PROJECT SUMMARY/ABSTRACT Innate immune sensors detect nucleic acid from viral and bacterial infections to clear infection, and also recognize endogenous (self) nucleic acid from stressed or dying cells. Toll-like receptors have long been known to detect nucleic acid, while nucleic acid s ensors within the cytosol have only recently been discovered. Importantly, activation of cytosolic DNA sensor pathways has been shown to promote autoimmune disease. Endogenous sources of DNA within cells can activate these pathways, including oxidized, “damaged” DNA that accrues with aging and can escape degradation, as well as DNA derived from replication of endogenous retroelements within the human genome. Despite the importance of the cytosolic DNA sensor pathways, little is known about their role in cell types other than macrophages and dendritic cells. We now demonstrate an important role for these pathways in bone that may provide insight into the bone loss occurring with aging and in certain autoimmune diseases. Several cytosolic DNA sensors signal through an ER-associated protein stimulator of interferon genes (STING), the most important of which is cyclic GMP-AMP synthase (cGAS). Activation of STING results in the production of type I interferons and other mediators. The cytosolic DNA sensor AIM2 does not signal through STING, but instead coordinates the assembly of an inflammasome complex, resulting in the activation of IL-1β and IL-18. We demonstrate that the STING and AIM2 pathways differentially regulate bone: STING deficient mice develop an osteopenic phenotype, implicating STING as a protective pathway for bone during states of DNA challenge such as viral and bacterial infection, while AIM2 deficiency enhances cortical and trabecular bone mass. We hypothesize that the STING and AIM2 pathways differentially regulate OC differentiation/function through distinct mechanisms. In Aim 1 we will determine the cell-intrinsic role of the STING pathway in the inhibition of osteoclast differentiation and the role of type I interferon and downstream regulatory elements in this process. In Aim 2 we will test the hypothesis that cytosolic DNA regulates bone homeostasis through the STING pathway, and determine the specific role of the critical DNA sensor cGAS upstream of STING in the regulation of osteoclastogenesis. Aim 3 will determine the role of the AIM2 inflammasome in regulating OC differentiation/function and will define interactions between the AIM2 and STING pathways. This proposal addresses the entirely novel hypothesis that cytosolic DNA sensors and their ligands regulate bone remodeling ...