Interleukin-17 induces pyroptosis in osteoblasts through the NLRP3 inflammasome pathway in vitro

Abstract

Objective: Interleukin-17 (lL-17), a pro-inflammatory cytokine produced by Th17 cells, is also considered to play an important role in bone metabolism, but the exact mechanism of bone destruction remains unclear. In this study, we explored whether IL-17 could induce osteoblasts pyroptosis in vitro.

Methods: The murine primary osteoblasts were isolated from the calvarial bones of mice. The proliferation of osteoblasts was evaluated by cell counting kit-8 (CCK-8) assay. The mRNA levels of NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis associated speck like protein containing a card (ASC), caspase-1, gasdermin-D (GSDMD), IL-1β and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured by real-time quantitative PCR. Pyroptosis after IL-17 treatment was evaluated by lactate dehydrogenase (LDH) Release Assay Kit and the morphological characteristics of osteoblasts were observed via Scanning Electron Microscopy (SEM). Pyroptosis associated proteins, cleaved IL-1β and RANKL were evaluated through western blot. The release of IL-1β and RANKL was measured by ELISA. In addition, calcium nodule was tested by alizarin red staining.

Results: High concentration IL-17 (100 ng/mL) could affect the proliferation of osteoblasts, promote the gene expression of NLRP3, caspase-1, GSDMD, IL-1β and RANKL. In contrast to control group, osteoblasts treated with IL-17 had the appearance of numerous pores, swelling and rupture. Also, the release of LDH, IL-1β and RANKL increased in the presence of IL-17. However, inhibition of NLRP3 prevented activation of the NLRP3 inflammasome, thereby restoring osteoblasts morphology and function.

Conclusion: IL-17 induced osteoblasts pyroptosis, and the pyroptosis of osteoblasts may prompt the release of IL-1β and RANKL,which may further contribute to disruption of bone metabolism. Besides, the NLRP3 inflammasome pathway was involved in the pyroptosis of osteoblasts.

Keywords: Bone metabolism; IL-17; NLRP3; Primary osteoblasts; Pyroptosis.