Edited by Xiao-Fan Wang In bone remodeling, after a lifespan of ϳ2 weeks, osteoclasts undergo apoptosis in each bone turnover cycle, resulting in generation of a large number of apoptotic bodies (ABs). However, the biological roles of osteoclast-derived ABs (OC-ABs) in bone remodeling have not been investigated and remain unknown. In this study, we stimulated bone marrow macrophages with receptor activator of NF-B ligand (RANKL) to obtain both preosteoclasts and mature osteoclasts (mOCs). We then used alendronate to induce apoptosis in preosteoclasts and mOCs and generate the respective ABs and used flow cytometry and immunoblotting to characterize the sizes and immunogenic characteristics of the extracted ABs. We show that mOC-ABs are engulfed by preosteoblastic MC3T3-E1 cells and promote the viability of these cells. Among all osteoclast-derived extracellular vesicles, mOC-ABs had the highest osteogenic potency. We further observed that mOC-ABs had the highest vesicular receptor activator of NF-B (RANK) levels among all types of osteoclast-derived extracellular vesicles. Of note, masking of vesicular RANK by soluble RANKL strongly abolished the osteogenic potency of osteoclast-derived ABs. Mechanistically, we found that mOC-ABs induce osteoblast differentiation by activatingPI3K/AKT/mechanistic target of rapamycin (mTOR)/ ribosomal protein S6 kinase signaling. In conclusion, OC-ABs promote osteogenic differentiation by stimulating osteoblast differentiation via activation of RANKL reverse signaling. These findings provide important insights into the reversal phase between the bone resorption and formation stages during bone remodeling and identify an AB-dependent cellular signaling mechanism in osteoclast-osteoblast coupling.

The balance between bone formation and bone resorption is closely related to bone homeostasis. Osteoclasts, originating from the monocyte/macrophage lineage, are the only cell type possessing bone resorption ability. Osteoclast overactivity is thought to be the major reason underlying osteoclast‐related osteolytic problems, such as Paget's disease, aseptic loosening of prostheses and inflammatory osteolysis; therefore, disruption of osteoclastogenesis is considered a crucial treatment option for these issues. WKYMVm, a synthetic peptide, which is a potent FPR2 agonist, exerts an immunoregulatory effect. This peptide inhibits the production of inflammatory cytokines, such as (IL)‐1β and TNF‐α, thus regulating inflammation. However, there are only few reports on the role of WKYMVm and FPR2 in osteoclast cytology. In the current study, we found that WKYMVm negatively regulates RANKL‐ and lipopolysaccharide (LPS)‐induced osteoclast differentiation and maturation in vitro and alleviates LPS‐induced osteolysis in animal models. WKYMVm down‐regulated the expression of osteoclast marker genes and resorption activity. Furthermore, WKYMVm inhibited osteoclastogenesis directly through reducing the phosphorylation of STAT3 and NF‐kB and indirectly through the CD9/gp130/STAT3 pathway. In conclusion, our findings demonstrated the potential medicinal value of WKYMVm for the treatment of inflammatory osteolysis.

Vitamins B are co-enzymes participating in energy metabolic pathways. While some vitamins B are known affecting bone homeostasis, the effects of vitamin B1 (thiamine) on bone health remains unclear. In our study, we used cell counting kit-8, tartrate-resistant acid phosphatase stain, actin cytoskeleton stain, and pit formation assay to evaluate the effect of thiamine on osteoclast differentiation, formation, and function, respectively. Then we used dichlorodihydro-fluorescein diacetate assay to investigate reactive oxygen species (ROS) generation and removal. Osteoporosis model by ovariectomy was established for animal experiments. We found that thiamine had inhibitory effect on osteoclast differentiation. And its inhibitory role on osteoclast differentiation is in a dosedependent way. Mechanistically, ThDP suppresses intracellular ROS accumulation and unfolded protein response signaling during osteoclastogenesis via inhibiting Rac-Nox1/2/4 and intracellular inositol-requiring protein-1α/X-boxbinding protein pathways, respectively. Osteoporotic mice treated with thiamine rich dietary showed better bone strength relative to thiamine deficient dietary. Our study explored the non-coenzyme inhibitory functions of B1 vitamin in receptor activator of nuclear factor κB ligand induced osteoclastogenesis and uncovered the significance of B1 vitamin in bone health. K E Y W O R D Snon-coenzyme, osteoclast, osteoporosis, thiamine, thiamine phosphate

Background: α-Klotho (Klotho) plays a wide range of roles in pathophysiological processes, such as lowturnover osteoporosis observed in klotho mutant mice (kl/kl mice). However, the precise function and underlying mechanism of klotho during osteoclastogenesis are not fully understood. Here, we investigated the effects of klotho on osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL). Methods:The effects of klotho deficiency on osteoclastogenesis were explored using kl/kl mice both in vivo and in vitro. In in vitro experiments, lentivirus transfection, real-time quantitative PCR (RT-qPCR) analysis, western blot analysis, immunostaining, RNA-seq analysis, differential pathway analysis, Energy-based protein docking analysis and co-immunoprecipitation were used for deeply investigating the effects of klotho on RANKL-induced Osteoclastogenesis and the underlying mechanism.Results: We found that klotho deficiency impaired osteoclastogenesis. Furthermore, in vitro studies revealed that klotho facilitated osteoclastogenesis and upregulated the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) during osteoclastogenesis. Mechanistically, we confirmed that klotho co-localized with nuclear factor kappa B (RANK) and facilitated the interaction between activated RANK and TNFR-associated factor 6 (TRAF6), thus klotho exerts its function in osteoclastogenesis through the activation of the NF-κB signaling pathway.Conclusions: Klotho promotes RANKL-induced osteoclastogenesis through upregulating the interaction between RANK and TARF6, Targeting on klotho may be an attractive therapeutic method for osteopenic diseases.

Purpose Previous studies have revealed that the receptor-binding domain (RBD) of the spike protein is immunogenic in both mice and healthy volunteers, and the humoral immune response plays key roles in RBD-mediated protection. In this study, we evaluated the immunodominant humoral response of RBD with different adjuvants and different immune routes in inducing neutralizing antibodies and immunodominant epitopes in RBD. Methods In this study, we investigated the protective efficacy of immunization with RBD plus three different adjuvants (Al(OH)3, ASO3 or AddaVax) and two different routes (intramuscular immunity or intranasal immunity) in a mouse model. Results The results showed that RBD-mediated protection was altered in response to different adjuvants; even with the same adjuvant, RBD-mediated protection was altered in different immune routes. Using antisera from immunized mice, we identified six B-cell immunodominant epitopes in the RBD, including 2 novel epitopes (RBD1 − 18 and RBD49 − 66) in intramuscular immunity and 3 novel epitopes (RBD31 − 48, RBD61 − 78, RBD97 − 114) in intranasal immunity. The B-cell immunodominant epitopes identified from mice immunized with RBD plus different adjuvants were also different from each other, which may explain the differences in protective immunity observed in each immunized group. Conclusions This study indicate that adjuvants and immune pathways largely affect the immunodominance of epitopes and the protective efficacy of RBD, which may guide further adjuvant screening for vaccine development and optimization.