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MicroRNA-146a Negatively Regulates the Immunoregulatory Activity of Bone Marrow Stem Cells by Targeting Prostaglandin E2 Synthase-2 This information is current as of September 30, 2021. Mariola Matysiak, Maria Fortak-Michalska, Bozena Szymanska, Wojciech Orlowski, Anna Jurewicz and Krzysztof Selmaj J Immunol 2013; 190:5102-5109; Prepublished online 15 April 2013; Downloaded from doi: 10.4049/jimmunol.1202397 http://www.jimmunol.org/content/190/10/5102 http://www.jimmunol.org/ References This article cites 45 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/190/10/5102.full#ref-list-1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MicroRNA-146a Negatively Regulates the Immunoregulatory Activity of Bone Marrow Stem Cells by Targeting Prostaglandin E2 Synthase-2

Mariola Matysiak, Maria Fortak-Michalska, Bozena_ Szymanska, Wojciech Orlowski, Anna Jurewicz, and Krzysztof Selmaj

The molecular mechanisms that regulate the immune function of bone marrow–derived mesenchymal stem cells (BMSCs) are not known. We have shown previously that freshly isolated BMSCs when induced to express neuronal stem cell markers lose immunoregulatory function when transferred into mice sensitized to develop experimental autoimmune encephalomyelitis. Re- cently, microRNAs (miRs) have been shown to be involved in the regulation of several immune responses in both innate and

acquired immunity. We now show that among several differentially expressed miRs, miR-146a was strongly upregulated in Downloaded from neuronally differentiated when compared with miR-146a expression in freshly isolated BMSCs or control BMSCs cultured in parallel but in nondifferentiating medium. Inhibition of miR-146a with a selective antagomir restored the immunoregulatory activity of nBMSCs. We mapped miR-146a to its multiple predicted target mRNA transcripts and found that miR-146a was predicted to block PGE2 synthase (ptges-2). We then showed that Ptges-2 was directly targeted by miR-146a using a luciferase reporter assay. Furthermore, increased expression of miR-146a in BMSCs correlated with inhibition of PGE synthase-2 and inhibition of PGE2 release. Accordingly, inhibition of miR-146a restored synthesis of PGE2. These data support the conclusion that http://www.jimmunol.org/ miR-146a plays a critical role in the control of the immunoregulatory potential of BMSCs. The Journal of Immunology, 2013, 190: 5102–5109.

ultiple sclerosis (MS) is a chronic inflammatory de- have also been shown to modulate immune responses by influ- myelinating condition of the human CNS charac- encing some T cell, B lymphocyte, NK, and dendritic cell func- M terized by recurrent episodes of immune-mediated tions (13–15). The mechanisms underlying these effects are largely demyelination and axonal loss (1). Among recently proposed unknown but are likely to be mediated either by direct cell–cell therapeutic strategies, transplantation of myelin-forming precursor contact and/or soluble factors such as TGF-b1 (16), hepatocyte by guest on September 30, 2021 cells or stem cells (SC) has generated both interest and reserva- growth factor (17), NO (18), and PGE2 (19–21). tions (2–4). Bone marrow–derived mesenchymal stem cells (BMSCs) MicroRNAs (miRs) have become recognized as important gene provide the structural and functional support for the generation of regulators in eukaryotic organisms, including SCs (22). miRs blood cell lineages from hematopoietic SCs (5). BMSCs expanded are a class of nonprotein-coding small RNAs that have a spe- in vitro can generate progeny that differentiate into multiple cell cific secondary stem-loop hairpin structure within their primary lineages (6). Several recent reports have demonstrated that under transcripts with a higher minimal folding free energy index (23). specific experimental conditions, BMSCs can differentiate into miRs negatively regulate gene expression at the posttranscrip- bone cells (7), fat cells (8), hepatocytes (9), glia, and neurons (10, tional level through direct mRNA cleavage (24), translational 11). The neuron-differentiation potential of BMSCs has attracted repression (25), or mRNA decay mediated by miR deadenylation intense interest in the possible application of BMSCs in cell and (26). Although many endogenous miRs have been identified in gene therapy strategies for neurologic diseases (12). BMSCs mammals, specific functions have remained largely undefined. However, recent studies indicate that the expression profiles of miRs in SCs are different from other tissues (27). This suggests Department of Neurology, Medical University of Lodz, 90-153 Lodz, Poland that miRs may play an essential role in SC self-renewal, differ- Received for publication August 27, 2012. Accepted for publication March 19, 2013. entiation, and other functions (28). With respect to immunoreg- This work was supported by Medical University of Lodz Grant 502-11-741 and ulation, miRs have been shown to be involved in the regu- Polish Ministry of Science and Higher Education Grant N N401 031637. lation of several immune responses in both innate and acquired The sequences presented in this article have been submitted to Gene Expression immunity. Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE43621. In this study, we analyzed the expression profile of miRs in Address correspondence and reprint requests to Dr. Krzysztof Selmaj, Department BMSCs as a correlate of their immunoregulatory potential. We of Neurology, Medical University of Lodz, Kopcinskiego Street 22, 90-153 Lodz, Poland. E-mail address: [email protected] found that several miRs were significantly upregulated, including miR-146a, in neuronally differentiated BMSCs that had lost im- Abbreviations used in this article: bFGF, basic fibroblast growth factor; BMSC, bone marrow–derived mesenchymal stem cell; cBMSC, cultured nondifferentiated Lin2 munoregulatory activity in mice with experimental autoimmune 1 Sca-1 bone marrow-derived mesenchymal stem cell; cPGES, cytosolic PGE syn- encephalomyelitis (EAE), an animal model for MS. We found thase; EAE, experimental autoimmune encephalomyelitis; fBMSC, freshly isolated Lin2Sca-1+ bone marrow–derived mesenchymal stem cell; miR, microRNA; mPGES, that miR-146a targeted PGE2-synthase 2, which resulted in microsomal PGE synthase; MSC, mesenchymal stem cell; nBMSC, neural-differenti- decreased secretion of PGE . Because PGE was shown to con- 2 1 2 2 ated Lin Sca-1 bone marrow–derived mesenchymal stem cell; SC, stem cell; UTR, tribute to the immunosuppressive activity of BMSCs (19–21), we untranslated region. analyzed whether the functional relation between miR-146a Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 and BMSC-induced immunosuppression depended on PGE2. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202397 The Journal of Immunology 5103

Materials and Methods BMSC cultures during differentiation and in nondifferentiated medium (on Isolation of BMSCs 4 and 7 d) or in 24-h culture of fBMSCs. Supernatants were frozen and analyzed according to the manufacturer’s instructions. Standard, control, Normal SJL mice, 6–8 wk old, were used for the isolation of BMSC. Bone and test samples were added to each well and incubated for appropriate marrow cells were obtained from the femurs and tibias of euthanized mice time with adequate primary Ab and conjugate. After washing, substrate by flushing with PBS. Cells were washed twice in sorting medium that solution was added to each well for 30 min at room temperature in the consisted of PBS supplemented with 0.5% BSA (Sigma-Aldrich, St. Louis, dark. Finally, Stop Solution was added and the OD of each well was de- MO), and subjected to negative magnetic sorting using the Lineage Cell termined within 30 min using a microplate reader (wavelength 450 nm). Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Depletion of cells expressing lineage Ags by mAbs and magnetic beads resulted in Western blotting 2 2 a pure fraction of Lin cells. Purity of the Lin fraction was invariably Cell lysates were obtained from cultures of fBMSCs (24 h), nBMSCs, and . 98%, as assessed by flow cytometry using Lineage Cocktail (anti-CD3e, cBMSCs. Cells were pelleted and lysed in lysing buffer (20 mM Tris-HCl anti-CD11b, anti-CD45R/B220, anti-Ly6G and Ly-6C, and anti–TER-119; [pH 7.4], 0.15 M NaCl, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM BD Biosciences, San Jose, CA) (29). Cells retained differentiation capacity Na VO , 1 mM PMSF, 1 mg/ml aprotinin and leupeptin). Lysates were and were characterized by lack of CD34, CD11b, and CD45. Isolation of 3 4 2 + 2 + centrifuged at 14,000 rpm for 5 min. An equal amount of protein from each murine bone marrow Lin Sca-1 cells (Lin Sca-1 BMSCs) was per- cell lysate was separated by SDS-PAGE electrophoresis and transferred to formed using MACS Sca-1 MultiSort Kit (Miltenyi Biotec., Bergisch PVDF membranes (Immobilon, Millipore, MA). Membranes were blocked Gladbach, Germany). After enrichment, Sca-1 expression was up to 20%, with 5% dried milk in TBS-TBS (Sigma Aldrich) overnight, followed by , 2 compared with 1% in Lin BMSCs, and in this population, c-kit was incubation for 1–2 h with PGE synthase-1, PGE synthase-2, and PGE , 2 2 2 2%. Sca-1 defines pluripotent capacity of SC population (30, 31). In synthase (cytosolic) (rabbit 1:500; Cayman Chemical, Ann Arbor, MI), and addition, examining these cells gene expression profile with Affymetrix GAPDH (Abcam, Cambridge, U.K.). After washing with TBS, blots were GeneChip, we found numerous mesenchymal SC (MSC)–specific markers, incubated with peroxidase-coupled secondary anti-rabbit Ab (1:20,000; such as cd44, alcam, casp3, eng, itgav, pdgfrb, prom1, vcam1, and thy1. Sigma-Aldrich). Proteins were detected by ECL–ECL (Amersham Bio- Downloaded from Moreover, several other genes associated with MSCs were also identified: sciences). Densitometry was performed for comparison of Western blot anxa5, bglap, ctnnb1, gtf3a, hgf, icam1, itgb1, nudt6, pigs, ptprc, tnf, data (Alpha Innotech, San Leandro, CA). vegfa, vim,andvwf. These data indicate that isolated lineage-negative murine bone marrow cell population enriched with Sca-1 possesses dif- miR analysis ferentiation potential and meets criteria for MSCs (32). Total RNA samples were isolated from fBMSCs and nBMSCs by using Neural differentiation of SCs the miRVana RNA Isolation kit (Ambion, Austin, TX) according to the manufacturer’s protocol. Reverse transcription was performed with miR- http://www.jimmunol.org/ BMSCs were cultured for 3 d in medium (DMEM: F12 [1:1], L-glutamine [1.9 146a–specific primer, mmu miR-146a (TaqMan MicroRNA Assays; Ap- mM], insulin [4.7 mg/ml], putrescine dihydrochloride [15.3 mg/ml], proges- plied Biosystem, Foster City, CA). Real-time PCR with TaqMan probes terone [58 ng/ml], apo-transferrin [94 mg/ml], sodium selenite [38 ng/ml], was normalized by small nucleolar RNA levels. To make cDNA for each L-thyroxine [380 ng/ml], tri-iodo-L-thyronine [317 ng/ml], basic fibroblast TaqMan miR assay, we incubated 10 ng total RNA with 0.15 ml dNTPs growth factor [bFGF; 10 ng/ml], and gentamicin [47 mg/ml]). The differ- (100 nM), 1.5 ml103 reaction buffer, 0.19 ml RNase inhibitor, 1 ml re- entiation of neural SCs was induced by removing the bFGF-containing verse transcriptase, and 3 ml gene-specific primer in a 15-ml reaction. Real- medium and resuspending cells in fresh bFGF-free medium. Examining these time PCR was performed using an Applied Biosystems 7500 Sequence cells gene expression profile with Affymetrix GeneChip, we found expression Detection System. Quantification of relative expression levels was per- of neural SC markers (abcg2, bmi-1, cxcr4, notch-1, slain1,andvimentin), neural formed using standard curves for target genes and the endogenous control. lineage markers (alcam, thy 1, l1cam, latexin, vamp-1, tle3,andpax6), and glial

The value of each control sample was set at 1 and was used to calculate the by guest on September 30, 2021 b lineage markers (cd44, claudin-11, fgfr4, fgfr1, gmf- , mbp, meteorin,andngfr). fold change in target genes. As a control, BMSCs cultured in nondifferentiated medium supple- For miRCURY LNA Array miR, RNA samples were concentrated to at mented with bFGF (cBMSC) were used. All experiments were performed least 1 mg/ml. The Exiqon miRCURY LNA array version 10.0 (http://www. after 4 and 7 d of differentiation. exiqon.com) (33) was used for data analysis in this study. All array data Induction and treatment of EAE have been deposited in the Gene Expression Omnibus database (http:// www.ncbi.nlm.nih.gov/geo) under accession number GSE43621. Female SJL mice, 6–8 wk old, were obtained from the Animal Care De- partment, Medical University of Lodz. All animals were housed in Anti-miR treatments pathogen-free conditions and were treated according to the guidelines of A 22-oligonucleotide, anti–miR-146a (antagomir; 59-AACCCAUGGAA- the local Animal Ethics Committee. Mice were immunized s.c. according UUCAGUUCUCA-39) was used at 10-nM concentration complexed with to a previously published protocol using 0.15 mg PLP139–151 in IFA (Difco the transfection reagent Lipofectamine 2000 (2 ml/cell; Invitrogen, Carlsbad, Laboratories), supplemented with 4 mg/ml Mycobacterium tuberculosis. CA). Anti–miR-146a (or Lipofectamine control) was used in nBMSCs cul- Immunization with PLP peptide was followed by i.v. administration of 400 turedin24/wells(0.53 106 cells/well). After 4 d of culture, cells were used ng pertussis toxin (Sigma-Aldrich), on days 0 and 2. for the transplantation procedure. After 48 and 96 h posttransfection, Freshly isolated Lin2Sca-1+ BMSCs (fBMSCs), neural-differentiated 2 + 2 + samples were analyzed by RT-PCR for miR-146a expression (as described Lin Sca-1 BMSCs (nBMSCs), and cultured nondifferentiated Lin Sca-1 earlier). 3 6 BMSCs (cBMSCs), 2 10 in 200 ml PBS were transferred i.v. into EAE mice Reverse transcription with miR-146a–specific primer, mmu-miR-146a at the peak of disease. Control mice with EAE received a sham injection of the (TaqMan MicroRNA Assays; Applied Biosystem), was performed to as- same volume (200 ml) of PBS. The clinical course of EAE was evaluated sess miR expression after antagomir transfection. To make cDNA for each daily on a 0 to 5 scale (0 = healthy, 1 = limp tail, 2 = ataxia and/or paresis of TaqMan miR assay, we incubated 10 ng total RNA with 0.15 ml dNTPs hind limbs, 3 = paralysis of hind limbs and/or paresis of forelimbs, 4 = tet- (100 nM), 1.5 ml103 reaction buffer, 0.19 ml RNase inhibitor, 1 ml re- raparalysis, 5 = moribund). Mice were observed for 90 d after immunization. verse transcriptase, and 3 ml gene-specific primer in a 15-ml reaction. TaqMan MicroRNA Assay is a target-specific stem-loop, reverse-transcriptase Proliferation assay primer highly specific only for mature miR and resulted in longer reverse Spleen cells were obtained 2 wk after immunization for EAE and cultured (2 3 transcription amplicon in range 50–150 bp according to manufactured spec- 105 cells/well) in triplicate for 72 h in medium supplemented with 10 mg/ml ification. PCR fragments were resolved using MultiNA Microchip Electro- phoresis System on gel for visualization of miR expression (Shimadzu PLP139–151. BMSCs irradiated in vitro (30 Gy) were cultured with spleno- cytes from EAE mice at a ratio 1:5. For the final 16 h, 1 mCi [3H]thymidine Biotech, Milton Keynes, U.K.). (TdR; Amersham Biosciences, Buckinghamshire, U.K.) was added to each well. At the end of the culture period, incorporation of [3H]thymidine was Luciferase reporter transfection and dual luciferase assay determined in a Wallac Betaplata liquid scintillation counter (Perkin Elmer 4 In 39-untranslated region (UTR) assay, 5 3 10 HEK293 cells were Life Sciences, Wellesley, MA). Results were expressed as cpm. transfected with 500 ng of the UTR reporter (pMir-Report) with inserted ELISA PGES-2 (420 bp), 20 pmol pre–miR-miR (miR-146a), and pre-miR (negative control; Ambion, Austin, TX), and 2 ml Lipofectamine 2000 Quantitative analysis of PGE2 was performed by ELISA, using commer- (Invitrogen). Lysates were harvested 24 h after transfection, and reporter cially available kits (R&D Systems). Supernatants were derived from activity was measured with Dual Luciferase Reporter Assay (Promega). 5104 miR-146a REGULATES THE IMMUNOREGULATORY ACTIVITY OF BMSC

Statistical analysis the microarray data, we performed RT-PCR, which detected an in- All data are expressed as the mean 6 SD, and differences between groups crease of miR-146a expression up to 500-fold in nBMSCs compared were determined using the Student t test and Mann–Whitney U test; p with fBMSCs (Fig. 1B). values ,0.05 were considered significant. MiR-146a inhibition reversed the loss of immunoregulatory function of nBMSCs Results miR expression profile in BMSCs in relation to their To confirm further the role of miR-146a in loss of immunoregu- immunoregulatory potential for EAE latory function of nBMSCs in EAE, we applied a selective miR- 146a antagonist (antagomir). Transfection of nBMSCs with miR- In a previous study, we demonstrated that mice transplanted i.v. 146a antagomir resulted in stable decreased expression of up to with fBMSCs showed significantly lower clinical scores and 90% of miR-146a (Fig. 2A, 2B). Transplantation of these nBMSCs faster recovery compared with control mice with EAE (34). How- 2 + that had been transfected with the miR-146a antagomir resulted in ever, transplantation of Lin Sca-1 BMSCs that had been in- amelioration of EAE comparable with that obtained with fBMSCs duced to differentiate into cells expressing GFAP, NeuN, and O4 (Fig. 2C). These results indicate that miR-146a represents a critical (nBMSCs) resulted in a diminished immunoregulatory effect on regulatory element in molecular mechanisms involved in the im- the course of EAE compared with the transplantation of fBMSCs. munosuppressive activity of BMSCs in the EAE model. Therefore, in this study, we tested with unbiased approach both BMSC populations, fBMSCs and nBMSCs, for miR expression miR-146a inhibition restored the regulatory activity of profiles to search for miR associated with their immunoregulatory differentiated nBMSCs on PLP-induced proliferation Downloaded from potential. When clustering samples and genes, 30 of 757 miRs In Fig. 3, we show that the immunoregulatory activity of fBMSCs passed the filtering criteria on variation across samples (SD . 1.0) in EAE correlated with an inhibitory effect on PLP-induced T cell (35) (Table I). Hence, these 30 miRs were used in the two-way proliferation (p , 0.013), whereas nBMSCs, which were not ef- hierarchical clustering analysis of genes. Analysis of log2(Hy3/ ficient in EAE immunoregulation, did not show an effect on PLP- Hy5) ratios showed that the most upregulated miR during BMSCs induced T cell proliferation. However, nBMSCs transfected with cultured under neural differentiation conditions was miR-146a. the miR-146a antagomir showed an inhibitory effect on prolifer- http://www.jimmunol.org/ The heat map diagram shows the result of the two-way hierarchi- ation of PLP-activated T cells (p , 0.0274) that was comparable cal clustering of miRs and samples of nBMSCs differentiated in with fBMSCs (Fig. 3). culture for 4 (nBMSC4d) or 7 (nBMSC7d) days versus fBMSCs (Fig. 1A). Nineteen miRs were found to be upregulated and 11 downregulated during the neuronal differentiation process. Among the differentially expressed miRs, miR-146a expression was increased most significantly in the nBMSCs that lack immunoregulatory function in EAE, compared with fBMSCs. To confirm by guest on September 30, 2021

Table I. miR expression using MiRCURY array

Log Median Ratios

nBMSC4d nBMSC7d fBMSC SD mmu-miR-451 25.56 24.81 1.44 3.84 mmu-miR-146a 0.45 0.69 24.98 3.21 mmu-miR-99b 0.89 0.51 24.65 3.09 mmu-miR-22 0.39 0.51 24.77 3.02 mmu-miR-144 23.62 24.00 1.14 2.86 mmu-miR-125a-5p 0.54 0.27 23.87 2.47 mmu-miR-155 0.56 1.21 23.30 2.44 mmu-miR-21 0.74 1.04 23.23 2.38 mmu-miR-146b 0.35 0.68 23.42 2.28 mmu-miR-342-3p 1.34 0.34 22.43 1.95 mmu-miR-29c 0.97 1.16 22.00 1.77 mmu-miR-222 0.63 0.61 22.12 1.58 mmu-miR-20a 21.81 21.19 1.13 1.55 mmu-miR-221 0.59 0.53 22.08 1.52 mmu-miR-652 0.96 0.71 21.55 1.38 FIGURE 1. miR expression during BMSC neural differentiation. (A) mmu-miR-24 0.72 0.42 21.80 1.38 The heat-map diagram shows the result of the two-way hierarchical mmu-miR-19a 21.54 20.63 1.15 1.37 clustering of miRs and samples. Each row represents an miR, and each mmu-miR-27b 0.29 0.55 21.93 1.36 column represents a sample. The miR clustering tree is shown on the left, mmu-miR-23b 0.54 0.44 21.83 1.34 and the sample clustering tree appears at the top. The color scale shown at 2 mmu-miR-23a 0.59 0.43 1.75 1.31 the bottom illustrates the relative expression level of an miR across all 2 2 mmu-miR-20b 1.81 1.08 0.71 1.30 samples: red color represents an expression level above mean, and blue mmu-miR-17 21.61 21.03 0.81 1.26 color represents expression lower than the mean. The clustering is per- mmu-miR-20b 21.77 21.14 0.65 1.26 mmu-miR-763 21.58 21.44 0.59 1.22 formed on log2(Hy3/Hy5) ratios, which passed the ﬁltering criteria on mmu-miR-19b 21.29 20.56 1.07 1.21 variation across samples; SD . 1.0. (B) The relative level of miR-146a mmu-miR-29b 0.52 0.72 21.37 1.16 increased after the neuronal differentiation of BMSCs for 4 and 7 d when mmu-miR-18a 21.37 20.61 0.87 1.14 compared with fBMSC and BMSC culture under nondifferentiating con- mmu-miR-27a 0.14 0.47 21.59 1.11 ditions (cBMSC). For the TaqMan real-time RT-PCR, small nucleolar RNA mmu-miR-106a 21.08 20.45 0.89 1.00 was used as an internal normalization control. Representative results (mean 6 2 mmu-miR-29a 0.24 0.52 1.33 1.00 SD) from three independent experiments are shown. The Journal of Immunology 5105

FIGURE 3. Transfer of fBMSCs effectively suppressed PLP-reactive T cell proliferation compared with nBMSCs. Transfection of nBMSCs with miR-146a antagomir effectively restored inhibition of T cell proliferation. Proliferation of PLP-reactive T cells was measured by [3H]thymidine uptake. Bars represent mean cpm (6 SD) from three independent experiments. Signiﬁcant difference in T cell proliferation between EAE and fBMSCs (*p , 0.013) or antagomiR-146a nBMSC-transferred mice (**p , 0.0274). Downloaded from

not find prediction of miR-146a for other genes related to PGE2 synthesis: ptges, ptges3, ptgs1,orptgs2. Thus, PGE2 synthase-2 was selected for further testing in relation to miR-146a control of immunoregulatory function of BMSCs. To investigate whether ptges2 can be directly targeted by miR-146a, we engineered a luciferase reporter construct that contained a 420-bp fragment of the http://www.jimmunol.org/ ptges2 gene. In HEK293 cells, the luciferase reporter was cotrans- FIGURE 2. The miR-146a antagomir reverses the loss of the immuno- fected with the miR-146a precursor (pre–miR-146a) or a nonspe- suppressive effect on EAE of nBMSCs. (A) Transfection of nBMSCs with cific control pre-miR. The results showed that the pre–miR-146a miR-146a antagomir resulted in stable decreased expression of up to 90% significantly reduced luciferase activity, whereas the control pre- of miR-146a. Diagram shows percentage of miR-146a inhibition using miR did not (Fig. 4B). three antagomir concentrations during transfection with Lipofectamine. Representative results (mean 6 SD) of two independent TaqMan real-time Increased expression of miR-146a in nBMSCs correlates with RT-PCR experiments are shown. (B) Expression level of miR-146a was inhibition of PGE synthase-2 and inhibition of PGE2 release confirmed using electrophoresis separation with MultiNA Microchip fBMSCs, which are capable of inhibiting EAE, showed high by guest on September 30, 2021 Electrophoresis system and fluorescence visualization. (C) EAE course was levels of PGE production, whereas nBMSCs that lack EAE im- analyzed by measure of mean clinical score. i.v. injection at the height of 2 clinical disease (arrow) of fBMSCs ameliorated PLP-induced EAE com- munoregulatory function showed significantly lower secretion of pared with untreated animals sensitized for EAE (p , 0.005) and animals PGE2 (Fig. 5A). PGE2 is synthesized by three different PGE syn- transferred with nBMSCs (p , 0.02). Similarly, EAE mice treated with thases: microsomal PGE synthase (mPGES)-1 and -2, and cytosolic cBMSCs showed ameliorated EAE compared with nBMSCs (p , 0.037). PGE synthase (cPGES). Using specific Abs for all PGE2 synthases, However, transfer of nBMSCs transfected with miR-146a antagomir we detected expression of all three syntheses in fBMSCs, but in resulted in amelioration of EAE with a clinical course comparable with nBMSCs, expression of mPGES-2 coded by ptges2 was signifi- fBMSCs (p , 0.5). Each point represents the mean of the clinical score cantly decreased compared with fBMSCs (Fig. 5B). We did not obtained from five to eight animals in each group in three independent observe differences in expression levels of known miR-146a tar- , experiments. SD was 15%. gets, IL-1R–associated kinase 1 and TNFR-associated factor 6, between fBMSCs and nBMSCs (Fig. 5C). MiR-146a is predicted to regulate the ptges2 gene Antagonism of miR-146a restores PGE synthase-2 expression We next mapped miR-146a to its multiple predicted target mRNA and PGE2 production in nBMSCs transcripts in relation to their role in BMSC immunoregulatory To establish the role of miR-146a in regulation of PGE2 produc- function. We used several miR target software (www.mirbase.org tion in BMSCs, we assessed the effect of the miR-146a antagomir [36], www.targetscan.org, and www.microRNA.org) and con- on PGE2 release. The results showed that nBMSCs transfected sistently found that miR-146a was predicted to block PGE2 with miR-146a antagomir had decreased expression of miR-146a synthase-2. It was of a particular interest that the ptges2 gene for (∼90%) compared with control BMSCs cultured under the same PGE2 synthase-2 was found to be high in the miR-146a predicted conditions but not transfected with antagomiR-146a (Fig. 6A). Most pool of genes, because PGE2 has been shown to maintain the im- importantly, in nBMSCs transfected with miR-146a antagomir, we munosuppressive function of BMSCs for allogeneic reactions (19– observed restoration of PGE2 production (Fig. 6B). Furthermore, 21). In all databases, miR-146a was shown to target ptges2 with PGE2 release in nBMSCs transfected with miR-146a antagomir total context score 20.06 (37) and aggregate PCT , 0.1 (38), correlated with increased expression of mPGES-2 (Fig. 6C). fBMSC which demonstrates high probability that ptges2 genes represent coculture with PLP-reactive spleen cells induced strong inhi- a conserved target for miR-146a. Fig. 4A indicates ptges2 se- bition of PLP-induced proliferation. nBMSCs showed a significantly quence target sites of conserved miRs defined by miRSVR (sup- milder inhibitory effect, but transfection with antagomiR-146a en- port vector regression) score # 20.1 (39). miR-146 was the only hanced inhibition of PLP-reactive T cells (p , 0.003; Fig. 6D). miR identified earlier in the Exiqon miRCURY LNA array of To correlate further miR-146a expression levels with PGE2 miRs in nBMSCs that might target ptges2 transcript. We also did synthesis, we performed the reverse experiment and attempted to 5106 miR-146a REGULATES THE IMMUNOREGULATORY ACTIVITY OF BMSC

FIGURE 4. miR-146a specifically interferes with ptegs2.(A) Conserved miR sequence target sites, defined by miR support vector regression score # 20.1, of ptegs2 transcript indicates interference site between miR-146a and ptges2 mRNA 39-UTR (www.microRNA.org). (B) Luciferase reporter assay. In HEK293 cells, the luciferase reporter was cotransfected with the miR-146a precursor and significantly reduced luciferase activity *p , 0.047 (data are calculated as [Sluc/Sgal]/[Cluc/Cgal], where

S = sample, C = control, luc = luciferase activity, Downloaded from and gal = b-galactosidase activity for transfection normalization). Error bar represents the SD from three independent experiments. http://www.jimmunol.org/

induce its expression in cBMSCs cultured in parallel with nBMSCs function similar to fBMSCs. cBMSCs transfected with pre–Mir-

but without the neuronal differentiation conditions. cBMSCs pro- miR-146a construct resulted in an increase in miR-146a expres- by guest on September 30, 2021 duced ample amounts of PGE2 and maintained immunoregulatory sion by 200-fold (Fig. 7A). Furthermore, cBMSCs transfected with pre–Mir-miR-146a showed a decrease in PGE2 production (Fig. 7B), which correlated with mPGES-2 synthesis inhibition (Fig. 7C). Proliferation assay revealed loss of immunosuppressive potential of cBMSCs transfected with pre–miR-146a (Fig. 7D). Thus, using both a loss-of-function approach with the miR-146a antagomir, as well as a gain of function with pre–Mir-miR-146a, we show that miR-146a controls PGE2 synthesis in BMSCs.

Discussion In a previous publication, we showed that freshly isolated Lin2 BMSCs enriched for Sca1 cells transplanted i.v. into mice sensitized for EAE ameliorated the course of disease (40). Freshly isolated Lin2Sca1+ cells expressed a wide panel of molecules characteristic for MSCs. The immunoregulatory activity of Lin- Sca1+ BMSC was dependent on a nondifferentiated state and re- quired secretion of PGE2 (34). In this study, we searched for molecular mechanisms that contributed to the immunoregulatory properties of freshly isolated nondifferentiated BMSCs. Based on recent progress in research identifying the role of miRs in the FIGURE 5. fBMSCs showed significantly higher PGE2 production com- dynamic processes involved in cell differentiation and prolifera- pared with nBMSCs. (A) BMSCs cultured under differentiation conditions for tion, we focused our studies on miRs (22). miR profiling of fBMSC 4and7d(p , 0.0012 and p , 0.0001, respectively) released significantly population, with high immunoregulatory activity, analyzed against less PGE2 than fBMSCs or BMSCs cultured with nondifferentiating media BMSCs differentiated toward a neuronal phenotype (nBMSC), (cBMSCs). Bars represent mean results (6 SD) from three experiments. (B) without immunoregulatory activity, showed a number of miRs that Western blot analysis using specific Abs for PGE2 synthases showed expression of all three isoforms in BMSC, but following differentiation to nBMSC mPGES- were differentially expressed. Nineteen miRs were upregulated 2 levels decreased markedly. (C) We did not observe differences in expression and 11 downregulated as a result of the neuronal differentiation levels of known miR-146a targets, IL-1R–associated kinase 1 and TNFR- process. The most highly differentially expressed miR was miR- associated factor 6, between fBMSCs and nBMSCs. In experiments, equal 146a, which in BMSCs cultured under neuronal differentiation amounts of protein level per sample were loaded. conditions for 4–7 d was increased 500-fold. Because nBMSCs The Journal of Immunology 5107

FIGURE 6. Antagonizing miR-146a during BMSC neuronal differentiation restores specifically PGE synthase-2 expression and PGE2 secretion. nBMSC transfection with miR-146a antagomir significantly decreased miR-146a expression assessed with RT-PCR (A) and restored secretion of PGE2 as measured by ELISA (B). (C) Restoration of PGE2 secretion in nBMSCs transfected with miR-146a antagomir correlated with increased expression of mPGES-2, but not with cPGES or mPGES-1. (D) Proliferation of PLP-reactive T cells was measured by [3H]thymidine uptake. Bars represent mean cpm (6 SD) from three independent experiments. In Western blot experiments, equal amounts of protein level per sample were loaded. fBMSC coculture with PLP-reactive spleen cells induced strong inhibition of PLP-induced proliferation. nBMSCs showed a significantly milder inhibitory effect, but transfection Downloaded from with antagomiR-146a enhanced inhibition of PLP-reactive T cells (*p , 0.003). http://www.jimmunol.org/ expressing high miR-146a lost immunoregulatory function, we activity of BMSCs, we mapped miR-146a to its multiple predicted hypothesized that miR-146a might negatively regulate this property target mRNA transcripts using three independent miRs databases. of BMSCs. We verified this assumption by experiments in which Interestingly, miR-146a was found to interfere with the ptges2 weselectivelyinhibitedmiR-146ainnBMSCsbytransfection gene that codes for PGE2 synthase-2, in all three databases. PGE2 with a specific anti–miR-146a antagomir. nBMSCs transfected in earlier studies was found to maintain the immunosuppressive with the miR-146a antagomir showed a reduction in miR-146a activity of BMSCs toward allogeneic reactions. The immuno- expression and restoration of immunoregulatory activity in EAE. suppressive function of MSCs was noted in the absence of direct

The miR-146 family is composed of two members, miR-146a cell contact, indicating a role of soluble mediators like PGE2 in by guest on September 30, 2021 and miR-146b. To date, a role for miR-146 has been demon- dendritic cell maturation and inhibition of activated T lymphocyte strated in the development of multiple cancers (41) and in the proliferation (21); PGE2 also contributed to the modulation of negative regulation of inﬂammation induced via the innate im- cytokine production by T cells (20). MSC-mediated immunoreg- mune response (42). miR-146a expression has been shown to be ulation by PGE2 was implicated in inhibition of TNF-a and IFN-g induced in response to LPS, TNF-a, or IL-1b activation of TLR-2, production by activating dendritic cells and T cells (19). Of par- -4, or -5 (43, 44). Searching for miR-146a targets that might be of ticular interest was that, in our previous study, we found that im- signiﬁcance in the negative regulation of the immunoregulatory munoregulation of EAE by BMSCs was dependent on PGE2

FIGURE 7. Transfection of cBMSCs with pre–miR-146 leads to an increase in miR-146

and a decrease in PGE2. cBMSC transfection with pre–Mir-miR-146a construct resulted in an increase in miR-146a expression by 200-

fold (A) and decreased production of PGE2 in cBMSCs assessed by ELISA (B). Results were compared with transfection with pre- Mir–control. Error bar represents the SD from three independent experiments. (C)

Decreased PGE2 secretion in pre–miR-146a transfected cBMSCs was correlated with mPGES-2 synthesis inhibition assessed by Western blot. (D) Proliferation assay revealed loss of immunosuppressive potential of cBMSCs transfected with pre–miR-146a (p , 0.0066). Proliferation of PLP-reactive T cells was measured by [3H]thymidine uptake. Bars represent mean cpm (6 SD) from three independent experiments. In Western blot experiments, equal amounts of protein level per sample were loaded. 5108 miR-146a REGULATES THE IMMUNOREGULATORY ACTIVITY OF BMSC secretion (34). Freshly isolated BMSCs produced high amounts of 9. Petersen, B. E., W. C. Bowen, K. D. Patrene, W. M. Mars, A. K. Sullivan, N. Murase, S. S. Boggs, J. S. Greenberger, and J. P. Goff. 1999. Bone marrow as PGE2 and suppressed EAE development, whereas neuronal dif- a potential source of hepatic oval cells. Science 284: 1168–1170. ferentiated BMSCs lost PGE2 secretion and were not able to 10. Brazelton, T. R., F. M. Rossi, G. I. 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