Genome wide expression profiling and mutagenesis studies reveal an absolute TLR4 and MD-2 requirement for LPS responsiveness dependent upon intermolecular ionic interactions
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE31078 | GEO | 2011/07/31
SECONDARY ACCESSION(S): PRJNA144725
REPOSITORIES: GEO
Similar Datasets
Project description:This SuperSeries is composed of the following subset Series: GSE31066: Lipopolysaccharide (LPS) response in macrophages from TLR4-deficient mice GSE31067: Lipopolysaccharide (LPS) response in macrophages from MD-2-deficient mice Refer to individual Series
2011-07-30 | E-GEOD-31078 | biostudies-arrayexpress
Project description: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.
| S-EPMC3178671 | biostudies-literature
Project description:The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2's hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4'phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamine's carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2's hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases.
| S-EPMC3127813 | biostudies-literature
Project description:Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)- and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS.
| S-EPMC4763747 | biostudies-literature
Project description:The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.
| S-EPMC4039514 | biostudies-literature
Project description:Lipopolysaccharide (LPS) activates innate immune responses through TLR4.MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4.MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV(A), a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV(A) activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV(A) activation. Computational studies suggested that unique ionic interactions exist between lipid IV(A) and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4'-phosphate on lipid IV(A) interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys(367) and Arg(434)) at the dimerization interface. When replaced with their negatively charged human counterparts Glu(369) and Gln(436), mouse TLR4 was no longer responsive to lipid IV(A). In contrast, human TLR4 gained lipid IV(A) responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV(A) species specificity. Thus, using lipid IV(A) as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.
| S-EPMC2838292 | biostudies-literature
Project description:To address the role of Toll-like receptor 4 (TLR4) single nucleotide polymorphisms (SNP) in lipopolysaccharide (LPS) recognition, we generated mice that differed only in the sequence of TLR4. We used a bacterial artificial chromosome (BAC) transgenic approach and TLR4/MD-2 knockout mice to specifically examine the role of human TLR4 variants in recognition of LPS. Using in vitro and in vivo assays we found that the expression level rather than the sequence of TLR4 played a larger role in recognition of LPS, especially hypoacylated LPS.
| S-EPMC5636155 | biostudies-literature
Project description:The cell surface interaction between bacterial lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4) and MD-2 is central to bacterial sepsis syndromes and wound healing. We have shown that a generation (G) 3.5 polyamidoamine (PAMAM) dendrimer that was partially glycosylated with glucosamine inhibits TLR4-MD-2-LPS induced inflammation in a rabbit model of tissue scaring. However, it was a mixture of closely related chemical species because of the polydispersity of the starting PAMAM dendrimer. Generation 2 triazine dendrimers with single chemical entity material status are available at low cost and at the kilogram scale. PAMAM dendrimer can be synthetically grafted onto this triazine core dendrimer to make new triazine-PAMAM hybrid dendrimers. This led us to examine whether molecular modelling methods could be used to identify the key structural design principles for a bioactive lead molecule that could be synthesized and biologically evaluated. We describe our computer aided molecular studies of several dendrimer based constructs and the key design principles identified. Our approach should be more broadly applicable to the biologically focused, rational and accelerated design of molecules for other TLR receptors. They could be useful for treating infectious, inflammatory and malignant diseases.
| S-EPMC3176918 | biostudies-literature
Project description:ObjectivesMicrobial-specific factors are likely critical in determining whether bacteria trigger preterm labor. Structural variations in lipopolysaccharide (LPS), a component of gram-negative bacteria, can determine whether LPS has an inflammatory (agonist) or anti-inflammatory (antagonist) effect through Toll-like receptor 4 (TLR4). Our objective was to determine whether amniochorion could discriminate between LPS variants in a nonhuman primate model. We also cloned Macaca nemestrina TLR4 and MD-2 and compared this complex functionally to the human homologue to establish whether nonhuman primates could be used to study TLR4 signaling in preterm birth.Study designAmniochorion explants from M. nemestrina were stimulated with a panel of LPS variants for 24 h. Supernatants were analyzed for IL-1beta, TNF-alpha, IL-6, IL-8 and prostaglandins E2 and F2alpha. Tissue expression of TLR1, 2, 4, 6, MyD88 and NF-kappaB was studied by RT-PCR. M. nemestrina TLR4 and MD-2 genes were cloned and compared with their human counterparts in a recombinant TLR4 signaling system to determine LPS sensitivity.ResultsLPS variants differentially stimulated cytokines and prostaglandins, which was not related to transcriptional changes of TLR4 or other TLRs. Nearly all elements of LPS binding and TLR4 leucine-rich repeats were conserved between humans and M. nemestrina. TLR4/MD-2 signaling complexes from both species were equally sensitive to LPS variants.ConclusionsLPS variants elicit a hierarchical inflammatory response within amniochorion that may contribute to preterm birth. LPS sensitivity is similar between M. nemestrina and humans, validating M. nemestrina as an appropriate model to study TLR4 signaling in preterm birth.
| S-EPMC2934902 | biostudies-literature
Project description:Although lipopolysaccharide (LPS) stimulation through the Toll-like receptor (TLR)-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for "humanized" TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with "humanized" TLR4/MD-2 transgenic mice.
| S-EPMC3469661 | biostudies-literature