Pyroptotic cell-derived factors promote osteoclast differentiation

Abstract The NLRP3 D301N substitution, the murine ortholog of the human D303N variant, causes NLRP3 constitutive activation (NLRP3 CA ) and inflammasome assembly. Here, we show that priming signals induced by lipopolysaccharide (LPS) are sufficient to trigger GSDMD-dependent pyroptosis in NLRP3 CA -expressing bone marrow-derived macrophages (BMDMs), but not in wild- type (NLRP3 WT ) cells. NLRP3 CA mice exhibit elevated IL-1β secretion and LDH release in bone marrow supernatants under both basal and LPS-challenged conditions. Other cytokines (e.g., TNF-α) increased comparably between genotypes, indicating that NLRP3 CA specifically amplifies inflammasome-dependent responses. Conditioned medium (CM) from LPS-treated NLRP3 CA BMDMs significantly enhanced osteoclast (OC) differentiation in vivo and in vitro compared to NLRP3 WT CM, as did bone marrow supernatants from NLRP3 CA mice. This osteoclastogenic activity was largely independent of IL-1β, as demonstrated by experiments using IL-1β-deficient NLRP3 CA BMDMs, IL-1β-neutralizing antibodies, and genetic deletion of the IL-1 receptor. The osteoclastogenic factors were primarily soluble proteins, as heat inactivation and proteinase K digestion markedly reduced OC formation. Extracellular vesicles (EVs) from NLRP3 CA CM modestly promoted OC differentiation, but EV-depleted supernatants retained full activity, indicating that the primary mediators are soluble proteins not carried within EVs. Together, these findings establish NLRP3 CA as a well-controlled model for studying pyroptosis and demonstrate that pyroptotic cells release soluble proteins that drive osteoclastogenesis independently of IL- 1β.