microrna-663 induces immune dysregulation by β1 production

ARTICLE

MicroRNA-663 induces immune dysregulation byinhibiting TGF-β1 production in bone marrow-derivedmesenchymal stem cells in patients with systemic lupuserythematosus

Linyu Geng1,6, Xiaojun Tang1,6, Kangxing Zhou1,6, Dandan Wang1, Shiying Wang1, Genhong Yao1,Weiwei Chen1, Xiang Gao2, Wanjun Chen3, Songtao Shi4, Nan Shen5, Xuebing Feng1 andLingyun Sun1

Mesenchymal stem cells (MSCs) are critical for immune regulation. Although several microRNAs (miRNAs) havebeen shown to participate in autoimmune pathogenesis by affecting lymphocyte development and function, theroles of miRNAs in MSC dysfunction in autoimmune diseases remain unclear. Here, we show that patients withsystemic lupus erythematosus (SLE) display a unique miRNA signature in bone marrow-derived MSCs (BMSCs)compared with normal controls, among which miR-663 is closely associated with SLE disease activity. MiR-663inhibits the proliferation and migration of BMSCs and impairs BMSC-mediated downregulation of follicular T helper(Tfh) cells and upregulation of regulatory T (Treg) cells by targeting transforming growth factor β1 (TGF-β1).MiR-663 overexpression weakens the therapeutic effect of BMSCs, while miR-663 inhibition improves theremission of lupus disease in MRL/lpr mice. Thus, miR-663 is a key mediator of SLE BMSC regulation and mayserve as a new therapeutic target for the treatment of lupus.Cellular and Molecular Immunology advance online publication, 26 March 2018; doi:10.1038/cmi.2018.1

Keywords: immune dysregulation; mesenchymal stem cells; miR-663; systemic lupus erythematosus; transforminggrowth factor β1

INTRODUCTION

Mesenchymal stem cells (MSCs), originally isolated from thebone marrow stroma, exhibit potent immunoregulatory func-tion by inhibiting the proliferation and function of majorimmune cells, including T and B lymphocytes, natural killercells and dendritic cells.1 MSCs have been shown to have greatpotential for clinical applications in the treatment of immunedisorders. Systemic lupus erythematosus (SLE) is a prototypicautoimmune disease characterized by systemic autoantibodyproduction and inflammatory cell infiltration in target organs.2

Previously, we have shown that bone marrow-derived MSCs(BMSCs) from SLE patients exhibit abnormalities in immunemodulation,3–7 and allogeneic normal MSCs show therapeuticeffects in both lupus mice and severe refractory SLE patients.8–12

However, despite ample evidence for the therapeutic potential ofMSCs in SLE, the mechanisms by which MSCs exert theirimmunomodulatory effects are incompletely understood.

MicroRNAs (miRNAs) are a novel class of endogenous,non-coding small RNAs of ~ 19–25 nucleotides in length andhave been recognized as important negative modulators of

1Department of Rheumatology and Immunology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China; 2KeyLaboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing 210000, China; 3Mucosal Immunology Section,OPCB, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda 20892-2190, MD, USA; 4Department of Anatomy andCell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6004, PA, USA and 5Joint Molecular Rheumatology Laboratory ofthe Institute of Health Sciences and Shanghai Renji Hospital, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and ShanghaiJiaotong University School of Medicine, Shanghai, China

Correspondence: Dr Xuebing Feng or Lingyun Sun, Department of Rheumatology and Immunology, The Affiliated Drum Tower Hospital of Nanjing UniversityMedical School, 321 Zhongshan Road, Nanjing 210008, China.E-mail: [email protected] or [email protected]

6These authors contributed equally to this work.

Received: 20 July 2017; Revised: 17 December 2017; Accepted: 22 December 2017

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genes in eukaryotic organisms. SLE patients have uniquemiRNA signatures in peripheral blood cells, body fluid andtarget tissues, such as kidney when compared with normalcontrols or patients with other diseases.13–15 Several miRNAs,including miR-155, miR-146a and miR-126, have been shownto affect the functions of T and B cells, thereby modulatingautoimmune pathogenesis.16,17 Recently, several studies haveidentified critical roles for miRNAs in the proliferation,migration and differentiation of MSCs, suggesting that theymight have an impact on the acquisition of reparative MSCsphenotypes.18 However, the roles of miRNAs in MSCs duringautoimmune pathogenesis remain to be elucidated.

Follicular T helper (Tfh) cells aid effector B cells andaugment autoimmunity.19 In contrast, regulatory T (Treg) cellsappear to inhibit B-cell responses by suppressing the activitiesof Tfh cells, and by doing so, prevent lupus development.20,21

The imbalance between T cell subsets is important in auto-immune disease including SLE pathogenesis,22 but the under-lying mechanisms remain unclear. In this study, usingcomparative miRNAs profilings in BMSCs, we identifiedmiR-663 as a candidate miRNA for SLE pathogenesis.MiR-663 downregulated the secretion of transforming growthfactor β1 (TGF-β1) by BMSCs, and consequently increased thepercentage of Tfh cells but decreased the frequency of Treg cellsin vitro. Importantly, the therapeutic effect of BMSC trans-plantation in MRL/lpr mice was substantially improved andimpaired after the inhibition and overexpression of miR-663 inBMSCs, respectively. Thus, miR-663 in MSCs may serve as anew target for autoimmune diseases associated with a Tfh/Tregcell imbalance.

MATERIALS AND METHODS

Patients and controlsA total of 13 SLE patients (mean age of 34.9± 7.6 years) wereincluded in the BMSC study. BMSCs from 4 SLE patients wereused for miRNA array analysis, and RT-PCR was performedwith BMSCs from another 9 SLE patients. All patients fulfilled4 or more criteria according to the revised 1997 AmericanCollege of Rheumatology criteria for SLE46 and had SLEDAI(Systemic Lupus Erythematosus Disease Activity Index) scoresgreater than 6 at the time of bone marrow extraction.46 For theBMSC study, 10 normal controls (mean age of 38± 6 years)were recruited as normal controls, and 3 patients with primarySjögren’s syndrome (SS, mean age of 42± 7 years) wererecruited as disease controls with efforts to match age andgender. In total, for PBMCs and serum RT-PCR analysis, 39normal controls (mean age of 34± 5 years), 22 SS patients(mean age of 39± 8 years), 28 SLE patients (mean age of 37± 7years), and 9 other types of connective tissue diseases (meanage of 41± 9 years) were included in the analysis. Thedemographic and clinical characteristics of these patientsamples are presented in Supplementary Table 3. All partici-pants provided written consent to participate in the study,which was approved by the Ethics Committee of the AffiliatedDrum Tower Hospital of Nanjing University Medical School.

BMSC cultureBone marrow mononuclear cells were isolated from bonemarrow obtained from the iliac crest from all the patients andnormal controls by using 1.073 g/ml Ficoll separation medium(TBD, China), and resuspended at a density of 2 × 107 cells per25 cm2 dish in low glucose Dulbecco’s Modified Eagle’sMedium (L-DMEM) (Gibco, USA) supplemented with 10%heat inactivated fetal bovine serum (FBS) (Invitrogen, USA)and 1% antibiotic-antimycotic solution for adherent screeningculture at 37 °C in a humidified 5% CO2 incubator (MCO--15AC, SANYO, Japan). Medium containing non-adherentcells was replaced after 48 h and then every 3 days. Cells grownto 90% confluency were recovered with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco, USA) andreplated at a density of 1 × 106 per 25 cm2. At passage 4, cellswere identified by flow cytometry (FCM) based on positivestaining (495%) for CD29, CD44, CD90, CD73 and CD166and negative staining (o2%) for CD45, CD34, CD19, HLA-DR and CD133 (Supplementary Fig. 1).

MRL/lpr miceFemale MRL/lpr mice were purchased from the Model AnimalResearch Center of Nanjing University. All mice were fedstandard chow diet at all ages and maintained in atemperature-controlled room with a 12-h light/dark cycleaccording to the approved protocol by the Affiliated DrumTower Hospital of Nanjing University Medical School Com-mittee for the use and care of experimental animals. To observethe in vivo effects of miR-663, BMSCs were transfected withmiR-663-C, miR-663-M, and miR-663-I eukaryotic expressionvectors and intravenously injected (1× 106) into 16-week-oldMRL/lpr mice, with the FLS injection group (1× 106) as thebackground control as described previously.27,28

Eight weeks later, serum samples and urine from eachgenotype of female mice were collected. The mice were thenkilled by cervical dislocation, and kidney and lymph node werecollected and spleen was weighed. The value (mg) was dividedby the body weight (g) and then multiplied by 10 to determinethe splenic index. Lumbar vertebrae and limbs were excised toobtain mononuclear cells from bone marrow, and then mouseBMSCs (mBMSCs) were cultured in vitro to prepare for thenext proliferation and migration study by CFSE staining andthe trans-well assay, respectively, as described below. Total IgG,IgG anti-ds-DNA and ANA were measured using a commercialELISA kit (R&D Systems, Inc., MN) according to the manu-facturer’s protocol. Cytokine levels were detected usingenzyme-linked immunosorbent assay kits. Proteinuria in freshurine was examined using the Coomassie blue staining assay(Bayer, Elkhart, IN).

ImmunohistochemistryHalf of the kidney was fixed in 10% formaldehyde andembedded in paraffin. Three-micrometer-thick sections ofkidney tissue were cut and observed for various morphologicallesions after hematoxylin-eosin (H&E) staining. Glomerularpathology was evaluated by assessing 20 glomerular cross-

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sections (gcs) per kidney and scored for each glomerulus on asemi-quantitative scale (0–3).47 The other half of the kidneywas embedded in Tissue-Tec OCT medium, frozen in liquidnitrogen, and stored at − 70 °C until sectioning. Five-micrometer-thick frozen sections were fixed with 4% paraf-ormaldehyde and blocked with 2% BSA. Subsequently, slideswere stained with goat anti-mouse IgG (1:150 dilutions;Abcam, Cambridge, UK) or goat anti-mouse complement C3(1:150 dilutions; Abcam, Cambridge, UK). The sections werethen stained with FITC-labeled anti-goat antibodies anddigitally photographed using a fluorescence microscope fittedwith a digital camera (Cannon Power shot G10, Cannon, Inc.).The fluorescence intensity within the peripheral glomerularcapillary walls and mesangial region were scored on a scalefrom 0 to 3 (0=none; 1=weak; 2=moderate; 3= strong).47

At least 10 glomeruli/section were analyzed.Lymph node samples were fixed for 2 h in 4% paraformal-

dehyde on ice, incubated in six changes of sucrose bufferovernight and embedded in Tissue-Tek OCT compound(Sakura Finetek). Sections were blocked with a Streptavidinand Biotin Blocking Kit (Vector Laboratories) and then stainedwith an antibody against mouse Foxp3 (Abcam, Cambridge,UK), followed by donkey anti-mouse antibody labeled withfluorescein isothiocyanate (FITC) (Abcam, Cambridge, UK).Then, the sections were stained with a biotinylated antibodyagainst mouse Bcl6 (Santa Cruz) followed by streptavidin-HRPconjugates and TSA tetramethylrhodamine. Next, the sectionswere stained with 4 ′,6-diamidino-2-phenylindole (DAPI,Sigma, USA) for 3–5min. Finally, the stained sections weredigitally photographed using a fluorescence microscope fittedwith a digital camera (Cannon Power shot G10, Cannon, Inc.)using a × 20 objective.

MicroRNA array analysisTotal RNA containing small RNA was extracted from BMSCsusing a mirVana miRNA Isolation Kit (Ambion, Austin, TX,USA). The purity and concentration of the RNA weredetermined from OD260/280 readings using a spectrophot-ometer (NanoDrop ND-1000). The RNA integrity and con-centration were determined by capillary electrophoresis usingthe RNA 6000 Nano Lab-on-a-Chip kit and the Bioanalyzer2100 (Agilent Technologies, Santa Clara, CA, USA). Only RNAextracts with RNA integrity number values ⩾ 6 underwentfurther analysis. MiRNA profiling was performed using aGeneChip miRNA Array (Affymetrix, Santa Clara, CA, USA).The array comprised 7815 probe sets, of which 6703 providedmiRNA coverage of human, mouse, rat, canine and others (71organisms) from the Sanger miRNA database (V.11) and anadditional 922 encompassed human snoRNAs and scaRNAs(from the Ensembl database and snoRNABase). Control targetswere also included in the array containing 95 backgroundprobe sets, 22 oligonucleotide spike-in control probe sets and10 identical probes for human 5.8s rRNA, and hybridizationcontrol probe sets. Microarray experiments were conductedaccording to the manufacturer's instructions. Briefly, 1 μg totalRNA was labeled with the Biotin FlashTag Biotin Labeling Kit

(Affymetrix). The labeling reaction was hybridized on themiRNA Array in an Affymetrix Hybridization Oven 640(Affymetrix) at 48 °C for 16 h. The arrays were stained with aFluidics Station 450 using fluidics script FS450_0003 (Affyme-trix) and then scanned on a GeneChip microarray scanner(Affymetrix).

For the microarray data analysis, after removing the adaptorsequence, miRNA probe outliers were defined according to themanufacturer's instructions (Affymetrix) and further analyzedfor data summarization, normalization and quality controlusing the miRNA QC Tool software (www.affymetrix.com). Todetermine the significant differentially expressed genes, Sig-nificance Analysis of Microarrays (SAM, version 3.02) wasperformed. To select differentially expressed genes, we usedthreshold values ⩾ 2, a ⩽− two fold change and an FDRsignificance level of o5%. The data were Log2-transformedand median-centered by gene using the adjust data function inthe CLUSTER 3.0 software and then analyzed by hierarchicalclustering with average linkage. Finally, tree visualization wasperformed using Java Treeview (Stanford University School ofMedicine, Stanford, CA, USA).

Quantitative real-time polymerase chain reaction (RT-PCR)analysisTotal RNAs in passage 4 BMSCs or PBMCs were extractedusing TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) accord-ing to the manufacturer’s instructions. MicroRNAs from eachserum sample were extracted using the mirVana miRNAIsolation Kit (Ambion, Austin, TX, USA) according to themanufacturer's protocol. RNA integrity was determined usingformaldehyde denaturalization agarose gel electrophoresis.RNA concentrations were measured using a smartspecTM plusspectrophotometer (BIO-RAD, Hercules, CA, USA). cDNAwas generated using SuperScript III First Strand SynthesisSuperMix (Takara, Dalian, China), a TaqMan MicroRNAReverse Transcript Kit (Applied Biosystems), or the MultiplexRT pool set (Systems Biosciences). TaqMan probes forindividual miRNAs were purchased from Applied Biosystems.Internal housekeeping controls were β-actin，U6 or miR-16.Quantification of mRNA and mature miRNA was performedon an ABI 7500 FAST real-time PCR detection system (AppliedBiosystems, USA) as previously described. Specific primeroligonucleotides (TaKaRa, Dalian, China) were used, and therelative expression of target genes was calculated with the2−△△Ct method.

FCM analysisThe following antibodies were applied in this study: fluoresceinisothiocyanate (FITC)-conjugated anti-human CD4, HLA-DR(BD Biosciences), CD34 and CD44 (BD eBioscience), phycoer-ythrin (PE)-conjugated anti-human CD4 (BD Biosciences),CD45, CD29, CD166, CD138 and Foxp3 (eBioscience), allophy-cocyanin (APC)-conjugated anti-human CD25 (BD Biosciences),CD19 (eBioscience), B220 (eBioscience), phycoerythrin-Cy7-conjugated anti-human IFN-γ (eBioscience) and their isotype-matched control antibodies (mouse IgG1, mouse IgG2a).


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Western blot analysis and ELISA assayWe used antibodies recognizing human Smad2/3, p38/MAPK,Akt and their phosphorylation forms, p-Smad2/3, p-p38/MAPK, p-Akt and GAPDH (Cell Signaling Technology Inc.,1:1000) to examine the concentrations of proteins in MSClysates. We detected the amounts of active and total TGF-β1 inthe conditioned medium and/or human serum with ELISA kits(eBioscience or BioLegend) according to the manufacturers’instructions.

Plasmid construction, transfection and reporter assayPlasmid vectors (Supplementary Fig. 2a, b) containing miR-663negative control (miR-663-C), pri-miR-663 (miR-663-M) andinhibitor-miR-663 (miR-663-I) were purchased from Invitro-gen (Life Technologies, USA) using the following sequences(Supplementary Table 6). Transient transfection of theseplasmids into BMSCs was performed by electroporation usingthe 4D-nucleofector system (Lonza, Germany) according to themanufacturer’s instructions.

DNA fragments of TGF-β1 with or without 3′-UTR (144 bp)extracted from the cDNA library were cloned into the PGL3luciferase vector (Promega, USA) via the XbaI 1934 site(Supplementary Fig. 3e). Primers for TGF-β1 without the3′-UTR were as follows: forward primer, 5′-CCGGAATTCGCCACCATGCCGCCCTCCGGGCTG-3′; reverse primer, 5′-CTAGTCTAGATCAGCTGCACTTGCAGGAG-3′. Primers for full-length TGF-β1 were: forward primer, 5′-CCGGAATTCGCCACCATGCCGCCCTCCGGGCTG-3′; reverse primer, 5′-CTAGTCTAGACCGCAGTCCTCTCTCCATC-3′. Transfection ofHEK293T cells with miRNAs was performed using Lipofecta-mine 2000 (Invitrogen, USA). HEK293T cells were co-transfected with the luciferase reporter constructs (200 ng)and appropriate miRNA plasmids. After 36 h, the cells werewashed and lysed with passive lysis buffer (Promega, USA), andf-luc and renilla luciferase (r-luc) activities were determinedusing the dual-luciferase reporter assay system (Promega,USA). Relative reporter activity was obtained by normalizationto the r-luc activity.

Cell proliferation and apoptosis assayBriefly, for the proliferation assay, 106 cells/ml (BMSCs orpurified T cells) were incubated with 3 μM carboxyfluoresceindiacetate succinimidyl ester (CFSE, Invitrogen, Camarillo, CA,USA). For the apoptosis assay, miR-663-transfected BMSCs(1× 106/well) were stained with the Annexin V/7AAD apopto-sis detection kit Annexin V (BD Biosciences). After culturingfor 4–5 days, cells were collected for examination by FCM, andthen they were analyzed using Flowjo v10.0.7.

Trans-well migration assayBMSCs at a density of 5 × 105 cells/ml in 0.2 ml L-DMEM(Gibco, USA) without FBS (Invitrogen, USA) were added tothe upper chamber of a 6.5-mm-diameter trans-well insert(8 μM pore size, Millipore). The lower chamber in 24-wellplates contained 0.5 ml of L-DMEM (Gibco, USA) with 10%FBS. After incubation at 37 °C and 5% CO2 for 12 h, the upper

surface of the membrane was gently scraped to remove non-migrating cells. Cells on the lower surface of the membranethat had migrated into the lower compartment of the chamberwere then fixed in 4% paraformaldehyde for 10min andstained with Giemsa solution or DAPI (Sigma, USA) for 3–5min. The number of migrating cells was quantified usingImage Pro-Plus 6.0 in five random morphological fields perwell. The values were averaged and then multiplied by the ratioper microscopic field area to the bottom area per 24 wells.

BMSC-PBMC co-culturePBMCs were isolated from heparinized venous blood by Ficoll-Paque gradient centrifugation (Takara, Dalian, China) and co-cultured with or without pre-plated miR-663-transfectedBMSCs at a ratio of 10:1 in 96-well flat-bottomed plates inthe presence of soluble anti-human CD3 (1 μg/ml) and anti-human CD28 (1 μg/ml) antibodies in a final volume of 200 μlRM1640 medium (Gibco, USA). Recombinant human TGF-β1(5 ng/ml; R&D Systems, USA) or anti-human TGF-β1 antibody(10 μg/ml; R&D Systems, USA) was added to the culturesystem to determine the role of TGF-β1 in the miR-663-mediated effect on BMSCs. After 72 h, non-adherent cells werecollected for FCM analysis, and supernatants were collected forcytokine measurement. The adherent BMSCs were washed andlysed with TRIzol (Takara, Dalian, China) for RT-PCR analysis.To rule out possible cell–cell contact effects, a trans-well system(0.4 μM pore size, Millipore) was applied.

Differentiation assayPBMCs were isolated from peripheral blood using Ficolldensity-gradient centrifugation. CD4+CD25- T cell subsets ornaïve CD4+ T cells were isolated and purified using a humanCD4+CD25+ regulatory T cell isolation kit or naïve CD4+ T cellisolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany)according to the manufacturer’s instructions. Then, CD4+

CD25- T cells (1× 106/well) were cultured with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) antibodies, with theaddition of recombinant human TGF-β1 (10 ng/ml; R&DSystems, USA) and IL-2 (100 U/ml; Peprotech, USA) to induceTreg cell conversion. After culturing for 5– 6 days, the cells werecollected for the measurement of CD4+CD25+ percentagesby FCM.

In addition, isolated naive CD4+ T cells (1 × 106/well) werestimulated with soluble anti-CD3 (1 μg/ml) and anti-CD28(1 μg/ml) antibodies, with the addition of recombinant humanIL-2 (100 U/ml; Peprotech, USA), IL-6 (20 ng/ml; Peprotech,USA), anti-IL-4 (10 μg/ml; R&D Systems, USA), anti-IFN-γ(10 μg/ml; R&D Systems, USA) and anti-TGF-β (10 μg/ml;R&D Systems, USA). After 5–6 days, the cells were collected forthe measurement of CXCR5++PD-1++CD4+ T cell or CXCR5++PD-1++ Foxp3+CD4+ T cell percentages by FCM.

In vivo homing assayTo determine the in vivo homing capacity, BMSCs were labeledwith PKH26 (Sigma, USA) according to the manufacturer’sprotocol, with a labeling efficacy 498%. Labeled cells were


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infused into 24-week-old MRL/lpr mice, and the mice werekilled 24, 48 and 72 h after infusion, with organs includingheart, liver, spleen, lung, kidney and lymph nodes collected andwrapped in the dark at − 80 °C. To quantify BMSC engraftmentin organ tissue, samples were stained with Giemsa solution or4′,6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 3–5min, fixed in 4% paraformaldehyde (Electron MicroscopySciences, PA), and embedded in 30% sucrose/PBS and inTissue-Tek OCT Compound (Sakura Finetek, CA). Fifteensections per organ were analyzed using a fluorescence invertedmicroscope (Axio observer A1, ZEISS, Germany), and imageswere acquired with an objective magnification of × 10 (×40total magnification) using an Olympus DP30BW camera(Olympus, Japan).

Statistical analysisThe data are shown as the mean± SEM. Differences betweentwo groups were determined using an unpaired Student’s t-testif the variance was normally distributed. Comparisons amongthree or more groups were conducted using one-way ANOVA.Data were calculated using GraphPad Prism 5 software, and avalue of Po0.05 was considered statistically significant.

RESULTS

The miR-663 level is increased in SLE BMSCsTo determine whether there were differentially expressedmiRNAs in SLE BMSCs, we performed comparative miRNAscreenings between SLE patients (n= 4) and normal controls(NOR, n= 4) using human miRNAs arrays containing 194miRNAs (Supplementary Table 1). Compared with normalcontrols, 8 miRNAs were highly expressed and 4 miRNAs weredecreased in SLE BMSCs (Figure 1a, Supplementary Table 2).Next, the data from miRNAs arrays were confirmed by real-time PCR (RT-PCR) in another set of BMSCs from 9 SLEpatients and 6 age- and gender- matched normal controls. Ofthe verified miRNAs, 4 (miR-663, miR-638, miR-214 andmiR-574-3p) remained increased and 2 (let-7 f and miR-374a)remained reduced in SLE BMSCs (Figure 1b). It has previouslybeen reported that several miRNAs, including miR-155, miR--126, miR-125a, miR-146a, miR-150, miR-181a and miR-21, inperipheral blood or kidneys are linked to SLE patients.16

However, we found that none of these SLE-associated miRNAswas abnormally expressed in SLE BMSCs, except that themiR-125a level was decreased in the patient group (Figure 1c).Thus, SLE patients may present a distinct BMSC miRNAsignature compared with normal controls.

Among the 6 verified miRNAs (Figure 1b) in SLE BMSCs,miR-663 had previously been implicated to participate inimmune regulation.23 Our data showed that the miR-663 levelwas positively correlated with SLE clinical spectrums, includingthe SLE disease activity index (SLEDAI, Figure 1d) and theerythrocyte sedimentation rate (ESR) and levels of C-reactiveprotein (CRP) and complement 3 (C3, data not shown),suggesting that miR-663 is potentially involved in the patho-genesis of SLE. Consistent with the observations in BMSCs,miR-663 expression in SLE serum, but not in peripheral blood

mononuclear cells (PBMCs), was also increased and associatedwith the SLEDAI score (Figures 1e–g, Supplementary Table 3).Unlike SLE, patients with Sjögren's syndrome (SS) did notshow high levels of miR-663 in their BMSCs (Figure 1h).Consequently, miR-663 was selected for further investigation.

MiR-663 is involved in BMSC proliferation, apoptosis andmigrationTo investigate the biological functions of miR-663 in BMSCsfrom normal controls, eukaryotic expression vectors(Supplementary Fig. 2a, b; Supplementary Table 5) of pri-miR-663 (miR-663-M) and inhibitor-miR-663 (miR-663-I)were generated. Compared with the negative control vector(miR-663-C)-transfected BMSCs, miR-663 production wasincreased in miR-663-M-transfected BMSCs and decreased inthe miR-663-I group (Supplementary Fig. 2c, d). MiR-663-Mectopic expression significantly suppressed proliferation(Figures 2a and b), promoted apoptosis (Figures 2c and d)and decreased the migration capacity (Figures 2e and f) ofBMSCs in vitro. Conversely, inhibition of miR-663 bymiR-663-I transfection resulted in opposite effects (Figure 2).As increased apoptosis, reduced proliferation and migrationconstituted the characteristic abnormalities of SLE BMSCs,3,4,6

the increased expression of miR-663 in BMSCs may contributeto the pathogenesis of SLE.

MiR-663 inhibits the immunoregulatory effect of BMSCs onTfh/Treg cellsTo explore whether miR-663 could affect the immunoregula-tory function of BMSCs, BMSCs were co-cultured withperipheral blood mononuclear cells (PBMCs) of NOR at aratio of 1:10 for 3 days. Compared with miR-663-C-transfectedBMSCs, there was no alteration of the T helper (Th) 1, Th2and Th17 subsets after co-culturing with miR-663-M ormiR-663-I-transfected BMSCs (Figure 3a, Supplementary Fig.3a-b). However, the frequency of Treg cells (CD25+Foxp3+/CD4+ T cells, Figure 3b) was upregulated, while the frequencyof Tfh cells (CXCR5++PD-1++/CD4+ T cells, Figure 3c,Supplementary Fig. 3c) and plasma cells (CD19-CD138+ cells,Figure 3d) were downregulated in miR-663-I-transfectedBMSC co-cultures. In contrast, a decreased frequency of Treg

cells but increased frequency of Tfh cells, as well as plasma cellswere observed in miR-663-M-transfected BMSC co-cultures(Figures 3b–d, Supplementary Fig. 3c). Consequently, the ratioof Tfh/Treg was significantly increased in the miR-663-M groupsbut decreased in the miR-663-I groups (Figure 3i), suggestingthat miR-663 controls the BMSC-mediated Tfh/Treg cellbalance.

To understand how miR-663 in BMSCs interferes with Tfh/Treg cells, we co-cultured miR-663-transfected BMSCs withCD4+CD25+ and CD4+CD25- T cells isolated from normalhuman PBMCs. The proliferation rate and absolute number ofFoxp3+CD4+CD25+ T cells were elevated in the miR-663-Igroup, but were reduced in the miR-663-M group comparedwith miR-663-C co-cultures (Figures 3e and f; SupplementaryFig. 3d), suggesting that miR-663 suppresses BMSC-mediated


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natural (thymic) Treg cells (nTreg) growth. In addition, our datashowed that miR-663-I BMSCs increased the differentiation ofTreg cells (iTreg) from CD4+CD25- T cells, but miR-663-MBMSCs decreased Treg cell differentiation (Figure 3g). Since ithas been recently reported that Treg cells may be converted intoTfh cells,24 we next investigated whether miR-663 could also

control the phenotypic conversion of Treg cells to Tfh cells. Asexpected, miR-663-M-transfected BMSCs significantlyincreased the conversion of CD4+CD25+ T cells to Tfh cellscompared with miR-663-C-transfected BMSCs, while there wasa trend toward a decrease in Tfh cell conversion in themiR-663-I group (Figure 3h). Thus, miR-663 in BMSCs may

Figure 1 MiR-663 expression is increased in SLE BMSCs. (a) Screening of miRNAs in BMSCs from SLE patients and normal controls(NOR). (b) RT-PCR validation of miRNA expressions in BMSCs from 9 SLE patients and 6 NOR. (c) Expression of SLE-associated miRNAsin BMSCs. (d and g) Association of miR-663 expression in BMSCs (d) and serum (g) with the SLE disease activity index (SLEDAI) score.(e,f) MiR-663 expression in serum and peripheral blood mononuclear cells (PBMCs) from SLE patients, Sjögren's syndrome (SS) patientsand NOR. For others, patients with rheumatoid arthritis (RA) and dermatomyositis (DM) were included. (h) MiR-663 expression in BMSCsfrom patients with SS and umbilical cord-derived MSCs (UCMSCs). All data represent the mean±SEM. *Po0.05, **Po0.01,***Po0.001.


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downregulate the frequency of Treg cells not only by inhibitingnTreg cell proliferation and iTreg cell differentiation but also byconverting Treg cells toward a Tfh cell phenotype.

MiR-663 acts through targeted regulation of the TGF-β13’UTRIt has been widely recognized that miRNAs exert their functionsthrough the regulation of target genes. To identify candidatemiR-663 target genes, two computational methods, Targetscan(http://www.targetscan.org) and PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html), were applied, andCapitalBio Molecule Annotation System V3.0 was used toperform pathway analysis on the putative miR-663 target genes(http://bioinfo.capitalbio.com/mas3/). According to the predic-tion, several molecules, including TGF-β1 were proposed aspotential targets of miR-663 (Supplementary Fig. 4a).

Interestingly, among the predicted targets, only TGF-β1mRNA (Supplementary Fig. 4b) and protein levels

(Figure 4a) were significantly lower in SLE BMSCs thanthat in normal BMSCs, and TGF-β1 derived from BMSCswas involved in upregulating Treg cells (Figure 4b) andpromoting BMSC migration (Figure 4c). In addition, bothactive and total protein levels of TGF-β1 secreted by BMSCswere further elevated in the miR-663-I group (Figure 4d).MiR-663-I transfection markedly increased TGF-β1 mRNAexpression in BMSCs but had no effect on TNF-α, IFN-α,IL-1β, JUND, GRID2D and EPHB3 (Supplementary Fig. 4c).To further confirm the role of TGF-β1 in miR-663-mediatedBMSC dysfunction, exogenous human recombinant TGF-β1and anti-TGF-β1 antibody were applied. Consistently,miR-663-M-modified dysregulation of Treg cell differentiationand dysfunction of BMSC migration capacity were restored byrecombinant TGF-β1, while the increased Treg cell differentia-tion (Figure 4e) and BMSC migration capacity (Figure 4f) bymiR-663-I transfected BMSCs were abolished by anti-TGF-β1antibody.

Figure 2 MiR-663 affects BMSC proliferation, apoptosis and migration in vitro. (a and b) Proliferation status of normal BMSCs (miR-663-N), normal BMSCs transfected with control vector (miR-663-C), pri-miR-663 (miR-663-M) or inhibitor-miR-663 (miR-663-I) by the CFSEassay. (c and d) Apoptotic status of BMSCs among the 4 groups using the Annexin V and 7-AAD staining assay. Dot plots showing thefrequency of apoptosis (Annexin V+7-AAD- early apoptotic cells and Annexin V+7-AAD+ late apoptotic cells) of BMSCs among differentgroups. (e and f) Migrated BMSC numbers per field in the 4 groups. The yellow arrow refers to the BMSC nucleus stained with Giemsasolution. All data represent the mean±SEM. n=6, *Po0.05, **Po0.01.


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http://www.targetscan.org

http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html

http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html

http://bioinfo.capitalbio.com/mas3/

Figure 3 MiR-663 downregulates the immunoregulatory effects of BMSCs in vitro. (a–d) Percentages of Th17 cells (IL-17 A+/CD4+ T cells,(a), Treg cells (CD25+Foxp3+/CD4+ T cells, (b), Tfh cells (CXCR5++PD-1++/CD4+ T cells, (c), and plasma cells (CD19-CD138+ cells, (d) afterco-culturing of pre-stimulated naïve T cells with miR-663-related vector-transfected BMSCs. (e) The percentage of proliferated Foxp3+ cellsin sorted CD4+CD25+ T cells after co-culture with different BMSCs. (f) The absolute number of Foxp3+CD4+CD25+ T cells increased in thepresence of miR-663-I BMSCs. (g and h) The effects of miR-663-related BMSCs on Treg cell differentiation and the conversion of Treg cellsto Tfh cells. (i) The ratio of Tfh (CXCR5++PD-1++/CD4+ T cells) to Treg (CD25+Foxp3+/CD4+ T cells) after co-culturing of pre-stimulatednaïve T cells with miR-663-related BMSCs. All data represent the mean±SEM. n=6, *Po0.05, **Po0.01, ***Po0.001.


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Figure 4 TGF-β1 is a direct target of miR-663. (a) SLE BMSCs produce fewer active and total TGF-β1 proteins than normal BMSCs afterculturing for 24 hours. (b and c) The role of TGF-β1 in the immunoregulatory effect of BMSCs on Treg cells (b) and the BMSC migrationcapacity (c). (d) Active and total TGF-β1 protein levels in cultured supernatants of various miR-663 related BMSCs. (e and f) The role ofmiR-663 in the immunoregulatory effect of BMSCs on Treg cells (e) and BMSC migration capacity (f) acting through the regulation of TGF-β1. (g and h) The effect of miR-663 and TGF-β1 on the Akt, p38/MAPK and Smad2/3 pathways in BMSCs and UCMSCs. (i) The role ofAkt inhibitor (GSK690693), p38/MAPK inhibitor (SB203580) and Smad inhibitor (SB431532) in the migration capacity of miR-663-related BMSCs. (j) Five potential miR-663 binding sites predicted in TGF-β1 3′-UTR. (k) The TGF-β1 3′-UTR is involved in miR-663-regulated gene expression. All data represent the mean±SEM. n=6, *Po0.05, **Po0.01, ***Po0.001.


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Several signaling pathways have been reported to participatein TGF-β1-induced BMSC migration.25,26 Our data showedthat miR-663-M significantly suppressed the phosphorylationof Akt and p38/MAPK in BMSCs, which could be abrogated byexogenous human recombinant TGF-β1, while miR-663-I ledto an opposite effect (Figures 4g and h). When treated with Aktinhibitor (GSK690693), p38/MAPK inhibitor (SB203580) and

Smad inhibitor (SB431532), only GSK690693 and SB203580significantly decreased the migration capacity of miR-663-IBMSCs (Figure 4i), suggesting that Akt and p38/MAPKsignaling pathways play a critical role in miR-663-mediatedBMSC dysfunction.

Bioinformatics analyses revealed five potential miR-663binding sites in the TGF-β1 3′-untranslated regions (3′-UTR,

Figure 5 MiR-663 overexpression impairs the treatment effects of BMSCs in MRL/lpr mice. (a–e) Lymph node size (a), spleen index (b),total IgG (c), anti-ds-DNA levels (d), and proteinuria (e) levels among different groups. (f–h) Representative images of renal pathology andimmunohistochemical analysis of MRL/lpr mice after treatment with miR-663-related BMSCs. All data represent the mean±SEM. n=8 foreach group at the beginning of the experiment. For detection at 24 weeks, n(FLS)=6, n(miR-663-M)=7, n(for other groups)=8.*Po0.05, **Po0.01, ***Po0.001.


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Figure 6 MiR-663 contributes to the Tfh/Treg cell imbalance in vivo. (a) MiR-663-related human BMSC homing capacity to different organsin MRL/lpr mice at 24 h after infusion. (b and c) Migration and proliferation capacity of mouse BMSCs (mBMSCs) from MRL/lpr mice at8 weeks after treatment of different human BMSCs. (d–i) Percentages of Treg (d,f), Tfh (e,g) and expression levels of Bcl6 mRNA (h) aswell as the ratio of Tfh/Treg (i) in mononuclear cells from the spleen of MRL/lpr mice after treatment with different BMSCs. (j and k) Theimmunofluorescence intensity ratio of Bcl6+/Foxp3+ cells in the germinal center (GC) of lymph nodes from MRL/lpr mice after treatmentwith different BMSCs. Foxp3+ cells (green) were present in the Bcl6+ germinal center area (red) in MRL/lpr mice. All data represent themean±SEM. n=3 for each group in (a), n=6 for each group in (b,c); for other images, n(FLS)=6, n(miR-663-M)=7, n(for othergroups)=8. *Po0.05, **Po0.01, ***Po0.001.


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Figure 4j). To experimentally validate whether the bindingbetween miR-663 and TGF-β1 3′-UTR is necessary for thetranscription of TGF-β1, luciferase reporters containing thewild type or mutated 3′-UTR of human TGF-β1(Supplementary Fig. 4d) were co-transfected with miR-663-Cor miR-663-M into HEK293T cell lines. In cells expressing aluciferase construct containing the wild-type TGF-β1 3′-UTR,miR-663-M transfection markedly reduced luciferase activityand miR-663-I transfection significantly increased luciferaseactivity (Figure 4k). However, in cells with the mutated TGF-β1 3′-UTR, co-transfection of either miR-663-M or miR-663-Idid not alter the reporter activity, supporting that binding tothe TGF-β1 3′-UTR is crucial for the regulation of TGF-β1expression by miR-663.

MiR-663 impairs the therapeutic effects of BMSCs in MRL/lpr miceSystemic infusion of BMSCs has been shown to be beneficialfor lupus mice.10,11 Since miR-663 participated in the immu-noregulatory effect of BMSCs, we next investigated whetherinhibition of miR-663 could augment the therapeutic effects ofBMSCs in MRL/lpr mice. Treatment with miR-663-I-transfected BMSCs significantly reduced the size of spleensand lymph nodes (Figures 5a and b) and decreased serumlevels of total immunoglobulin (IgG) and IgG anti-double-stranded DNA antibodies (anti-ds-DNA) (Figures 5c and d) inMRL/lpr mice compared with those treated with miR-663-C-transfected BMSCs. Renal impairments were also amelioratedin the miR-663-I group, as shown by lower proteinuria(Figure 5e), reduced glomerular enlargement and hypercellu-larity (Figure 5f) and less IgG and complement 3 (C3)deposition in the peripheral capillary loops (Figures 5g andh). In contrast, the therapeutic effect of BMSCs in MRL/lprmice was almost abolished when BMSCs were transfected withmiR-663-M, with serological alterations and organ involvementsimilar to the mice treated with fibroblast-like synoviocytes(FLS), which were used as negative controls based on previousstudies.27,28 Taken together, these in vivo findings demonstratethat miR-663 overexpression impairs the therapeutic effects ofMSCs in MRL/lpr mice.

Inhibition of miR-663 restores the Tfh/Treg cell balancein vivoNext, we studied the mechanisms of the in vivo effect ofmiR-663 on BMSCs. To examine the homing capacity ofBMSCs in MRL/lprmice, miR-663-M or miR-663-I-transfectedBMSCs were labeled with PKH26 and then infused intrave-nously. After 24 h, massively trapped BMSCs within the lungand liver rather than inflammatory organs, including kidney,lymph nodes and spleen were present in the miR-663-Mgroup. In contrast, a significantly enhanced BMSC homingcapacity to kidney, lymph node and spleen was observed in themiR-663-I group (Figure 6a; Supplementary Fig. 5-10). Asimilar effect was detected at 48 and 72 h (data not shown).

To determine the role of miR-663 in BMSC migration,BMSCs from MRL/lpr mice (mBMSCs) were collected 8 weeks

after BMSC or FLS treatment (Supplementary Fig. 11a). Wefound the migration capacity of mBMSCs increased aftertreatment with miR-663-I-transfected BMSCs by the trans-well assay but decreased in the miR-663-M group comparedwith mice treated with miR-663-C-BMSCs (Figure 6b,Supplementary Fig. 11b). Thus, downregulation of miR-663could not only enhance the homing capacity of infused BMSCsbut also facilitate the migration of host mBMSCs. In additionto the migration capacity, the proliferation rate of mBMSCswas also increased after infusion of miR-663-I-transfectedBMSCs (Figure 6c).

The interaction between activated Tfh cells and long-livedplasma cells would promote autoantibody production, which isinvolved in lupus onset.19 Consistent with the in vitro data, notonly the mRNA level of B-cell lymphoma/leukemia 6 (Bcl6), awell-recognized transcription factor defining the Tfh lineagebut also the frequencies of Tfh cells and plasma cells wereelevated in the miR-663-M group compared with the miR-663-C group (Figures 6d–h; Supplementary Fig. 11g, j). Similarly,the ratio of Tfh/Treg increased in the miR-663-M group anddecreased in the miR-663-I group (Figure 6i). To determinethe location of T cell subsets in germinal centers (GC), Tfh cells(Bcl6+) and Treg cells (Foxp3+) were stained using immuno-fluorescence histochemistry in lymph nodes from MRL/lprmice. The intensity ratio of Bcl6+/Foxp3+ cells was significantlylower in the miR-663-I group compared with the miR-663-Cgroup (Figures 6j and k), supporting the role of miR-663 in theregulation of Tfh/Treg cells. Similar to the in vivo findings, therewere no differences in Th1, Th2 and Th17 cells in PBMCs andprotein levels of IFN-γ, IL-4 and IL-17A in the serum amongthese groups (Supplementary Fig. 11c–e, 12c,e,f). However, thetotal TGF-β1, IL-10 and IL-21 level remained upregulated8 weeks after the infusion of miR-663-I-transfected BMSCs(Supplementary Fig. 12a,b,d). Thus, miR-663 serves as a vitalfactor in the MSC-regulated Tfh/Tfr cell balance.

DISCUSSION

The bone marrow microenvironment, especially BMSCs, iscritical for the acquisition and maintenance of immunologicalbalance.29 MiRNAs, which target messenger RNA for cleavage ortranslational repression, have recently been shown to play animportant role in the regulation of MSC functions.18,30 Although,miRNA profiling in PBMCs has been performed in individualautoimmune diseases,31,32 the role of MSC-derived miRNAs inautoimmune pathogenesis is largely unknown. Here, we identi-fied 8 highly expressed miRNAs in BMSCs from patients withSLE, a prototypical autoimmune disease, among which 4miRNAs (miR-663, miR-638, miR-214 and miR-574-3p)remained upregulated after verification. These data differ fromthose in other tissues, in which miR-155, miR-l26 and miR-146ahave received extensive attention,13,16,33 suggesting a distinctmiRNA signature in SLE BMSCs.

The characterization of miRNAs present in BMSCs maybe relevant not only as a signature of the cell type but alsofor understanding their biological activities. We further char-acterized miR-663 because this miRNA is potentially associated


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with immune functions23 and relevant to SLE disease activity(Figure 1d). MiR-663 has been demonstrated to play a criticalrole in anti-malignancies34–36 because it induces the differen-tiation but suppresses the proliferation and invasion of tumorcells. Moreover, miR-663 is important for the key eventsinduced in endothelial cells by stress agents and oxidizedlipids,37 and thus it may have a potential role in thedevelopment of atherosclerosis. However, the effect ofmiR-663 in MSCs remains largely unknown. In this study,we identified that miR-663 is not only associated with theapoptosis, proliferation and migration capacity of BMSCs, butit is also involved in the immunoregulatory function of BMSCsin the Tfh/Treg cell balance both in vitro and in vivo.

Many miR-663 target genes have been predicted by bioin-formatics analysis, among which several have been confirmedin specific cell types. MiR-663 may decrease endogenousactivator protein-1 (AP-1) activity, at least in part by directlytargeting the JUNB and JUND transcripts in THP-1 cells.23

Moreover, myosin light chain 9 (MYL9) in human vascularsmooth muscle cells,38 TGF-β1 in A549 cells,39 and heparinsulfate proteoglycan 2 (HSPG2) in human breast cancer cells40

have been identified as downstream targets of miR-663. Here,we evaluated the effects of miR-663 on the expression ofpredicted targets (TGF-β1, JUND, MYL9, GRID2D, EPHB3)and several well-known pro-inflammatory cytokines, includingTNF-α, IFN-α and IL-1β, in BMSCs. Our data showed that inaddition to MYL9, only TGF-β1 expression was regulated bymiR-663 via binding to the 3’UTR.

TGF-β1, the most abundant isoform of the TGF-β familyinvolved in immune regulation, can be produced by bonemarrow cells. TGF-β1 is an important multifunctional cytokinethat is involved in the control of several biological processes,including cell proliferation, differentiation, migration, apopto-sis and, most importantly, immune modulation.41 While therole of TGF-β1 in Treg cell generation is widely accepted,42 theunderlying molecular mechanism remains unclear. In thisstudy, we showed that miR-663 participated in BMSC-regulated Treg differentiation through the modulation ofTGF-β1 production, which might be related to the p38/MAPKand Akt signaling pathways.

Tfh cells have recently been highlighted for their crucial rolein humoral immunity as well as their abnormal control leadingto the induction of autoimmune diseases.19 Multiple cytokines,signaling molecules and transcription factors have beenreported to be involved in Tfh cell differentiation.19 A recentstudy has illustrated that TGF-β can induce miR-10a expres-sion in Treg cells, which helps attenuate the phenotypicconversion of inducible Treg cells into Tfh cells,24 implicatinga possible link between TGF-β1 and Tfh cells. MSCs may alsointerfere with the viability and differentiation of follicularlymphoma-infiltrating Tfh cells, yet the underlying mechanismremains unclear. The present data support that the imbalancebetween Treg and Tfh cells in SLE patients may be linked tooverexpression of miR-663 in their BMSCs. Inhibition ofmiR-663 in BMSCs could restore the production of Treg andTfh cells through the secretion of TGF-β1. To further support

this notion, we showed that in MRL/lpr mice, the therapeuticeffect of BMSCs transplantation was augmented after treatmentwith miR-663-I-transfected BMSCs, together with elevatedTGF-β1 expression and a reversed frequency of Tfh andTreg cells.

Tfr cells, a specialized subset of Treg cells that share featuresof Tfh and Treg cells, have been found to localize in follicularregions of GC and dampen GC responses by limiting thenumbers of both Tfh and B cells.43,44 The balance between Tfhand Tfr cells in the GC environment likely represents a keyfactor in the generation of both high-affinity protectiveantibodies and pathogenic autoantibodies.20,45 Our datashowed that similar to Treg cells, the percentage of Tfr cells(Foxp3+CXCR5++PD-1++/CD4+) was downregulated in themiR-663-M group (Supplementary Fig. 13a). Moreover, thenumbers of Tfr cells (Foxp3+Bcl6+) in lymph nodes and theexpression levels of CTLA-4 (cytotoxic T-lymphocyte-asso-ciated protein 4), a direct mediator of Tfr function,

44 were alsodecreased in spleen and GC in MRL/lpr mice after treatmentwith miR-663-M-transfected BMSCs (Supplementary Fig. 13b-d), suggesting that Tfr cells may represent a main Treg cellsubset regulated by miR-663.

In addition to the direct role on Treg cell dysregulation andthe Tfh/Treg cell balance, transplanted MSCs may also actthrough the regulation of host MSCs to exert their functions, asrecent studies have indicated that the exchange of miRNAsthrough microvesicles between neighboring cells is an integralpart of MSC communication with tissue-injured cells.18 Con-sequently, we observed the alterations of the migration capacityand proliferation rate of mouse BMSCs (mBMSCs) 8 weeksafter the infusion of miR-663-M or miR-663-I-transfectednormal BMSCs. Thus, infused BMSCs may act directly orthrough the modulation of host BMSCs to exert theirimmunoregulatory functions. The miR-663-TGF-β1-Tfh/Tregcould help not only to elucidate the cause of abnormalities inSLE BMSCs but also to explain the therapeutic function ofnormal allogeneic MSCs in refractory SLE patients.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTS

The work was supported by the Major International (Regional)Joint Research Project (No.81720108020), National Natural ScienceFoundation of China (No. 81373199, 81501347 and 81370730,81273304), National Natural Science Foundation of Jiangsu(BK20150098), and Jiangsu Province Major Research andDevelopment Program (BE2015602) and Jiangsu Province 333Talant Grant (BRA2016001). WC was supported by theIntramural Research Program of NIH, NIDCR.

AUTHOR CONTRIBUTIONSLS designed, coordinated and supervised the study. LG carried outmost of the experiments, performed the data acquisition and analysis,and wrote the manuscript. XF contributed to the data interpretationand manuscript drafting. KZ, XG, WC, SS and NS participated in the


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study design. SW, GY, XT, WC and DW participated in humansample collection and breeding of MRL/lpr mice. All authors read andapproved the final manuscript.

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Supplementary Information for this article can be found on the Cellular &Molecular Immunology website (http://www.nature.com/cmi)


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microrna-663 induces immune dysregulation by β1 production

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