Background Extracellular vesicles (EVs) are submicron-sized carriers of a variety of biomacromolecules, cytokines, autoantigens, enzymes, pathogen- and damage-associated molecular patterns besides DNA and RNA. Exosomes are the smallest EVs, their size range is about 50-100 nm whereas the diameter of microvesicles (MVs) is between 100 and 1000 nm. EVs are likely participants during intercellular comunication. Thus far, their role on human osteoclast differentiation and activation is unknown.
Objective To investigate the effect of EVs on human osteoclast differentation and gene expression.
Methods Monocytes were isolated from PBMCs using positive selection (EasySep, StemCell) and treated with 50 ng/ml recombinant human macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Samples were treated with either blood- or U.937 monocyte cell line-derived MVs, exosomes; or with soluble immune complexes (sIC, generated from purified human immunoglobulin G and Staphylococcal Protein A); or were left untreated. On day 8, total RNA was isolated or the samples were fixed and stained for tartrate resistant acid phosphatase (TRAP) expression. TRAP positive cells with a minimum of 3 nuclei were counted. The expression of calcitonin receptor (CALCR), c-Fos, cathepsin K, M-CSF receptor (c-Fms), transmembrane 7 superfamily member 4 (DC STAMP), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), osteoclast associated immunglobuline-like receptor (OSCAR), RANK, TRAP and Src like adaptor protein 2 (SLAP-2) (IDT) were analyzed by quantitative real-time polymerase chain reaction.
Results Immune complexes (n=8) profoundly inhibited the osteoclast differentiation. Blood- and cell line-derived MVs (n=7) did not alter the number of mature osteoclasts. By contrast, exosomes isolated from plasma and U937-derived supernatant (n=7) significantly (p<0.05) decreased the number of the TRAP positive multinucleated cells. Both U.937-exosome and sIC treatment (n=4) increased the c-Fos, c-Fms, OSCAR expression and decreased the CALCR expression.
Conclusions Our present data suggest that exosomes, or their cargo, may have a role in the regulation of osteoclastogenesis. This provides a novel insight into the regulation of osteoclast development in the context of inflammatory arthritis in which exosomes have been detected.
Disclosure of Interest None declared
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