CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain–containing adapter-inducing IFN-β (TRIF)-mediated signaling pathways. Recently, Zanoni et al. (2011) Cell 147(4):868–880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-β in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 “pocket,” completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14.Toll-like receptor 4 (TLR4) signaling plays a crucial role in host defense against Gram-negative bacteria by recognizing the outer membrane component, lipopolysaccharide (LPS) (1–3). TLR4 signaling is initiated by transfer of an LPS monomer from LPS binding protein (LBP) to cluster of differentiation 14 (CD14) (GPI-linked or soluble). In turn, CD14 transfers monomeric LPS to myeloid differentiation factor 2 (MD-2), a protein that associates noncovalently with TLR4 (4). Appropriate ligand binding to MD2 results in dimerization of two TLR4/MD2 complexes (4). TLR4 is unique in that it is the only TLR that activates both myeloid differentiation primary response 88 (MyD88) and TIR-domain–containing adapter-inducing IFN-β (TRIF)-dependent signaling pathways (5, 6). MyD88-mediated, TLR4 signaling occurs mainly at plasma membranes and involves IL-1R–associated kinases phosphorylation, association of TNF-receptor–associated factor 6, and downstream signaling that results in NF-κB activation and induction of proinflammatory mediators such as TNF-α and IL-6 (7). In contrast, TRIF-mediated signaling in response to LPS occurs at the endosomal membrane after internalization of the TLR4 that, in turn, activates IFN regulatory factor 3 (IRF3), resulting in production of IFN-β, IP-10, and other IRF-3–dependent genes, as well as delayed NF-κB activation (8). Recent studies have shown that the endocytosis of TLR4 is tightly controlled by several molecules. Rab11a, ARF6, and p120-catenin have been implicated in Escherichia coli/LPS-induced TLR4 endocytosis and IRF3 activation (9–11). Zanoni et al. showed that CD14 plays critical roles in translocation of TLR4 into endosomes and in activation of IRF3 that are dependent upon the enzymatic activities of PLCγ2 and Syk (12). However, CD14-independent TLR4 endocytosis and TRIF signaling have not been reported.UT12 is a monoclonal antibody (MAb) with specificity for the mouse TLR4/MD2 complex and mediates LPS-like signaling (13). It has been shown that UT12 induces endotoxic shock-like symptoms in mice including augmentation of TNF-α and IL-6. Furthermore, UT12 induced long-term tolerance and protection against LPS-induced lethal shock in mice (14). However, the ability of UT12 to induce translocation of TLR4/MD2 into endosomes, as well as its potential for mediating TRIF-dependent signaling, has not been reported. Recently, a group of substituted pyrimido5,4-b]indoles, synthetic ligands for TLR4 that activate NF-κB that act in a CD14-independent manner, were discovered by high-throughput screening (15). These synthetic ligands induced IL-6 and IP-10 in a TLR4/MD2-dependent, but CD14-independent manner (16). They, too, have not been tested for TLR4 endocytosis and TRIF-dependent intermediates.In this study, we report, for the first time to our knowledge, CD14-independent translocation of TLR4 to endosomes and TRIF signaling by UT12 and small synthetic TLR4 ligands (1Z105). A TLR4 antagonist, Eritoran, that binds to a deep hydrophobic pocket in MD2 and blocks signaling induced by LPS, UT12, and 1Z105, blocked only TLR4 internalization and dimerization induced by LPS and 1Z105. Despite TLR4/MD2 internalization, endotoxin-tolerized macrophages fail to activate TRIF-mediated signaling. These findings reveal previously unidentified insights into the possible role of CD14 in LPS-mediated TLR4 endocytosis and signaling and demonstrate that TLR4 endocytosis and signaling are dissociable processes.