NEUTROPHIL-SECRETED LACTOFERRIN BLUNTS OXIDATIVE STRESS SENSITIVITY OF SUBCHONDRAL BONE TO ACCELERATE OSTEOARTHRITIS PROGRESSION

This study aims to investigate the role of neutrophils in aberrant subchondral bone remodeling and identify the mechanistic association during OA progression. Destabilized medial meniscus (DMM) surgery was performed to induce OA. Safranin O-Fast green staining, immunostaining of Osterix (Osx) and Ly6g (neutrophil marker) and TRAP staining were utilized to reveal aberrant bone remodeling and neutrophil expresssion in the tibial subchondral bone of OA mice. Intraperitoneal injection of neutrophil-neutralizing antibody was conducted in DMM mice and flow cytometry was further applied to confirm the clearance efficiency of neutrophils in peripheral blood and bone marrow. Van Frey test, open field test, micro-CT analysis and histological staining were respectively used to evaluate joint pain, locomotor activity, subchondral bone parameters and OA severity of DMM mice after eliminating neutrophils. Proteomics analysis of bone marrow supernatant from Gfi1 -/- mice, which was characterized by congenital neutropenia, screened out the key protein-Lactoferrin (LTF) secreted by neutrophils. The role of LTF in OA subchondral bone remodeling was further evaluated in LTF overexpressed mice via hydrodynamic injected plasmids and in mice with genetic deletion of LTF in neutrophils. The downstream molecular mechanism of neutrophil-secreted LTF was further revealed by RNA-seq. As the exacerbation of subchondral osteosclerosis, neutrophils accumulated in the subchondral bone of mice with OA. After eliminating neutrophils, joint pain, locomotor activity, subchondral osteosclerosis and OA progression was significantly improved in DMM mice. The key protein-LTF secreted by neutrophils was screened via proteomics analysis. LTF was highly co-localized to neutrophils and observed upregulation in the subchondral bone of both OA mice and patients. LTF overexpression significantly aggravated subchondral sclerosis and OA progression. In contrast, genetic deletion of LTF in neutrophils ameliorated subchondral sclerosis and OA progression. Mechanistically, RNA-seq revealed that the role of neutrophil-secreted LTF in aggravating OA subchondral sclerosis was associated with its anti-oxidation effect via upregulating glutathione (GSH) level and further validated in preosteoblast and osteoclast under oxidative stress environment stimulated by tert-butyl hydroperoxide (TBHP) and in OA mice. Neutrophil-secreted LTF blunts oxidative stress sensitivity of subchondral bone to accelerate OA progression by upregulating GSH level.