金黄色葡萄球菌外囊泡通过调控Toll样受体通路激活破骨细胞驱动感染性骨破坏的研究OA

Background Staphylococcus aureus,a common pathogen in bone infections,can excessively activate osteoclasts through various pathways,leading to osteolytic destruction.However,whether its secreted outer membrane vesicles play a pivotal pathogenic role in this process remains unclear,and the underlying mechanisms warrant further investigation.Objective To systematically define the osteoclast-activating effects and phenotypic remodeling induced by Staphylococcus aureus extracellular vesicles,and explore the dose-dependent association between extracellular vesicle burden and bone destruction,together with the underlying biological mechanisms.Methods Staphylococcus aureus extracellular vesicles were isolated using a tangential flow filtration system combined with ultracentrifugation,and their morphology,size distribution,and zeta potential were characterized by transmission electron microscopy and nanoparticle tracking analysis.Bone marrow-derived monocytes/macrophages were isolated from mice and induced to differentiate into osteoclasts in the presence of macrophage colony-stimulating factor(M-CSF)and receptor activator of nuclear factor-κB ligand(RANKL).Cells were treated with different concentrations of Staphylococcus aureus extracellular vesicles(0,0.6,6,and 60 μg/mL)or vehicle control.Osteoclast differentiation and cellular viability were evaluated by tartrate-resistant acid phosphatase(TRAP)staining and CCK-8 assays,respectively,while the expression of osteoclast-related functional genes was assessed by quantitative real-time PCR.Transcriptomic profiling was performed to compare the 6 μg/mL extracellular vesicle-treated group with the control group,followed by pathway enrichment analysis.Furthermore,an in vivo mouse model was established by intramedullary injection of extracellular vesicles into the femur,with phosphate-buffered saline as a control.Bone destruction and osteoclast activation were evaluated using micro-computed tomography and cathepsin K(CTSK)immunofluorescence staining.Results Staphylococcus aureus extracellular vesicles with typical vesicular morphology were successfully isolated,exhibiting a size distribution predominantly ranging from 100 to 400 nm and a zeta potential between-30 and-10 mV,indicating good colloidal stability.In vitro experiments demonstrated that,compared with the control group,extracellular vesicle treatment significantly promoted osteoclast differentiation and multinucleation,with the number and area of TRAP-positive cells increasing in a concentration-dependent manner and peaking at 6 μg/mL(P＜0.01).CCK-8 assays revealed that extracellular vesicles at concentrations of 0.6-6 μg/mL significantly enhanced osteoclast viability(P＜0.01),whereas this stimulatory effect was attenuated at 60 μg/mL.RT-qPCR analysis showed that the mRNA expression levels of Acp5,Dc-stamp,Ctsk,and Mmp9 were significantly upregulated following extracellular vesicle treatment,with the highest expression observed in the 6 μg/mL group(all P＜0.01).Transcriptomic analysis identified 161 differentially expressed genes,with upregulated genes predominantly enriched in inflammatory responses,Toll-like receptor signaling pathways,and osteoclast differentiation-related pathways.In vivo,extracellular vesicle-treated mice exhibited a significant reduction in femoral bone mineral density(23%),accompanied by decreases in trabecular number(30%)and trabecular thickness(20%)compared with controls(all P＜0.001).Consistently,CTSK-positive signals in bone tissue were markedly increased in the extracellular vesicle-treated group(P＜0.001),indicating markedly elevated osteoclast activity.Conclusion Staphylococcus aureus extracellular vesicles can establish a localized pro-inflammatory microenvironment by activating the Toll-like receptor pathway,thereby driving the activation and differentiation of osteoclasts and leading to the development of infectious bone destruction.