Interleukin-17 promotes osteoclastogenesis and periodontal damage via autophagy in vitro and in vivo.

ABSTRACT

OBJECTIVE: Because of its potent pro-inflammatory properties, interleukin-17 (IL-17) contributes to the pathogenesis of various inflammatory diseases. This study explored the effects of IL-17 on osteoclastogenesis in an osteoclast monoculture and osteoblast-osteoclast co-culture system, as tools to investigate the molecular mechanisms underlying the interactions between osteoclastogenesis and autophagy. METHODS: Various ratios of calvarial osteoblasts (OB) and osteoclast precursor cells (mouse macrophage cell line RAW264.7, hereinafter referred to as OC) were tested. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the optimum osteoblastsosteoclasts ratio. IL-17 was added to the co-culture system to test its effects on multinucleated osteoclast formation and osteoclast-related proteins. We assessed the effects of IL-17 on receptor activator of nuclear factor-kappa B ligand (RANKL) expression in osteoblasts, and determined if IL-17 alone could modulate osteoclast formation in an osteoclast monoculture. Administration of exogenous RANKL combined with IL-17 was employed to stimulate RAW264.7 cells osteoclastogenesis and to determine production of osteoclasts and autophagy-related proteins. We knocked down Beclin1 expression in RAW264.7 cells and examined the expression of autophagy-related and osteoclast-related proteins in RAW264.7 cells and the co-culture system, and the TAK1-binding protein 3 (TAB3)/ extracellular signal regulated kinase (ERK) pathway. RESULTS: A ratio of 20 OB 1 OC yielded the highest rate of osteoclast formation. Low IL-17 concentrations increased osteoclastogenesis in co-cultures significantly, but high levels of IL-17 had the opposite effect. IL-17 alone could not induce formation of TRAP+ multinucleated cells in RAW264.7 cells. Low IL-17 concentrations promoted osteoclast differentiation and autophagy in RAW264.7 cells induced by exogenous RANKL, but high IL-17 concentrations inhibited this process. Knockdown of Beclin1 reversed the enhanced effects of 0.1 ng/mL IL-17 on osteoclastogenesis and autophagy in RAW264.7 cells. The TAB3/ERK pathway was also blocked after autophagy inhibition. CONCLUSION: In the co-culture model used in this study, a ratio of 20 OB1 OC proved to be the optimal ratio to facilitate osteoclast formation. IL-17 regulated RANKL-induced osteoclastogenesis via autophagy. The Beclin1/TAB3/ERK pathway was involved in osteoclast autophagy.