Activated synovial macrophages (M[phi]s) are major producers of pathogenic cytokines, and play a key role in RA pathogenesis. The long term goals of this project are to understand mechanisms that regulate the balance between activating and feedback inhibitory signaling pathways that are activated in human M[phi]s by inflammatory factors that drive RA synovitis. An associated goal is to use this knowledge to therapeutically modulate balance and crosstalk between signaling pathways to promote resolution of inflammation. Important activators of RA synovial M[phi]s include autocrine TNF, immune complexes, T cell factors, and damage-associated molecular patterns (DAMPs) generated by tissue damage and cell death. DAMPs that activate Toll-like receptors (TLRs), which are amongst the most potent activators of M[phi]s and inflammatory genes such as TNF, IL6, and IL1B, have emerged as important drivers of RA synovial M[phi] activation. RA synovium contains abundant DAMPs including nucleic acids (NAs) derived from dying cells that activate intracellular RNA and DNA sensors. Interestingly, RA synovium also abundantly expresses molecules that bind NAs and potentiate activation of endosomal NA-sensing TLRs (eTLRs: TLR3/7/8/9). Of the eTLRs, TLR7 and TLR8 are highly expressed in RA synovial macrophages, potently drive inflammatory cytokine production including in RA synovial explants, and have been most closely implicated in pathogenesis of RA and inflammatory arthritis models. Human TLR8 is selectively expressed in myeloid cells and potently drives inflammatory cytokine production, but has been relatively understudied as mouse TLR8 is non-functional; interestingly, BAC-transgenic mice expressing human TLR8 (hTLR8) exhibit increased arthritis associated with erosive synovitis, high TNF, and macrophage infiltration that models RA. In the previous project period we focused on regulation of the balance between activating and feedback inhibitory pathways downstream of TNF and TLR8 in human M[phi]. We found that the balance of TLR8 signaling was shifted towards superinduction of inflammatory genes including TNF and IL6 by CXCL4, which is highly expressed in RA synovium. CXCL4 was previously shown to augment eTLR-mediated IFN and cytokine production in DCs by binding NAs and promoting their delivery to endolysosomal compartments. We identified novel and complementary mechanisms whereby CXCL4 activated signaling and chromatin remodeling in human M[phi]s to boost TLR8 responses. Our overarching hypothesis is that CXCL4 and eTLRs like TLR8 cooperatively activate signaling and epigenetic mechanisms that result in synergistic inflammatory gene activation, and that these mechanisms are operative in activated RA synovial M[phi]s. We propose to investigate mechanisms underlying CXCL4-TLR8 synergy and their pathophysiological implications for inflammatory arthritis. We anticipate our studies will yield insights that can be used to attenuate pathologic M[phi] super-activat...