The Inhibition of Osteoblast Viability by Monosodium Urate Crystal-Stimulated Neutrophil-Derived Exosomes

Abstract

Background and ObjectiveBone erosion is common in patients with gout. The role of neutrophil-derived exosomes in gouty bone erosion remains elusive. This study aimed to investigate the functions of the neutrophil-derived exosomes in the development of bone erosion in gout.MethodsNeutrophil-derived exosomes were collected and assessed by transmission electron microscopy and nanoparticle tracking analysis. Cell counting kit-8 assay was applied to evaluate cell viability, and cell apoptosis was assessed by flow cytometry. In addition, quantitative Real-time PCR and Western blotting were used to determine the expression levels of alkaline phosphatase (ALP), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL). Neutrophil-derived exosomes were tagged with PKH67. The miRNA expression profiles of exosomes and human fetal osteoblasts (hFOB) were compared using high-throughput sequencing. Functional miRNAs transfected into hFOB after co-incubation with exosomes were selected and validated by preliminary qPCR.ResultsNeutrophil-derived exosomes were stimulated by monosodium urate (MSU). The exosomes could inhibit the viability of the hFOB, and the expression levels of ALP and OPG were down-regulated, while the expression level of RANKL was up-regulated. However, there was no significant difference in the viability of osteoclasts and the expression of nuclear factor of activated T cells 1. Exosomes were observed in the cytoplasm under a confocal microscopy, confirming that exosomes could be taken up by hFOB. In total, 2590 miRNAs were found, of which 47 miRNAs were differentially expressed. Among the delivered miRNAs, miR-1246 exhibited the highest level of differential expression. The viability of hFOB was reduced by miR-1246 mimics and increased by miR-1246 inhibitors. There was no significant difference in hFOB apoptosis rate between the miR-1246 mimic and miR-1246 inhibitor group. MiR-1246 overexpression decreased the expression levels of ALP and OPG, whereas increasing the expression level of RANKL. In contrast, miR-1246 inhibitor increased the expression levels of ALP and OPG, while decreasing the expression level of RANKL. Neutrophil-derived exosomes stimulated by MSU could increase the expression of miR-1246. ConclusionNeutrophil-derived exosomes stimulated by MSU could inhibit the viability of osteoblasts.

Osteogenic Potential of Monosodium Urate Crystals in Synovial Mesenchymal Stem Cells

“…Runx2 is a relevant biomarker, because medications such as allopurinol reduce osteoblast apoptosis, increase their viability, and reduce the risk of vascular calcification by decreasing Runx2 in animal models of hyperuricemia [31,32]. In a recent study by Naot et al [33], factors such as PGE2 and TNF-α, secreted by macrophages stimulated with MSU crystals, reduced the viability of osteoblasts in a dose-dependent manner in long-term cultures (13 days); however, the doses ranged from 100 to 500 µg/mL, which decreased the expression of Runx2, suggesting that bone erosion is a result of direct and indirect effects, such as the stimulation of exosomes derived from neutrophils by MSU crystals, which has a negative effect on the viability of osteoblasts [34].…”

The effects of preosteoblast‐derived exosomes on macrophages and bone in mice

“…Preosteoblast‐derived exosomes were tagged with PKH67 (PKH67 Green Fluorescent Cell Linker kit, (Sigma). MC4exo were mixed with diluted PKH67 and incubated at 37°C for 5 min 18 . Following incubation, exosome‐free medium containing FBS (10%) was added to stop the reaction.…”

“…MC4exo were mixed with diluted PKH67 and incubated at 37°C for 5 min. 18 Following incubation, exosome‐free medium containing FBS (10%) was added to stop the reaction. BMMΦ were seeded onto glass cover slips and placed in 6‐well plates.…”

Recent advances on neutrophil dysregulation in the pathogenesis of rheumatic diseases

“…It further emphasizes the pathogenic role of neutrophil activation in synovial inflammation. Moreover, in patients with gout, monosodium urate (MSU) stimulates neutrophil-derived exosomes that inhibit osteoblast viability, promoting bone erosion [33]. Notably, MSU also induces NETosis [34].…”