Adipose Tissue-Derived Mesenchymal Stem Cells Induce Expansion of Interleukin-10-Producing Regulatory B Cells and Ameliorate Autoimmunity in a Murine Model of Systemic Lupus Erythematosus

Mice

Female Roquin^san/san C57BL/6 mice between 15 and 20 weeks of age were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and subsequently bred under specific pathogen-free conditions in an animal facility with controlled humidity (55 ± 5%), light (12 h/12 h light/dark), and temperature (22 ± 1°C). The air in the facility was passed through a HEPA filter system designed to exclude bacteria and viruses. Animals were fed mouse chow and tap water ad libitum. The protocols used in this study were approved by the Animal Care and Use Committee of The Catholic University of Korea.

Isolation and Culture of MSCs

Mesenchymal stem cells used in this study were derived from a surplus of frozen cell stock for stem cell banking (RNLBio, Seoul, Korea) and no longer needed for that purpose. Fat samples were obtained from donors with written informed consent for research use. In brief, human abdominal subcutaneous fat tissues were obtained by simple liposuction from two male donors, 59 and 77 years old. MSCs were isolated from the fat stromal vascular fraction (SVF) by their adherence to plastic and were culture expanded. Cryopreserved stem cells, stored in liquid nitrogen vapor, were thawed and recultured in growth medium (RKCM; RNLBio) based on the injection schedule under Good Manufacturing Practice conditions (RNLBio). Harvested cells were anonymized and transported to the preclinical investigator under cold storage conditions (4,24).

Cell Cultures

Splenocytes isolated from C57BL/6 (wild type) or Roquin^san/san mice were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 20 mM HEPES (Gibco), 2 mM L-glutamine (Gibco), 5% heat-inactivated FBS (Gibco), 100 mM sodium pyruvate (Gibco), and 1% antibiotics [penicillin (100 U/ml)-streptomycin (100 mg/ml); Gibco] alone or with irradiated human adiposederived MSCs (20:1, 5:1, and 1:1 ratios, respectively) in the presence or absence of 10 μg/ml lipopolysaccharide (LPS; Sigma-Aldrich St. Louis, MO, USA) for 3 days. MSCs were irradiated (2,000 cGy) using a ¹³⁷Cs source (Gammacell^® 3000; MDS Nordion, Ottawa, ON, Canada) to inhibit mitosis.

Stem Cell Injection

Mice received lateral tail vein injections of 1 × 10⁶ MSCs in 0.2 ml phosphate-buffered saline (PBS; Gibco). The negative control group received PBS instead of cells.

FACS Analysis

Mononuclear cells were immunostained with various combinations of the following fluorescence-conjugated antibodies (0.2 μg/10⁶ cells): CD19, CD1d, CD5, CD21, CD23, CD4, CD25, CD44, CD95, CD138, GL-7, ICOS, and B220 (BD Biosciences, San Diego, CA, USA). These cells were also intracellularly stained with the following antibodies (0.25 μg/10⁶ cells): IL-4 (BD Biosciences), IL-10 (Biolegend, San Diego, CA, USA), IL-17 (eBioscience, San Diego, CA, USA), and Foxp3 (eBioscience). Before intracellular staining, the cells were restimulated for 4 h with 25 ng/ml PMA (Sigma-Aldrich) and 250 ng/ml ionomycin (Sigma-Aldrich) in the presence of GolgiSTOP (BD Biosciences). Intracellular staining was conducted using an intracellular staining kit (eBioscience) according to the manufacturer's protocol. Flow cytometry was performed using a FACSCalibur cytometer (BD Biosciences).

Assessment of Histopathology Scoring of Nephritis

Kidneys were harvested and fixed in 10% neutral-buffered formalin (Sigma-Aldrich). Formalin-fixed tissues were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin (Sigma-Aldrich), eosin (Muto Pure Chemical Co., Ltd., Tokyo, Japan), and/or Periodic acid–Schiff (PAS; Sigma-Aldrich) for histological examination. The severity of the nephritis score was graded on a 0-4 scale as follows: 0, normal; 1, mild increase in mesangial cellularity and matrix; 2, moderate increase in mesangial cellularity and matrix with thickening of the glomerular basement membrane (GBM); 3, focal endocapillary hypercellularity with obliteration of capillary lumina and a substantial increase in the thickness and irregularity of the GBM; and 4, diffuse endocapillary hypercellularity, segmental necrosis, crescents, and hyalinized end-stage glomeruli (17).

Measurement of IgG Titers and Anti-dsDNA Antibodies

Serum levels of IgG and IgG1 antibodies were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Bethyl Laboratories, Montgomery, TX, USA). The IgG anti-dsDNA antibody was measured by ELISA using serum samples diluted at 1:500 and alkaline phosphatase (AP)-conjugated goat antimouse IgG, secondary antibody at a 1:1,000 dilution.

Confocal Staining

Spleen tissue was collected, snap frozen in liquid nitrogen, and stored at −80°C. Tissue cryosections (7 μm thick) were fixed in 4% paraformaldehyde (Sigma-Aldrich) and stained using PE-labeled anti-IFN-r, IL-4, IL-17, or Foxp3 antibody (1:100 dilution; eBioscience) or FITC-labeled anti-CD4 antibody (1:200 dilution; Biolegend). After overnight incubation at 4°C, the stained sections were analyzed using a confocal microscopy system (LSM 510 Meta; Zeiss, Gottingen, Germany).

Statistical Analysis

Statistical analyses were performed using SAS ver. 9 software (SAS Institute, Cary, NC, USA). The experimental values are presented as means ± standard deviations (SD). Data were analyzed by one-way ANOVA using Tukey's post hoc test in multiple groups and by a two-tailed unpaired Student's t-test in cases in which two groups were compared. Values of p < 0.05 were considered significant.

Human Adipose-Derived MSCs Expand IL-10-Producing B Cells In Vitro

We first examined whether human adipose-derived MSCs had an effect on the population of Bregs mice in vitro. Splenocytes isolated from C57BL/6 mice were cultured in medium alone or graded doses of irradiated human adipose-derived MSCs in the presence or absence of LPS for 72 h. IL-10-producing B cells, which were considered to be Bregs, were identified by flow cytometry. As shown in Figure 1A, in vitro treatment with human adipose-derived MSCs expanded the IL-10-producing B cells in a dose-dependent manner. Cotreatment with LPS augmented the Breg-expanding effect of human adipose-derived MSCs. The percentage of IL-10-producing cells in B cells was also increased by human adipose-derived MSCs (Fig. 1B).

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Figure 1.

Human adipose-derived mesenchymal stem cells (MSCs) expand IL-10-producing B cells in a dose-dependent manner in vitro. (A) Splenocytes (1 × 10⁶) isolated from C57BL/6 mice were cultured in medium alone or with irradiated human adipose-derived MSCs (20:1, 5:1, and 1:1 ratio, respectively) in the presence or absence of LPS for 72 h. IL-10-producing B cells (IL-10-producing CD19⁺ cells) were identified by flow cytometry as described in the Materials and Methods section. The representative result is shown. (B) The percentage of IL-10-producing cells in CD19⁺ B cells is shown. Data are expressed as the mean ± SD. *p < 0.05. (C) The population of CD1d^hiCD5⁺ B cells obtained splenocytes from Roquin^san/san mice is shown in the left panel. Almost all B10 B cells expressed CD19, but only a small proportion of B10 B cells produced IL-10 without LPS stimulation (right panel). The representative results are shown. (D) Splenocytes (1 × 10⁶) isolated from Roquin^san/san mice were cultured in medium alone or with irradiated human adipose-derived MSCs (20:1, 5:1, and 1:1 ratio, respectively) for 72 h. The percentage of CD1d^hiCD5⁺ B cells in CD19⁺ B cells was determined by flow cytometry. *p < 0.05. (E) Splenocytes isolated from Roquin^san/san mice were cultured in medium alone or with irradiated human adipose-derived MSCs (20:1, 5:1, and 1:1 ratio, respectively) in the presence or absence of LPS for 72 h. IL-10-producing CD1d^hiCD5⁺ B cells were determined by flow cytometry as described in the Materials and Methods section. The representative result is shown in the left panel. The percentage of IL-10-producing B10 B cells is shown in the right panel. Data are expressed as the mean ± SD. *p < 0.05.

Human Adipose-Derived MSCs Expand the Population of CD1d^hiCD5⁺ B Cells In Vitro

Among various Breg phenotypes, CD1d^hiCD5⁺ B cells are the most well-characterized Bregs in mice (21,32). Therefore, we investigated the effect of human adipose-derived MSCs on CD1d^hiCD5⁺ B cells from Roquin^san/san mice in vitro. As shown in Figure 1C, the proportion of CD1d^hiCD5⁺ B cells among CD19+ B cells from Roquin^san/san mice was small without stimulation, and a minority of CD1d^hiCD5⁺ B cells produced IL-10. In vitro treatment with human adipose-derived MSCs increased the percentage of CD1d^hiCD5⁺ B cells (Fig. 1D).

We also examined the effect of human adipose-derived MSCs on IL-10-producing CD1d^hiCD5⁺ B cells in vitro. Splenocytes isolated from Roquin^san/san mice were cultured in medium alone or graded doses of irradiated human adipose-derived MSCs in the presence or absence of LPS for 72 h. The population of IL-10-producing CD1d^hiCD5⁺ B cells was analyzed. The results showed that human adipose-derived MSCs expanded IL-10-producing CD1d^hiCD5⁺ B cells in a dose-dependent manner in vitro (Fig. 1E).

Effect of In Vivo Cell Therapy with Human Adipose-Derived MSCs on Renal Histopathology and Autoantibody Production

We next tested whether in vivo treatment with human adipose-derived MSCs might have beneficial effects on renal histopathology and autoantibody production in the animal model of SLE (Roquin^san/san mice). Beginning at age 17 weeks, we initiated a therapeutic protocol consisting of intravenous infusion of 1 × 10⁶ human adipose-derived MSCs or an equal volume of PBS, which was administered once weekly for 5 weeks. The following parameters were evaluated: histopathology of the kidney, serum levels of immunoglobulins (IgG, IgG1, IgM, and IgE), and IgG anti-dsDNA. The histopathologic analysis of the kidney revealed a significant improvement in mesangial proliferation and interstitial inflammatory cell infiltration in mice treated with human adipose-derived MSCs (Fig. 2A, B). Moreover, serum concentrations of immunoglobulins (IgG, IgG1, IgM, and IgE) and IgG anti-dsDNA decreased markedly following treatment with human adipose-derived MSCs (Fig. 2C, D). These results suggest that in vivo treatment with human adipose-derived MSCs ameliorate the lupus-like phenotype of Roquin^san/san mice.

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Figure 2.

In vivo treatment with human adipose-derived MSCs ameliorates the lupus-like phenotype of Roquin^san/san mice. (A) Representative images of kidney sections stained with hematoxylin and eosin (H&E) and Periodic acid–Schiff (PAS) from Roquin^san/san mice treated intravenously with 1 × 10⁶ human adipose-derived MSCs once weekly for 5 weeks or phosphate-buffered saline (PBS). Scale bar: 50 μm. (B) Scores of nephritis severity in Roquin^san/san mice that received saline or MSCs. Data are represented as mean ± SD in bar graphs. (C and D) Serum levels of IgG, IgG1, IgM, IgE, and IgG anti-double-stranded DNA (anti-dsDNA) antibodies in Roquin^san/san mice treated with human adipose-derived MSCs or PBS, as determined by ELISA. The combined data from two independent experiments (n = 6 per group) are shown. Data are expressed as the mean ± SD. *p < 0.05.

In Vivo Treatment with Human Adipose-Derived MSCs Decreases Th1, Th17, and Tfh Cells but Increases Tregs in Roquin^san/san Mice

We also tested the effect of in vivo cell therapy with human adipose-derived MSCs on histopathology of the spleen in Roquin^san/san mice. We found that treatment with human adipose-derived MSCs significantly decreased the number of lymphoid follicles in spleens compared with those treated with PBS (Fig. 3A).

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Figure 3.

Human adipose-derived MSCs decrease Th1, Th17, and Tfh cells but increase regulatory T cells in Roquin^san/san mice. (A) Roquin^san/san mice (n = 6) were injected intravenously with 1 × 10⁶ hMSCs every week for 5 weeks. Disease control mice (n = 6) received intravenous injections of an equal volume of PBS at the same time points. Representative images of spleen sections stained with H&E from each group of mice. Scale bar: 50 μm. Bar graph indicates the number of lymphoid follicles of spleens from each group of mice. The lymphoid follicles were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented in the form of histogram. (B) Representative confocal microscopic images of spleen sections from Roquin^san/san mice treated with human adipose-derived MSCs or PBS. Cells were stained with fluorescence-tagged antibodies to identify CD4 (white), ICOS (red), B220 (blue), and CD44 (green). CD44⁺ICOS⁺CD4⁺ cells, which are considered Tfh cells, are densely located near the B-cell zone (yellow arrows), especially in the spleen of the PBS-treated mice. Roquin^san/san mice treated with human adipose-derived MSCs showed markedly decreased size of the germinal center. (C) Representative flow cytometric contour plots and graphical analysis of CD4⁺ICOS⁺CD44⁺ T cells (follicular helper T cells, Tfh cells). Bar graph indicates the percentage of Tfh cells in CD4⁺ T cells in spleens from each group of mice (right panel). *p < 0.05. (D) Representative flow cytometric contour plots and graphical analysis of CD4⁺IFN-γ T cells (Th1 cells), CD4⁺IL-17⁺ T cells (Th17 cells), and CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) (left panel). Bar graphs indicate the percentage of Th1 cells, Th17 cells, and Tregs in CD4⁺ T cells in spleens from each group of mice (right panel). The combined data from two independent experiments (n = 6 per group) are shown. *p < 0.05.

We analyzed the population of Th1 cells, Th17 cells, Tregs, and Tfh cells in spleens from mice treated with human adipose-derived MSCs or PBS by flow cytometry to determine the effect of in vivo cell therapy with human adipose-derived MSCs on various effector T helper cell subsets. Tfh cells, a recently discovered effector T helper cell subset, are deeply implicated in GC formation, which is an essential step in the humoral immune response (6,30).

A confocal microscopic analysis of spleen tissues revealed that mice treated with human adipose-derived MSCs showed markedly decreased GC size compared with mice treated with PBS (Fig. 3B). The population of CD4⁺ICOS⁺CD44⁺ T cells, which are considered Tfh cells, decreased profoundly in mice treated with human adipose-derived MSCs (Fig. 3C). The population of CD4⁺IFN-γ T cells (Th1 cells) and CD4⁺ IL-17⁺ T cells (Th17 cells) also decreased in mice treated with human adipose-derived MSCs. In contrast, spleen tissues from mice treated with human adipose-derived MSCs showed increased numbers of Foxp3⁺ Tregs, which suppress autoimmune processes and maintain peripheral tolerance (22), compared with that of mice treated with PBS (Fig. 3D).

Effect of In Vivo Cell Therapy with Human Adipose-Derived MSCs on B-cell Subsets in Roquin^mn/san Mice

We investigated the effect of in vivo cell therapy with human adipose-derived MSCs on various B-cell subsets in Roquin^san/san mice. Confocal microscopic analysis of spleen tissues revealed that mice treated with human adipose-derived MSCs showed markedly decreased expression of GL-7, the major surface marker of GC B cells (Fig. 4A). The population of GC B cells (GL-7⁺ CD95⁺ B220⁺ B cells) decreased markedly in mice treated with human adipose-derived MSCs (Fig. 4B).

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Figure 4.

Human adipose-derived MSCs decrease germinal center (GC) B cells and plasma cells but increase Breg cells in Roquin^san/san mice. (A) Representative confocal microscopic images of spleen sections from Roquin^san/san mice treated with human adipose-derived MSCs or PBS. Cells were stained with fluorescence-tagged antibodies to identify GL-7 (green), CD138 (red), B220 (blue), and CD4 (white). Roquin^san/san mice treated with human adipose-derived MSCs showed markedly decreased numbers of GL-7⁺ B cells. (B) Flow cytometric analysis shows the proportion of GL-7⁺CD95⁺B220⁺ B cells (germinal center B cells, GC B cells) in the spleen tissues from each group of mice. The percentage of GC B cells in B220⁺ B cells from each group of mice is shown in the right panel. *p < 0.05. (C) Representative flow cytometric contour plots and graphical analysis of CD1d^hiCD5⁺ B cells (B10 B cells). Bar graphs indicate the percentage of CD1d^hiCD5⁺ B cells in CD19⁺ B cells of spleen from each group of mice (upper panel). IL-10-producing CD1d^hiCD5⁺ B cells were determined by flow cytometry as described in the Materials and Methods section. Bar graphs indicate the percentage of IL-10-producing CD1d^hiCD5⁺ B cells in CD19⁺ B cells in spleens from each group of mice (lower panel). *p < 0.05. (D) Representative flow cytometric contour plots and graphical analysis of B220⁺CD23^highCD21^low B cells (follicular B cells, FOB cells) and B220⁺CD23^lowCD21^high B cells (marginal zone B cells, MZB cells) from each group of mice are shown in the upper panel. Representative flow cytometric contour plots and graphical analysis of plasma cells (B220^-CD138⁺IgD^- cells) from each group of mice are shown in the lower panel. The combined data from two independent experiments (n = 6 per group) are shown.*p < 0.05.

To our surprise, spleen tissues from mice treated with human adipose-derived MSCs showed increased numbers of both CD1d^hiCD5⁺ B cells and IL-10-producing CD1d^hiCD5⁺ B cells compared with those of mice treated with PBS (Fig. 4C), suggesting that treatment with human adipose-derived MSCs expands the population of Bregs from mice both in vitro and in vivo.

We also analyzed the other B-cell subsets, including B220⁺CD23^highCD21^low B-cells (FOB cells), B220⁺ CD23^lowCD21^high B-cells (MZB cells), and plasma cells (B220-CD138⁺IgD^- cells). The results revealed that mice treated with human adipose-derived MSCs had decreased numbers of FOB cells and plasma cells and increased numbers of MZB cells (Fig. 4D).