Spatiotemporal regulation of rp105 subcellular localization shapes endosomal tlr4 signaling

Innate immune sensing of microbial and endogenous ligands by Toll-like receptor (TLR) 4 drives inflammation and antimicrobial mechanisms. While critical for host defense against pathogens, unchecked TLR4 activation causes organ damage and chronic disease. Molecular mechanisms that drive versus turn off TLR4 activity are being pursued as potential targets for preventing and treating infections and inflammation-driven pathologies. Mechanisms of the temporal and spatial regulation of TLR4 signaling originating from the cell surface are well established. In contrast, the regulation of TLR4 activation originating from endosomal compartments is not well known. Here we show that the TLR-related receptor, Radioprotective 105 kDa (RP105), localizes to TLR4-containing endosomal compartments in murine macrophages. RP105 has been proposed as a negative regulator of TLR4-MyD88 signaling originating from the cell surface, but whether it modulates endosomal TLR4 signaling is unknown. Our data shows that RP105-deficiency reduces LPS- and E. coli-induced type I IFN responses, suggesting that RP105 facilitates endosomal TLR4-TRIF signaling. We employed high-resolution microscopy and AI-guided quantitative analyses to interrogate the spatiotemporal relationship between RP105 and endosomal compartments. Cellular activation led to a transient reduction of RP105 from endosomal compartments. Our results suggest TLR4 activation via TRIF signaling mediates this process, but not MyD88. Moreover, our data identify phosphatidylinositol 3-kinase p110-delta and Bruton’s tyrosine kinase as critical regulators of RP105 endosomal localization. Collectively, our data identify RP105 as a positive regulator of endosomal TLR4 signaling. AAI Careers in Immunology Fellowship, 2022-2023