By Reduced CCL2 Expression Marrow Mesenchymal Stem Cells Is

Impaired B Cell Inhibition by Lupus Bone Marrow Mesenchymal Stem Cells Is Caused by Reduced CCL2 Expression

This information is current as Nan Che, Xia Li, Lu Zhang, Rui Liu, Haifeng Chen, Xiang of September 25, 2021. Gao, Songtao Shi, Wanjun Chen and Lingyun Sun J Immunol 2014; 193:5306-5314; Prepublished online 22 October 2014; doi: 10.4049/jimmunol.1400036

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Supplementary http://www.jimmunol.org/content/suppl/2014/10/19/jimmunol.140003 Material 6.DCSupplemental http://www.jimmunol.org/ References This article cites 41 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/193/10/5306.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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Impaired B Cell Inhibition by Lupus Bone Marrow Mesenchymal Stem Cells Is Caused by Reduced CCL2 Expression

Nan Che,*,1 Xia Li,*,1 Lu Zhang,* Rui Liu,* Haifeng Chen,* Xiang Gao,† Songtao Shi,‡ Wanjun Chen,x and Lingyun Sun*

Mesenchymal stem cells (MSC) from healthy human and normal mice can inhibit normal B cell proliferation, differentiation, and Ab secretion in vitro. However, it remains unknown whether MSC from lupus-like mice and patients with systemic lupus erythematosus (SLE) exhibit the same immunoregulatory activity as normal MSC for B cell inhibition and, if not, what the underlying molecular mechanism would be. In this study, we showed that bone marrow–derived MSCs from lupus-like mice and

SLE patients had an impairment in suppressing normal B cell proliferation and differentiation, which was caused by the reduction Downloaded from of CCL2 levels. Knockdown of CCL2 in normal MSC damaged their suppressive capacity for B cells. Conversely, overexpression of CCL2 in lupus MSCs restored their immunoregulatory ability for B cells in vitro and ameliorated the pathology of lupus nephritis and serological changes in MRL/lpr mice in vivo. Mechanistically, MSC-mediated B cell inhibition was dependent on matrix metalloproteinase proteolytic processing of CCL2. These ﬁndings reveal a novel function of CCL2 in B cell regulation by MSCs and suggest that CCL2 manipulation on MSCs may serve as a potential pathway for developing the more effective MSC- based therapy in autoimmune diseases associated with B cell activation, such as SLE. The Journal of Immunology, 2014, 193: http://www.jimmunol.org/ 5306–5314.

esenchymal stem cells (MSCs) are the microenviron- macrophages (6). The interaction between MSCs and B cells is ment components in bone marrow and other tissues, recently gaining interest. Presently, the few published articles M and are capable of differentiating into mesenchymal studying the effect of MSCs on B cell proliferation and differ- lineages including adipose, bone, cartilage, muscle, and myelo- entiation have shown the disparities in their approaches and supportive stroma (1, 2). In addition, MSCs exhibit immunoreg- results. However, for mature B cells, human bone marrow MSCs ulatory activities for multiple immune cells. The impacts of MSCs (BMMSCs) have been reported to inhibit B cell proliferation, on T cells have been widely studied (3), and there is also evidence differentiation, and chemotaxis (7). We have previously shown by guest on September 25, 2021 that MSCs can suppress NK cells (4), dendritic cells (5), and that normal human umbilical cord MSCs markedly suppressed the proliferation and differentiation of mouse splenic B cells as + *Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, demonstrated by the decreased number of CD138 cells and re- Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu duced levels of IgM and IgG (8). However, the underlying † 210008, People’s Republic of China; Model Animal Research Center, Nanjing mechanisms by which MSCs suppress B cell function remain University, Nanjing, Jiangsu 210093, People’s Republic of China; ‡Center for Cra- niofacial Molecular Biology, Herman Ostrow School of Dentistry of USC, University largely unknown. x of Southern California, Los Angeles, CA 90033; Mucosal Immunology Section, Multiple reports support the idea that BMMSCs regulate a va- Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892 riety of T cell responses by cell-contact–dependent (9, 10) and –independent ways mediated by some soluble factors including 1N.C. and X.L. are co-first authors. TGF-b (11), PGE (12), indolamine 2,3-dioxygenase (13), NO Received for publication January 8, 2014. Accepted for publication September 17, 2 2014. (14), heme oxygenase-1 (15), and HLA-G (16). Data from Fran- This work was supported by the National Natural Science Foundation of China Grant cesca Schena (17) showed that BMMSCs inhibited Ag-dependent 81273304, Major International (Regional) Joint Research Project 81120108021, the proliferation and differentiation to plasma cells of both follicular Jiangsu Province Kejiao Xingwei Program, and the Intramural Research Program of B cells and marginal zone B cells in vitro, and this inhibitory the National Institutes of Health, National Institute of Dental and Craniofacial Re- search (to W.C). effect was dependent on IFN-g and mediated by cell–cell contact, Address correspondence and reprint requests to Prof. Lingyun Sun, Department of which involved the interactions between program death-1 and PD Rheumatology and Immunology, Nanjing Drum Tower Hospital, Affiliated Hospital ligand-1. CCL2 produced by MSCs also suppresses the production of Nanjing University Medical School, No. 321, Zhongshan Road, Nanjing, Jiangsu, of Ig by plasma cells through the inactivation of STAT3 and in- 210008 People’s Republic of China. E-mail address: [email protected] duction of paired box protein 5 (18). In addition, MSC-conditioned The online version of this article contains supplemental material. medium inhibited experimental autoimmune encephalomyelitis– Abbreviations used in this article: ANA, anti-nuclear Ab; BMMSC, bone marrow derived CD4+ T cell activation by suppressing STAT3 phosphory- MSC; CCL2-OE, CCL2 overexpression; HC, healthy control subject; hMMP1, human MMP1; HMSC, MSC from healthy human; LMSC, MRL/lpr mice MSC; lation via MSC-derived CCL2 (19). These data suggest that CCL2, LMSCT, LMSC transplantation; MMP, matrix metalloproteinase; mpCCL2, matrix although a potent chemoattractant for monocytes and macrophages metalloproteinase–processed CCL2; MSC, mesenchymal stem cell; MSCT, MSC transplantation; NC, normal control; NMSC, C57BL/6 MSC; NMSCT, NMSC trans- to areas of inflammation, when secreted by MSCs, may be involved plantation; OE, overexpression; rCCL2, recombinant CCL2; sCD40L, soluble in B cell or T cell inhibition. CD40L; siRNA, small interfering RNA; SLE, systemic lupus erythematosus; SMSC, Systemic lupus erythematosus (SLE) is a prototypical multi- MSC from SLE patients. system disease characterized by dysfunction of T cell and poly- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 clonal B cell activation (20). B cells, being the cellular source of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400036 The Journal of Immunology 5307 characteristic anti-nuclear Abs (ANA), especially anti-dsDNA (Miltenyi Biotec) according to the manufacturer’s instructions. After Abs, play a crucial role in the pathogenesis of SLE (21). Defects staining for B220 (eBioscience), B cells were sorted with a FACSCalibur . of bone marrow hematopoietic stem cells have been suggested (Becton Dickinson). The purity of the sorted cells was 95%. To monitor the effector functions of activated B cells, we cultured normal to contribute to the development of SLE (22), but the role of mouse B cells in RPMI 1640 medium (Life Technologies) supplemented BMMSCs in the pathogenesis of lupus remains unclear. We and with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin in the absence others have shown that lupus BMMSCs grow slowly with early or presence of the following stimuli: 2.5 mg/ml CpG 2395 synthetic signs of senescence, and exhibit some disorders in cytoskeleton and oligonucleotide (CpG 2395; Hycult Biotech), 100 ng/ml soluble CD40L (sCD40L; R&D Systems), 2 mg/ml F(ab9)2 anti-mouse IgM (Jackson ultrastructure (23–25). In addition, accumulating evidence has in- Immunoresearch), and 10 ng/ml IL-4 (R&D Systems). dicated that allogeneic normal MSCs, rather than autologous MSC transplantation (MSCT), are more effective in treating lupus in Normal human B cell isolation, culture, and activation preclinical animal studies and SLE patients (26–29), which suggests Normal human B cells were isolated from the peripheral blood of the that lupus MSCs have a deficiency in suppressing SLE inflamma- healthy volunteers obtained from Nanjing Red Cross Blood Center by tion. However, whether lupus MSCs are functionally impaired in positive selection with the use of CD19 microbeads (Miltenyi Biotec) inhibiting normal B cells remains elusive. according to the manufacturer’s instructions. After staining for CD19 (eBioscience), B cells were sorted with a FACSCalibur (Becton Dick- In this article, we provided evidence that lupus BMMSCs pre- inson). The purity of the sorted cells was .95%. sented functional defect for normal B cell inhibition compared with To study the effector function of activated B cells, human B cells were normal BMMSCs, and this impairment is in great part attributed to also cultured in RPMI 1640 medium (Life Technologies) supplemented their reduction of CCL2 levels by lupus BMMSCs. with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin in the absence or presence of the following stimuli: 2.5 mg/ml CpG 2395 synthetic oligonucleotide (CpG 2395; Hycult Biotech), 3 mg/ml sCD40L (R&D Systems), Downloaded from Materials and Methods 5 mg/ml F(ab9)2 anti-human IgM (anti-IgM; Jackson Immunoresearch), and Mice 50 ng/ml IL-4 (R&D Systems). Female C3MRL-Faslpr/J (MRL/lpr) and background matched C57BL/6 Proliferation assay mice were purchased from Chinese Academy of Military Medical Scien- ces Animal Laboratory. All animal experiments were performed under an Purified normal mouse or human B cells were cocultured in 96-well flat- bottom plates (Costar) with human or mouse BMMSC in the presence institutionally approved protocol for the use of animal research. http://www.jimmunol.org/ of four stimuli (CpG 2395, sCD40L, anti-IgM, and IL-4) in a total volume of Mice BMMSC isolation and culture 0.2 ml RPMI 1640 medium per well in triplicate. For neutralizing Ab assay, anti-CCL2 Ab (anti-mouse and anti-human Abs are from eBioscience) was Bone marrow cells were flashed out from bone cavity of femurs and tibias added to coculture system of BMMSCs and B cells. After 3 d, 60 mM BrdU with heat-inactivated 10% FBS (Life Technologies) in PBS. All nuclear (Sigma-Aldrich) was added into the cultures for another 3 h. After the 6 cells (10–20 3 10 ) were seeded into 100-mm culture dishes (Corning) and incorporation of BrdU, nonadherent cells were washed with PBS con- initially incubated for 24 h at 37˚C in a 5% CO2 incubator. The cultures taining 0.5% BSA (Sigma-Aldrich) and fixed overnight with 1% parafor- were washed with PBS twice to eliminate the nonadherent cells. The at- maldehyde in PBS containing 0.1% Tween 20. After fixation, cells were tached cells were cultured for 14–16 d. Colony-forming attached cells washed and resuspended in Ca2+ and Mg2+ containing PBS with 100 were passed once to use for further experiments. BMMSCs were cultured Kunitz U/ml bovine pancreatic DNase I (Sigma-Aldrich). After digestion, with DMEM-F12 (Invitrogen) supplemented with 15% FBS and antibiotics the cells were washed and resuspended in PBS supplemented with BSA by guest on September 25, 2021 (100 U/ml penicillin and 100 mg/ml streptomycin; Life Technologies) (26). and Tween 20. Then 5 ml FITC-coupled anti-BrdU (eBioscience) was added. After an hour of incubation, cells were washed and resuspended in Patients and healthy control subjects PBS. Data were acquired on a FACSCalibur flow cytometer (Becton Study protocol was reviewed and approved by the Ethics Committee of the Dickinson) using the Cell Quest software program (Becton Dickinson). A Affiliated Drum Tower Hospital of Nanjing University Medical School minimum of 10,000 events/tube was collected for analysis. where all subjects were recruited excluding those with a current infection. Differentiation assay All patients and healthy volunteers provided written, informed consents to participate in the study. Enrollment criteria for SLE patients included the Purified normal mouse or human B cells were cocultured in 96-well flat- fulfillment of the 1997 revised criteria of the American College of bottom plates with human or mouse BMMSCs in the presence of CpG Rheumatology and positive serum ANA levels (titer .1:100 dilution) at the 2395, sCD40L, anti-IgM, and IL-4 in a total volume of 0.2 ml RPMI 1640 study entry. Healthy volunteers were recruited as healthy control subjects medium per well in triplicate. For neutralizing Ab assay, anti-CCL2 Ab was (HCs) with an effort to match the age and sex of SLE patients. added to the coculture system of BMMSCs and B cells. After 4 d, nonadherent cells were washed and resuspended in PBS; then 5 ml PE-coupled Human BMMSC isolation and culture anti-CD138 mAb (anti-mouse Ab from Becton Dickinson and anti-human Human bone marrow aspirated from iliac of three SLE patients and their Ab from eBioscience) was added. After half an hour of incubation, cells relatives were diluted with PBS containing heparin (1250 U/ml) and were washed and resuspended in PBS. Data were acquired on a FACSCa- separated using Ficoll-Hypaque (density 1.077 g/ml) (Tianjin Haoyang libur flow cytometer using the Cell Quest software program. A minimum Biological Manufacture, Tianjin, China) by centrifuge at 2000 rpm for 30 of 10,000 events/tube was collected for analysis (8). 3 6 min to obtain mononuclear cells. The single-cell suspensions (5 10 /ml) ELISA were cultured in 25-cm2 flasks (Corning) with DMEM (Life Technologies) supplemented with 15% FBS and antibiotics at 37˚C. After 3 d of culture, Detection of Ig production. Purified normal mouse or human B cells were nonadherent cells were removed and the medium was changed twice cocultured in 96-well flat-bottom plates with human or mouse BMMSCs in weekly thereafter. Once 80% confluence was reached, adherent cells were the presence of CpG 2395, sCD40L, anti-IgM, and IL-4 in a total volume of replaced at a density of 104 cells/cm2 for expansion (26, 29). 0.2 ml RPMI 1640 medium per well in triplicate. After 6 d, supernatants After two passages, cells were harvested. BMMSC were then characterized were collected for the test of IgM and IgG levels by ELISA kit (anti-mouse in accordance with International Society of Cellular Therapy recommenda- and anti-human Abs are all from R&D Systems) according to instructions. tions (30). Flow cytometric analysis confirmed the expression of CD73, CD105, Quantitation of serum levels of anti-dsDNA Abs, ANA, IL-10, and TGF-b1. CD90, CD29 (at least .95%), and the absence of CD45, CD34, CD14, Serum from treated or untreated mice was harvested and immediately frozen CD79, and HLA-DR (,2%) on these cells. The capacity of BMMSCs dif- at 280˚C until use. The levels of anti-dsDNA Abs, ANA, IL-10, and TGF- ferentiating into adipogenic and osteogenic lineages was also assayed. b1 were measured by commercial kits according to the recommendations of the manufacturer (Shibayagi, Gunma, Japan). Mouse B cell isolation, culture, and activation Array analysis for cytokines To avoid inadvertent activation of B cells, we isolated normal mouse B cells from the spleen of the 6- to 8-wk-old C57BL/6 female mice (Chinese Supernatant cytokines of BMMSC culture from different MRL/lpr or Academy of Military Medical Experimental Animal Center, Beijing, China) C57BL/6 mice were analyzed using a mouse cytokine array kit (R&D by negative selection (CD43 depletion) with the use of CD43 microbeads Systems; QAH-TH17-1, RayBiotech, Norcross, GA) according to the 5308 IMPAIRED B CELL INHIBITION BY LUPUS BMMSC manufacturer’s specification. Each sample was prepared in triplicate. An complex deposits, we analyzed sections by the avidin-biotin-peroxidase Axon scanner 4000B with GenePix software was used to collect fluores- method, using biotin-labeled polyclonal goat anti-mouse IgG or IgM Ab cence intensities. (Organon Teknica, Scarborough, CA). Preimmune biotin-labeled goat serum served as a negative control. The relative intensity for IgM deposition Generation of matrix metalloproteinase–processed CCL2 and for IgG deposition was scored separately using a scale of 0–4, where in vitro 0 = no apparent staining as compared with the isotype control, 1 = detectable staining, 2 = moderate staining intensity, 3 = severe staining To generate matrix metalloproteinase (MMP)–processed CCL2 (mpCCL2) intensity, and 4 = maximum staining intensity (17). Two experienced in vitro, we added 10 ng pure recombinant human MMP1 (hMMP1) di- evaluators who were blinded with regard to the experimental group eval- rectly to 50 mg pure recombinant CCL2 (rCCL2) for a period of 4 h at uated the samples. 37˚C. mpCCL2 was directly used in assays according to the published The glomerular cellularity was evaluated under light microscopy by protocols (18, 19). counting the total number of nuclei. Numbers of glomerular cells for each RNA isolation and real-time PCR mouse were quantified as counts of positive-stained nuclei in hematoxylin stain, performed in a double-blinded manner by two independent observers. Total RNA was extracted from different human or mouse BMMSC using A total of 50 sequential glomeruli from the superior, middle, and inferior TRIzol reagent (Invitrogen) according to the manufacturer’s recom- cortices of each kidney were scored for the presence of proliferating glo- mendations. RNA was reverse-transcribed and quantified by real-time PCR meruli and were then expressed as the percentage of proliferating glomer- using the PrimeScript RT-PCR and SYBR Premix Ex Taq kit (TaKaRa uli of the total number of glomeruli analyzed. Biotechnology). The relative expressions of CCL2 or MMP mRNA were determined and normalized to the expression of the internal housekeeping CCL2 digestion and mass spectrometry data acquisition and gene GAPDH. Relative quantification was calculated using the compara- identification tive cycle threshold method. Primer sequences were described as follows:

A total of 100 mg peptide from the supernatants of three-passage BMMSCs Downloaded from human CCL2: 59-GCTCATAGCAGCCACCTTCATTC-39 (forward), 59-GG- 3 6 ACACTTGCTGCTGGTGATTC-39 (reverse); human GAPDH: 59-GCACC- of SLE patients or HCs (5 10 cells) was reduced with 10 mM DTT at GTCAAGGCTGAGAAC-39 (forward), 59-TGGTGAAGACGCCAGTGGA- 60˚C for 1 h and then alkylated with 55 mM iodoacetic acid at 37˚C for 39 (reverse); mouse CCL2: 59-GCATCCACGTGTTGGCTCA-39 (forward), 40 min. Tryptic peptides were desalted and dried in vacuo (Speed Vac, 59-CTCCAGCCTACTCATTGGGATCA-39 (reverse); mouse GAPDH: 59-AA- Eppendorf). The peptides were analyzed on the LTQ-Orbitrap instrument (Thermo Fisher) connecting to a Nano ACQUITY UPLC system via ATGGTGAAGGTCGGTGTGAAC-39 (forward), 59-CAACAATCTCCA- a nanospray source. Liquid chromatography–tandem mass spectrometry CTTTGCCACTG-39 (reverse). was operated in positive ion model as described previously (1, 2). The Western blot analysis analytical condition was set at a linear gradient from 0 to 60% of buffer B http://www.jimmunol.org/ (CH3CN) in 150 min and flow rate of 200 nl/min. For analysis of proteins Human or mouse BMMSC were pelleted, washed twice with PBS, and lysed from human placenta, one full MS scan was followed by five MS/MS scans in ice-cold lysis buffer (140 mM NaCl, 10 mM EDTA, 10% glycerol, 1% on those five highest peaks, respectively. The MS/MS spectra acquired Nonidet P-40, 20 mM Tris pH 7.0, 1 mM pepstatin, 1 mg/ml aprotinin, 1 mg/ml from precursor ions were submitted to Maxquant (version 1.2.2.5) using leupeptin, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulphonyl the following search parameters: the database for search was Uniprot fluoride). Samples were centrifuged at 13,000 rpm at 4˚C for 10 min, and the proteome (version 20140405); the enzyme was trypsin (full cleavage); supernatants were collected into new tubes. Cell extracts were resuspended dimethylation labeling for quantification; the dynamic modifications were and separated using NaDodSO4 gel electrophoresis. After transfer to poly- set for oxidized Met (+16); carbamidomethylation of cysteine was set as vinylidene fluoride membranes (Millipore), filters were blocked for 1 h in static modification; MS/MS tolerance was set at 10 ppm; the minimum 10 mM Tris pH 7, 150 mM NaCl, 0.1% Tween 20 (TBST) plus 1% BSA, and peptide length was 6; and the false detection rates for peptides were all set by guest on September 25, 2021 then incubated with the corresponding Ab. Membranes were incubated with ,0.01 (31). rabbit anti-CCL2 (1:1000) or anti-GAPDH (1:1000) (Cell Signaling Tech- nology). After washing with TBST, filters were incubated with HRP- Statistical analysis conjugated secondary Abs for 30 min and the bands were visualized in a Data are expressed as means 6 SD. For comparison between groups, the p luminol-based detection system with -iodophenol enhancement. Student t test was performed. All statistical analyses were performed using , Transfection of CCL2 expression plasmid or small interfering GraphPad Prism software (Graph-Pad, San Diego, CA). A p value 0.05 was considered significantly different. RNA The expression plasmid of CCL2 and small interfering RNA (siRNA) Results targeting CCL2 mRNA (CCL2 siRNA; sequence 59-AUCUGGCUGC- MRL/lpr mice BMMSCs showed defect in suppressing B cell CAACACGUGGAUG-39) were purchased from Invitrogen. Stealth RNAi Low GC Duplex (Invitrogen) and mock vehicle was used as negative responses in vivo 3 control siRNA. BMMSCs (1 3 10 ) were seeded into upper wells of We first investigated whether the transplantation of MRL/lpr mice transwell plates in BMMSC growth medium supplemented with 10% FBS without antibiotics. After 24 h, the expression plasmid of CCL2 or siRNA MSCs (LMSCs) had the same therapeutic effects as C57BL/6 MSC was transfected into BMMSCs using Lipofectamine in Opti-MEM I (NMSC) in suppressing B cells. We injected LMSC, NMSC, or according to the protocol recommended by the manufacturer (Invitrogen). PBS (as normal controls [NCs]) into MRL/lpr mice at the age of 18 wk (n = 10) and examined the serum levels of both ANA and Allogenic mouse BMMSC transplantation into MRL/lpr mice anti-dsDNA Abs in the recipients at 22 wk of age. Although both Under general anesthesia, original or modified BMMSCs from MRL/lpr NMSC (NMSCT) and LMSC transplantation (LMSCT) signifi- 6 or C57BL/6 mice (0.1 3 10 cells per 10 g body weight) and PBS were cantly reduced the serum levels of ANA and anti-dsDNA Abs infused into MRL/lpr mice via tail vein at age of 18 wk (n = 10). All mice were sacrificed at 22 wk of age for further analysis. compared with PBS treatment, LMSC treatment was significantly less effective in decreasing serum Abs in lupus mice than was Urinary protein concentration assessments NMSC treatment (Fig. 1A). Consistent with the changes in serum To compare renal function, we measured urinary protein of mice treated Abs, LMSCT failed to effectively suppress the frequency and 2 + with different BMMSCs derived from MRL/lpr or C57BL/6 mice. Twenty- number of CD19 CD138 plasmablasts in the spleen, and did four-hour urine was collected using metabolism cages before sacrifice. not lower IgG or IgM deposition in the kidney of the recipients Urinary protein concentration was measured by Coomassie brilliant blue. compared with NMSCT (Fig. 1B). Immunohistopathologic analysis Published studies reported increased IL-10 and decreased TGF- b1 in SLE patients and lupus mice (32, 33). Next, we examined The kidneys were cut into small pieces and fixed in 10% formalin for 24 h at 4˚C to assess pathologic kidney changes in MRL/lpr mice after different the levels of IL-10 and TGF-b1 in the serum in MSC-treated lupus BMMSCs from MRL/lpr or C57BL/6 mice transplantation. Paraffin sec- mice. Both NMSCT and LMSCT significantly decreased the se- tions (4 mm) of renal tissues were stained with H&E. To detect immune rum IL-10 levels in lupus mice compared with the NC group, but The Journal of Immunology 5309 Downloaded from http://www.jimmunol.org/

FIGURE 1. LMSCT resulted in more impaired inhibition in serum Ab levels and numbers of CD192CD138+ plasmablasts of MRL/lpr mice than NMSCT. (A) Serum levels of ANA and anti-dsDNA Ab. (B) Plasmablasts were identiﬁed using CD19 and CD138 Abs. Frequencies and numbers of CD192 CD138+ plasmablasts in spleen and the amounts of IgG or IgM deposition in kidney of lupus mice. IgG and IgM deposition was detected using immunohistochemical method. (C) Serum IL-10 and TGF-b1 levels in MRL/lpr mice. Data are representative of three independent experiments. *p , 0.05.

NMSCT showed more profound effect on the reduction of IL-10 by NMSCs or LMSCs at passage 3 with a mouse cytokine array kit. than LMSCT (Fig. 1C). In contrast, only NMSCT signiﬁcantly The search revealed that CCL2 levels were substantially decreased in

increased serum TGF-b1 levels in the lupus mice, whereas LMSC supernatants compared with NMSC supernatants (Fig. 3A). by guest on September 25, 2021 LMSCT showed no effects on serum TGF-b1 levels (Fig. 1C). We further confirmed that CCL2 transcription and protein expression Taken altogether, the data indicate that LMSCs have defects for byPCRandWesternblotassaydecreasedinLMSCsoflupusmice their immunoregulatory activity for B cells. at 18 wk of age (onset; Fig. 3B), although there was no significant difference in CCL2 protein expression between passage 3 NMSCs LMSC deficiency in B cell suppression in vitro and LMSCs in 9-wk-old lupus mice (premorbid; Supplemental Fig. 2). Because the aforementioned data suggested that LMSCT had de- To investigate whether decreased CCL2 expression was indeed fective suppression of B cells in lupus mice, we next investigated responsible for LMSC deficiency in B cell inhibition, we cocultured whether this phenomenon could be directly observed in vitro. As normal mouse activated B cells with NMSCs in the presence and expected, we determined that NMSCs significantly reduced the absence of anti-CCL2 Abs in a transwell system. The addition of frequency of both BrdU+ B cells and CD138+ plasmablasts, as well neutralizing anti-CCL2 Ab substantially blocked NMSC-mediated as IgM and IgG production in coculture supernatant at the ratios of inhibitory effect on B cell proliferation and differentiation (Fig. 3C). 1:1 and 1:10 (MSC/B cells; Supplemental Fig. 1A). In addition, To investigate the underlying mechanisms through which CCL2 NMSCs significantly suppressed B cell proliferation and differen- inhibits B cells, we cultured normal mouse activated B cells with tiation in a transwell culture system, in which B cells were physi- rCCL2 and/or hMMP1 (equal to mouse MMP13) in the presence and cally separated from NMSCs at a 1:1 ratio (Supplemental Fig. 1B), absence of anti-CCL2 Ab. We showed that the frequencies of BrdU+ which suggests that soluble factors play a key role in B cell sup- BcellsandCD138+ plasmablasts were markedly decreased when pression by NMSCs. Next, we cocultured NMSCs or LMSCs with activated B cells were treated concurrently with rCCL2 and hMMP1 normal mouse B cells at 1:1 ratios in the presence of CpG 2395, (means MMP1-processed rCCL2), and that the addition of CCL2- sCD40L, anti-IgM, and IL-4 as stimuli for 72 h, 96, h or 6 d. As neutralizing Ab reversed this effect. Addition of rCCL2 or hMMP1 shown in Fig. 2, although LMSCs had some inhibitory effect on alone had no impact on B cell proliferation and differentiation B cell proliferation (Fig. 2A) and differentiation (Fig. 2B), as well (Fig. 3D), suggesting that CCL2 function depends on MMP1. as Ig (IgG/IgM) production (Fig. 2C), in both cell–cell contact and To further confirm the causative effects of CCL2 expression in transwell culture systems, their suppressive function was signifi- B cell suppression by MSCs, we overexpressed CCL2 with a CCL2 cantly weaker than NMSCs. Thus, LMSCs also are defective in expression vector (CCL2 overexpression [CCL2-OE]) or knocked their immunoregulatory activity to B cells in vitro. down CCL2 with CCL2 siRNA in LMSCs or NMSCs, respectively (Supplemental Fig. 3A). CCL2 gene OE or knockdown did not CCL2 was required for B cell suppression by BMMSCs significantly affect the cell-surface markers, viability, and prolif- MSCs suppress B cells through many soluble factors. To determine eration of LMSCs and NMSCs (Supplemental Fig. 3B, 3C). We which factor(s) cause LMSC impairment in B cell suppression, we then examined the impact of these modified MSCs on B cell compared the levels of soluble factors in the supernatants produced proliferation and differentiation. In the cocultures of activated 5310 IMPAIRED B CELL INHIBITION BY LUPUS BMMSC

FIGURE 2. LMSCs had a weakened inhibition of B proliferation and differentiation in vitro compared with NMSCs. (A) The percentage of BrdU+ B cells in the coculture system of NMSCs or LMSCs and activated B cells in the condition of cell– cell contact or transwell system. (B) The percentage of CD138+ plasmablasts in the coculture system of NMSCs or LMSCs and activated B cells. (C) Supernatant IgG/IgM levels in the coculture system of NMSCs Downloaded from or LMSCs and activated B cells. Data are representative of three independent experiments. *p , 0.05. CT, cell–cell contact; TW, transwell system. http://www.jimmunol.org/

B cells and MSCs, CCL2 knockdown in NMSCs reversed their munosuppressive activity of LMSCs (Fig. 4). The data altogether suppression of the numbers of BrdU+ B cells and CD138+ plas- indicate that CCL2 expression in MSCs is crucial for their sup- by guest on September 25, 2021 mablasts, and the levels of IgG/IgM in the culture supernatants. pression of B cells, and that decreased CCL2 expression is a major Conversely, OE of CCL2 in LMSCs corrected the impaired im- mechanism for LMSC defect in B cell suppression.

FIGURE 3. Impaired LMSC suppression of B cells may be associated with lower CCL2 expressions. (A) Soluble factors analysis secreted by LMSCs or NMSCs using a mouse cytokine array kit. (B) CCL2 transcription and expressions on LMSCs or NMSCs by real-time PCR, Western blot. (C) The percentage of BrdU+ B cells or CD138+ plasmablasts in the coculture system of NMSCs and activated B cells in the presence of neutralized anti-CCL2 Ab. (D) The percentage of BrdU+ B cells or CD138+ plasmablasts in the coculture system of NMSCs and activated B cells in the presence of CCL2 or MMPs. Data are representative of three independent experiments. *p , 0.05, **p , 0.01. CT, cell–cell contact; TW, transwell. The Journal of Immunology 5311

To further understand the mechanism, we next detected peptides significant effects among all the treatments in lupus mice (Fig. 5C). derived from CCL2 after processing with MMP in normal human Thus, OE of CCL2 corrected the suppressive defect of LMSCs for and SLE MSCs using mass spectrometry method. We showed that B cells in vivo. there was only N-terminal peptide of CCL2 in the supernatants of Human SLE BMMSCs had lower CCL2 expression and normal human MSCs compared with N-terminal and C-terminal exhibited impaired B cell suppression peptide of CCL2 in the supernatants of SLE MSCs. The data suggest that the CCL2 from SLE MSCs is less and differentially To study whether BMMSCs from SLE patients had similar im- processed compared with normal human MSCs (Supplemental pairment with MRL/lpr mice–derived BMMSCs in B cell sup- Fig. 4). pression, we cocultured activated normal human B cells with BMMSCs from healthy human (HMSC) or SLE patients (SMSC). Overexpression of CCL2 in LMSCs suppressed circulating In both cell–cell contact and transwell culture systems, SMSC autoantibody levels in MRL/lpr mice showed a much weaker suppression of B cell proliferation and The restoration of the suppressive function in LMSCs with OE of differentiation than did HMSC (Fig. 6A). Importantly, SMSC CCL2 in vitro encouraged us to study whether CCL2 gene-modified exhibited significantly lower levels of CCL2 transcription and LMSCs exhibited similar effects on B cells in vivo. The same protein expression compared with HMSC (Fig. 6B). To confirm numbers of LMSC–negative-OE (the mock vector coded with that CCL2 expression was indeed involved in HMSC-mediated scrambled gene) or LMSC–CCL2-OE were injected into 18-wk- B cell suppression, we added anti-CCL2 Ab in the cocultures of old MRL/lpr mice, and the mice were sacrificed 4 wk later. human activated B cells and HMSC. We showed that anti-CCL2

Transplantation of LMSC–CCL2-OE signiﬁcantly inhibited B cell Ab blocked the suppression of B cell proliferation and differen- Downloaded from function in lupus mice as evidenced by reduced serum levels of tiation mediated by HMSC (Fig. 6C). Thus, CCL2 is also required ANA and anti-dsDNA Ab (Fig. 5A), decreased frequency and for human B cell suppression mediated by MSCs. number of splenic CD138+ plasmablasts, and reduction of de- posited amounts of IgG/IgM in kidney (Fig. 5B), when compared Discussion with control LMSC–negative-OE treatment. Although both In this study, we showed that BMMSC from lupus-like MRL/lpr

LMSCs and LMSC–CCL2-OE decreased IL-10 and increased mouse or SLE patients are defective in the immunomodulatory http://www.jimmunol.org/ TGF-b1 levels in the serum, LMSC–CCL2-OE showed the most activity for normal B cells. More importantly, we have uncovered by guest on September 25, 2021

FIGURE 4. Effects of LMSCs or NMSCs transfected, respectively, with CCL2 expression vector or CCL2 siRNA on B cells. (A) The percentage of BrdU+ B cells in the coculture system of activated B cells and different sources of MSCs (LMSCs, LMSCs with CCL2-OE, NMSCs or NMSCs with CCL2-OE). (B) The percentage of CD138+ plasmablasts in the coculture system of activated B cells and different sources of MSCs (LMSCs, LMSCs with CCL2-OE, NMSCs or NMSCs with CCL2-OE). (C) Supernatant IgG/IgM levels in the coculture system of activated B cells and different sources of MSCs (LMSCs, LMSCs with CCL2-OE, NMSCs or NMSCs with CCL2-KD). Data are representative of three independent experiments. *p , 0.05. CCL2-KD, CCL2 knockdown; Neg-KD, negative knockdown; Neg-OE, negative OE. 5312 IMPAIRED B CELL INHIBITION BY LUPUS BMMSC Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. LMSCT with CCL2-OE led to more decreased serum autoantibody levels in MRL/lpr mice than original LMSCs. (A) Serum levels of ANA and anti-dsDNA Abs. (B) Plasmablasts were identified using CD19 and CD138 Abs. Frequencies and numbers of CD192CD138+ plasmablasts in spleen and the amounts of IgG or IgM deposition in kidney of lupus mice. IgG and IgM deposition was detected using immunohistochemical method. (C) Serum IL-10 and TGF-b1 levels in MRL/lpr mice. Data are representative of three independent experiments. *p , 0.05. that this impaired B cell inhibition was caused by lower expression notion that LMSCs did possess a defect in immune suppression, of CCL2 in lupus BMMSC. We further elucidated that CCL2- especially in B cell suppression. mediated suppressive effect on B cells required MSC-secreted To further confirm that B cell inhibition was impaired by MMP cleavage. LMSCs, we investigated the effect of LMSCs on B cell prolifer- As a novel therapeutic approach, we and others have demon- ation and differentiation in vitro. It is generally believed up to now strated that allogeneic BMMSC or umbilical cord–derived MSC that MSCs from healthy human and normal mice can inhibit B cell transplantation could significantly improve the pathology of lupus proliferation, differentiation, and Ab secretion in vitro, although nephritis and serological changes in MRL/lpr mice (27, 34). More the published data are not unanimous, which might be because importantly, we have also found reduced proteinuria, ANA, and of different experimental systems and conditions (7, 8, 36, 37). Our anti-dsDNA Abs in refractory human SLE after MSC infusion results are in line with the data that NMSCs are inhibitory for (28, 29). We recently showed that BMMSCs derived from B cells in a dose-dependent way and mediated by soluble factors. (NZB/NZW)F1 mice after onset of lupus disease failed to decrease However, LMSCs displayed impaired suppressive capacity for glomerular IgG deposits and splenic CD19+CD21+ B cells num- B cells in vitro and in vivo. bers (35), suggesting that lupus-derived BMMSCs may have Studies performed on MSC secretome revealed the presence of immunomodulatory disability. In this article, we show that normal a wide range of cytokines and chemokines including CCL2. As BMMSC (NMSC) transplantation could ameliorate lupus renal shown in Fig. 3A, we found that the expression of other chemo- pathology and decrease serum Ab levels, as well as the number of kines such as CXCL1, CXCL10, and CXCL11 were also reduced peripheral CD138+ plasmablasts, whereas these effects mediated in BMMSCs from lupus mice. Because of obvious differences of by LMSCs were weakened. These findings also forwarded the CCL2 from NMSCs and LMSCs, we focus in this article on CCL2 The Journal of Immunology 5313

+ + FIGURE 6. SLE-derived MSCs had impaired B cell suppression via lower CCL2 expression. (A) The percentage of BrdU B cells or CD138 plas- Downloaded from mablasts and supernatant IgG/IgM levels in the coculture system of HMSC or SMSC and normal human B cells. (B) CCL2 transcription and expressions on SMSC or HMSC detected by real-time PCR and Western blot. (C) The percentage of BrdU+ B cells or CD138+ plasmablasts in the coculture system of HMSC and normal human activated B cells in the presence of neutralized anti-CCL2 Ab. Data are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. CT, cell–cell contact; H, HMSC; S, SMSC; TW, transwell.

biological function in this process, although we believe this is not Taken altogether, we have uncovered a novel function of CCL2 http://www.jimmunol.org/ specific for CCL2. CCL2, a member of the CC chemokine family, in MSC-mediated B cell immunoregulation. Our data suggest that is a potent chemoattractant for monocytes and macrophages to CCL2 manipulation on MSCs may serve as a potential pathway areas of inflammation. It has been documented that CCL2 plays for developing more effective MSC-based therapy in autoimmune a critical role in inflammatory diseases, and enhanced levels of this diseases associated with B cell activation, such as SLE. chemokine are commonly observed in some autoimmune disorders (38). Previous studies have shown that CCL2 was pathogenic for Disclosures kidney injury in mouse lupus nephritis, and significantly associated The authors have no financial conflicts of interest. with human SLE nephritis, and urine CCL2 has been regarded as a candidate biomarker for SLE renal flare (39). A previous report References by guest on September 25, 2021 showed that MSC-conditioned medium inhibited experimental 1. Prockop, D. J. 1997. Marrow stromal cells as stem cells for nonhematopoietic + autoimmune encephalomyelitis–derived CD4 T cell activation by tissues. Science 276: 71–74. suppressing STAT3 phosphorylation via MSC-derived CCL2 (19), 2. Pittenger, M. F., A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simonetti, S. Craig, and D. R. Marshak. suggesting a novel immunoregulatory effect of CCL2. We have 1999. Multilineage potential of adult human mesenchymal stem cells. 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FIGURE 1. NMSC suppressed B cell activation viathe soluble factors. (A) The percentage of Brdu+B cells or CD138+ plasmablasts and supernatant lgG/IgM levels in the coculture system of NMSC and B cells at the NMSC/B cells ratio of 1:1 and

1:10. (B) The percentage of Brdu+B cells or CD138+ plasmablasts and supernatant lgG/IgM levels in the coculture system of NMSC and B cells in the condition of cell-cell contact or transwell system.

LMSC：MRL/lpr mice-derived MSC, NMSC: C57BL/6 mice-derived MSC, CT: cell-cell contact, TW: transwell system. *P < 0.05, **P < 0.01. Data are representative of three independent experiments.

FIGURE 2. CCL2 expression of P3 NMSC and LMSC from 9-week lupus mice

(premorbid) by western blot analysis.

N: NMSC, L: LMSC, LMSC: MRL/lpr mice-derived MSC, NMSC: C57BL/6 mice-derived MSC.

FIGURE 3. CCL2 expression and characteristics analysis of modified MSC

(LMSC with CCL2-OE or NMSC with CCL2-KD). (A) CCL2 expression on modified MSC. (B) the proliferation analysis of modified MSC. C, surface marker expressions of modified MSC.

LMSC: MRL/lpr mice-derived MSC, NMSC: C57BL/6 mice-derived MSC,

CCL2-OE: CCL2 over-expression, CCL2-KD: CCL2 knock-down. *P < 0.05. Data are representative of three independent experiments.

FIGURE 4. CCL2 in SLE MSC was less effective processed than that in normal human MSC. (A) Amino acid sequences of full-length (CCL2 precursor) or MMP processed CCL2 (mpCCL2). The underline sequences are tested by mass spectrometry, and the red sequences are the differences between full-length and processed sequences. (B) There are only N-terminal peptide of CCL2 in normal human MSC supernatants. (C) There are N-terminal and C-terminal peptide of CCL2 in SLE MSC supernatants.

Data are representative of three independent experiments.