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.

No abstract

Osteoblasts produce an array of immune molecules following bacterial challenge that could recruit leukocytes to sites of infection and promote inflammation during bone diseases, such as osteomyelitis. Recent studies from our laboratory have shed light on the mechanisms by which this cell type can perceive and respond to bacteria by demonstrating the functional expression of members of the Toll-like family of cell surface pattern recognition receptors by osteoblasts. However, we have shown that bacterial components fail to elicit immune responses comparable with those seen following challenge with the intracellular pathogens salmonellae and Staphylococcus aureus. In the present study, we show that UV-killed bacteria and invasion-defective bacterial strains elicit significantly less inflammatory cytokine production than their viable wild-type counterparts. Importantly, we demonstrate that murine osteoblasts express the novel intracellular pattern recognition receptors Nod1 and Nod2. Levels of mRNA encoding Nod molecules and protein expression are significantly and differentially increased from low basal levels following exposure to these disparate bacterial pathogens. In addition, we have shown that osteoblasts express Rip2 kinase, a critical downstream effector molecule for Nod signaling. Furthermore, to begin to establish the functional nature of Nod expression, we show that a specific ligand for Nod proteins can significantly augment immune molecule production by osteoblasts exposed to either UVinactivated bacteria or bacterial lipopolysaccharide. As such, the presence of Nod proteins in osteoblasts could represent an important mechanism by which this cell type responds to intracellular bacterial pathogens of bone.While the primary roles of osteoblasts are to synthesize the components of bone matrix and to control the bone-resorbing activity of osteoclasts, recent studies have revealed an additional function during bone diseases, the initiation and maintenance of inflammatory immune responses. Previous studies by our laboratory have shown that osteoblasts exposed to salmonellae and Staphylococcus aureus, the two most common causative agents in bone and joint diseases (23, 32), are a significant source of an array of soluble inflammatory mediators (2-4, 10, 26). Furthermore, we have recently described the surprising ability of osteoblasts to activate T lymphocytes by antigen presentation in the context of major histocompatibility complex II molecules (33) and to express the key costimulatory molecule CD40 (34) following exposure to bacteria. Such a pattern of immune molecule expression is one that can promote the recruitment of leukocytes to sites of bacterial infection and could serve to initiate and sustain inflammatory responses during inflammatory bone diseases, such as osteomyelitis.The recent demonstration of members of the Toll-like family of cell surface pattern recognition receptors in osteoblasts may represent a means by which these cells recognize bacterial pathogens. Kichuchi and coworkers (21) repo...

No abstract

No abstract