In rheumatoid arthritis (RA) inflammatory cytokines produced by synovial (joint) cells drive disease pathogenesis by activating cells in inflamed joints and promoting recruitment of immune cells. The long-term goals of this project are to understand how cytokines and inflammatory factors expressed in RA synovium activate cells to drive disease pathogenesis, and to elucidate mechanisms that regulate production of pathogenic cytokines in the context of RA synovitis. An associated goal is to use this knowledge to develop safer and effective therapies that target disease-related mechanisms of cytokine production and function. This project has focused on two key synovial cell types important in RA pathogenesis, fibroblast-like synoviocytes (FLS) and synovial macrophages (Ms; previously termed type A synoviocytes). These two cell types interact closely in RA synovium. RA synovial macrophages express gene signatures reflecting inflammatory NF-B and Jak-STAT signaling and produce inflammatory cytokines. RA FLS produce cytokines and inflammatory factors such prostaglandins (PGs), and exhibit tissue-destructive properties. In the previous project period, we defined a role for FLS in regulating inflammatory M phenotype by modulating TNF responses to suppress IFN-stimulated gene (ISG) expression while promoting synergistic expression of inflammatory genes and growth factors that in turn increased FLS invasive behavior. A large fraction of the FLS-modulated TNF response in Ms was mediated by prostaglandin E2 (PGE2) and accordingly was almost completely recapitulated by exogenous PGE2, working via G protein- coupled receptors (GPCRs) and cAMP signaling. PGE2-cAMP signaling suppressed TNF and ISG expression, but cooperated with TNF to induce distinct inflammatory `synergy genes'. Strikingly, the gene signature commonly induced by FLS+TNF and PGE2+TNF was expressed in RA synovial macrophages, including a subset identified by single cell RNA sequencing that also expresses inflammatory and IFN signatures. These results highlight the importance of GPCR and downstream cAMP signaling in the RA synovial macrophage phenotype. GPCR-cAMP signaling in RA synovium can be activated by various factors including PGs, acidosis and neuropeptides. Thus, it is important to understand the impact of GPCR-cAMP signaling on macrophage inflammatory responses. Our overarching hypothesis is that the impact of PGE2- and GPCR-cAMP signaling on macrophage inflammatory responses can be selectively modulated to more effectively suppress TNF and ISGs, while avoiding induction of potentially pathogenic `synergy genes' such as those expressed by RA macrophages. In this project we will dissect signaling and chromatin-mediated mechanisms by which cAMP signaling regulates inflammatory responses in human macrophages. We anticipate that our studies will provide knowledge that can be used to shift GPCR-cAMP signaling towards increased suppression of inflammation.