Aging-induced decrease in progenitor B cells enhances the osteoclastogenesis of bone marrow macrophages via ROS-activated Fos/CCL3 signaling‌ pathway.

Senile osteoporosis (SOP) is becoming a critical public health burden particularly in aging societies due to escalating fracture susceptibility among elderly populations. Despite advances in SOP research, the mechanistic role of immune cell alterations in senile osteoporosis remains unclear. As a validated SOP model, senescence-accelerated mouse prone 6 (SAMP6) mice were used for mechanistic studies, comparing with age- and gender-matched controls-senescence-accelerated mouse resistant 1 (SAMR1) mice. We combined micro-CT, molecular biology and histological techniques to assess the bone mineral density in femurs. To investigate dynamic interactions and differential pathways of immune cells within the bone marrow microenvironment, we employed a multi-omics integration strategy including single-cell RNA sequencing, transcriptome sequencing and flow cytometry. Furthermore, we conducted multicolor immunofluorescence and multiplex cytokine analysis to verify the above results. Moreover, we utilized BaF3 and RAW 264.7 cell lines to study the cell interactions during osteoclastogenesis and BX471(a CCR1 antagonist) was used to block this process. SAMP6 mice showed a marked decline in bone mass accompanied by elevated reactive oxygen species (ROS) level in 3-month-old. The single-cell RNA sequencing of bone marrow revealed that a prominent depletion of B lymphocytes, with pro-B cells exhibiting the most pronounced reduction. Multi-omics integration analysis of pro-B cells suggested a significant activation of the Fos/Jun (AP-1) transcription complex accompanied by upregulation of chemokine CCL3 in the SAMP6 group. In vitro assays showed that H2O2-induced oxidative stress in BaF3 cells triggers Fos/Ccl3 axis activation and promoted osteoclastogenesis in RAW 264.7 cells. Moreover, blocking CCR1 with BX471 could suppress this effect. Our findings suggest that the elevation of ROS levels within bone marrow microenvironment accelerates immune-senescence of B lymphocytes, characterized by a significant reduction in progenitor B-cell populations, which subsequently activates the Fos/Jun-mediated stress signaling pathway. The cascade ultimately results in the overexpression of CCL3, further promoting osteoclastogenesis of macrophage through CCL3-CCR1 axis. CCR1 antagonist could alleviate pro-inflammatory osteoclast formation, revealing a potential therapeutic target for senile osteoporosis.