L-ornithine alleviates inflammatory bone loss by suppressing osteoclastogenesis

Background: Inflammatory arthritis-associated bone loss is a social burden and remains a great clinical challenge. Current treatment strategies remain limited in reversing osteoclast-mediated bone destruction. L-ornithine, a non-proteinogenic amino acid involved in the urea cycle, has been implicated in various cellular biochemical and metabolic processes, but its role in bone remodeling remains poorly understood. Our previous work demonstrated that L-arginine supplementation significantly attenuated joint inflammation and bone loss in murine models of rheumatoid arthritis. Metabolomic analysis further revealed that L-arginine is metabolized into L-ornithine and its downstream products, raising the hypothesis that L-ornithine itself may play an important role in regulating inflammation-induced bone loss. To date, however, this possibility has not been explored. Methods: We utilized a serum transfer-induced arthritis (SIA) model and a collagen-induced arthritis (CIA) murine model where oral L-ornithine (35g/L in drinking water) was administrated to determine its effects on joint inflammation, bone erosion as well as systemic bone loss. Micro-CT analysis, and tartrate resistant acid phosphatase (TRAP) staining were used to assess bone mass and osteoclasts number and size. In vitro, primary mouse bone marrow cells were induced to differentiate into osteoclasts by receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF) in the presence or absence of L-ornithine (12.5 or 25 mM) and TNFα (40ng/mL), the stimulator to mimic an inflammatory condition. TRAP staining and bulk RNA-sequencing were performed to evaluate the impact on osteoclastogenesis and underlying gene expression profiles. Results: L-ornithine supplementation did not alter the joint inflammation but significantly attenuated bone erosion and systemic bone loss. L-ornithine reduced osteoclast numbers in vivo. In vitro, L-ornithine suppressed osteoclast differentiation and fusion in response to RANKL without affecting classical osteoclastogenic genes expression. Transcriptomic analysis revealed a subset of differentially expressed genes that were consistently regulated by L-ornithine regardless of TNFα stimulation, which were enriched in sodium ion transmembrane transport pathway. Integration of transcriptional regulatory analysis predicted several potential upstream transcription factors, among which NFAT5 emerged as the top one candidate, with the highest NES (normalized enrichment score) score and regulating the largest number of target genes. Conclusion: Our findings support that L-ornithine is a negative regulator of osteoclast differentiation and bone loss in inflammatory arthritis. We propose a testable hypothesis of a mechanistic model involving an “ornithine-ion homeostasis-NFAT5-Nrf2” axis, although further validation is required.