The IRE1‐XBP1s Axis Drives Inflammatory Osteolysis by Regulating a 5‐HT Dependent Endogenous Anti‐Autophagy Mechanism

Inflammatory osteolysis arises from pro-inflammatory cytokine-driven osteoclast activation and disrupted bone remodeling equilibrium. The IRE1-XBP1s axis, a major unfolded protein response pathway, regulates cellular homeostasis, but its role in inflammatory osteoclastogenesis remained unexplored. Single-cell RNA-seq showed increased osteoclast precursor cells and activated IRE1-XBP1s in LPS-induced osteolysis. Inhibition of this axis reduced osteoclastogenesis and bone loss in vitro and in vivo. RNA-seq indicated that blocking IRE1-XBP1s suppressed Slc6a4 transcription, with gene set enrichment analysis confirming its role in 5-HT transport. Dual-luciferase assays and ChIP-PCR demonstrated XBP1s' direct transcriptional regulation targeting the Slc6a4 promoter. The 5-HT transporter inhibitor escitalopram also inhibited osteoclastogenesis, highlighting the IRE1-XBP1s-Slc6a4 axis's importance. Notably, untargeted metabolomics suggested 5-HT inhibited intracellular 3-methyladenine (3MA) metabolism, a compound previously considered unnatural. HPLC-MS confirmed the presence of 3MA metabolism in inflammatory osteoclasts, and 3MA supplementation attenuated 5-HT-induced autophagy and osteoclast differentiation. Blocking the IRE1-XBP1s-Slc6a4 axis reduced pro-osteoclastogenic effects in inflammatory bone disease patient-derived PBMCs. This study demonstrates that IRE1-XBP1s inhibition alleviates inflammatory osteoclastogenesis and osteolysis via a 5-HT-dependent anti-autophagy mechanism, proposing this pathway as a therapeutic target for inflammatory bone loss.