IL-3, even at high concentrations (Fig. 3F). Fig. 3Grepresents the average data of three independent experiments of

the CD4+CD25lowFoxp32 population. Collectively, these resultssuggest that IL-3 induces development of Treg cells indirectlythrough secretion of IL-2 by non-Treg cells.

IL-3 synergies with IL-2 for activation of STAT5

We next investigated the mechanism of stimulatory effect of IL-3on Treg cell development. IL-3R signaling is primarily mediatedthrough activation of JAK2 with subsequent phosphorylation andactivation of STAT5 (15). Also, STAT5 promotes Treg cell dif-ferentiation by regulating expression of Foxp3 (25). Therefore, weexamined the effect of IL-3 on STAT5 phosphorylation in T cellsin the absence and presence of IL-2. We first performed the ti-tration for both cytokines IL-2 and IL-3 independently for theireffects on phosphorylation of STAT5. CD4+CD252 T cells werestimulated for 20 min independently with different concentrationsof IL-2 or IL-3. Cells were stained for intracellular pSTAT5 and

all above experiments are representative of three independent experiments. F, CD4+CD252 T cells were stimulated with anti-CD3ε and anti-CD28 mAbs for

4 d in the presence of TGF-b1 with or without different concentrations of IL-3. After 4 d, cells were activated with PMA (50 ng/ml) and ionomycin (500 ng/

ml) for 5–6 h before being analyzed for the secretion of IL-2 by intracellular staining. Gating was performed for two cell populations, CD4+CD25high and

CD4+CD25low, and cells were analyzed for the expression of Foxp3 and IL-2. G, Average data of three independent experiments of CD4+CD25lowFoxp32

population in experiment F. Values are expressed as mean 6 SEM. *p , 0.05 versus control.

FIGURE 4. IL-3 synergies with IL-2 for activation of STAT5. A, Splenic CD4+CD252 T cells were stimulated for 20 min independently with or without

different concentrations of IL-2 or IL-3. Cells were washed, fixed-permeabilized, and stained with PE-labeled p-STAT5 mAb as per the manufacturer’s

instruction and analyzed by FACS. Data are averages of three independent experiments. Values are expressed as mean6 SEM. *p, 0.05 versus control. B,

Splenic CD4+CD252 T cells were stimulated for 20 min with IL-2 (5 ng/ml) or IL-3 (30 ng/ml) alone or in the presence of both. Cells were stained with

PE-labeled p-STAT5 mAb and analyzed by FACS. C, Average data of three independent experiments of B showing percentage of pSTAT5. Values are

expressed as mean 6 SEM. *p , 0.05 versus control, **p , 0.05 versus IL-2 or IL-3.

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analyzed by FACS. Both IL-2 and IL-3 independently increasedthe phosphorylation of STAT5 in a dose-dependent manner (Fig.4A). The data in Fig. 4A show the average percentage of STAT5phosphorylation from three independent experiments (p , 0.05).To further check the synergistic effect of IL-2 and IL-3 on

phosphorylation of STAT5, we stimulated CD4+CD252 T cells for20 min with low concentrations of IL-2 (5 ng/ml) or IL-3 (30 ng/ml) alone or in the presence of both. We found that there was15.13 and 15.64% phosphorylation of STAT5 by IL-2 and IL-3,respectively. Phosphorylation of STAT5 was increased to 34.67%when both of the cytokines were added together, which was more

than that of the additive effect of both cytokines (Fig. 4B). Fig. 4Cshows the average percentage of STAT5 phosphorylation fromthree independent experiments (p , 0.05). Thus, IL-3 activatesSTAT5 in T cells and synergizes with IL-2 for enhanced activationof STAT5.

IL-3 reduces severity of arthritis by enhancing the percentageof Foxp3+ Treg cells in vivo in CIA mice

To investigate the in vivo role of IL-3 in regulation of Treg cells, weused a well-established CIA mouse model of human RA. DBA/1Jmice were primed on day 0 with CII and then boosted on day 21. Inthe treatment group, mice were injected for 15 d with rmIL-3 (1.5mg/d i.p. in two divided doses at 12-h intervals) from day 21 at thetime of booster. Arthritis was manifested by redness and swellingof the paws, including digits. In PBS-injected control mice, nosigns of inflammation were seen, whereas CIA mice developedsevere inflammation as evidenced by marked swelling and ery-thema of the hind paws. In contrast, mice treated with IL-3 dis-played a significant reduction in paw thickness (Fig. 5A) and meanarthritic score (Fig. 5B). Photographs of hind paws in Fig. 5Cshow significant reduction of inflammation in mice treated withIL-3. In the presence of IL-3, thickness of the inflammatory softtissues was also decreased when examined by radiological soft x-rays (Fig. 5D). RA is a chronic inflammatory disorder that ulti-mately leads to the destruction of joint architecture. By radio-logical examination we observed that IL-3 treatment preventeddamage to articular cartilage (Fig. 5D, enlarged regions). CIAmice had markedly enlarged spleen and inguinal lymph nodes,which were normal in size in IL-3–treated mice (Fig. 5E). Byhistological examinations of knee joints on day 36, we observedno infiltration of inflammatory cells and no damage to the articularcartilage of control mice. The knee joints in CIA mice showedmassive infiltration of polymorphonuclear and other inflammatorycells, and there was multiple superficial cartilage erosion. In con-trast, mice treated with IL-3 showed infiltration of few inflam-matory cells, and erosion of articular cartilage was not observed(Fig. 5F).We also assessed trabecular structure of tibiae by m-CT. In CIA

mice there was significant loss of trabecular and cortical bones,which was prevented in IL-3–treated mice (Fig. 6A). Also, a sig-nificant increase in trabecular thickness, trabecular number, bonevolume fraction (bone volume/tissue volume), connectivity den-sity, and cortical thickness was observed in IL-3–treated mice withrespect to CIA mice (Fig. 6B). Bone architecture denoted by thestructure model index was not altered. These results suggest thatIL-3 maintains normal bone structure in mice. Thus, IL-3 treat-ment reduces arthritic score and inflammation and prevents dam-age to bone and cartilage tissues in knee joints in CIA mice.To investigate the in vivomechanism of IL-3 action in prevention

of CIA, we analyzed the effect of IL-3 on Foxp3+ Treg cell de-velopment. Mice were sacrificed on day 36 and total lymphocytepopulations derived from lymph nodes, spleen, and thymus tissueswere analyzed for percentage of CD4+Foxp3+ Treg cells by FACS.As compared with control mice, the percentage of Foxp3+ Tregcells in CIA mice was drastically decreased in thymus, lymphnodes, and spleen. There was a .50% decrease in percentage ofFoxp3+ Treg cells in lymph nodes and an ∼70% decrease in spleenand thymus. Interestingly, in IL-3–treated mice the percentage ofCD4+Foxp3+ Treg cells in all the three tissues was restored to nearnormal levels (Fig. 7A). Fig. 7B represents the average percentageof Foxp3+ Treg cells in lymph nodes, spleen, and thymus of micetreated with or without IL-3. These results suggest that IL-3 alsohas a potential to augment the percentage of Foxp3+ Treg cellsin vivo.

FIGURE 5. IL-3 suppresses inflammation and protects joint destruction

in CIA mice. CIA was induced in 8- to 10-wk-old DBA/1J mice by CII

emulsion as described in Materials and Methods. Control mice were

injected s.c. with PBS on day 0 and on day 21. In another group mice were

injected s.c. with CII prepared in CFA (200 mg/mice) on day 0 followed by

s.c. injection on day 21 with a booster dose of CII prepared in IFA (200

mg/mice). In the third group mice were treated with IL-3 i.p. (1.5 mg/

mouse/d in two divided doses) for 15 d starting from day 21 along with

booster dose of CII. Two independent blinded observers assessed the se-

verity of arthritis every fifth day after booster injection for hind paw

thickness (A) and clinical arthritic score (B). Data are represented as mean6SEM (n = 5 mice/group). C, Photographs of paw swelling in DBA/1J mice

showing front and side views of hind paws, and x-rays of soft tissues

showing inflammation of surrounding tissue. D, X-ray radiographs of knee

joints showing bone and cartilage structure. E, Appearance of spleen and

lymph nodes from control, CIA, and IL-3 treated mice. F, Mice were

sacrificed on day 36, and knee joints were dissected, fixed, decalcified, and

processed for histopathology. Five-micrometer sections were stained for

H&E. Original magnification 310. Similar results were obtained in two

independent experiments.

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IL-3 decreases production of proinflammatory cytokines andincreases anti-inflammatory cytokines in CIA mice

The pathogenic events that lead to the development of human RAare not fully understood, although the pivotal role of proin-flammatory cytokines such as TNF-a, IL-1b, and IL-6 in the in-duction and maintenance of RA is well documented (26). Thesecytokines promote the deleterious imbalance in bone metabolismand contribute to enhanced bone destruction. Treg cells in activeRA are defective in controlling production of proinflammatorycytokines (11). Treg cells suppress immune response through nu-merous mechanisms, including the production of anti-inflam-matory cytokines, direct cell to cell contact, and by modulatingthe activation state and function of APCs (27). Therefore, we fur-ther examined the effect of IL-3 on the levels of various proin-flammatory and anti-inflammatory cytokines in the serum of con-trol, CIA, and IL-3–treated mice. In CIA mice there was a sig-nificant increase in production of IL-6, IL-17A, TNF-a, IL-1, andIFN-g (p , 0.05) and decrease in secretion of IL-5 and IL-10.IL-2 production was also decreased in CIA mice. Surprisingly, we

observed that IL-3 significantly increases the levels of IL-10, IL-2,IL-5, and IFN-g and decreases the levels of IL-6, IL-17A, TNF-a,and IL-1 (Fig. 8). These results suggest that IL-3 has a potential toinhibit production of proinflammatory cytokines and induce anti-inflammatory cytokines.

DiscussionTreg cells represent an important immune mechanism for induc-ing tolerance to both self and foreign Ags to prevent autoimmunityand unwanted inflammation. Dysfunctional Treg cells are a causeof various autoimmune diseases, allergy, and immunopathology(28). However, it remains to be determined how these cells areinvolved in regulating the pathophysiology of common immuno-logical disease such as RA. Also, Treg cells potently inhibitosteoclastogenesis and bone resorption. Importantly, the capacityof a single Treg cell to block differentiation of 100 monocytes intoosteoclasts underlines the powerful role of Treg cells in regulationof bone resorption (24). Thus, Treg cells appear to be an attractivetarget cell with substantial therapeutic potential in RA, and they

FIGURE 6. IL-3 prevents bone loss in CIA mice.

Mice were sacrificed on day 36 and tibias were

analyzed for bone parameters. A, m-CT recon-

structions showing trabecular and cortical bone

structures of representative tibias in control, CIA,

and IL-3–treated CIA mice. B, Morphological

measurements of trabecular and cortical bone in-

dices such as bone volume fraction (bone volume/

tissue volume, or BV/TV), trabecular number (Tb.

N), trabecular thickness (Tb. Th), structure model

index (SMI), connective density (Conn. D.), and

cortical thickness (Ct. Th.) were computed from

m-CT reconstructions of control, CIA, and IL-3–

treated CIA mice tibias. Similar results were

obtained in two independent experiments.

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may act as an important link between the immune and bonesystem. However, therapeutic applications of Treg cells are limitedbecause of their scarcity. Therefore, development of Treg cellsin vitro and in vivo is an essential requirement for their effectiveclinical intervention. In this study, we investigated the role of IL-3

in regulation of Treg cell development in both in vitro and in vivoconditions.IL-3 exerts its biological activities by binding to specific high-

affinity receptors expressed by hematopoietic stem cells, endo-thelial cells, and monocytes (29). Previous reports have demon-

FIGURE 7. IL-3 prevents CIA by enhancing the

percentage of Foxp3+ Treg cells in vivo. A, Mice

were sacrificed on day 36 and total cells derived

from lymph nodes, spleen, and thymus tissues were

analyzed for percentage of CD4+Foxp3+ Treg cells

by FACS. Isotype controls of Foxp3 for each group

are shown. B, Average percentage of CD4+Foxp3+

cells from lymph nodes, spleen, and thymus tissues

of three mice. Values are expressed as mean 6SEM. p , 0.05 in all the groups.

FIGURE 8. Effects of IL-3 on production of various cytokines in CIA mice. Serum samples of mice were analyzed on day 36 for secretion of various

proinflammatory and anti-inflammatory cytokines by CBA. Data are represented as mean 6 SEM (n = 5). Similar results were obtained in two independent

experiments.

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strated that IL-3–sensitive T cell clones activated by CD3 expressIL-3Ra, whereas T cell clones that were insensitive to the growth-enhancing effect of IL-3 were negative for IL-3Ra expression(30, 31). These differences in reactivity to IL-3 among differentT cell clones might be due to differences in the methods used forestablishing such T cell clones (31). To our knowledge, we re-port in this study for the first time that both natural and inducedTreg cells show IL-3Ra expression at both the gene and proteinlevels. Sustained expression of Foxp3, a key distinguishing featureof Treg cells, is required for the differentiation, suppressor func-tion, maintenance of cell phenotype, and metabolic fitness of Tregcells (32). Mice and humans harboring a loss-of-function mutationin the Foxp3 gene are affected by fatal early onset lymphoproli-ferative immune-mediated disease affecting a variety of organsand tissues (33). Interestingly, in our system IL-3 significantlyincreases the percentage of Foxp3+ natural and induced Tregcells, and these cells suppress the proliferation of effector T cells.Thus, IL-3–treated Treg cells are endowed with the classicalsuppressive function ascribed to naturally occurring Treg cells. Wedemonstrate that IL-3 promotes development of Treg cells in-directly through secretion of IL-2 by non-Treg cells. IL-2 is anindispensable cytokine for the peripheral maintenance of naturalTreg cells. However, natural Treg cells do not produce this cyto-kine, and the exact source of IL-2 has been a matter of intenseresearch in the last decade (9, 34). Our results are consistent withearlier findings by other groups that the main source of IL-2 isCD4+CD25low non-Treg cells and not CD4+CD25high Treg cells(35). IL-3 signaling involves activation of STAT5 in hematopoieticstem cells (15). Also, STAT5 activation by IL-2 is critical for Tregcell development and Foxp3 induction (25). Deletion of STAT5 inCD4+ T cells results in marked reduction of Foxp3+ Treg cells inboth thymus and periphery, whereas enhanced STAT5 activationpromotes Treg cell differentiation (25, 36). We report in this studythat IL-3 synergizes with IL-2 for enhanced activation of STAT5in T cells.Deficiency of Treg cells leads to breakdown of tolerance in

various human autoimmune diseases including type 1 diabetes andRA (11). Also, depletion of Treg cells exacerbates various ex-perimental autoimmune diseases, including CIA (37). Foxp3+

Treg cells constitute 5–15% of peripheral CD4+ T cells (38, 39),and this proportion appears to be reduced in mice geneticallyprone to autoimmune diseases such as diabetes and CIA (37, 40).In our studies, we found that therapeutic treatment of CIA micewith IL-3 led to a significant rise in the percentage of both naturaland peripheral Foxp3+ Treg cells in thymus, spleen, and lymphnodes. An increased percentage of Treg cells improved clinicalsigns of arthritis and suppressed local and systemic bone de-struction. Also, we observed that IL-3–treated Treg cells are morepotent in suppressing osteoclast formation in vitro when comparedwith Treg cells generated with TGF-b and IL-2 (R. Srivastava,unpublished observations). Thus, the increased percentage ofFoxp3+ Treg cells in CIA mice by IL-3 may lead to the inhibitionof osteoclastogenesis and bone loss, thereby attenuating CIA.Similar to human RA, various proinflammatory cytokines, in-

cluding TNF-a and IL-1b, are involved in CIA, a rodent model ofhuman RA. IL-3 significantly suppresses the production of mostproinflammatory cytokines (namely, IL-6, IL-17A, TNF-a, andIL-1) and induces the production of various anti-inflammatorycytokines (namely, IL-10, IL-2, IL-5, and IFN-g) in vivo. Be-cause inflammation and bone loss are two frequently occurringand tightly linked disorders, we next determined the effect of IL-3on bone loss with the help of m-CT analysis. Interestingly, m-CTmeasurements of tibias revealed that IL-3 prevents bone loss inCIA mice. Thus, our study clearly demonstrates that IL-3 en-

hances the percentage of Foxp3+ Treg cells in vitro indirectlythrough secretion of IL-2, and it also restores the loss of Foxp3+

Treg cells in vivo. Also, IL-3–generated Treg cells have the po-tential to cure CIA in mice. Furthermore, our data suggest thatIL-3 may represent a novel therapeutic strategy to reduce immuneresponses, inflammation, and bone loss in autoimmune diseases.Cellular therapy based on ex vivo development of Treg cells

and their subsequent transfer to patients is currently the focus of in-tense research in the treatment of various autoimmune diseases(28). Also, transfer of Treg cells has already been shown to pre-vent a wide range of experimental autoimmune diseases, includ-ing diabetes, experimental encephalomyelitis, and colitis (40–42).Several, preclinical studies have shown that either freshly isolatedor ex vivo expanded Treg cells can prevent both local and sys-temic organ and tissue destruction (43). Two broad therapeuticapproaches have been considered: first, to expand Treg cells invitro with the intension of infusing these cells into patients; andsecond, to manipulate the immune system in vivo resulting in anincrease in Treg cells. Importantly, IL-3 appears to increase thepercentage of Treg cells in both in vitro and in vivo conditions.Moreover, inhibition of most proinflammatory cytokines by IL-3may results in an increase in percentage of Treg cells. Thus, to ourknowledge, we reveal for the first time that IL-3 may be useful toexpand Treg cells under both in vitro and in vivo conditions, and itmay act as an important therapeutic agent to treat RA and otherautoimmune diseases.

AcknowledgmentsWe thank Hemangini Shikhare and Swapnil for FACS analysis.

DisclosuresThe authors have no financial conflicts of interest.

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