Mesenchymal stem cells inhibit Th17 cell differentiation by IL-10 secretion
2012; Elsevier BV; Volume: 40; Issue: 9 Linguagem: Inglês
10.1016/j.exphem.2012.05.006
ISSN1873-2399
AutoresXuebin Qu, Xingxia Liu, Kai Cheng, Rongcun Yang, Robert Chunhua Zhao,
Tópico(s)Cancer Immunotherapy and Biomarkers
ResumoRecent findings indicate that mesenchymal stem cells (MSCs) may act as a regulator of Th17 cell differentiation, however, the underlying mechanism is still under debate. To investigate the underlying mechanisms of MSCs' regulatory effect, mouse bone marrow–derived MSCs were cocultured with mouse CD4+CD25lowCD44lowCD62Lhigh T cells in vitro, and the proportion of induced Th17 cells, cytokines secretion, and transcription factors expression were examined by flow cytometry, enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction, and Western blotting. For the first time, our results showed that bone marrow–derived MSCs were able to inhibit Th17 cell differentiation via interleukin (IL)-10 secretion as the Th17 cell proportion was significantly regained when IL-10 was neutralized, or expression of IL-10 by bone marrow–derived MSCs was downregulated by RNA interference technique. Furthermore, IL-10 may suppress expression of Rorγt, the key transcription factor for Th17 cells, both by activating suppressor of cytokine signaling 3 through signal transducers and activators of transcription 5 phosphorylation, and decreasing signal transducers and activators of transcription 3 binding, which is at the promoter of Rorγt. Thus, our results demonstrate the inhibitory effect of MSCs on Th17 cells differentiation, and suggest increased IL-10 secretion might be the key factor. Recent findings indicate that mesenchymal stem cells (MSCs) may act as a regulator of Th17 cell differentiation, however, the underlying mechanism is still under debate. To investigate the underlying mechanisms of MSCs' regulatory effect, mouse bone marrow–derived MSCs were cocultured with mouse CD4+CD25lowCD44lowCD62Lhigh T cells in vitro, and the proportion of induced Th17 cells, cytokines secretion, and transcription factors expression were examined by flow cytometry, enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction, and Western blotting. For the first time, our results showed that bone marrow–derived MSCs were able to inhibit Th17 cell differentiation via interleukin (IL)-10 secretion as the Th17 cell proportion was significantly regained when IL-10 was neutralized, or expression of IL-10 by bone marrow–derived MSCs was downregulated by RNA interference technique. Furthermore, IL-10 may suppress expression of Rorγt, the key transcription factor for Th17 cells, both by activating suppressor of cytokine signaling 3 through signal transducers and activators of transcription 5 phosphorylation, and decreasing signal transducers and activators of transcription 3 binding, which is at the promoter of Rorγt. Thus, our results demonstrate the inhibitory effect of MSCs on Th17 cells differentiation, and suggest increased IL-10 secretion might be the key factor. Bone marrow–derived mesenchymal stem cells (BM-MSCs) exhibit a profound immunosuppressive activity that can be exploited to prevent and treat immune disorders. MSCs and their secreted cytokines can inhibit T-lymphocyte activation and proliferation induced by mitogens, recall Ags, and allo-Ags in vitro [1Di Nicola M. Carlo-Stella C. Magni M. et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2588) Google Scholar, 2Krampera M. Glennie S. Dyson J. et al.Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide.Blood. 2003; 101: 3722-3729Crossref PubMed Scopus (1359) Google Scholar, 3Majumdar M.K. Keane-Moore M. Buyaner D. et al.Characterization and functionality of cell surface molecules on human mesenchymal stem cells.J Biomed Sci. 2003; 10: 228-241Crossref PubMed Google Scholar, 4Ren G. Zhang L. Zhao X. et al.Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.Cell Stem Cell. 2008; 2: 141-150Abstract Full Text Full Text PDF PubMed Scopus (1462) Google Scholar]. In addition, MSCs can regulate cytokine production by T-cell subsets Th1 and Th2 [5Aggarwal S. Pittenger M.F. Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105: 1815-1822Crossref PubMed Scopus (3523) Google Scholar] and increase the proportion of CD4+CD25+ regulatory Th (Treg) cells [6Augello A. Tasso R. Negrini S.M. Cancedda R. Pennesi G. Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis.Arthritis Rheum. 2007; 56: 1175-1186Crossref PubMed Scopus (480) Google Scholar] to exert immunomodulatory effects both in vitro and in vivo. Th17 cell is a novel subset of Th cell, and can secrete high level of interleukin (IL)-17 and express key transcription factor Rorγt [7Aggarwal S. Ghilardi N. Xie M.H. de Sauvage F.J. Gurney A.L. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17.J Biol Chem. 2003; 278: 1910-1914Crossref PubMed Scopus (1456) Google Scholar, 8Ivanov I.I. McKenzie B.S. Zhou L. et al.The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (3805) Google Scholar]. Th17 cells play a detrimental role in experimental murine models of inflammatory diseases, such as multiple sclerosis, rheumatoid arthritis, and experimental autoimmune encephalomyelitis (EAE), as well as in human inflammatory bowel disease and psoriasis [9Murphy C.A. Langrish C.L. Chen Y. et al.Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation.J Exp Med. 2003; 198: 1951-1957Crossref PubMed Scopus (1356) Google Scholar, 10Langrish C.L. Chen Y. Blumenschein W.M. et al.IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3142) Google Scholar, 11Amadi-Obi A. Yu C.R. 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Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice.J Immunol. 2003; 171: 6173-6177PubMed Google Scholar]. Consistently, therapeutic neutralization of IL-17 secreted from Th17 cells could ameliorate clinical symptoms of EAE [16Hofstetter H.H. Ibrahim S.M. Koczan D. et al.Therapeutic efficacy of IL-17 neutralization in murine experimental autoimmune encephalomyelitis.Cell Immunol. 2005; 237: 123-130Crossref PubMed Scopus (350) Google Scholar]. Thus, Th17 cells are a major contributor of autoimmune inflammation in various autoimmune diseases, and inhibiting the differentiation of Th17 cells may be a good target for treatment of some autoimmune diseases. Several reports displayed that MSCs could suppress proinflammatory Th17 cells for therapy of some autoimmune diseases in which Th17 cells are recognized as key participants [17Zhang J. Li Y. Chen J. et al.Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice.Exp Neurol. 2005; 195: 16-26Crossref PubMed Scopus (309) Google Scholar, 18Zappia E. Casazza S. Pedemonte E. et al.Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy.Blood. 2005; 106: 1755-1761Crossref PubMed Scopus (1186) Google Scholar, 19Rafei M. Campeau P.M. Aguilar-Mahecha A. et al.Mesenchymal stromal cells ameliorate experimental autoimmune encephalomyelitis by inhibiting CD4 Th17 T cells in a CC chemokine ligand 2-dependent manner.J Immunol. 2009; 182: 5994-6002Crossref PubMed Scopus (283) Google Scholar, 20Ghannam S. Pene J. Torcy-Moquet G. Jorgensen C. Yssel H. Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype.J Immunol. 2010; 185: 302-312Crossref PubMed Scopus (416) Google Scholar], while on the contrary, Guo et al. showed that MSCs promoted expansion of Th17 cells [21Guo Z. Zheng C. Chen Z. et al.Fetal BM-derived mesenchymal stem cells promote the expansion of human Th17 cells, but inhibit the production of Th1 cells.Eur J Immunol. 2009; 39: 2840-2849Crossref PubMed Scopus (64) Google Scholar]. Thus, there is contradiction about the immunoregulatory roles of MSCs on Th17 cells, and the precise molecular mechanisms remain largely unknown. Finding out the immunomodulatory effects of MSCs on Th17 cells makes it appealing for MSC-based therapy of Th17-mediated autoimmune diseases. MSCs are known to exert immunosuppressive function via producing an array of anti-inflammatory cytokines such as IL-10, transforming growth factor–β, prostaglandin E2 (PGE2), indoleamine-2,3-dioxygenase (IDO), and nitric oxide (NO) [4Ren G. Zhang L. Zhao X. et al.Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.Cell Stem Cell. 2008; 2: 141-150Abstract Full Text Full Text PDF PubMed Scopus (1462) Google Scholar, 22Oh J.Y. Kim M.K. Shin M.S. et al.The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury.Stem Cells. 2008; 26: 1047-1055Crossref PubMed Scopus (298) Google Scholar], among which MSC-derived PGE2 and IDO have been demonstrated to inhibit Th17 differentiation [23Duffy M.M. Pindjakova J. Hanley S.A. et al.Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor.Eur J Immunol. 2011; 41: 2840-2851Crossref PubMed Scopus (165) Google Scholar, 24Tatara R. Ozaki K. Kikuchi Y. et al.Mesenchymal stromal cells inhibit Th17 but not regulatory T-cell differentiation.Cytotherapy. 2011; 13: 686-694Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]. IL-10 plays a particularly prominent role in attenuating immunomediated inflammation in the context of infection, allergy, and autoimmunity [25Jankovic D. Kugler D.G. Sher A. IL-10 production by CD4+ effector T cells: a mechanism for self-regulation.Mucosal Immunol. 2010; 3: 239-246Crossref PubMed Scopus (158) Google Scholar, 26Groux H. Cottrez F. The complex role of interleukin-10 in autoimmunity.J Autoimmun. 2003; 20: 281-285Crossref PubMed Scopus (136) Google Scholar, 27Sun J. Madan R. Karp C.L. Braciale T.J. Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10.Nat Med. 2009; 15: 277-284Crossref PubMed Scopus (438) Google Scholar, 28Roers A. Siewe L. Strittmatter E. et al.T cell-specific inactivation of the interleukin 10 gene in mice results in enhanced T cell responses but normal innate responses to lipopolysaccharide or skin irritation.J Exp Med. 2004; 200: 1289-1297Crossref PubMed Scopus (237) Google Scholar, 29Stehr M. Greweling M.C. Tischer S. et al.Charles River altered Schaedler flora (CRASF) remained stable for four years in a mouse colony housed in individually ventilated cages.Lab Anim. 2009; 43: 362-370Crossref PubMed Scopus (45) Google Scholar]. MSC-derived IL-10 can mediate suppression of T-cell proliferation [1Di Nicola M. Carlo-Stella C. Magni M. et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2588) Google Scholar, 30Tse W.T. Pendleton J.D. Beyer W.M. Egalka M.C. Guinan E.C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation. 2003; 75: 389-397Crossref PubMed Scopus (1263) Google Scholar] and infiltration [31Burchfield J.S. Iwasaki M. Koyanagi M. et al.Interleukin-10 from transplanted bone marrow mononuclear cells contributes to cardiac protection after myocardial infarction.Circ Res. 2008; 103: 203-211Crossref PubMed Scopus (131) Google Scholar]. Min et al. showed that IL-10–transduced MSCs could attenuate the severity of acute graft-vs-host disease after experimental allogeneic stem cell transplantation [32Min C.K. Kim B.G. Park G. Cho B. Oh I.H. IL-10-transduced bone marrow mesenchymal stem cells can attenuate the severity of acute graft-versus-host disease after experimental allogeneic stem cell transplantation.Bone Marrow Transplant. 2007; 39: 637-645Crossref PubMed Scopus (97) Google Scholar]. However, there is no evidence implicating the immunoregulatory roles of MSC-derived IL-10 on Th17 cell differentiation. Here, we found that mouse BM-MSCs inhibited the Th17 cell differentiation through IL-10. Further results showed that IL-10 might suppress the expression of Rorγt through upregulating the expression of suppressor of cytokine signaling (SOCS) 3 via signal transducers and activators of transcription (STAT) 5 activation; decreasing the binding quantity of STAT3 at the promoter of Rorγt. BALB/c mice were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, PR China). All mice were bred and maintained under specific pathogen-free conditions. Animal use and experimental procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences. Mouse BM-MSCs were prepared according to an established protocol [33Anjos-Afonso F. Siapati E.K. Bonnet D. In vivo contribution of murine mesenchymal stem cells into multiple cell-types under minimal damage conditions.J Cell Sci. 2004; 117: 5655-5664Crossref PubMed Scopus (260) Google Scholar]. Briefly, bone marrow cells were collected by flushing the femurs, tibias, and iliac crests from 5- to 6-week-old BALB/c mice with D-Hanks media supplemented with 2% fetal calf serum (StemCell Technologies Inc., Vancouver, BC, Canada). Red blood cell–depleted bone marrow mononuclear cells were plated in murine mesenchymal medium with murine mesenchymal supplements (StemCell Technologies Inc.). Nonadherent cells were eliminated by a half medium change at the 3rd day, and all the medium was replaced weekly with fresh medium. The cells were grown for 2 to 3 weeks until almost confluent. The entire adherent fraction was then detached by trypsinization and replated using a 1:3 dilution factor. After the 3rd passage, cells were used for experimentation. Cell growth and cell cycle were analyzed as described previously [34Zhang B. Liu R. Shi D. et al.Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population.Blood. 2009; 113: 46-57Crossref PubMed Scopus (245) Google Scholar]. Flow cytometric analysis for MSCs and T cells were performed as described previously [34Zhang B. Liu R. Shi D. et al.Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population.Blood. 2009; 113: 46-57Crossref PubMed Scopus (245) Google Scholar]. Briefly, the cells were incubated for 15 min at 4°C with an antibody specific for CD16/CD32 (BD Biosciences, Franklin Lakes, NJ, USA) at a concentration of 1 μg/106 cells per 100 μL to block Fc receptors. Cells were washed twice with ice-cold phosphate-buffered saline (pH 7.2) containing 0.1% NaN3 with 0.5% bovine serum albumin and were incubated with 1 μg fluorescent antibodies for 30 min at 4°C. Cells were then washed twice and resuspended in 500 μL phosphate-buffered saline. To detect intracellular Ags, the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) was used. The fluorescent antibodies used in the study included the following: fluorescein isothiocyanate–conjugated anti-mouse Sca-1, CD4, CD14, CD19, CD31, CD34, CD44, CD45, CD106, CD117, H-2Kd and H-2Kb; phycoerythrin-conjugated anti-mouse CD25, CD73, CD184, Ia, TER-119 (BD Biosciences), and Rorγt (eBioscience, San Diego, CA, USA); allophycocyanin-conjugated anti-mouse CD4, IL-17, CD9, CD62L, and CD90 (eBioscience). For each antibody, IgG of the same isotype from the same species was used as the isotype control. Analysis was performed on Accuri C6 Flow Cytometers with CFlow software (Accuri Cytometers, Inc., Ann Arbor, MI, USA). For adipogenic differentiation, BM-MSCs were induced for 3 weeks in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 0.1 mM ascorbic acid. Cells were then stained with fresh Oil-Red-O solution (Sigma-Aldrich, St Louis, MO, USA) to visualize lipid droplets in differentiated cells. For osteogenic differentiation, cells were induced in osteogenic medium (Dulbecco's modified Eagle's medium supplemented with 0.1 μM dexamethasone, 10 mM β-glycerophosphate, 0.05 mM ascorbic acid, and 10% fetal bovine serum) for 3 weeks. The cells were then stained for alkaline phosphatase to assess osteogenic differentiation. Splenocytes from 7- to 8-week-old BALB/c mice were used to purify CD4+ T cells according to manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD4+ T cells was consistently >98%. CD4+ T cells were cocultured with MSCs at a ratio of 10:1 (CD4+ to MSC) under Th17-cell polarizing conditions: RPMI-1640 containing 10% fetal calf serum, 1 mM glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids (Sigma-Aldrich), anti-CD3 plus anti–CD28-coated beads (Invitrogen, Carlsbad, CA, USA), 5 ng/mL IL-2 (R&D Systems, Minneapolis, MN, USA), 20 ng/mL IL-6, 5 ng/mL transforming growth factor−β, 10 ng/mL IL-23, 2 ug/mL anti–IL-4 (BD Pharmingen) and 2 ug/mL anti–interferon-γ (BD Pharmingen). In some experiments, neutralizing antibodies (NA) for IL-10 (10 μg/mL) or 10 μM CGP 41251 (a phospho-STAT5 inhibitor; Merck, Darmstadt, Germany) were used. In addition, carboxyfluorescein diacetate succinimidyl ester (Sigma) labeled CD4+ T cells were cocultured with BM-MSCs and subsequently counted by fluorescence-activated cell sorting to determine the effect of MSCs on T-cell proliferation. Levels of IL-10, IL-17A, IL-17F, IL-21, and IL-22 in the supernatants were assayed by enzyme-linked immunosorbent assay kits according to manufacturer's instructions (BD Technologies). To measure cytokines secreted from T cells cocultured with BM-MSCs, nonadherent T cells were washed away slightly from MSCs monolayer that adhered to the bottom of plate, and then cultured alone in basal medium for 4 h. The experiments were performed in duplicate to ensure reproducibility. Total RNA was extracted using TRIZOL (Invitrogen) and was then treated with RNase-Free DNase I (Promega, Madison, WI, USA) to eliminate any residual genomic DNA; 1 μg total RNA from each sample was reverse-transcribed using a QuantScript RT kit (Tiangen, Beijing, PR China) in a final volume of 20 μL. Polymerase chain reaction products were analyzed using electrophoresis on a 2% agarose gel and were photographed after ethidium bromide staining. Gene expression was examined using a SYBR Green real-time PCR kit (Takara, Liaoning, PR China) by an IQ5 System (Bio-Rad, Hercules, CA, USA). Data were normalized to the reference gene β-actin. The primers used are: Rorγt, 5′-TGCAAGACTCATCGACAAGG-3′, 5′-AGGGGATTCAACATCAGTGC-3′; IL-10, 5′-ACTCCAGGACCTAGACAGA-3′, 5′-GCCAGCCTTAGAATAGAA-3′; β-actin, 5′-GAGACCTTCAACACCCCAGCC-3′, 5′-AATGTCACGCACGATTTCCC-3′. Cells were lysed in ice-cold radio immunoprecipitation assay lysis buffer (Beyotime, Jiangsu, PR China). Equal quantities of extracts (30 μg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidine difluoride membranes. After blocking, membranes were sequentially incubated with primary antibodies diluted 1/1000 in 3% bovine serum albumin. Horseradish peroxidase–labeled IgG was used as the secondary antibody (1/3000) for detection. All of the antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Three IL-10 siRNAs (TTCCACUUCUGAAAGAAAGUU, TTGUCCUGAAAUUCCCAAUGA and TTCGGUUCGGAAUAGCCUUUA) and negative control (UUCUCCGAACGUGUCACGUTT) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, PR China). Small interfering RNAs (siRNAs) were transfected into cells with Lipofectamine 2000 (Invitrogen) according to the instructions. Chromatin immunoprecipitation was performed using an EZ-ChIP kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Four micrograms chromatin immunoprecipitation–grade antibody specific for STAT3 were obtained from Millipore. Primers for PCR are: Rorγt, 5′-ACTGGGAAAGAAAGAACTGG-3′, 5′-TGAATAGGAAGGCGGAGA-3′; IL-17A, 5′-CACCTCACACGAGGCACAAG-3′, 5′-ATGTTTGCGCGTCCTGATC-3′; IL-17F, 5′-CCCACAAAGCAACACTCTTGTC-3′, 5′-ACTGCATGACCCGAAAGCA-3′. All experiments were performed at least three times. Results were expressed as the mean ± standard deviation. Paired t tests were used for intergroup comparison, and p values <0.05 were considered statistically significant. BM-MSCs were obtained from BALB/c mice, and showed a spindle-shaped morphology (Fig. 1A), a typical growth curve (Fig. 1B), and a high percentage of cells at G0/G1 phase (Fig. 1C). The cultured BM-MSCs expressed CD9, CD73, CD44, Sca-1, H-2Kd, CD90, CD184 and Ia surface antigens, while lacking the lineage markers CD14, CD19, CD31, CD34, CD45, CD117, Ter, and H-2Kb (Fig. 1D). Moreover, we demonstrated that these cells could differentiate into osteoblasts and adipocytes under specific differentiation induction conditions (Fig. 1E, F). The BM-MSCs were further characterized by their capability to inhibit T-cell proliferation by carboxyfluorescein diacetate succinimidyl ester incorporation method after a 3-day coculture (Fig. 1G), consistent with previous results [1Di Nicola M. Carlo-Stella C. Magni M. et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2588) Google Scholar, 2Krampera M. Glennie S. Dyson J. et al.Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide.Blood. 2003; 101: 3722-3729Crossref PubMed Scopus (1359) Google Scholar, 4Ren G. Zhang L. Zhao X. et al.Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.Cell Stem Cell. 2008; 2: 141-150Abstract Full Text Full Text PDF PubMed Scopus (1462) Google Scholar].Figure 1Characteristics of mouse BM-MSCs. (A) Spindle-shaped morphology of BM-MSCs. Bar: 100 μm. (B) Growth curve of BM-MSCs. (C) Cell cycle analysis of BM-MSCs. (D) Phenotype analysis of BM-MSCs by fluorescence-activated cell sorting (FACS). The gray line represented isotype controls. Representative data from one of three independent experiments were shown. (E) Osteogenic differentiation of BM-MSCs. Blue color represents the positive staining of alkaline phosphatase (ALP). Bar: 100 μm. (F) Adipogenic differentiation of BM-MSCs. Red color displays the lipid droplets by Oil Red O staining. Bar: 100 μm. (G) Carboxyfluorescein diacetate succinimidyl diester–labeled CD4+ T cells were cultured for 72 h, and then analyzed by FACS. The labeled T cells were used as the control group (Con). Data were representative of three separate experiments. ⋆p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has been reported that MSCs could regulate the Th17 cells [19Rafei M. Campeau P.M. Aguilar-Mahecha A. et al.Mesenchymal stromal cells ameliorate experimental autoimmune encephalomyelitis by inhibiting CD4 Th17 T cells in a CC chemokine ligand 2-dependent manner.J Immunol. 2009; 182: 5994-6002Crossref PubMed Scopus (283) Google Scholar, 20Ghannam S. Pene J. Torcy-Moquet G. Jorgensen C. Yssel H. Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype.J Immunol. 2010; 185: 302-312Crossref PubMed Scopus (416) Google Scholar, 21Guo Z. Zheng C. Chen Z. et al.Fetal BM-derived mesenchymal stem cells promote the expansion of human Th17 cells, but inhibit the production of Th1 cells.Eur J Immunol. 2009; 39: 2840-2849Crossref PubMed Scopus (64) Google Scholar], where the underlying mechanisms need further discussion, thus, we chose a classic differentiation system of CD4+ T cells to Th17 cells, and observed BM-MSCs' effects in it. The isolated CD4+ T cells expressed high level of CD62L (>85%), and low levels of CD25 (<4%) and CD44 (<3%) (Fig. 2A), so we designated this CD4+ T-cell population as CD4+CD25lowCD44lowCD62Lhigh T cells, and induced them to differentiate into Th17 cells. After 3 days coculture with BM-MSCs, the percentage of IL-17+Rorγt+ Th17 cells induced from the CD4+CD25lowCD44lowCD62Lhigh T cells decreased significantly compared with the CD4+ T cells cultured alone (Fig. 2B). Accordingly, T cells in the coculture group showed a decreased transcription of Rorγt (Fig. 2C), and secreted lower levels of IL-17A and IL-17F (Fig. 2D). These results suggest that BM-MSCs might inhibit the production of Th17 cells.Figure 2BM-MSCs inhibit the production of Th17 cells. (A) Expression levels of CD25, CD44, and CD62L on the isolated CD4+ T cells were analyzed by fluorescence-activated cell sorting (FACS). Representative data from one of three independent experiments were shown. (B–D) CD4+CD25lowCD44lowCD62Lhigh T cells were cultured with BM-MSCs (MSCs+) or without BM-MSCs (MSCs−) under Th17-cell polarizing conditions. The induced T cells were collected to analyze the IL-17+Rorγt+ cells at 72 h (B), messenger RNA level of Rorγt (C) and cytokines measurement of IL-17A and IL-17F (D) at different time points. Data are expressed as the means ± standard deviation of triplicate assays. ⋆p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Duffy et al. showed that when cocultured at a high MSC to T cell ratio (≥1:30), MSCs still inhibited Th17 production even after separating them by Transwell [23Duffy M.M. Pindjakova J. Hanley S.A. et al.Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor.Eur J Immunol. 2011; 41: 2840-2851Crossref PubMed Scopus (165) Google Scholar]. Consistently, we also found that separation of BM-MSCs and T cells by Transwell during coculture did not prevent inhibition of Th17 cell differentiation (data not shown). We screened the key cytokines in coculture supernatant to explore the possible factors underlying this inhibitory effect. We found that the level of IL-10, which has been reported as one of the most important cytokine secreted by MSCs and playing anti-inflammatory roles [1Di Nicola M. Carlo-Stella C. Magni M. et al.Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.Blood. 2002; 99: 3838-3843Crossref PubMed Scopus (2588) Google Scholar, 30Tse W.T. Pendleton J.D. Beyer W.M. Egalka M.C. Guinan E.C. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation.Transplantation. 2003; 75: 389-397Crossref PubMed Scopus (1263) Google Scholar, 31Burchfield J.S. Iwasaki M. Koyanagi M. et al.Interleukin-10 from transplanted bone marrow mononuclear cells contributes to cardiac protection after myocardial infarction.Circ Res. 2008; 103: 203-211Crossref PubMed Scopus (131) Google Scholar, 32Min C.K. Kim B.G. Park G. Cho B. Oh I.H. IL-10-transduced bone marrow mesenchymal stem cells can attenuate the severity of acute graft-versus-host disease after experimental allogeneic stem cell transplantation.Bone Marrow Transplant. 2007; 39: 637-645Crossref PubMed Scopus (97) Google Scholar], was significantly increased when BM-MSCs existed than that of T cells cultured alone (Fig. 3A). To test whether IL-10 was responsible for BM-MSCs' inhibitory effect, we added neutralizing antibody against IL-10 (NA–IL-10) to the coculture system. When cocultured with BM-MSCs, CD4+CD25lowCD44lowCD62Lhigh T cells derived Th17 cells were limited to 2.0%, while IL-17+ cells counted up to 2.3%, 6.0%, and 12.3% of induced cells with 1 μg/mL, 5 μg/mL, and 10 μg/mL NA–IL-10, respectively, in a dose-dependent manner (Fig. 3B). Titrated concentrations of NA–IL-10 showed that 10 μg/mL was sufficient to neutralize IL-10 in coculture medium. The addition of sufficient NA–IL-10 to coculture system not only completely restored the proportion of IL-17+Rorγt+ T cells (Fig. 3C) and blocked the inhibition of Th17 cell production caused by BM-MSCs (Fig. 3C), but also upgraded secretion levels of IL-17A and IL-17F (Fig. 3D). These were further confirmed by the expression level of Rorγt, the key transcription factor of Th17 cells was significantly increased after NA-IL-10 addition (Fig. 3E). These results suggest that IL-10 may play an important role in suppre
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