Abstract: BACKGROUND: Recent studies have found that immune cells play an important role in bone metabolism. Myeloid-derived suppressor cells, as a type of immunosuppressive cell, play a significant role in tumor development, but their role in primary osteoporosis remains unclear.
OBJECTIVE: To investigate the osteoclastogenic potential of myeloid-derived suppressor cells in naturally aged and ovariectomy-induced osteoporosis mouse models.
METHODS: (1) Myeloid-derived suppressor cells and bone marrow-derived macrophages were isolated from 6-8-week-old female C57BL/6 mice. Both cell types were induced for osteoclast differentiation. After 5 days of induction, osteoclast formation was detected by tartrate-resistant acid phosphatase staining. After 3 days of induction, mRNA expression of nuclear factor of activated T-cells 1 and osteoclast-associated immunoglobulin-like receptor was detected by qRT-PCR. (2) 6-8-week-old female C57BL/6 mice (young group, n=6) and 18-month-old female C57BL/6 mice (naturally aged group, n=6) were taken. Bone microstructure of the distal femur was analyzed by Micro-CT. Bone marrow cells were collected from both groups, and the proportion of myeloid-derived suppressor cells was detected by flow cytometry. Myeloid-derived suppressor cells were isolated from both groups and induced for osteoclast differentiation. After 5 days of induction, osteoclast formation was detected by tartrate-resistant acid phosphatase staining. After 3 days of induction, mRNA expression of nuclear factor of activated T-cells 1 and osteoclast-associated immunoglobulin-like receptor was detected by qRT-PCR. (3) 6-8-week-old female C57BL/6 mice were randomly divided into a sham-operated group (n=6) and an ovariectomized group (n=6). Eight weeks after ovariectomy, bone microstructure of the distal femur was analyzed by Micro-CT. Bone marrow cells were collected from both groups, and the proportion of myeloid-derived suppressor cells was detected by flow cytometry. Serum levels of tumor necrosis factor-α and interleukin-6 were detected by ELISA. Myeloid-derived suppressor cells were isolated from both groups and induced for osteoclast differentiation. After 5 days of induction, osteoclast formation was detected by tartrate-resistant acid phosphatase staining. After 3 days of induction, mRNA expression of nuclear factor of activated T-cells 1 and osteoclast-associated immunoglobulin-like receptor was detected by qRT-PCR.
RESULTS AND CONCLUSION: (1) Tartrate-resistant acid phosphatase staining and qRT-PCR showed that the osteoclastogenic potential of myeloid-derived suppressor cells was stronger than that of bone marrow-derived macrophages. (2) Micro-CT analysis showed that compared with the young group, the naturally aged group had a lower bone mineral density, bone volume fraction, and trabecular number (P < 0.05), and higher trabecular separation (P < 0.05). The proportion of myeloid-derived suppressor cells in the naturally aged group was higher than that in the young group (P < 0.05). Tartrate-resistant acid phosphatase staining and qRT-PCR showed that the osteoclastogenic potential of myeloid-derived suppressor cells in the naturally aged group was stronger than that in the young group. (3) Micro-CT analysis showed that compared with the sham-operated group, the ovariectomized group had lower bone mineral density, bone volume fraction, and trabecular number (P < 0.05), and higher trabecular separation (P < 0.05). The proportion of myeloid-derived suppressor cells and the serum levels of tumor necrosis factor-α and interleukin-6 in the ovariectomized group were higher than those in the sham-operated group (P < 0.05). Tartrate-resistant acid phosphatase staining and qRT-PCR showed that the osteoclastogenic potential of myeloid-derived suppressor cells in the ovariectomized group was stronger than that in the sham-operated group. These findings indicate that the increased proportion and enhanced osteoclastogenic potential of myeloid-derived suppressor cells under natural aging and estrogen-deficient conditions may be involved in the occurrence and development of osteoporosis.

Key words: osteoporosis, myeloid-derived suppressor cells, osteoclast activation, naturally aged mice, ovariectomy, tartrate-resistant acid phosphatase staining, inflammatory cytokines