title: Genome-wide expression profiling and mutagenesis studies reveal that lipopolysaccharide responsiveness appears to be absolutely dependent on TLR4 and MD-2 expression and is dependent upon intermolecular ionic interactions.
abstract: Lipid A (a hexaacylated 1,4' bisphosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. In this article, we define the role of TLR4 and MD-2 in LPS signaling by using genome-wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulation with peptidoglycan-free LPS and synthetic Escherichia coli lipid A. Of the 1396 genes significantly induced or repressed by any one of the treatments in the wild-type macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin and that both are required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. According to our murine TLR4/MD-2-activation model, the two phosphates on lipid A were predicted to interact extensively with the two positively charged patches on mouse TLR4. When either positive patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were nearly abrogated. However, the MyD88-dependent and -independent pathways were impaired to the same extent, indicating that the adjuvant activity of monophosphorylated lipid A most likely arises from its decreased potential to induce an active receptor complex and not more downstream signaling events. Hence, we concluded that ionic interactions between lipid A and TLR4 are essential for optimal LPS receptor activation.
authors.affiliation: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA. jianmin.meng@umassmed.edu
authors.forename: Jianmin
authors.initials: J
authors.lastname: Meng
authors.forename: Mei
authors.initials: M
authors.lastname: Gong
authors.forename: Harry
authors.initials: H
authors.lastname: Björkbacka
authors.forename: Douglas T
authors.initials: DT
authors.lastname: Golenbock
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D055106:Genome-Wide Association Study; D015151:Immunoblotting; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000276:immunology*; D051218:Lymphocyte Antigen 96 / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000737:chemistry* / Q000276:immunology
chemical_list: D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4