Inhibition of Osteoclast Differentiation and Activation by Leukotriene B4 Loaded in Microspheres

Abstract

AbstractAimLeukotriene B4 (LTB4) is a labile inflammatory lipid mediator important for host defense. We hypothesised that sustained delivery of LTB4 would be a therapeutic strategy to prevent osteoclast cell differentiation in bone resorption in inflammatory diseases. Therefore, the aim of this study was to investigate the role of LTB4 in differentiation of monocytic lineage cells into osteoclasts after stimulation with LTB4 loaded in microspheres (MS).DesignLTB4-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process. Sterility, LPS contamination, characterization and efficiency of LTB4 encapsulation were investigated. J774A.1 cells were cultured in the presence of monocyte colony stimulating factor (M-CSF) and ligand for receptor activator of nuclear factor kappa B (RANKL) and then stimulated with LTB4-MS. Cytotoxicity was determined by lactate dehydrogenase assay, osteoclast formation by means of the activity of tartrate-resistant acid phosphatase enzyme and gene expression was measured by quantitative reverse transcription polymerase chain reaction to investigate regulation of Alox5, Alox5ap, Acp5, Mmp9, Calcr and Ctsk.ResultsWe found that 5-lipoxygenase pathway is involved in the osteoclastic differentiation hematopoietic lineage cells and that exogenous addition of LTB4-MS inhibited osteoclastogenesis induced by M-CSF and RANKL. The mechanism of LTB4-MS involved induction of Mmp9 gene expression and inhibition of Calcr and Ctsk, without changing Acp5.ConclusionLTB4-MS inhibited differentiation of macrophages into an osteoclastic phenotype and cell activation under M-CSF and RANKL stimulus shedding light on a potential therapeutic strategy to prevent osteoclast differentiation.