Pyroptotic cell-derived factors promote osteoclast differentiation.

The NLRP3 D301N substitution, the murine ortholog of the human D303N variant, causes NLRP3 constitutive activation (NLRP3CA) and inflammasome assembly. Here, we show that priming signals induced by lipopolysaccharide (LPS) are sufficient to trigger GSDMD-dependent pyroptosis in NLRP3CA-expressing bone marrow-derived macrophages (BMDMs), but not in wild-type (NLRP3WT) cells. NLRP3CA 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 NLRP3CA specifically amplifies inflammasome-dependent responses. Conditioned medium (CM) from LPS-treated NLRP3CA BMDMs significantly enhanced osteoclast (OC) differentiation in vivo and in vitro compared to NLRP3WT CM, as did bone marrow supernatants from NLRP3CA mice. This osteoclastogenic activity was largely independent of IL-1β, as demonstrated by experiments using IL-1β-deficient NLRP3CA 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 NLRP3CA 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 NLRP3CA as a well-controlled model for studying pyroptosis and demonstrate that pyroptotic cells release soluble proteins that drive osteoclastogenesis independently of IL-1β.