Adipose-Derived Mesenchymal Stem Cells Prevent Systemic Bone Loss in Collagen-Induced Arthritis

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammatory synovitis leading to joint destruction and systemic bone loss. The inflammation-induced bone loss is mediated by increased osteoclast formation and function. Current antirheumatic therapies primarily target suppression of inflammatory cascade with limited or no success in controlling progression of bone destruction. Mesenchymal stem cells (MSCs) by virtue of their tissue repair and immunomodulatory properties have shown promising results in various autoimmune and degenerative diseases. However, the role of MSCs in prevention of bone destruction in RA is not yet understood. In this study, we investigated the effect of adipose-derived MSCs (ASCs) on in vitro formation of bone-resorbing osteoclasts and pathological bone loss in the mouse collagen-induced arthritis (CIA) model of RA. We observed that ASCs significantly inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in both a contact-dependent and -independent manner. Additionally, ASCs inhibited RANKL-induced osteoclastogenesis in the presence of proinflammatory cytokines such as TNF-α, IL-17, and IL-1β. Furthermore, treatment with ASCs at the onset of CIA significantly reduced clinical symptoms and joint pathology. Interestingly, ASCs protected periarticular and systemic bone loss in CIA mice by maintaining trabecular bone structure. We further observed that treatment with ASCs reduced osteoclast precursors in bone marrow, resulting in decreased osteoclastogenesis. Moreover, ASCs suppressed autoimmune T cell responses and increased the percentages of peripheral regulatory T and B cells. Thus, we provide strong evidence that ASCs ameliorate inflammation-induced systemic bone loss in CIA mice by reducing osteoclast precursors and promoting immune tolerance.

Copyright © 2015 by The American Association of Immunologists, Inc.

Figures

FIGURE 1.

Culture-expanded murine ASCs exhibit multilineage differentiation and immunosuppressive potential. ASCs were isolated from s.c. adipose tissue of DBA/1J mice and culture expanded as described in Materials and Methods. (A) Cells of passage 2 were analyzed for their clonogenic potential by CFU-F assay. (B) Surface phenotyping for the expression of mesenchymal and hematopoietic markers by flow cytometry. Numbers indicate percentage expression of the marker with respect to their isotype controls. (C–E) Osteogenic, adipogenic, and chondrogenic differentiation of ASCs was induced in respective differentiation media. Matrix mineralization by differentiated osteoblasts was assessed by Alizarin red S staining. Adipocytes were characterized by Oil Red O staining. Chondrogenic differentiation shows cells in lacunae in H&E-stained sections of micromass pellet cultures (original magnification ×10). (F) Suppression of activated CD4+ T cell proliferation by ASCs or (G) ASC-CM was assessed by coculturing splenic CD4+ T cells (2.5 × 105 cells/ml) with ASCs (2.5 × 103 cells/ml) or ASC-CM (50% of culture volume) under activated or unactivated conditions for 72 h. T cell activation Dynabeads were used for activating T cells, and proliferation was measured by a [3H]thymidine incorporation assay. Bar graphs are expressed as mean ± SEM of three to five replicates per group. ***p ≤ 0.001. (A)–(E) are representative of more than five batches of ASCs. (F) and (G) are representative of two independent experiments.

FIGURE 2.

ASCs inhibit RANKL-induced osteoclast differentiation in vitro. M-CSF–dependent OCPs derived from bone marrow of DBA/1J mice were cultured in the presence of M-CSF (M, 30 ng/ml) and RANKL (R, 40 ng/ml) with or without ASCs. (A) OCPs (1 × 105 cells/well) were cultured in 48-well plates with graded ratios of ASCs, from 500:1 (OCP/ASC) to 10:1 and number of osteoclasts were counted as TRAP+ MNCs (three or more nuclei). The p values are with respect to M-CSF and RANKL group without ASCs. (B) Representative images of coculture at an OCP/ASC ratio of 50:1, stained for TRAP and counterstained with hematoxylin (original magnification ×10). (C) Markers of osteoclast formation and function, namely TRAP, CTR, DC-STAMP, integrin β3, MMP9, and cathepsin K (Cat K), were measured at the mRNA level by real-time PCR. (D) RANK and c-fms expression was also measured by quantitative PCR. Results are representative of two independent experiments. ###p ≤ 0.001 compared with M-CSF group; *p ≤ 0.05, ***p ≤ 0.001 compared with M-CSF and RANKL group. AM, M-CSF with ASCs; AMR, M-CSF and RANKL with ASCs; M, M-CSF; MR, M-CSF and RANKL.

FIGURE 3.

ASCs inhibit RANKL-induced osteoclast differentiation in a contact-independent manner. OCPs from mouse bone marrow were cultured in the presence of M-CSF and RANKL with or without ASCs in a contact-independent manner. (A) Representative photographs of TRAP+ MNCs of Transwell cultures of osteoclast differentiation with OCPs in the lower chamber and ASCs in culture inserts. Original magnification ×10. Representative images (original magnification ×10) (B) and number of osteoclasts (C) in cultures where OCPs (5 × 104 cells/well) were differentiated into osteoclasts in 96-well plates in the presence or absence of ASC-CM (S) for 3–4 d are shown. ASC-CM was added at two different concentrations (25% and 50%). Gene expression analysis of markers of osteoclasts (D) and receptors of OCPs (E) is shown. Results are representative of three (A–C) and two (D and E) independent experiments. (F and G) OCPs were differentiated in 96-well plates coated with a calcium phosphate film, with or without 50% ASC-CM (S) for 8 d. Cells were washed off and resorption of the calcium phosphate film by osteoclasts was assessed by bright-field microscopy. Original magnification ×10. Data are shown as representative images (F) and percentage resorption (G) of two independent experiments with eight replicates in each. ###p ≤ 0.001 compared with M-CSF group; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with M-CSF and RANKL group. M, M-CSF; MR, M-CSF and RANKL; MRS, M-CSF and RANKL with ASC-CM; MS, M-CSF with ASC-CM.

FIGURE 4.

ASC-CM potently inhibits osteoclastogenesis. M-CSF–dependent OCPs were differentiated into osteoclasts using M-CSF (30 ng/ml) and RANKL (40 ng/ml). ASC-CM was added at three different time points along the course of differentiation, that is, on days 0, 1, and 2. Cultures were fixed after 4 d and stained for TRAP. Representative images (A) and numbers (B) of osteoclasts in the cultures are shown (original magnification ×10). ###p ≤ 0.001 compared with M-CSF group; ***p ≤ 0.001 compared with M-CSF and RANKL group. (C and D) OCPs were cultured using M-CSF and RANKL (10 ng/ml) with or without proinflammatory cytokines TNF-α, IL-17, and IL-1β at a concentration of 30 ng/ml. ASC-CM was added to these cultures at 50% volume. Cultures were terminated after 3 d and stained for TRAP (original magnification ×10). Numbers are represented as mean ± SEM of five replicates in each group. Data are shown as representative images (C) and numbers of osteoclasts (D) of two independent experiments. M, M-CSF; MR, M-CSF and RANKL; MRS, M-CSF and RANKL with ASC-CM.

FIGURE 5.

ASCs decrease the disease severity and bone destruction in CIA. CIA was induced in 8- to 10-wk-old DBA/1J mice as described in Materials and Methods. On day 22, 2 × 106 ASCs in 100 μl PBS were injected i.p. to one group and an equal volume of PBS to the other group of CIA mice. Severity of arthritis was evaluated by measuring mean arthritic score of four limbs per animal (A) and hindpaw thickness (B) until day 36. Data are presented as mean ± SEM (n = 6–7/group). Significance was calculated by two-way ANOVA followed by a Bonferroni multiple comparisions test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. Similar results were obtained in four independent experiments. Representative images (C) and radiographs (D) of hindpaws of mice on day 36 are shown. Yellow arrows in (D) show bone density at the metatarsophalangeal joints. (E) Sections of knee joints were stained with H&E and histologically evaluated for cellular infiltration and pannus formation (n = 4–6 mice/group). Original magnification ×10. Significance was calculated by a Student t test between CIA plus PBS and CIA plus ASC groups. Similar results were obtained in three independent experiments. Hindlimbs along with ankle joints were dissected out and analyzed for histomorphometric measurements using μCT. (F) Representative three-dimensional images of distal tibiae from control (nonarthritic), PBS-treated, and ASC-treated CIA mice. (G) Trabecular bone indices, including volumetric bone mineral density (trabecular BMD [Tb. BMD]), BV/TV, Tb. Th, Tb. Sp, Tb. N, and Conn. D, were quantified from μCT reconstructions. Data are pooled from three independent experiments and are presented as mean ± SD of 14–18 mice per group. Significance was calculated by a one-way ANOVA with a Bonferroni multiple comparisons test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. ##p ≤ 0.01, ###p ≤ 0.001 with respect to control group; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 with respect to CIA plus PBS group.

FIGURE 6.

ASCs protect from periarticular and systemic bone loss in CIA mice. CIA mice were injected i.p. with PBS or 2 × 106 ASCs on day 22 of primary immunization and sacrificed on day 36. (A) Representative three-dimensional images of distal femur from control nonarthritic, PBS-treated, and ASC-treated CIA mice. (B) Trabecular bone indices, including Tb. BMD, BV/TV, Tb. Th, Tb. Sp, Tb. N, and Conn. D, were quantitated from μCT reconstructions. Data are pooled from three independent experiments and are presented as mean ± SD of 14–18 mice per group. Representative two-dimensional images (C) and quantitative measurements (D) of femur mid-diaphysis Ct. BMD and Cs. Th are shown. Data are pooled from two independent experiments and are presented as mean ± SD of 9–11 mice per group. Representative three-dimensional images (E) and trabecular parameters (F) of the fifth lumbar vertebra (L5) are shown. Data are pooled from three independent experiments and are presented as mean ± SD of 14–18 mice per group. Significance was calculated by a one-way ANOVA with a Bonferroni multiple comparisons test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 with respect to control group; *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001 with respect to CIA plus PBS group.

FIGURE 7.

ASCs decrease RANKL-induced osteoclastogenesis in CIA mice by reducing osteoclast precursors. CIA mice were injected i.p. with PBS or 2 × 106 ASCs on day 22 of the first immunization and sacrificed on day 36. Bone marrow was harvested and M-CSF–dependent OCPs were differentiated into osteoclasts in the presence of M-CSF with or without two different concentrations of RANKL (25 or 50 ng/ml). Representative images (A) and numbers of osteoclasts (B) in cultures are shown as a measure of osteoclastogenesis (original magnification ×10). (C) Percentage of osteoclast precursors (CD11b+c-fms+) in bone marrow. (D) The amount of RANK expressed on CD11b+ cells of bone marrow. Data are presented as mean ± SEM of seven mice per group in (B), four to five mice per group in (C), and five mice per group in (D). Significance was calculated by a one-way ANOVA with a Bonferroni multiple comparisons test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. Similar results were obtained in three independent experiments. #p ≤ 0.05, ###p ≤ 0.001 with respect to control group; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 with respect to CIA plus PBS group. M, M-CSF; MR, M-CSF and RANKL.

FIGURE 8.

ASCs reduce CII-specific T cell proliferation and induce regulatory lymphocytes in arthritic mice. CIA mice were injected with PBS or 2 × 106 ASCs on day 22 of initial immunization. On day 36, mice were sacrificed and analyzed for immune parameters. (A) dLN cells were cultured in the presence of CII (40 μg/ml) or plate-bound anti-CD3ε (2 μg/ml) for 72 h and proliferation was measured by [3H]thymidine incorporation assay. Stimulation indices (S.I.) values are calculated using the formula: cpm in response to Ag/cpm in absence of Ag. The inset in (A) shows the representation of decreased size of dLNs in ASC-treated CIA mice compared with PBS-treated controls. (B) Magnetically sorted splenic CD4+ T cells (5 × 105 cells/well) from DBA/1J mice were cultured with or without ASCs (5 × 103 cells/well) in 48-well plates for 72 h. Cells were stained for Foxp3 and IL-17A and analyzed by flow cytometry. Plots are representative of two independent experiments. (C) Representative plots and average percentages of CD4+Foxp3+ Tregs in the peritoneal cavity, synovium, peripheral blood, dLNs, and spleens of control, arthritic, and ASC-treated arthritic mice. (D) Percentages of CD1dhiCD5+ Bregs from CD19-gated population of splenocytes. (E) Anti-CII IgG titers were measured in serum by ELISA. Data are presented as mean ± SEM of six to seven mice per group in (A), five to seven mice per group in (C), three to six mice per group in (D), and four mice per group in (E). Significance was calculated by a one-way ANOVA with a Bonferroni multiple comparisons test between 1) control versus CIA plus PBS groups and 2) CIA plus PBS versus CIA plus ASC groups. Similar results were observed in two independent experiments. ##p ≤ 0.01, ###p ≤ 0.001 with respect to control group; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 with respect to CIA plus PBS group.