Abstract

Modulating the immune microenvironment has become an emerging strategy for promoting functional bone regeneration, identifying key therapeutic targets remains challenging. Our previous work showed that Nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5 (NLRC5) is involved in bone destruction associated with periodontitis, but its potential and mechanism in regulating bone tissue repair and regeneration have not been fully elucidated. A monocortical bone defect model was established in the mouse femur to assess the impact of NLRC5 on in situ bone healing and regeneration. Mouse bone marrow-derived mesenchymal stem cells (BMSCs) were isolated to evaluate the effects of NLRC5 on osteogenic differentiation, proliferation, and migration. RNA sequencing was used to explore the direct regulatory mechanism of NLRC5 on osteogenic differentiation of mouse BMSCs. Mass cytometry was employed to examine the effect of NLRC5 on the bone marrow immune microenvironment, followed by in vitro validation experiments. Loss of NLRC5 impaired the healing and regeneration of femoral bone defects in mice, and led to a high inflammatory state in the early stage of healing. The absence of NLRC5 inhibited the osteogenic differentiation ability of BMSCs, and could be restored by NLRC5 overexpression, which was achieved through activation of the phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT) signaling pathway. Mass cytometry data revealed that NLRC5 may serve as an important factor in maintaining the differentiation and maturation of regulatory T cells (Tregs). By modulating the levels of inflammatory cytokines, NLRC5 further influences the osteogenic differentiation of BMSCs. NLRC5 serves as a key regulator and promising candidate for bone repair. NLRC5 contributes to bone regeneration through a dual mechanism: it promotes BMSCs osteogenic differentiation, at least in part via the PI3K/AKT/β-catenin signaling pathway, and in