Cellular & Molecular Immunology (2017) 14, 895–908 & 2017 CSI and USTC All rights reserved 2042-0226/17 $32.00 www.nature.com/cmi RESEARCH ARTICLE Mesenchymal stem cells ameliorate B-cell-mediated immune responses and increase IL-10-expressing regulatory B cells in an EBI3-dependent manner Kyung-Ah Cho1, Jun-Kyu Lee1, Yu-Hee Kim1, Minhwa Park1, So-Youn Woo1 and Kyung-Ha Ryu2 Effector B cells are central contributors to the development of autoimmune disease by activating autoreactive T cells, producing pro-inflammatory cytokines and organizing ectopic lymphoid tissue. Conversely, IL-10-producing regulatory B (Breg) cells have pivotal roles in maintaining immunological tolerance and restraining excessive inflammation in autoinflammatory disease. Thus, regulating the equilibrium between antibody-producing effector B cells and Breg cells is critical for the treatment of autoimmune disease. In this study, we investigated the effect of human palatine tonsil-derived mesenchymal stem cells (T-MSCs) on estradiol (E2)-induced B-cell responses in vivo and in vitro. Transplantation of T-MSC into E2-treated mice alleviated B-cell-mediated immune responses and increased the population of IL-10-producing Breg cells. T-MSCs regulated the B-cell populations by producing Epstein–Barr virus (EBV)-induced 3 (EBI3), one of the two subunits of IL-35 that is the well-known inducer of Breg cells. We demonstrate a critical role of EBI3 (IL-35) in vitro by depleting EBI3 in T-MSCs and by adding exogenous IL-35 to the culture system. Taken together, our data suggest that IL-35-secreting MSCs may become an attractive therapeutic to treat B-cell-mediated autoimmune diseases via expanding Breg cells. Cellular & Molecular Immunology (2017) 14, 895–908; doi:10.1038/cmi.2016.59; published online 2 January 2017 Keywords: autoimmune disease; B cells; IL-35; mesenchymal stem cells INTRODUCTION cell function is interleukin (IL)-10,9 which inhibits the produc- B cells have a central role in the adaptive immune response to tion of pro-inflammatory cytokines and supports regulatory 10 antigens via their differentiation into plasma cells for antibody T(Treg)-cell differentiation. Therefore, modulating B-cell production or into memory B cells for enhanced recall activation and inducing sufficient Breg cells may be effective response to an antigen.1,2 Their role as antibody-producing therapeutic strategies to treat autoimmune diseases. cells in autoimmune diseases like systemic lupus erythematosus Mesenchymal stem cells (MSCs) are multipotent adult stem (SLE) and rheumatoid arthritis3 is well accepted.4–6 Besides cells that have been shown to possess immunomodulatory and producing antibodies, B cells perform a variety of immuno- tissue regeneration properties. These properties, together with logical functions, including antigen presentation, production of their low immunogenic potential, make them a promising multiple cytokines and regulation of lymphoid tissue architec- new treatment for severe refractory autoimmune diseases.11 ture. Certain B cells, referred to as regulatory B (Breg)cells,can Originally, MSCs were collected from the bone marrow (BM) also negatively regulate immune responses by producing of patients, which required a highly invasive procedure; regulatory cytokines and directly interacting with pathogenic however, now, they can also be isolated from the umbilical T cells via cell-to-cell contact. Breg cells contribute to the cord (UC) or other tissues, such as palatine tonsils. Given the maintenance of fine equilibrium that is required for tolerance. immunomodulatory properties of MSCs, their effects on a Moreover, they restrain excessive inflammatory responses that variety of immune cells, including T cells, dendritic cells (DCs) 7,8 12 occur in autoimmune diseases. The central mediator of Breg and natural killer cells, have been widely studied. However, 1Department of Microbiology, School of Medicine, Ewha Womans University, Seoul 07985, Republic of Korea and 2Department of Pediatrics, School of Medicine, Ewha Womans University, Seoul 07985, Republic of Korea Correspondence: Professor S-Y Woo, MD, PhD, Department of Microbiology, School of Medicine, Ewha Womans University, Seoul 07985, Republic of Korea. E-mail: [email protected] or Professor K-H Ryu, MD, PhD, Department of Pediatrics, School of Medicine, Ewha Womans University, Seoul 07985, Republic of Korea. E-mail: [email protected] Received: 30 July 2016; Revised: 29 October 2016; Accepted: 29 October 2016 MSCs regulate B-cell response by expressing EBI3 K-A Cho et al 896 the interactions between MSCs and B cells, and the mechan- Enzyme-linked immunosorbent assay isms that regulate these interactions, remain to be determined. To detect circulating IgG1, IgM and IgA, serum was collected We previously established human palatine tonsil-derived from 5-month-old female or male C57BL/6 mice. Antibody MSCs (T-MSCs) as a new source of MSC,13 and we demon- levels in serially diluted serum were analyzed with HRP- fi strated that T-MSCs have suppressive effects on T cells, DCs conjugated antibodies speci c for mouse IgG1 and IgG2a using and B cells.14,15 These results provided evidence that T-MSCs mouse immunoglobulin isotyping enzyme-linked immunosor- can exert potent immunosuppressive actions. On the basis of bent assay (ELISA) kit (BD Biosciences, San Jose, CA, USA). these findings, in this study, we investigated the impact of The serum IgG1 and IgG2a from control, E2-treated and T-MSCs on B cells in an estrogen-triggered, immune-activated T-MSC-transplanted E2-treated mice, were detected as state in vivo and in vitro, following treatment of mice with described above. To detect IgG from mouse splenic B cells, estradiol (E2). Women are generally more susceptible than single-cell suspensions of splenocytes from female C57BL/6 5 men to many autoimmune diseases.16 A gender difference in mouse were seeded at a density of 5 × 10 per well in 96-well disease incidence exists for several autoimmune disorders, plates with phenol red-free Roswell Park Memorial Institute including: rheumatoid arthritis (female-to-male ratio is medium (Welgene, Korea) supplemented with BAFF (4 ng/ml), μ 2–4:1), multiple sclerosis (2–5:1) and lupus (9:1).17,18 More- anti-CD3 Ab (1 g/ml, Biolegend, San Diego, CA, USA), anti- μ over, studies have shown that the sex hormone estrogen is, in CD28 Ab (2 g/ml, Biolegend), recombinant IL-2 (50 ng/ml, part, responsible for the higher occurrence of autoimmune Biolegend) and recombinant IL-4 (10 ng/ml, Biolegend). Then the cells were cultured for 1 week in the presence or absence of disorders in females.19,20 Thus, we investigated whether E2 EBI3 knocked down-T-MSCs (5 × 104 per well), E2 (10 nM) or itself could trigger B-cell activation in non-autoimmune-prone the combination of T-MSCs and E2. Cells were. After 1 week, mice, and we assessed the therapeutic effects of T-MSCs on the cell culture supernates were collected, and IgG titers were E2-treated mice. Finally, to better understand immuno- measured using an IgG Mouse ELISA kit (Abnova, Taipei City, modulatory mechanisms of MSCs on B cells, we attempted Taiwan). to identify crucial mediators by which MSCs modulate E2-induced B-cell responses. Reverse transcription PCR and quantitative RT-PCR analysis For analysis of estrogen receptor-1 (Esr1) expression in various MATERIALS AND METHODS organs of the mice (5-month-old female C57BL/6 mice and 5- Mice and E2 treatment month-old male C57BL/6 mice), brains, spinal cords, livers, We purchased 8-week-old female C57BL/6 mice from Orient lungs, kidneys, small intestines, large intestines, BM, muscles, Bio (Emsung, Korea). All animals were maintained under spleens (SPs) and dLN, were isolated and homogenized in pathogen-free conditions on a 12-h light/dark cycle with free TRIzol (Invitrogen, Carlsbad, CA, USA). Complementary DNA access to food and water. All procedures were approved by the was synthesized using the First-Strand cDNA Synthesis Kit Animal Care and Use Committee at the Ewha Womans (TOYOBO, Osaka, Japan) according to the manufacturer’s University School of Medicine (ESM 15-0311) and all experi- instructions. Esr1 (202 bp) was amplified using the following ments were performed in accordance with the approved primers: 5′-TGCCGTGTGCAATGACTATG-3′ (forward) and guidelines and regulations. Eight-week-old, female mice 5′-TTTCATCATGCCCACTTCGT-3′ (reverse). Esr2 (129 bp) β received injections of 17- estradiol (E2, Sigma Aldrich, was amplified using the following primers, 5′-CTGTGCCTCT St Louis, MO, USA) at 5 mg/kg in olive oil vehicle subcuta- TCTCACAAGGA-3′ (forward) and 5′-TGCTCCAAGGGTAG neously for 12 consecutive days. For the T-MSC-transplanted GATGGAC-3′(reverse). The expression levels of B-cell activat- groups, T-MSCs were intravenously injected on the first, third ing factor (Baff), a proliferation-inducing ligand (April), and seventh days of the 12 days of E2 treatment. Control mice osteopontin (Opn) and interleukin-21 (IL-21)indLNs,SPs, were injected subcutaneously with olive oil vehicle alone. Four BMs and small intestines were compared using the following days after the final injection of E2, mice were killed. primers: 5′-GCTACCGAGGTTCAGCAACA-3′ (forward) and 5′-TCTGCAATCAGCTGCAGACA-3′ (reverse) for Baff Cell culture (271 bp), 5′-TGGCAACCAGTACTTAGGCG-3′ (forward) and fi B cells were puri ed from mouse spleen and draining lymph 5′-TAGGCACGGTCAGGATCAGA-3′ (reverse) for April nodes (dLNs) via magnetic isolation (Miltenyi Biotec, GmbH, (212 bp), 5′-CTCGATGTCATCCCTGTTGC-3′ (forward) and Gladbach, Germany). Single-cell suspensions of B cells were 5′-AGCTTGTCCTTGTGGCTGTG-3′ (reverse) for Opn seeded at a density of 5 × 105 per well in 96-well plates with (236 bp), 5′-AGCCATCAAACCCTGGAAAC-3′ (forward) and phenol red-free Roswell Park Memorial Institute medium 5′-GTACCCGGACACAACATGGA-3′ (reverse) for IL-21 (Welgene, Korea) containing 4 ng/ml BAFF to support survival. (234 bp). In B cells, Cd1d and Cd5 were amplified with the For co-culture with T-MSCs, 5 × 104 T-MSCs were added to following primers: 5′-CCATCAAAGTGCTCAACGCT-3′ (forward) establish a 10:1 ratio of B cells:T-MSCs.