title: CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201;
authors.forename: Rajesh
authors.initials: R
authors.lastname: Rajaiah
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201;
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Perkins
authors.affiliation: Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD 20993.
authors.forename: Derek D C
authors.initials: DD
authors.lastname: Ireland
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201; svogel@som.umaryland.edu.
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004187:Disaccharides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism; D008024:Ligands; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013403:Sugar Phosphates / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D004187:Disaccharides; D050838:Interferon Regulatory Factor-3; D016899:Interferon-beta; D008024:Ligands; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D017346:Protein Serine-Threonine Kinases; D013403:Sugar Phosphates; D051197:Toll-Like Receptor 4; C494233:Irf3 protein, mouse; C476286:Ticam1 protein, mouse; C493487:Tlr4 protein, mouse; C512420:eritoran; C498588:Tbk1 protein, mouse
keywords: Eritoran; TLR4 endocytosis; agonistic antibody; endotoxin tolerance; small-molecule ligands