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.
B cells have a central role in the adaptive immune response to antigens via their differentiation into plasma cells for antibody production or into memory B cells for enhanced recall response to an antigen.1, 2 Their role as antibody-producing cells in autoimmune diseases like systemic lupus erythematosus (SLE) and rheumatoid arthritis3 is well accepted.4, 5, 6 Besides producing antibodies, B cells perform a variety of immunological functions, including antigen presentation, production of multiple cytokines and regulation of lymphoid tissue architecture. Certain B cells, referred to as regulatory B (Breg) cells, can also negatively regulate immune responses by producing regulatory cytokines and directly interacting with pathogenic T cells via cell-to-cell contact. Breg cells contribute to the maintenance of fine equilibrium that is required for tolerance. Moreover, they restrain excessive inflammatory responses that occur in autoimmune diseases.7, 8 The central mediator of Breg cell function is interleukin (IL)-10,9 which inhibits the production of pro-inflammatory cytokines and supports regulatory T (Treg)-cell differentiation.10 Therefore, modulating B-cell activation and inducing sufficient Breg cells may be effective therapeutic strategies to treat autoimmune diseases.
Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have been shown to possess immunomodulatory and tissue regeneration properties. These properties, together with their low immunogenic potential, make them a promising new treatment for severe refractory autoimmune diseases.11 Originally, MSCs were collected from the bone marrow (BM) of patients, which required a highly invasive procedure; however, now, they can also be isolated from the umbilical cord (UC) or other tissues, such as palatine tonsils. Given the immunomodulatory properties of MSCs, their effects on a variety of immune cells, including T cells, dendritic cells (DCs) and natural killer cells, have been widely studied.12 However, the interactions between MSCs and B cells, and the mechanisms that regulate these interactions, remain to be determined.
We previously established human palatine tonsil-derived MSCs (T-MSCs) as a new source of MSC,13 and we demonstrated that T-MSCs have suppressive effects on T cells, DCs and B cells.14, 15 These results provided evidence that T-MSCs can exert potent immunosuppressive actions. On the basis of these findings, in this study, we investigated the impact of T-MSCs on B cells in an estrogen-triggered, immune-activated state in vivo and in vitro, following treatment of mice with estradiol (E2). Women are generally more susceptible than men to many autoimmune diseases.16 A gender difference in disease incidence exists for several autoimmune disorders, including: rheumatoid arthritis (female-to-male ratio is 2–4:1), multiple sclerosis (2–5:1) and lupus (9:1).17, 18 Moreover, studies have shown that the sex hormone estrogen is, in part, responsible for the higher occurrence of autoimmune disorders in females.19, 20 Thus, we investigated whether E2 itself could trigger B-cell activation in non-autoimmune-prone mice, and we assessed the therapeutic effects of T-MSCs on the E2-treated mice. Finally, to better understand immunomodulatory mechanisms of MSCs on B cells, we attempted to identify crucial mediators by which MSCs modulate E2-induced B-cell responses.
Materials and methods
Mice and E2 treatment
We purchased 8-week-old female C57BL/6 mice from Orient Bio (Emsung, Korea). All animals were maintained under pathogen-free conditions on a 12-h light/dark cycle with free access to food and water. All procedures were approved by the Animal Care and Use Committee at the Ewha Womans University School of Medicine (ESM 15-0311) and all experiments were performed in accordance with the approved guidelines and regulations. Eight-week-old, female mice received injections of 17-β estradiol (E2, Sigma Aldrich, St Louis, MO, USA) at 5 mg/kg in olive oil vehicle subcutaneously for 12 consecutive days. For the T-MSC-transplanted groups, T-MSCs were intravenously injected on the first, third and seventh days of the 12 days of E2 treatment. Control mice were injected subcutaneously with olive oil vehicle alone. Four days after the final injection of E2, mice were killed.
B cells were purified from mouse spleen and draining lymph nodes (dLNs) via magnetic isolation (Miltenyi Biotec, GmbH, Gladbach, Germany). Single-cell suspensions of B cells were seeded at a density of 5 × 105 per well in 96-well plates with phenol red-free Roswell Park Memorial Institute medium (Welgene, Korea) containing 4 ng/ml BAFF to support survival. For co-culture with T-MSCs, 5 × 104 T-MSCs were added to establish a 10:1 ratio of B cells:T-MSCs. Cells were treated with E2 (working concentration: 10 nM) for 48 h to detect B-cell activation markers and for 72 h to observe induction of Breg cells.
Enzyme-linked immunosorbent assay
To detect circulating IgG1, IgM and IgA, serum was collected from 5-month-old female or male C57BL/6 mice. Antibody levels in serially diluted serum were analyzed with HRP-conjugated antibodies specific for mouse IgG1 and IgG2a using mouse immunoglobulin isotyping enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Jose, CA, USA). The serum IgG1 and IgG2a from control, E2-treated and T-MSC-transplanted E2-treated mice, were detected as described above. To detect IgG from mouse splenic B cells, single-cell suspensions of splenocytes from female C57BL/6 mouse were seeded at a density of 5 × 105 per well in 96-well plates with phenol red-free Roswell Park Memorial Institute medium (Welgene, Korea) supplemented with BAFF (4 ng/ml), anti-CD3 Ab (1 μg/ml, Biolegend, San Diego, CA, USA), anti-CD28 Ab (2 μg/ml, Biolegend), recombinant IL-2 (50 ng/ml, Biolegend) and recombinant IL-4 (10 ng/ml, Biolegend). Then the cells were cultured for 1 week in the presence or absence of EBI3 knocked down-T-MSCs (5 × 104 per well), E2 (10 nM) or the combination of T-MSCs and E2. Cells were. After 1 week, cell culture supernates were collected, and IgG titers were measured using an IgG Mouse ELISA kit (Abnova, Taipei City, Taiwan).
Reverse transcription PCR and quantitative RT-PCR analysis
For analysis of estrogen receptor-1 (Esr1) expression in various organs of the mice (5-month-old female C57BL/6 mice and 5-month-old male C57BL/6 mice), brains, spinal cords, livers, lungs, kidneys, small intestines, large intestines, BM, muscles, spleens (SPs) and dLN, were isolated and homogenized in TRIzol (Invitrogen, Carlsbad, CA, USA). Complementary DNA was synthesized using the First-Strand cDNA Synthesis Kit (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. Esr1 (202 bp) was amplified using the following primers: 5′-TGCCGTGTGCAATGACTATG-3′ (forward) and 5′-TTTCATCATGCCCACTTCGT-3′ (reverse). Esr2 (129 bp) was amplified using the following primers, 5′-CTGTGCCTCTTCTCACAAGGA-3′ (forward) and 5′-TGCTCCAAGGGTAGGATGGAC-3′(reverse). The expression levels of B-cell activating factor (Baff), a proliferation-inducing ligand (April), osteopontin (Opn) and interleukin-21 (IL-21) in dLNs, SPs, BMs and small intestines were compared using the following primers: 5′-GCTACCGAGGTTCAGCAACA-3′ (forward) and 5′-TCTGCAATCAGCTGCAGACA-3′ (reverse) for Baff (271 bp), 5′-TGGCAACCAGTACTTAGGCG-3′ (forward) and 5′-TAGGCACGGTCAGGATCAGA-3′ (reverse) for April (212 bp), 5′-CTCGATGTCATCCCTGTTGC-3′ (forward) and 5′-AGCTTGTCCTTGTGGCTGTG-3′ (reverse) for Opn (236 bp), 5′-AGCCATCAAACCCTGGAAAC-3′ (forward) and 5′-GTACCCGGACACAACATGGA-3′ (reverse) for IL-21 (234 bp). In B cells, Cd1d and Cd5 were amplified with the following primers: 5′-CCATCAAAGTGCTCAACGCT-3′ (forward) and 5′-ACATGACACACCAGCTGCCT-3′ (reverse) for Cd1d (202 bp) and 5′-AGGGTATTCGTCACATGCCA-3′ (forward) and 5′-CAATCCACTGACGCTGCTTT-3′ (reverse) for Cd5 (180 bp). The expression of Cd1d and Cd5 on B cells was confirmed by the quantitative RT-PCR on a StepOnePlus instrument (Applied Biosystems, Foster City, CA, USA) using SYBR green (TOYOBO).
For the normalization of all genes in RT-PCR, the internal control gene Gapdh (123 bp) was amplified using the following primers: 5′-AGGTCGGTGTGAACGGATTTG-3′ (forward) and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (reverse). The band pixel densities of genes were divided by the pixel densities of the corresponding Gapdh bands for quantitation using UN-SCAN-IT-gel 6.1 software (Silk Scientific, Orem, UT, USA).
Preparation of conditioned medium
To generate MSC-conditioned medium (MSC-CM), BM-derived MSCs (BM-MSCs), adipose tissue-derived MSCs (AT-MSCs) and T-MSCs (at passages 7–8) were grown to 80–90% confluence in 100-mm tissue culture plates. The T-MSCs were obtained and maintained as we previously reported.13 The AT-MSCs were generously provided by RNLBio (Seoul, Korea), and the BM-MSCs were purchased from the Severance Hospital Cell Therapy Center (Seoul, Korea). At 80–90% confluence, the cells were washed twice with phosphate-buffered saline, and the medium was replaced with serum-free Dulbecco’s modified Eagle’s medium to generate CM. The medium was collected after 48 h of culture, centrifuged at 1300 r.p.m. for 5 min and passed through a 0.2-μm filter. The CM was concentrated to 20-fold of the original concentration by centrifugal filtration (cut-off of 3K, Amicon Ultra-15, Millipore, Bedford, MA, USA). The concentrated CM were frozen and stored at −80 °C for future use. As a negative control, the above mentioned serum-free culture medium was processed in the same manner. Additionally, conditioned media from murine MSC line, C3H10T1/2 cells (ATCC no. CCL-226, Manassas, VA, USA) were collected as described above.
Equal amounts of CM from each type of human MSCs (BM-MSCs, AT-MSCs and T-MSCs) were loaded per lane, and the blotted membranes were incubated overnight with a primary antibody against EBI3 (G-4, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-12p35 (H-197, Santa Cruz Biotechnology) and IL-27 (ab56576, Abcam, Cambridge, UK). For T-MSCs, a primary antibody against β-actin was used for normalization of EBI3 expression. After intensive washing, the membranes were incubated with the corresponding secondary antibodies (anti-mouse IgG, Sigma Aldrich) and detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Walthan, MA, USA). The secretion of EBI3 and IL-12p35 in C3H10T1/2 cells was observed as described above.
To reduce endogenous Ebi3 expression, T-MSCs were transfected with Ebi3-specific siRNA oligonucleotides (Santa Cruz Biotechnology) using Lipofectamine 2000 reagent (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Non-targeted siRNA oligonucleotides (Santa Cruz Biotechnology) were used as negative controls. At 48 h post-transfection, the cells were collected for protein extraction. Ten micrograms of protein were loaded per sample to measure EBI3 knockdown using western blot. Endogenous Ebi3 expression of C3H10T1/2 cells was downregulated by transfection with mouse Ebi3-specific siRNA oligonucleotides (Santa Cruz Biotechnology) as described above.
SPs from control, E2-treated and T-MSC-transplanted E2-treated mice were fixed with 4% formaldehyde and embedded in paraffin. Sections (5-μm thickness) were mounted on slides and stained with hematoxylin and eosin (H&E) for histological evaluation.
Paraffin-embedded spleen sections were de-paraffinized and dehydrated. After blocking with 1% bovine serum albumin in 0.02% Tween-20 in PBS (PBST), slides were incubated overnight with primary antibodies anti-mouse CD4 (MT310, Santa Cruz Biotechnology) or anti-mouse B220 (RA3-6B2, Santa Cruz Biotechnology) at 4 °C. On the following day, slides were washed three times in PBS for 5 min and incubated with secondary antibodies, FITC-conjugated anti-mouse IgG (ab6785, Abcam) and Cy3-conjgated anti-Rat IgG (ab6953, Abcam) for 1 h at room temperature in the dark. Following three, 5-min washes in PBS, slides were mounted using DAPI (4′,6-diamidino-2-phenylindole) mounting solution (Vector Laboratories, Youngstown, OH, USA) per the manufacturer’s instructions. Immunofluorescence was detected by confocal microscopy (LSM 5 Pascal Microscope, Carl Zeiss, Oberkochen, Germany).
Control, E2-treated and T-MSC-transplanted E2-treated mice were killed, and the germinal center (GC) B cells, plasma cells and IL-10-expressing B cells were investigated by flow cytometric analysis. GC B cells were detected by labeling with FITC-conjugated anti-mouse B220 (RA3-6B2, Biolegend) and PerCP-conjugated anti-mouse class II MHC (M5/114.15.2, Biolegend) antibodies. Plasma cells were detected by labeling with APC-conjugated anti-mouse CD138 (281-2, Biolegend) and FITC-conjugated anti-mouse B220 antibodies. For detection of IL-10-expressing B cells, intracellular IL-10 was analyzed after gating on CD19+ cells. A FITC-conjugated anti-mouse IL-10 antibody (JES5-16E3, BD Biosciences) was used for staining intracellular IL-10 in B cells. To compare the expression of surface Esr1 on B cells from spleens and dLN in female and male mice, cells were labeled with mouse anti-Estrogen receptor alpha Ab (ab16460, Abcam), followed by incubation with FITC-secondary Ab against mouse IgG (ab6785). To analyze the surface expression of CD80, CD86, MHC II, CD40, CD69, CCR7 and CD19 on B cells from normal C57BL/6 female mice, cells were labeled with FITC-conjugated anti-mouse CD80 (16-10A1, BD Biosciences), PE-conjugated anti-mouse CD86 (GL1, BD Biosciences), PerCP-conjugated anti-mouse MHC II (M5/114.15.2, Biolegend), FITC-conjugated anti-mouse CD40 (3/23, BD Biosciences), PE-conjugated anti-mouse CD69 (H1.2F3, BD Biosciences), PE-conjugated anti-mouse CCR7 (4B12, R&D Systems, Minneapolis, MN, USA) or PE-conjugated anti-mouse CD19 (6D5, Biolegend) antibodies. Lastly, surface expression of PD-L1, CD1d and CD5 on the CD19-gated B cells was detected using FITC anti-CD19 (Biolegend), PerCP-Cy anti-mouse CD1d (1B1, Biolegend), PE anti-mouse CD5 (53-7.3, Biolegend) and APC anti-mouse PD-L1 (10F.9G2, Biolegend) antibodies. The level of non-specific staining was evaluated using each corresponding isotype control antibody. Each sample was acquired on a FACSCalibur system (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Data are presented as mean±standard error of the mean (s.e.m.). The statistical significance was analyzed by one-way ANOVA or Student’s t-test using GraphPad PRISM 7 software (GraphPad Software, San Diego, CA, USA). For all analyses, a P<0.05 was considered as statistically significant.
Female mice have higher steady-state immunological activation than male mice
Considering that the female gender appears to be a risk factor for autoimmunity, we wondered whether there are intrinsic differences in immunological features between female and male mice. Therefore, we compared steady-state levels of circulating IgG1, IgM and IgA between 5-month-old male and female C57BL6 mice. Interestingly, there were significantly higher levels of circulating immunoglobulins, including IgG1, IgM and IgA, in female mice than in male mice (Figures 1a–c). Because estrogen is one of the crucial factors that contributes to gender bias in the development and progression of autoimmune disease, we next investigated the pattern of estrogen receptor-1 (Esr1) expression in various organs of female and male mice. Although Esr1 was ubiquitously expressed in the organs of female mice, it was expressed at lower levels in the kidney, small intestine, muscle, spleen and dLN of male mice (Figures 1d and e). Esr2 was also evenly distributed over the organs in both female and male mice, but male mice showed higher Esr2 expression in the liver and kidney tissue. Instead, lymphoid tissues, including BM, SP and dLN, revealed lower expression of Esr2 in male mice (Supplementary Figures 1a and b). Surface Esr1 on B cells from SP or dLN showed a consistent expression pattern, but levels were significantly higher in female mice than in male mice (Supplementary Figures 1c and d).
Next, we determined the expression pattern of genes that are known to promote B-cell survival, proliferation and differentiation. In female mice, B-cell activating factor (Baff), a proliferation-inducing ligand (April), osteopontin (Opn) and interleukin-21 (IL-21) were all detected in lymphoid tissues, including SPs, BMs and dLNs. However, these genes were expressed at lower levels in the SPs of male mice, and IL-21 was not even detectable in male dLNs (Figure 1f). Furthermore, Baff, April, Opn and IL-21 were also notably expressed lower in male, compared with female, in small intestines (Figure 1g), which is consistent with the lower expression of Esr1 that we observed in small intestines from male mice. Altogether, these observations suggest that the sex hormone estrogen is associated with intrinsic differences in immunological features that affect B-cell activation.
E2 treatment leads to immune activation involving a B-cell response, which is ameliorated by T-MSC transplantation
To identify the effects of estrogen on immune responses, particularly B-cell activation, we examined the effect of E2 exposure in female C57BL/6 mice that had been treated with E2 or olive oil for 12 consecutive days. E2 treatment induced aberrant enlargement of follicles in the SP, accompanied by an increase in B220+ cells, which was not observed in control mice (Figure 2a). E2 also caused a significant increase in the levels of self IgG1 and IgG2a compared with those in the vehicle-treated mice (Figures 2b and c). To determine whether T-MSC transplantation affects these E2-induced immune responses, mice were intravenously injected with T-MSCs on the first, third and seventh days of the 12-day E2 exposure. The T-MSC-transplanted mice showed normal architecture in most follicles of the SP and a decrease in B220+ cells, like those of control mice (Figure 2a). Moreover, T-MSC transplantation attenuated the increased IgG production that was observed in the mice stimulated with E2 alone (Figures 2b and c).
T-MSCs alter the E2-induced disturbance of B-cell populations in mice
To evaluate the B-cell compartment after exposure to E2, we analyzed germinal center (GC) B cells, plasma cells and IL-10-secreting B cells by flow cytometry. GC B cells were identified by their expression of the surface markers B220 and MHC II, whereas plasma cells were identified by their expression of the surface markers B220 (low expression) and CD138, which is a receptor for A proliferation-inducing ligand (APRIL).21 APRIL is a survival factor that is involved in homing plasma cells to the BM and regulating the differentiation of plasma cells into long-lived cells.22 As shown in Figure 3a, the splenic GC B-cell population was increased by E2 treatment, and this increase was abrogated in the T-MSC-transplanted group. In the dLNs, there was a modest increase in the number of GC B cells in the E2-treated mice, but this increase was dampened by T-MSC transplantation. Similarly, the CD138+/B220low plasma cells were increased in the E2-treated group, but this increase was abrogated by T-MSC transplantation (Figure 3b). Finally, we analyzed intracellular levels of IL-10 in B cells, which were identified based on their expression of the cell surface marker CD19. Interestingly, T-MSC transplantation increased the IL-10-secreting B-cell population two–threefold compared with the mice stimulated with E2 alone (Figure 3c). Altogether, these data indicate that T-MSCs are capable of ameliorating B-cell activation induced by hormonal stimulation, and increasing the population of immunosuppressive IL-10-secreting Breg cells.
T-MSCs regulate E2-induced B-cell activation in vitro
Next, we tested the effect of T-MSCs on B cells, which were derived from the SPs and dLNs of C57BL/6 female mice, in the presence of 10 nM E2 in vitro. For the co-culture of T-MSCs and B cells, we seeded T-MSCs before seeding B cells at a ratio of 1:10. To assess B-cell activation, we analyzed the surface molecules CD80, CD86, MHC II, CD40, CD69 and CCR7, which are B-cell activation markers, by flow cytometry. E2 stimulation of B cells for 48 h resulted in the upregulation of B-cell activation markers, whereas E2 stimulation of B cells co-cultured with T-MSCs showed attenuated expression of these markers (Figures 4a and b). Because we observed an increase in the number of IL-10+ B cells following T-MSC transplantation in vivo, we speculated that T-MSC addition in vitro would also increase the population of IL-10-expressing Breg cells. Indeed, flow cytometric analysis of B cells co-cultured with T-MSCs showed an increase in the number of IL-10-expressing CD19+ cells, even without further supplementation with E2 (Figure 4c). Consistent with our in vivo data, our in vitro data show that the T-MSCs negatively regulate E2-mediated B-cell activation, and simultaneously, they positively regulate IL-10-expressing Breg cells.
T-MSCs modulate E2-induced B-cell responses in an EBI3-dependent manner
Next, we investigated candidate soluble factors produced by T-MSCs that could modulate B-cell activation. IL-35 is the most recently discovered member of the IL-12 family of heterodimeric cytokines and is composed of EBI3, a β-chain subunit encoded by the EBV-induced gene 3 (Ebi3), and IL-12p35, a subunit encoded by IL12A.23 An early study showed that IL-35 is mainly produced by natural Treg cells, and it contributes to their suppressive activities.24 However, a recent study showed that IL-35 also induces Breg cells that function to suppress autoimmune diseases.25 This prompted us to test whether T-MSCs constitutively produce both chains EBI3 and IL-12p35. In addition, EBI3 (or IL-27B) is a component of a heterodimeric cytokine IL-27, which is formed by EBI3 association with an IL-27p28 (or IL-30) subunit. Thus, we checked the expression of IL-27p28 to distinguish, which cytokine contributes to this mechanism. As shown in Figure 5a, EBI3, IL-12p35 and IL-27p28 were all detected in supernates from all tested types of MSCs (BM-MSCs, AT-MSCs and T-MSCs). Particularly, T-MSCs showed strong expression IL-12p35 rather than IL-27p28, indicating that IL-35 would likely have a more dominant role. Next, we transfected T-MSCs with a siRNA targeting Ebi3 or, as a negative control, we transfected T-MSCs with a scrambled, non-specific siRNA, and we demonstrated effective knockdown of endogenous EBI3 in T-MSCs (Figures 5b and c). Then, we cultured B cells isolated from mouse SPs and dLNs in the presence of E2, with or without transfected T-MSCs. As expected, the control-transfected T-MSCs ameliorated the E2-induced expression of B-cell activation markers, whereas the Ebi3-depleted T-MSCs were unable to modulate the expression of B-cell activation markers (Figure 5d). Importantly, this decreased activity in Ebi3-depleted T-MSCs was rescued by exogenous supplementation with rhIL-35. Of note, single treatments with rhIL-35 or T-MSCs also decreased the E2-induced expression of surface activation markers on B cells. As exogenous E2 increased IgG levels in vivo (Figures 2b and c), we evaluated IgG from splenic B cells in several experimental conditions. E2-treated splenocytes produced significantly higher quantities of IgG, but T-MSC supplementation stifled IgG production from splenocytes. This activity was EBI3-dependent because Ebi3-depleted T-MSCs had no effect (Figure 5e).
MSCs induce IL-10-expressing Breg cells in an EBI3-dependent manner
Because co-culturing T-MSCs with B cells also increased the number of Breg cells, even without further stimulation (Figure 4c), we next assessed the effects of T-MSC-derived EBI3 on the population of IL-10-expressing B cells. Therefore, we co-cultured B cells with T-MSCs transfected with Ebi3-specific siRNA or scrambled control siRNA, without E2 stimulation. The B cells co-cultured with control siRNA-transfected T-MSCs showed increased expression of Cd1d and Cd5, which are the signature molecules associated with Breg cells,26, 27 similar to levels observed in B cells stimulated with rIL-35, a known inducer of Breg cells (Figures 6a–c). Conversely, the B cells co-cultured with Ebi3 siRNA-transfected T-MSCs showed no change in Cd1d or Cd5 levels; however, addition of rIL-35 to this treatment group augmented expression of Cd1d and Cd5 similar to levels observed in the control group (Figures 6a–c). Finally, the co-culture of B cells with the control T-MSCs resulted in an increased percentage of IL-10+/CD19+ Breg cells, which was not observed in the EBI3-depleted T-MSC/B-cell co-cultures (Figure 6d). Interestingly, recent reports have shown that E2 treatment induces regulatory B cells, including CD1dhi, CD5+, PD-L1+ and IL-10-producing Breg cells, which have protective roles in experimental autoimmune encephalomyelitis (EAE).28, 29, 30 Thus, we examined the surface expression of CD1d, CD5 and PD-L1 in splenic B cells treated with E2 in the presence or absence of T-MSCs. As shown in Supplementary Figure 2, E2 itself did not increase the number of CD1d+CD5+ or PD-L1+CD5+ cells. Instead, T-MSCs induced those population regardless of E2 treatment. This result implies that Breg induction by E2 stimulation may require a more complex inflammatory milieu.
Because human IL-35 bears ~60% homology with mouse IL-35, next we examined the effect of murine MSCs on Breg cell induction using C3H10T1/2 cells. C3H10T1/2s are murine multipotent MSCs for which we previously verified immunosuppressive properties in a mouse model of graft-versus-host disease.31 Single-chain EBI3 and IL-12p35 were observed in the supernates of C3H10T1/2 cells; thus, we suppressed EBI3 expression through transfection of mouse Ebi3 siRNA (Figures 7a and b). In line with our previous findings in T-MSCs, C3H10T1/2 cells also augmented expression of critical molecules, Cd1d or Cd5, on B-cell surfaces, and this augmentation was dependent on EBI3 (Figures 7c–e). On treatment with recombinant human IL-35, recombinant mouse IL-35 (rmIL-35) or murine MSCs, Cd1d, but not Cd5, expression changed significantly (Figures 7f and g). Although co-culture with C3H10T1/2 cells or treatment with rmIL-35 induces mouse Breg cells more robustly than co-culture with T-MSCs or treatment with rhIL-35, T-MSCs alone are sufficient to induce Breg cells via IL-35 activity (Figure 7h). Collectively, our data suggest that MSCs directly induce Breg cells in an IL-35-dependent manner.
In this study, we used T-MSCs and murine MSCs to demonstrate the immunomodulatory effects of MSCs on B-cell-associated immune responses induced by E2 treatment. Furthermore, we showed that these MSCs express and secrete IL-35, an anti-inflammatory cytokine consisting of EBI3 and IL-12p35. Importantly, we showed that MSCs abrogate E2-induced B-cell activation and, simultaneously, induce Breg cells, and that both of these effects on B cells require MSC-derived EBI3, a critical component of IL-35.
Because MSCs have been shown to modulate several effector immune functions by interacting with innate and adaptive immune cells,32, 33 cell therapy using ex vivo-expanded MSCs may be an attractive approach for the treatment of autoimmune disease. Our laboratory previously established T-MSCs, which are palatine tonsil cells derived from tonsillectomy, as a new source of adult stem cells. For example, we showed that T-MSCs have an immunosuppressive effect on mouse BM-derived DCs,14 and that T-MSCs inhibit proliferation of human-peripheral blood-derived B cells.15 Furthermore, in mouse models of liver disease, we found that T-MSCs can migrate into sites of inflammation or injury, differentiate and provide anti-inflammatory signals, demonstrating that T-MSCs also have promising therapeutic potential in regenerative medicine.34, 35
MSCs can ameliorate autoimmune disease by regulating Treg cells as demonstrated by a recent study that showed UC MSCs up-regulate Treg cells and downregulate Th17 cells through the regulation of TGF-β and PGE2 in lupus patients.36 However, the effect of MSCs on B cells is still poorly understood. Moreover, the effects of MSCs on Breg cells, a recently identified effector B-cell subset that inhibits the excessive inflammatory responses that occur during the development of autoimmune disease, remain undetermined. In this study, we demonstrated the effects of MSCs on regulating B-cell activation and inducing Breg cells. Furthermore, even though previous studies have shown that interactions between B cells and T cells regulate B-cell responses, we demonstrated that T-MSCs may regulate B-cell-mediated immune responses via IL-35 signaling, without acting on T cells.
Many autoimmune diseases are more prevalent in women than in men. More specifically, it has been shown that women are more affected by autoimmune diseases that are more frequent and appear later in life. Also, female gender appears to be a risk factor for poly-autoimmunity, a condition that involves more than one autoimmune disease co-existing in a single patient.37 Therefore, the sex hormone estrogen is one of the critical factors in determining gender bias in autoimmune disease. For instance, a hyperestrogenic state is associated with SLE flare-ups and is believed to trigger SLE in predisposed individuals.38 In this study, we showed that steady-state immunological features differ between female and male mice. More specifically, we showed that female mice had a significantly higher number of immunoglobulins in circulation than male mice. Also, the tissue distribution of Esr1 expression differed between female and male mice, and was correlated with several genes that promote B-cell proliferation, differentiation and activation in multiple organs, including lymphoid tissues. These data suggest that tissues with higher estrogen activity may prime B cells and, ultimately, render an immune-activated state preferentially in females rather than in males. Thus, we propose that the B-cell immune responses triggered by E2 are valid therapeutic targets in the treatment of autoimmune disease. In this study, the perturbed follicular architecture, increase of B220+ cells and increased levels of IgG displayed in E2-treated mice were significantly abrogated by T-MSC transplantation. Furthermore, our in vitro observations of the effects of T-MSCs on E2-induced B-cell activation and production of IgG further support the therapeutic potential of T-MSCs on B-cell-mediated immune responses. We also showed that co-culturing T-MSCs with B cells led to an increase in the number of IL-10-expressing, CD19+ Breg cells. Although a previous study showed that AT-MSCs are able to induce Breg cells and ameliorate symptoms in a mouse model of SLE,39 the underlying mechanism remained elusive. In this study, we suggest the factor IL-35 from MSCs may modulate the B-cell-mediated immune response by inducing Breg cells.
IL-12 family of cytokines has emerged as a critical regulator of immunity in infectious and autoimmune diseases.23 Although some members, such as IL-12 and IL-23, are associated with the pathogenesis of chronic inflammatory diseases, others, such as IL-27 and IL-35, mitigate autoimmune diseases.25, 40 IL-12 cytokines comprise heterodimeric subunits, which include one of three α subunits (IL-12p35, IL-23p19 and IL-27p28) and one of two β subunits (IL-12p40 and EBI3). Interestingly, the pairing of an alpha chain with IL-12p40 appears to generate IL-12 cytokines that promote inflammation, whereas dimerization with EBI3 gives rise to members that suppress inflammation and autoimmune diseases. Moreover, mice deficient in p35 or EBI3, the two subunits of IL-35, exhibit an exacerbation of experimental autoimmune encephalomyelitis specifically in B cells.41 In addition, culture of B cells in the presence of IL-35 induces an increase in the B-cell subpopulation expressing IL-35, called IL-35+ regulatory B cells, and half of them express IL-10. Similarly, in in vivo experiments, although IL-35 inhibits the proliferation of conventional B cells, it selectively induces the expansion of CD19+ CD5+ B220low B10 regulatory B cells.25 On the basis of these data, we hypothesized that IL-35 could be the mediator of T-MSCs effects on B-cell immune responses.
To investigate whether IL-35 could be the regulatory factor from MSCs that alleviated B-cell activation and induced Breg cells, we tried to detect secreted IL-35, including the single-chain components EBI3 and IL-12p35, via western blot analysis of cell culture supernates. We found that four types of MSCs, including BM-MSCs, AT-MSCs, T-MSCs and murine MSCs, C3H10T1/2 cells constitutively produced and secreted EBI3, IL-12p35 even in the absence of any stimulation. Moreover, T-MSCs secreted a higher amount of EBI3 in the supernates than BM-MSCs. Immunosuppressive capacity of MSCs is highly plastic in response to dynamic changes that occur within the inflammatory niche, including alterations in the concentration of inflammatory cytokines, the kinds of cytokines and the presence of immunosuppressive agents.32, 42 These ideas would imply that the therapeutic efficacy of MSCs after infusion would vary among individuals, and that certain pretreatments of MSCs may be necessary to enhance their immunosuppressive activity before transplantation. However, our result showing the intrinsic expression of IL-35 in T-MSCs is promising as it may enable the simple use of T-MSCs for infusion without any specific preconditioning. Moreover, the constitutive production and secretion of IL-35 expression by T-MSCs could result in consistent therapeutic effects regardless of individual variations in the inflammatory microenvironment.
Further, we demonstrated that EBI3 expression in MSCs was critical for abrogating E2-induced upregulation of B-cell activation markers using siRNA-mediated Ebi3 knockdown in MSCs. This result suggests that T-MSCs and C3H10T1/2 cells signal directly to B cells via IL-35 to regulate the immune-activated state induced by E2. We showed that MSCs increased the intracellular levels of IL-10 in B cells, and increased the levels of Cd1d and Cd5, key molecules of Breg cells, and that both effects required MSC-derived EBI3. We found E2 alone was not sufficient to increase IL-10+ or CD1d+ CD5+PD-L1+ B cells. However, E2 is capable of inducing Breg cells in a more complex inflammatory milieu, such as that seen in EAE.28, 29, 30 Above studies suggest a protective role for the sex hormone E2 when cells encounter an aberrant immune response.
Our findings provide the basis for powerful, new strategies for treating B-cell-mediated immune diseases, including autoimmune diseases such as SLE. Furthermore, we believe T-MSCs will also be beneficial in the prevention of immune rejection in transplantation by targeting B cells. In the past few decades, studies in the transplantation field have focused mostly on T cell-directed therapy. However, B cells are also important players in transplant rejection, especially in chronic rejection. B cells infiltrate allografts and locally stimulate effector T cells. Indeed, ectopic germinal centers, called tertiary lymphoid tissues, have been identified in transplanted tissues.43, 44 The most deleterious role for B cells in transplantation is because of their differentiation into plasma cells, which produce high levels of alloantibodies.45
In conclusion, our findings suggest that T-MSCs alleviate the B-cell-involved immune response and augment production of Breg cells. Thus, T-MSCs could become a therapeutic target for transplant rejection or autoimmune diseases. Further, T-MSCs might be an attractive therapeutic target to treat B-cell-mediated aberrant immune activation.
This research was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government (2012M3A9C6049823). In addition, this work was supported by RP-Grant 2016 of Ewha Womans University.
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Supplementary Information for this article can be found on the Cellular & Molecular Immunology website (http://www.nature.com/cmi)
Cellular and Molecular Life Sciences (2019)