Induced T regulatory cells suppress osteoclastogenesis and bone erosion in collagen-induced arthritis better than natural T regulatory cells

Abstract

Background: Although natural regulatory T cells (nTregs) can suppress osteoclastogenesis, the role of TGF-β-induced CD4+Foxp3+ Tregs (iTregs) in osteoclastogenesis remains unknown.

Objective: To determine the effects of iTregs on osteoclastogenesis in vitro and on bone erosion in vivo in collagen-induced arthritis (CIA).

Methods: Osteoclastogenesis was induced in bone marrow CD11b+ cells with receptor activator of nuclear factor κ B (NF-κB) ligand (RANKL) and macrophage colony stimulating factor. Graded doses of Tregs were added to inhibit osteoclastogenesis. Transwell and antibody blockade experiments were performed to assess the roles for cell contact and soluble cytokines. NF-κB activation was determined by western blot. iTregs or nTregs were adoptively transferred to mice with CIA to assess in vivo effects on disease incidence and bone erosion, the latter determined by CT scanning.

Results: Both nTregs and iTregs greatly suppressed osteoclastogenesis in vitro, but only iTregs sustained this effect when interleukin-6 was present. iTregs, but not nTregs, significantly suppressed development of CIA. Bone erosions in iTregs-treated mice were diminished compared with untreated mice or nTregs-treated mice. Treatment with iTregs, but not with nTregs, dramatically decreased NF-κB p65/p50 levels in osteoclasts in vitro and p65/50 and RANKL expression by synovial tissues in vivo.

Conclusion: iTregs may be therapeutically beneficial in rheumatoid arthritis and related diseases associated with bone erosions.

Figure 1

CD4⁺ iTregs inhibit osteoclastogenesis in vitro in a dose-dependent manner. OCPs were stimulated with M-CSF (30ng/ml) and RANKL (50ng/ml) and co-cultured with CD4⁺CD25-cells, nTregs,Tconts,or iTregs (2:1 ratio) for four days and stained for TRAP.TRAP⁺ cells were counted in high power of microscopy. A. Under 2:1 ratio, and data were mean of TRAP⁺ cells ± SEM of four independent experiments. B. TRAP⁺ cells under different ratios of Tregs to CD11b⁺ cells. C. TRAP⁺ cells of Tregs to CD11b⁺ cells (2:1) ± IL-6. D. Data were representative photomicrographs of TRAP⁺ cells from the cultures in panel C. **p=<0.01, ***p=<0.001 compared with baseline and Tconts or Tregs treatment.

Figure 2

CD4⁺ iTregs suppress bone erosion in vivo. A. CT imaging of feet and vertebral in CIA, Tcont-, nTreg- and iTregs-infused CIA in 56-day after CII/CFA immunization. 3×10⁶ various CD4⁺ cells were adoptively transferred to DBA/1J mice at 14-day after immunization. B. Incidences of CIA in DBA/1J mice after immunization or other therapeutic groups. C. Correlation of the clinical scores of each group and severities of bone destruction was shown. Each group had five mice. The results are from two independent experiments.

Figure 3

CD4⁺ iTregs blocks the osteoclastogenesis via a RANKL-mediated NF-κB P65/P50 pathway. A. OCPs were cultured or cocultured with CD4⁺ Tconts or iTregs as described above. After four days culture, cells were harvested for western blot analysis to determine the expression of P65 and P50 on non-T cells. B. Synovial tissues isolated from joints in different CIA mice or treated with Tregs and P65/P50 and RANKL expression was determined and quantified. All data in A and B were representative of three independent experiments.