Endocytosis of Toll-like receptor 4 (TLR4) is a significant regulatory mechanism in lipopolysaccharide (LPS)-driven cellular activation, curbing MyD88-mediated pro-inflammatory cytokine responses in favour of TRIF-dependent endosomal signalling and Type I interferon (IFN) expression. The molecular events underpinning TLR4 activation are well established, with LPS-binding protein (LBP) and CD14 vital for trafficking LPS to the TLR4 accessory protein MD-2. However, the full hierarchy, interplay and identity of the constituents of the molecular cascade that occurs between interaction of LPS with TLR4/MD-2 and engagement of endocytic machinery by the TLR4 complex is less defined.
Radioprotective 105 kDa (RP105) is a TLR family member closely related to TLR4. Observations of elevated LPS-induced pro-inflammatory cytokine production in RP105-deficient (Rp105-/-) macrophages and DCs have led to the prevailing view that RP105 negatively regulates myeloid cell TLR4 activation. Here we demonstrate a thus far unknown role for RP105 in promoting LPS-induced TLR4 endocytosis and Type I IFN expression. Rp105-/- primary mouse macrophages and CRISPR/Cas9-generated RP105-negative RAW264.7 macrophages exhibited impaired LPS-induced TLR4 internalisation, and downstream TBK-1 phosphorylation. LPS- and E. coli-induced IFN-β responses were reduced in RP105-deficient primary and RAW264.7 macrophages, and LPS-challenged Rp105-/- mice exhibited significantly reduced levels of serum IFN-β. Pharmacological and inhibitory antibody studies indicated that molecular events underpinning RP105-mediated TLR4 internalisation are distinct from established regulators of TLR4 endocytosis such as CD14, SYK, and PLCγ2. Together, these data identify RP105 as a novel driver of LPS-driven TLR4 endocytosis and Type I IFN expression, and further elucidate the molecular network that regulates TLR4 functions.