ALKBH5 enhances CX3CL1 RNA stability and governs macrophage polarization and bone integrity in glucocorticoid-induced osteonecrosis.

This study aims to investigate the role of C-X3-C motif chemokine ligand 1 (CX3CL1) in the pathogenesis of glucocorticoid-induced osteonecrosis of the femoral head (ONFH), particularly focusing on its impact on macrophage polarization and bone cell function. CX3CL1 was upregulated in the femoral head of mice with methylprednisolone (MPS)-induced osteonecrosis. CX3CL1 knockout in mice reduced improved bone morphology while reducing M1 polarization in femoral head tissues, whereas CX3CL1 knock-in led to inverse trends. In vitro, CX3CL1 knockdown in MPS-challenged MLOY4 cells enhanced cell viability and reduced M1 skewing and migration of the co-cultured RAW264.7 macrophages. The m6A modification in tissues and cells was increased following MPS challenge. AlkB homolog 5 (ALKBH5), an RNA demethylase, was found to modulate m6A demethylation of CX3CL1 and enhance its RNA stability. Inhibiting ALKBH5 levels using Indole-2,3-dione-5-carbaldehyde substantially reduced CX3CL1 levels and alleviated M1 polarization of macrophages, thus improving bone function in MPS-challenged mice. In conclusion, this study suggests that CX3CL1, upregulated upon m6A demethylation, impairs bone function in glucocorticoid-induced ONFH by recruiting and promoting pro-inflammatory skewing of macrophages. Targeting ALKBH5 or CX3CL1 may represent promising strategies for managing glucocorticoid-induced ONFH.