UNLABELLED: Bacterially induced osteoblast apoptosis may be a major contributor to bone loss during osteomyelitis. We provide evidence for the functional expression in osteoblasts of NLRP3, a member of the NLR family of cytosolic receptors that has been implicated in the initiation of programmed cell death. INTRODUCTION: Osteoblasts undergo apoptosis after exposure to intracellular bacterial pathogens commonly associated with osteomyelitis. Death of this bone-forming cell type, in conjunction with increased numbers and activity of osteoclasts, may underlie the destruction of bone tissue at sites of bacterial infection. To date, the mechanisms responsible for bacterially induced apoptotic osteoblast cell death have not been resolved. MATERIALS AND METHODS: We used flow cytometric techniques to determine whether intracellular invasion is needed for maximal apoptotic cell death in primary osteoblasts after challenge with Salmonella enterica. In addition, we used real-time PCR and immunoblot analyses to assess osteoblast expression of members of the nucleotide-binding domain leucine-rich repeat region-containing family of intracellular receptors (NLRs) that have been predicted to be involved in the induction of programmed cell death. Furthermore, we have used co-immunoprecipitation and siRNA techniques to confirm the functionality of such sensors in this cell type. RESULTS: In this study, we showed that invasion of osteoblasts by Salmonella is necessary for maximal induction of apoptosis. We showed that murine and human osteoblasts express NLRP3 (previously known as CIAS1, cryopyrin, PYPAF1, or NALP3) but not NLRC4 (IPAF) and showed that the level of expression of this cytosolic receptor is modulated after bacterial challenge. We showed that osteoblasts express ASC, an adaptor molecule for NLRP3, and that these molecules associate after Salmonella infection. In addition, we showed that a reduction in the expression of NLRP3 attenuates Salmonella-induced reductions in the activity of an anti-apoptotic transcription factor in osteoblasts. Furthermore, we showed that NLRP3 expression is needed for caspase-1 activation and maximal induction of apoptosis in osteoblasts after infection with Salmonella. CONCLUSIONS: The functional expression of NLRP3 in osteoblasts provides a potential mechanism underlying apoptotic cell death of this cell type after challenge with intracellular bacterial pathogens and may be a significant contributory factor to bone loss at sites of infection.