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IL-3 Attenuates Collagen-Induced by Modulating the Development of Foxp3 + Regulatory T Cells

This information is current as Rupesh K. Srivastava, Geetanjali B. Tomar, Amruta P. of September 28, 2021. Barhanpurkar, Navita Gupta, Satish T. Pote, Gyan C. Mishra and Mohan R. Wani J Immunol 2011; 186:2262-2272; Prepublished online 17 January 2011;

doi: 10.4049/jimmunol.1002691 Downloaded from http://www.jimmunol.org/content/186/4/2262

References This article cites 41 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/186/4/2262.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-3 Attenuates Collagen-Induced Arthritis by Modulating the 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 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 proliferation. Interestingly, IL-3 treatment significantly reduces the severity of arthritis and restores the loss of Foxp3+ Treg cells in , lymph nodes, and spleen in collagen-induced arthritis mice. Most significantly, we show that IL-3 decreases the production of proinflammatory IL-6, IL-17A, TNF-a, and IL-1 and Downloaded from 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 and other auto- immune diseases. The Journal of Immunology, 2011, 186: 2262–2272.

egulatory T (Treg) cells play a crucial role in controll- IL-3, a cytokine secreted by Th cells, stimulates the proliferation, http://www.jimmunol.org/ ing , and they prevent the development of differentiation, and survival of pluripotent hematopoietic stem cells R chronic inflammatory and autoimmune diseases by sup- (15). In previous studies, we have shown that IL-3 is a potent pressing autoreactive T cells (1). Treg cells generated in the thy- inhibitor of osteoclastogenesis and it inhibits both RANKL- and mus are called natural Treg cells. Treg cells could also be gen- TNF-a–induced mouse osteoclast formation (16, 17). We have erated outside the thymus from peripheral naive CD4+CD252 recently shown that IL-3 inhibits RANKL-induced osteo- T cells under both in vitro and in vivo conditions and are referred clasts derived from and cells of as adaptive or induced Treg cells (2, 3). The forkhead transcription osteoporotic individuals (18). IL-3 also inhibits TNF-a–induced

factor (Foxp3) is the master regulator for the development and pathological bone resorption in the presence of other proin- by guest on September 28, 2021 function of Treg cells (4, 5). The in vitro conversion of naive flammatory cytokines such as IL-1a, TGF-b1, TGF-b3, IL-6, and + 2 2 + + + CD4 CD25 Foxp3 T cells to CD4 CD25 Foxp3 Treg cells de- PGE2, and pretreatment with IL-3 prevents the development of pends on the presence of TGF-b1 (2, 6, 7) and of IL-2, which is inflammatory arthritis in mice, and protects cartilage and bone essential for TGF-b–mediated induction of Foxp3 (8, 9). Both destruction in the joint (19). These results suggest that IL-3 has an TGF-b1 and IL-2 play an important role in the induction, main- anti-inflammatory activity. tenance of Foxp3 expression, and function of Treg cells under In RA patients and in a collagen-induced arthritis (CIA) mouse in vitro and in vivo conditions (10). Treg cells in rheumatoid ar- model, Treg cells are defective and there is increased osteoclasto- thritis (RA) patients have a defect in their ability to suppress pro- genesis (11, 20–22). Decreased circulating Treg cell numbers are inflammatory cytokine production by activated T cells and mono- associated with increased osteoclast formation and bone resorption cytes (11, 12). Also, deficiency of Foxp3 results in the paucity of in (23). Also, Treg cells are potent inhibitor of osteoclast CD4+CD25+ Treg cells and leads to severe multiorgan autoim- differentiation and bone resorption, and they prevent development mune diseases in both mice and humans (13, 14). Thus, the de- of CIA (22, 24). Therefore, we hypothesized that the anti-in- velopment of functional Treg cells holds promise for the treatment flammatory activity of IL-3 may be through regulation of Treg of RA and other autoimmune diseases. cell development. In this study, we investigated the role of IL-3 in regulation of Treg cell differentiation in both in vitro and in vivo conditions. We demonstrate that IL-3 increases the percentage of National Centre for Cell Science, University of Pune Campus, Pune 411 007, India + 1 Foxp3 Treg cells indirectly through secretion of IL-2 by non-Treg G.C.M. and M.R.W. share senior authorship. cells. Importantly, IL-3 treatment decreases severity of arthritis and Received for publication August 9, 2010. Accepted for publication December 14, restores the loss of Foxp3+ Treg cells in lymph nodes, spleen, and 2010. thymus in CIA mice. Thus, we reveal the novel role of IL-3 in 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- modulation of Treg cell development and suggest its therapeutic tific and Industrial Research, New Delhi, India. potential in treatment of RA and other autoimmune diseases. Address correspondence and reprint requests to Dr. Mohan R. Wani, National Centre for Cell Science, University of Pune Campus, Pune 411 007, India. E-mail address: [email protected] Materials and Methods Abbreviations used in this article: CBA, cytometric bead array; CIA, collagen- Mice induced arthritis; CII, collagen type II; m-CT, microcomputed tomography; RA, rheumatoid arthritis; rm, recombinant murine; Treg, regulatory T. BALB/c mice 6–8 wk old and DBA/1J mice 8–10 wk old were obtained from the Experimental Animal Facility of the National Centre for Cell Science Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 (NCCS, Pune, India). Water and food were provided ad libitum. All pro- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002691 The Journal of Immunology 2263 cedures involving animals were conducted according to the requirements and Foxp3 staining was performed using allophycocyanin-conjugated anti- with the approval of the Institutional Animal Ethics Committee of NCCS. Foxp3 Ab in Foxp3 permeabilization buffer for 45 min on ice. Cells were washed thoroughly and analyzed by flow cytometry. Abs and reagents Intracellular staining of IL-2 production was performed by stimulating cells for 5–6 h with PMA (50 ng/ml) and ionomycin (500 ng/ml). After 1 h, The following reagents were purchased from BD Biosciences: purified anti- GolgiStop (1 mg/ml) was added to block cytokine secretion. Cells were CD3ε (145-2C11, NA/LE), purified anti-CD28 (37.51, NA/LE), PerCP-Cy5.5- surface stained with PerCP-Cy5.5-anti–CD4 and PE-Cy7-anti–CD25 Abs anti–CD4 (RM4-5), PE-Cy7-anti–CD25 (PC61), biotinylated anti-CD123 for 30 min on ice. Cells were washed thoroughly with wash buffer, fixed- (5B11), purified anti–IL-2 mAb (S4B6), PE-anti–pSTAT5 (pY694), stre- permeabilized with Foxp3 permeabilization buffer for 30 min on ice, and ptavidin-PE, biotin-rat IgG2a-k, mouse IgG1-k, GolgiStop, and recombinant stained intracellularly with allophycocyanin-conjugated anti-Foxp3 and murine (rm)IL-3. Allophycocyanin-anti–Foxp3 (FJK-16s), PE-anti–IL-2 PE-conjugated anti–IL-2 Abs in permeabilization buffer for 30 min on ice. (JES6-5H4), allophycocyanin-rat IgG2a-k, PE-rat IgG2b-k, Foxp3 staining Cells were washed thoroughly and analyzed by flow cytometry. buffer, and RBC lysis buffer were obtained from eBioscience. rmIL-2 For pSTAT5 labeling, T cells were stimulated and fixed with Lyse/Fix and recombinant human TGF-b1 were obtained from R&D Systems. CFSE buffer (BD Biosciences) for 30 min at 37˚C, washed and permeabilized was obtained from Molecular Probes. PMA and ionomycin were purchased with Perm Buffer III (BD Biosciences) for 30 min at 4˚C, washed with from Sigma-Aldrich. Serum-free medium X-Vivo 15 was obtained from FACS buffer, and stained with PE-pSTAT5 for 30 min at room tempera- Lonza. ture. Cells were washed and analyzed by FACS. Isolation of T Cytometric bead array flex 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 CD4 Levels of IL-1, IL-2, IL-5, IL-6, IL-10, IL-17A, TNF-a, and IFN-g in T cell enrichment mixture (Miltenyi Biotec), and untouched CD4+ T cells culture supernatants were assessed by fluorescent bead-based technology + using cytometric bead array (CBA) Flex sets according to the manu- were isolated using autoMACS (Miltenyi Biotec). The purified CD4 cells Downloaded from were further incubated with PE-anti–CD25 followed by incubation with facturer’s instruction (BD Biosciences). Fluorescent signals were read and anti-PE beads (Miltenyi Biotec) to isolate CD4+CD252 and CD4+CD25+ analyzed on a FACSCanto II flow cytometer (BD Biosciences) with the T cells, according to the manufacturer’s instructions. The purity of sorted help of BD FCAP Array software (BD Biosciences). + 2 + + . CD4 CD25 and CD4 CD25 T cells was typically 96%. Induction and assessment of CIA In vitro Treg cell development Native chicken collage type II (CII; Sigma-Aldrich) was dissolved in 50 mM + 2 6 acetic acid at 4˚C overnight and then emulsified with an equal volume of For in vitro Treg cell differentiation, CD4 CD25 T cells (10 cells/well of http://www.jimmunol.org/ 48-well plate) from splenocytes were stimulated for 4 d with plate-bound CFA (Chondrex, containing 4 mg/ml Mycobacterium ; strain anti-CD3ε (10 mg/ml) plus soluble anti-CD28 (2 mg/ml) mAbs in serum- H37Ra). Male DBA/1J mice 6–8 wk old were injected s.c. at the base of the free X-Vivo 15 medium. Where indicated, the medium was supplemented tail with 100 ml emulsion containing 200 mg CII. At day 21 after the primary with recombinant human TGF-b1 (5 ng/ml) and rmIL-2 (20 ng/ml) with or immunization, mice were boosted s.c. with 200 mg CII emulsified with an without different concentrations of IL-3. Cells were then harvested and the equal volume of IFA (Sigma-Aldrich). Treatment with rmIL-3 began with percentage of Foxp3+ Treg cells was analyzed by FACS. the secondary immunization, and cytokine was administered i.p. daily (1.5 mg/mouse/d in two divided doses) until day 35 after primary immunization. In vitro suppression assay Mice were observed by two independent blinded examiners every fifth day and monitored for signs of arthritis onset using two clinical parameters: paw The induced and natural Treg cells generated as described above were swelling and arthritic score. Paw swelling was assessed by measuring + + harvested and various cell ratios of CD4 CD25 Treg cells treated with or thickness of the affected hind paws with 0–10 mm calipers. Clinical arthritis by guest on September 28, 2021 without IL-3 were added to freshly isolated CFSE (2.5 mM)-labeled CD4+ was assessed using the following system: grade 0, no swelling; grade 1, 2 CD25 T cells (2.5 3 105/well) and activated with anti-CD3ε (1 mg/ml) slight swelling and erythema; grade 2, pronounced edema; and grade 3, joint for 72 h in the presence of irradiated (800 rad) syngenic T cell-depleted rigidity. Each limb was graded, giving a maximum possible score of 12 per splenocytes (5 3 104/well) in 96-well flat-bottom plates. CFSE-labeled animal. Inflammatory swellings of soft tissues were radiologically evaluated 2 2 CD4+CD25 T cells were also cocultured with unlabeled CD4+CD25 by taking soft x-ray photographs (Siemens). T cells serving as a “crowding control”. Proliferation of effector T cells was analyzed by FACS for CFSE dilution. Histology RNA extraction and analysis by RT-PCR For histological analysis, mice were killed by cervical dislocation on day 36. Whole knee joints were removed and fixed for 4 d in 10% formalin. After Expression of IL-3Ra and GAPDH was assessed by RT-PCR analysis. The decalcification in 5% formic acid, the specimens were processed for paraffin primer sequences used were: IL-3Ra forward, 59-GTTTACCCTCGGAAGC- embedding. Next, 5-mm sections were prepared and stained for H&E. TCAG9-3 and reverse, 59-GTTCCCACACGACGATCTC-39; and GAPDH forward, 59-GGTGCTGAGTATGTCGTGGA-39 and reverse, 59-CCTTCC- Microcomputed tomography ACAATGCCAAAGTT-39. RNA was isolated using TRIzol reagent (Invi- trogen). Total RNA (2 mg) was used for synthesis of cDNAs by reverse tran- Microcomputed tomography (m-CT) of the tibiae was performed using scription (cDNA synthesis kit; Invitrogen). The cDNAs were amplified using a SkyScan 1176 scanner (SkyScan). Scanning was done at 100 kV, 100 mA PCR for 35 cycles. Each cycle consisted of 30 s denaturation at 94˚C and using a 1-mm aluminum filter and exposure set to 590 ms. In total, 1800 30 s annealing at 55˚C and 30 s extension at 72˚C. GAPDH was used as an projections were collected at a resolution of 6.93 mm/pixel. Reconstruction internal control. of sections was achieved using a modified Feldkamp cone-beam algorithm with beam hardening correction set to 50%. CTAnalyzer software (version Flow cytometric analysis 1.02; SkyScan) was employed for morphometric quantification of trabec- ular bone indices such as bone volume fraction (bone volume/tissue vol- T cells in single-cell suspensions isolated from thymus, spleen, and lymph ume), trabecular number, trabecular thickness, structure model index, and nodes or in vitro-induced Treg cells were stained with PerCP-Cy5.5-anti– connectivity density. CD4 and PE-Cy7-anti–CD25 Abs for 30 min on ice. Cells were washed thoroughly with wash buffer and fixed-permeabilized with Foxp3 per- Statistical analysis meabilization buffer for 30 min on ice. Intracellular Foxp3 staining was performed using allophycocyanin-conjugated anti-Foxp3 Ab in per- The results were evaluated by using ANOVAwith subsequent comparisons meabilization buffer for 45 min on ice. Cells were washed thoroughly with by Student t test for paired or nonpaired data, as appropriate. Statistical 6 permeabilization buffer and analyzed by flow cytometry in a FACSCanto II significance was defined as p # 0.05. Values are reported as mean SEM. (BD Biosciences), and the data were analyzed using either BD FACSDiva or CellQuest Pro software (BD Biosciences). For analysis of IL-3Ra expression on Treg cells, cells were first surface Results stained with PerCP-Cy5.5-anti–CD4, PE-Cy7-anti–CD25, biotinylated IL-3Ra is expressed by Treg cells anti-CD123 (IL-3Ra), and streptavidin-PE Abs for 30 min on ice. Cells were washed thoroughly with buffer and further fixed-permeabilized with To evaluate the role of IL-3 in Treg cell development, we first Foxp3 fixation-permeabilization buffer for 30 min on ice. Intracellular determined the expression of IL-3Ra on both natural and induced 2264 IL-3 ATTENUATES COLLAGEN-INDUCED ARTHRITIS

Treg cells. We isolated CD4+CD25+ natural Treg cells from IL-3 enhances the percentage of both induced and natural Treg splenocytes of 6- to 8-wk-old BALB/c mice, and induced Treg cells in vitro and potently suppresses the proliferation of cells were generated from splenic CD4+CD252 T cells by stim- effector T cells ε ulating with plate-bound anti-CD3 (10 mg/ml) and soluble anti- We next examined whether IL-3 promotes conversion of CD4+ CD28 (2 mg/ml) mAbs in the presence of TGF-b1 (5 ng/ml) and CD252 T cells into Foxp3-expressing Treg cells. Splenic CD4+ IL-2 (20 ng/ml). Cells were incubated for 4 d and the expression CD252 T cells stimulated with anti-CD3ε and anti-CD28 mAbs of IL-3Ra (CD123) was checked at mRNA level by RT-PCR. We were incubated with TGF-b1 with or without IL-2 and in the observed that both natural and induced Treg cells show strong absence or presence of different concentrations of IL-3. After 4 d, expression of IL-3Ra (Fig. 1A). Expression of IL-3Ra was also cells were analyzed for Foxp3 expression by FACS. We found that analyzed by FACS on both natural and induced Treg cells by IL-3 in a dose-dependent manner increases the TGF-b1–induced surface labeling for CD4, CD25, and IL-3Ra, and cells were conversion of CD4+CD252Foxp32 T cells into CD4+CD25+Foxp3+ further stained intracellularly for Foxp3. Gating was done on + + T cells (Fig. 2A). Fig. 2B represents the average percentage of CD4 CD25 T cells for expression of Foxp3 and IL-3Ra. When CD4+CD25+Foxp3+ cells increased by IL-3. To further check the IL-3Ra+ cell population was plotted against Foxp3, we ob- + whether IL-3 also enhances the percentage of natural Treg cells, served that almost all the Foxp3 Treg cells expressed IL-3Ra splenic CD4+CD25+ T cells were incubated with TGF-b1 with or (Fig. 1B). These results demonstrate that both natural and induced without IL-2 and in the absence or presence of IL-3 (50 and 100 Treg cells express IL-3Ra at both the and levels. ng/ml). After 4 d, cells were analyzed for Foxp3 expression by FACS. We observed that IL-3 also increases the percentage of Foxp3+ natural Treg cells (Fig. 2C). Fig. 2D represents the average Downloaded from percentage of CD4+CD25+Foxp3+ cells in presence of IL-3. These results suggest that IL-3 enhances the expression of Foxp3 in both natural 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. http://www.jimmunol.org/ Freshly isolated, CFSE-labeled conventional CD4+CD252 T cells were 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. We found that IL-3–expanded in- duced and natural Treg cells inhibit the proliferation of responder by guest on September 28, 2021 T cells in a cell density-dependent manner (Fig. 2E). These results suggest that IL-3 promoted Treg cells are bona fide Treg cells.

IL-3 enhances percentage of Foxp3+ Treg cells indirectly through 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 cell differentiation in the absence of exogenous IL-2, we stimulated CD4+CD252 T cells with anti-CD3ε and anti-CD28 mAbs for 4 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 exogenous IL-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-CD28 mAbs for 4 d in TGF-b1 with or without IL-3 and in the absence or presence of anti–IL-2 mAb (10 mg/ml). Anti–IL-2 mAb completely neutralized the stimulatory effect of IL-3 on TGF-b1– FIGURE 1. IL-3Ra is expressed by both natural and induced Treg cells. induced Treg cells (Fig. 3B). Also, IL-2 secretion induced by IL-3 A, CD4+CD25+ natural Treg cells were isolated from splenocytes of 6- to was significantly inhibited by anti–IL-2 mAb (Fig. 3C). These 8-wk-old BALB/c mice. Induced Treg cells were generated from splenic results suggest that IL-3 induces development of Treg cells in- + 2 CD4 CD25 T cells stimulated with plate-bound anti-CD3ε (10 mg/ml) directly through secretion of IL-2 by non-Treg cells. This was and soluble anti-CD28 (2 mg/ml) mAbs and incubated for 4 d in the further confirmed by a dose-dependent effect of IL-3 on secretion presence of TGF-b1 (5 ng/ml) and IL-2 (20 ng/ml). Both cell populations of IL-2 by CBA. We observed that IL-3 induces secretion of IL-2 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 in a dose-dependent manner. There was a drastic increase in IL-2 cells by FACS by surface labeling for CD4, CD25, and IL-3Ra, and cells secretion at 100 and 300 ng/ml concentrations of IL-3 (Fig. 3D). were further stained intracellularly for Foxp3. Gating was done on CD4+ Also, the enhanced secretion of IL-2 was directly correlated with CD25+ T cells for expression of Foxp3 and IL-3Ra. Results in A and B are a simultaneous increase in expression of IL-2Ra (CD25) on these representative of three independent experiments. i, induced; n, natural. cells (Fig. 3E). The Journal of Immunology 2265 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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. 2266 IL-3 ATTENUATES COLLAGEN-INDUCED ARTHRITIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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 The Journal of Immunology 2267 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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 mean 6 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.

The induced Treg cell population contains a significant pro- the CD4+CD25lowFoxp32 population. Collectively, these results portion of Foxp32 cells, which are likely to produce IL-2. There- suggest that IL-3 induces development of Treg cells indirectly fore, to further check the source of IL-2 secretion we stimulated through secretion of IL-2 by non-Treg cells. splenic CD4+CD252 T cells with anti-CD3ε and anti-CD28 mAbs for 4 d in the presence of TGF-b1 with or without different con- IL-3 synergies with IL-2 for activation of STAT5 centrations of IL-3. After 4 d, cells were activated with PMA We next investigated the mechanism of stimulatory effect of IL-3 (50 ng/ml) and ionomycin (500 ng/ml) for 5–6 h before being ana- on Treg cell development. IL-3R signaling is primarily mediated lyzed for the secretion of IL-2 by intracellular staining by FACS. through activation of JAK2 with subsequent phosphorylation and Gating was done on two cell populations, CD4+CD25high activation of STAT5 (15). Also, STAT5 promotes Treg cell dif- and CD4+CD25low, and cells were analyzed for expression ferentiation by regulating expression of Foxp3 (25). Therefore, we of Foxp3 and IL-2. We found that IL-3 in a dose-dependent examined the effect of IL-3 on STAT5 phosphorylation in T cells manner increased the percentage of IL-2–producing CD4+ in the absence and presence of IL-2. We first performed the ti- CD25lowFoxp32 cells from 2.3 to 9.1% (Fig. 3F). However, no IL- tration for both cytokines IL-2 and IL-3 independently for their 2> was produced by CD4+CD25highFoxp3+ cells in the absence or effects on phosphorylation of STAT5. CD4+CD252 T cells were presence of IL-3, even at high concentrations (Fig. 3F). Fig. 3G stimulated for 20 min independently with different concentrations represents the average data of three independent experiments of of 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. 2268 IL-3 ATTENUATES COLLAGEN-INDUCED ARTHRITIS analyzed by FACS. Both IL-2 and IL-3 independently increased than that of the additive effect of both cytokines (Fig. 4B). Fig. 4C the phosphorylation of STAT5 in a dose-dependent manner (Fig. shows the average percentage of STAT5 phosphorylation from 4A). The data in Fig. 4A show the average percentage of STAT5 three independent experiments (p , 0.05). Thus, IL-3 activates phosphorylation from three independent experiments (p , 0.05). STAT5 in T cells and synergizes with IL-2 for enhanced activation To further check the synergistic effect of IL-2 and IL-3 on of STAT5. phosphorylation of STAT5, we stimulated CD4+CD252 T cells for IL-3 reduces severity of arthritis by enhancing the percentage 20 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 was of Foxp3 Treg cells in vivo in CIA mice 15.13 and 15.64% phosphorylation of STAT5 by IL-2 and IL-3, To investigate the in vivo role of IL-3 in regulation of Treg cells, we respectively. Phosphorylation of STAT5 was increased to 34.67% used a well-established CIA mouse model of human RA. DBA/1J when both of the cytokines were added together, which was more mice were primed on day 0 with CII and then boosted on day 21. In the treatment group, mice were injected for 15 d with rmIL-3 (1.5 mg/d i.p. in two divided doses at 12-h intervals) from day 21 at the time of booster. Arthritis was manifested by redness and swelling of the paws, including digits. In PBS-injected control mice, no signs of inflammation were seen, whereas CIA mice developed severe 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 mean Downloaded from arthritic score (Fig. 5B). Photographs of hind paws in Fig. 5C show significant reduction of inflammation in mice treated with IL-3. In the presence of IL-3, thickness of the inflammatory soft tissues 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- http://www.jimmunol.org/ logical examination we observed that IL-3 treatment prevented damage to articular cartilage (Fig. 5D, enlarged regions). CIA mice had markedly enlarged spleen and inguinal lymph nodes, which were normal in size in IL-3–treated mice (Fig. 5E). By histological examinations of knee joints on day 36, we observed no infiltration of inflammatory cells and no damage to the articular cartilage of control mice. The knee joints in CIA mice showed massive infiltration of polymorphonuclear and other inflammatory cells, and there was multiple superficial cartilage erosion. In con- by guest on September 28, 2021 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, bone volume fraction (bone volume/tissue volume), connectivity den- sity, and cortical thickness was observed in IL-3–treated mice with respect to CIA mice (Fig. 6B). Bone architecture denoted by the structure model index was not altered. These results suggest that IL-3 maintains normal bone structure in mice. Thus, IL-3 treat- FIGURE 5. IL-3 suppresses inflammation and protects joint destruction ment reduces arthritic score and inflammation and prevents dam- in CIA mice. CIA was induced in 8- to 10-wk-old DBA/1J mice by CII age to bone and cartilage tissues in knee joints in CIA mice. emulsion as described in Materials and Methods. Control mice were To investigate the in vivo mechanism of IL-3 action in prevention 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 of CIA, we analyzed the effect of IL-3 on Foxp3 Treg cell de- s.c. injection on day 21 with a booster dose of CII prepared in IFA (200 velopment. Mice were sacrificed on day 36 and total mg/mice). In the third group mice were treated with IL-3 i.p. (1.5 mg/ populations derived from lymph nodes, spleen, and thymus tissues mouse/d in two divided doses) for 15 d starting from day 21 along with were analyzed for percentage of CD4+Foxp3+ Treg cells by FACS. booster dose of CII. Two independent blinded observers assessed the se- As compared with control mice, the percentage of Foxp3+ Treg verity of arthritis every fifth day after booster injection for hind paw cells in CIA mice was drastically decreased in thymus, lymph thickness (A) and clinical arthritic score (B). Data are represented as mean 6 nodes, and spleen. There was a .50% decrease in percentage of SEM (n = 5 mice/group). C, Photographs of paw swelling in DBA/1J mice Foxp3+ Treg cells in lymph nodes and an ∼70% decrease in spleen showing front and side views of hind paws, and x-rays of soft tissues and thymus. Interestingly, in IL-3–treated mice the percentage of showing inflammation of surrounding tissue. D, X-ray radiographs of knee CD4+Foxp3+ Treg cells in all the three tissues was restored to near joints showing bone and cartilage structure. E, Appearance of spleen and lymph nodes from control, CIA, and IL-3 treated mice. F, Mice were normal levels (Fig. 7A). Fig. 7B represents the average percentage + sacrificed on day 36, and knee joints were dissected, fixed, decalcified, and of Foxp3 Treg cells in lymph nodes, spleen, and thymus of mice processed for histopathology. Five-micrometer sections were stained for treated with or without IL-3. These results suggest that IL-3 also H&E. Original magnification 310. Similar results were obtained in two has a potential to augment the percentage of Foxp3+ Treg cells independent experiments. in vivo. The Journal of Immunology 2269

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 Downloaded from 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 http://www.jimmunol.org/ 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. by guest on September 28, 2021

IL-3 decreases production of proinflammatory cytokines and observed that IL-3 significantly increases the levels of IL-10, IL-2, increases anti-inflammatory cytokines in CIA mice IL-5, and IFN-g and decreases the levels of IL-6, IL-17A, TNF-a, The pathogenic events that lead to the development of human RA and IL-1 (Fig. 8). These results suggest that IL-3 has a potential to are not fully understood, although the pivotal role of proin- inhibit production of proinflammatory cytokines and induce anti- flammatory cytokines such as TNF-a, IL-1b, and IL-6 in the in- inflammatory cytokines. duction and maintenance of RA is well documented (26). These cytokines promote the deleterious imbalance in bone Discussion and contribute to enhanced bone destruction. Treg cells in active Treg cells represent an important immune mechanism for induc- RA are defective in controlling production of proinflammatory ing tolerance to both self and foreign Ags to prevent autoimmunity cytokines (11). Treg cells suppress through nu- and unwanted inflammation. Dysfunctional Treg cells are a cause merous mechanisms, including the production of anti-inflam- of various autoimmune diseases, , and immunopathology matory cytokines, direct cell to cell contact, and by modulating (28). However, it remains to be determined how these cells are the activation state and function of APCs (27). Therefore, we fur- involved in regulating the pathophysiology of common immuno- ther examined the effect of IL-3 on the levels of various proin- logical disease such as RA. Also, Treg cells potently inhibit flammatory and anti-inflammatory cytokines in the serum of con- osteoclastogenesis and bone resorption. Importantly, the capacity trol, CIA, and IL-3–treated mice. In CIA mice there was a sig- of a single Treg cell to block differentiation of 100 monocytes into nificant increase in production of IL-6, IL-17A, TNF-a, IL-1, and osteoclasts underlines the powerful role of Treg cells in regulation IFN-g (p , 0.05) and decrease in secretion of IL-5 and IL-10. of bone resorption (24). Thus, Treg cells appear to be an attractive IL-2 production was also decreased in CIA mice. Surprisingly, we target cell with substantial therapeutic potential in RA, and they 2270 IL-3 ATTENUATES COLLAGEN-INDUCED ARTHRITIS

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. controls of Foxp3 for each group are shown. B, Average percentage of CD4+Foxp3+ cells from lymph nodes, spleen, and thymus tissues Downloaded from of three mice. Values are expressed as mean 6 SEM. p , 0.05 in all the groups. http://www.jimmunol.org/ by guest on September 28, 2021 may act as an important link between the immune and bone in regulation of Treg cell development in both in vitro and in vivo system. However, therapeutic applications of Treg cells are limited conditions. because of their scarcity. Therefore, development of Treg cells IL-3 exerts its biological activities by binding to specific high- in vitro and in vivo is an essential requirement for their effective affinity receptors expressed by hematopoietic stem cells, endo- clinical intervention. In this study, we investigated the role of IL-3 thelial cells, and monocytes (29). Previous reports have demon-

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. The Journal of Immunology 2271 strated that IL-3–sensitive T cell clones activated by CD3 express hances the percentage of Foxp3+ Treg cells in vitro indirectly IL-3Ra, whereas T cell clones that were insensitive to the growth- through secretion of IL-2, and it also restores the loss of Foxp3+ enhancing effect of IL-3 were negative for IL-3Ra expression Treg cells in vivo. Also, IL-3–generated Treg cells have the po- (30, 31). These differences in reactivity to IL-3 among different tential to cure CIA in mice. Furthermore, our data suggest that T cell clones might be due to differences in the methods used for IL-3 may represent a novel therapeutic strategy to reduce immune establishing such T cell clones (31). To our knowledge, we re- responses, inflammation, and bone loss in autoimmune diseases. port in this study for the first time that both natural and induced Cellular therapy based on ex vivo development of Treg cells Treg cells show IL-3Ra expression at both the gene and protein and their subsequent transfer to patients is currently the focus of in- levels. Sustained expression of Foxp3, a key distinguishing feature tense research in the treatment of various autoimmune diseases of Treg cells, is required for the differentiation, suppressor func- (28). Also, transfer of Treg cells has already been shown to pre- tion, maintenance of cell phenotype, and metabolic fitness of Treg vent a wide range of experimental autoimmune diseases, includ- cells (32). Mice and humans harboring a loss-of-function ing , experimental encephalomyelitis, and colitis (40–42). in the Foxp3 gene are affected by fatal early onset lymphoproli- Several, preclinical studies have shown that either freshly isolated ferative immune-mediated disease affecting a variety of organs or ex vivo expanded Treg cells can prevent both local and sys- and tissues (33). Interestingly, in our system IL-3 significantly temic organ and tissue destruction (43). Two broad therapeutic increases the percentage of Foxp3+ natural and induced Treg approaches have been considered: first, to expand Treg cells in cells, and these cells suppress the proliferation of effector T cells. vitro with the intension of infusing these cells into patients; and Thus, IL-3–treated Treg cells are endowed with the classical second, to manipulate the in vivo resulting in an suppressive function ascribed to naturally occurring Treg cells. We increase in Treg cells. Importantly, IL-3 appears to increase the Downloaded from demonstrate that IL-3 promotes development of Treg cells in- percentage of Treg cells in both in vitro and in vivo conditions. directly through secretion of IL-2 by non-Treg cells. IL-2 is an Moreover, inhibition of most proinflammatory cytokines by IL-3 indispensable cytokine for the peripheral maintenance of natural may results in an increase in percentage of Treg cells. Thus, to our Treg cells. However, natural Treg cells do not produce this cyto- knowledge, we reveal for the first time that IL-3 may be useful to kine, and the exact source of IL-2 has been a matter of intense expand Treg cells under both in vitro and in vivo conditions, and it

research in the last decade (9, 34). Our results are consistent with may act as an important therapeutic agent to treat RA and other http://www.jimmunol.org/ earlier findings by other groups that the main source of IL-2 is autoimmune diseases. CD4+CD25low non-Treg cells and not CD4+CD25high Treg cells (35). IL-3 signaling involves activation of STAT5 in hematopoietic stem cells (15). Also, STAT5 activation by IL-2 is critical for Treg Acknowledgments cell development and Foxp3 induction (25). Deletion of STAT5 in We thank Hemangini Shikhare and Swapnil for FACS analysis. CD4+ T cells results in marked reduction of Foxp3+ Treg cells in both thymus and periphery, whereas enhanced STAT5 activation Disclosures promotes Treg cell differentiation (25, 36). We report in this study The authors have no financial conflicts of interest. that IL-3 synergizes with IL-2 for enhanced activation of STAT5 by guest on September 28, 2021 in T cells. 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