Staphylococcus aureus and Salmonella are common causes of bone and joint infections in humans. Unfortunately, the pathogenesis of bacterial bone and joint infections are poorly understood. Bacteria, including S. aureus, can infect osteoblasts and survive intracellularly within these bone-forming cells which begins to explain how bone infections might be chronic, and how the host's immune response might have difficulty in eliminating these pathogens. While osteoblasts can internalize and harbor bacteria, it is not at all clear if infected osteoblasts contribute to the immune responses to the invasion. Recently, we have described the surprising ability of osteoblasts to secrete significant amounts of bioactive IL-12. These cells are stimulated to produce this key pro- inflammatory cytokine when exposed to bacteria previously demonstrated to reside intracellularly. Such a finding is particularly significant given the central role IL-12 plays in the preferential initiation of Th1-type, cell- mediated, immune responses. Such immune responses are essential for the successful elimination of intracellular pathogens. The ability of these non-leukocytic cells to produce this IL-12 may point to a previously unrecognized role for osteoblasts in the generation of protective inflammatory responses and the resolution of infection. In the present application, we propose to investigate the mechanisms responsible for inducing the production of this important cytokine by osteoblasts. We will utilize RT-PCR and immunofluorescent techniques to determine whether IL-12 induction in human and mouse osteoblasts occurs as either a direct or indirect consequence of bacterial invasion. Furthermore, we will determine whether the production of IL-12 seen in vitro is reproducible in vivo using an animal model developed in our laboratory. Finally we will attempt to determine the biological significance of IL-12 production by osteoblasts in infected tissues in vivo by monitoring bacterial burden and T-cell infiltration utilization immunofluorescence techniques. In this manner, we will examine whether IL-12 attenuates or exacerbates bacterial infection of osteoblasts and local inflammation, thereby expanding the recognized role of these cells to include being integral components in the host responses to intracellular pathogens at these sites.
