Macrophages Switch to an Osteo‐Modulatory Profile Upon RANKL Induction in a Medaka (Oryzias latipes) Osteoporosis Model

Abstract

In mammals, osteoclasts differentiate from macrophages in the monocyte lineage. Although many factors driving osteoclast formation are known, the detailed processes underlying precursor recruitment, differentiation, and interaction of macrophages with other cell types involved in bone remodeling are poorly understood. Using live imaging in a transgenic medaka osteoporosis model, where ectopic osteoclasts are induced by RANKL expression, we show that a subset of macrophages is recruited to bone matrix to physically interact with bone-forming osteoblast progenitors. These macrophages subsequently differentiate into cathepsin K-(ctsk-) positive osteoclasts. One day later, other macrophages are recruited to clear dying osteoclasts from resorbed bone by phagocytosis. To better understand the molecular changes underlying these dynamic processes, we performed transcriptome profiling of activated macrophages upon RANKL induction. This revealed an upregulation of several bone-related transcripts. Besides osteoclast markers, we unexpectedly also found expression of osteoblast-promoting signals in activated macrophages, suggesting a possible non-cell autonomous role in osteogenesis. Finally, we show that macrophage differentiation into osteoclasts is dependent on inflammatory signals. Medaka deficient for TNFα or treated with the TNFα-inhibitor pentoxifylline exhibited impaired macrophage recruitment and osteoclast differentiation. These results show the involvement of inflammatory signals and the dynamics of a distinct subset of macrophages during osteoclast formation.

Transcriptome Profiling of Osteoblasts in a Medaka (Oryzias latipes) Osteoporosis Model Identifies Mmp13b as Crucial for Osteoclast Activation

“…Fluorescent images of whole embryos were used for the quantification of osteoclast numbers following the method described previously ( Phan et al, 2020a ). For quantification of bone lesion sizes, four consecutive vertebral bodies were used for each analysis.…”

“…. mmp13b +/+ : N 6, mmp13b −/− : N 5. was conducted to examine transcription levels of several osteoclast differentiation genes, which we had earlier identified in medaka as upregulated under Rankl+ conditions (Phan et al, 2020a). These included mmp9, rank, ctsk, traf6, TRAP, and il10, as well as ECM-related genes, such as col6a1, col6a3, itga2.2, and itgb8 (Figure 4A).…”

Fish Models of Induced Osteoporosis

“…Transcriptome profiling of Rankl-activated macrophages revealed the differential expression of several osteoclast markers, e.g., trap, ctsk, siglec15, nfatc1, tgfb1, and dap12, a situation also observed in mammals (Phan et al, 2020 and references therein). Another feature common to both fish and mammalian OP models, is the similar response to genetic or chemical inhibition of tnf α, which improved bone loss in both models, by reducing ectopic osteoclast differentiation in a selective manner (Phan et al, 2020). The rescue of OP phenotype by bisphosphonates was also evaluated in adult medaka, and both alendronate and etidronate inhibited osteoclast activity in a dose-dependent manner upon rankl induction, thus contributing to the maintenance of bone homeostasis and integrity.…”

“…In rankl transgenic fish, active osteoclasts were formed ectopically and promoted bone resorption in mineralized arches and vertebral bodies, in a way that resembles findings in human osteoporosis. Besides phenotypical similarities, rankl -induced ectopic osteoclasts were shown to derive from a subset of macrophages that migrated and accumulated into the vertebral column, and then interacted with osteoblast progenitors before differentiating into bone resorbing osteoclasts ( Phan et al, 2020 ). Transcriptome profiling of Rankl-activated macrophages revealed the differential expression of several osteoclast markers, e.g., trap , ctsk , siglec15 , nfatc1 , tgfb1 , and dap12 , a situation also observed in mammals ( Phan et al, 2020 and references therein).…”

“…Besides phenotypical similarities, rankl -induced ectopic osteoclasts were shown to derive from a subset of macrophages that migrated and accumulated into the vertebral column, and then interacted with osteoblast progenitors before differentiating into bone resorbing osteoclasts ( Phan et al, 2020 ). Transcriptome profiling of Rankl-activated macrophages revealed the differential expression of several osteoclast markers, e.g., trap , ctsk , siglec15 , nfatc1 , tgfb1 , and dap12 , a situation also observed in mammals ( Phan et al, 2020 and references therein). Another feature common to both fish and mammalian OP models, is the similar response to genetic or chemical inhibition of tnf α, which improved bone loss in both models, by reducing ectopic osteoclast differentiation in a selective manner ( Phan et al, 2020 ).…”