NLRP3 inflammasome impairs fracture repair in Rheumatoid arthritis through RhoA/Rac1-IL1β axis-mediated suppression of osteoblast differentiation

Rheumatoid arthritis (RA) is often characterized by bone loss and fragility fractures and is a frequent comorbidity. The NLRP3 inflammasome drives inflammatory processes that fundamentally accompany the pathogenesis of RA. However, the role of NLRP3 inflammasome in RA fracture healing remains unclear. For in vivo analyses, we established tibial fractures in two murine RA models: TNF-transgenic (TNF Tg ) mice and collagen-induced arthritis (CIA). To address the contribution of NLRP3 inflammasome to fracture repair, we generated TNF Tg ; NLRP3 KO mice by deleting the NLRP3 gene in TNF Tg mice. The effects of TNFα overexpression on osteogenic differentiation were assessed using mesenchymal progenitor cells (MPCs) with or without MCC950. The role of MCC950 in RA fracture repair was investigated using CIA mice. TNF Tg mice exhibited delayed fracture healing, characterized by decreased callus bone volume and reduced bone mechanical strength. The NLRP3 inflammasome was excessively activated in TNF Tg mice, leading to elevated expression of NLRP3, pro-Caspase-1, Caspase-1 p20, pro-IL-1β and IL-1β. Moreover, NLRP3 deficiency in TNF Tg mice significantly mitigated the delayed fracture healing. Mechanistically, TNFα overexpression suppressed osteogenic differentiation of MPCs through NLRP3 inflammasome activation. This process involves RhoA/Rac1-dependent NF-κB signaling that triggers inflammasome assembly, ultimately leading to IL-1β secretion. Notably, MCC950 administration significantly attenuated these pathological effects. Lastly, in vivo MCC950 treatment rescued the delayed fracture healing by reducing NLRP3 inflammasome activation and promoting bone formation in CIA mice. Collectively, these findings suggest that NLRP3 inflammasome activation drives impaired fracture healing in RA through RhoA/Rac1‒IL-1β axis-mediated suppression of osteoblast differentiation, and pharmacologic inhibition with MCC950 effectively rescues delayed fracture healing in RA mouse model. This study provides novel insights into the mechanisms underlying delayed fracture healing in RA and highlights the potential therapeutic benefits of targeting the NLRP3 inflammasome.