C3aR dampens intracellular cAMP to control TRIF-mediated inflammation in activated macrophages

Abstract

Abstract Recent studies from our lab and others have shown that complement components can enhance cell-intrinsic inflammatory signaling pathways. Specifically, we have found that activation of complement receptor C3aR enhances TLR4 signaling in lipopolysaccharide (LPS)-treated macrophages via enhanced p-38 phosphorylation. We found C3aR−/− mice treated with systemic LPS have decreased inflammatory cytokines (IL-6 and TNF) compared to wild-type (WT) mice. Our results corroborate previous studies showing C3aR−/− mice display decreased systemic cytokines (IL-6 and TNF) and increased bacterial burden during Gram-negative (GN) infection compared to WT mice. These results suggest C3aR is a critical regulator of TLR4 signaling. TLR4-TRIF signaling leads to activation of IRF3 and expression of interferon-regulated genes. We find that primary bone marrow-derived macrophages (BMDMs) from C3aR−/− mice show significantly reduced TRIF-dependent ifnb expression and IFNb secretion following 24h LPS exposure. Additionally, WT BMDMs treated with a C3aR antagonist (C3aRi) show a similar reduction in ifnb expression after LPS-treatment. These data demonstrate C3aR is critical for TRIF-dependent signaling downstream of TLR4 activation. Here we find WT BMDMs treated with C3aRi and LPS show increased intracellular cAMP. These findings demonstrate C3aR activation leads to depleted cAMP, which is essential for TRIF-mediated signaling downstream of TLR4. We hypothesize that C3aR is working as an inhibitory GPCR to regulate inflammation via intracellular cAMP and enhancing TRIF signaling in LPS-treated macrophages. Our findings are the first to suggest C3aR reduces cAMP to control TRIF-mediated inflammation in activated macrophages.