Deciphering transcriptome alterations in bone marrow immune cells at single-cell resolution under denosumab treatment

Denosumab, an anti-RANKL antibody, effectively inhibits bone resorption, increases bone mass, and reduces fracture risk; however, its immunomodulatory effects raise concerns about possible immune imbalances during the treatment. Given the key role of bone marrows in immune cell development, this study aimed to evaluate alternations in the bone marrow osteoimmune microenvironment under denosumab treatment.

Adult female mice were administered 10 mg/kg anti-RANKL antibody via intraperitoneal injections twice weekly for 4 weeks. Bone marrow cells from both treated and control mice were analyzed via single-cell RNA sequencing (scRNA-seq) to characterize cell-type-specific changes. Shifts in cell populations were validated using single-cell flow cytometry and immunofluorescence.

Transcriptomic profiling identified 10 distinct bone marrow cell clusters under the anti-RANKL antibody treatment. The anti-RANKL antibody treatment resulted in an increase proportion of neutrophils and B lymphocytes, with a concomitant decrease in T lymphocytes. These changes were validated by single-cell flow cytometry and immunofluorescence. Among neutrophils, the Mmp8^hi mNeu subset showed the greatest expansion and activated neutrophil extracellular trap (NET) formation in the anti-RANKL antibody-treated group. Furthermore, elevated Mmp8^hi mNeu interacted with macrophages via the THBS1-CD36 signaling pathway, suggesting a role in modulating macrophage polarization under anti-RANKL antibody treatment.

Anti-RANKL antibody induces notable alterations in the bone marrow osteoimmune microenvironment, including expansion of Mmp8^hi mNeu and altered lymphocyte composition. These findings underscore the need for further investigation into the impact of Mmp8^hi mNeu and neutrophil-macrophage interactions on immune balance during denosumab treatment. This study presents a potential therapeutic target for mitigating the immune-related adverse effects in osteoporosis management.