Pyroptotic cell-derived factors promote osteoclast differentiation

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.