Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) 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. Here 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 stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype 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 are both absolutely 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. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.

Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) 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. Here 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 stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype 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 are both absolutely 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. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. Bone marrow-derived macrophages were pooled from four individual WT or TLR4-deficient mice and stimulated with either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 for 2 hours and compared to PBS-stimulated control cells. We also compared PBS-stimulated WT cells directly to PBS-stimulated TLR4-deficient cells to compare the basal expression of genes in the two genotypes. This experiment was repeated once in its entirety.

Project description:Toll-like receptors (TLRs), a family of pattern recognition receptors, bind microbial and host-derived molecules, leading to intracellular signaling and activation of host innate immune responses. TLR4 is unique in that ligand-mediated activation initiates two signaling cascades: the MyD88-dependent pathway, localized to the cell membrane, elicits rapid MAP kinase and NF-κB activation, and the TRIF-dependent pathway, localized to TLR4-containing endosomes, results in IRF-3 activation. Previous studies have associated adjuvanticity with the TRIF pathway. Gram-negative LPS is a potent TLR4 agonist, and structurally related molecules also signal through TLR4 to different extents. Herein, we compared two synthetic, non-toxic lipid A analogs used previously as vaccine adjuvants, monophosphoryl lipid A (PHAD®, sMPL) and E6020, for their capacity to activate TLR4-mediated innate immune responses. In primary mouse macrophages, high dose sMPL activates the MyD88-dependent signaling equivalently to E6020. In contrast to sMPL, E6020 exhibits robust activation of the TRIF pathway, including TLR4 internalization and IRF-3-dependent transcription, at all doses examined. Eritoran, a TLR4/MD2 antagonist, competitively inhibited sMPL more strongly than E6020. Despite these differences, sMPL and E6020 adjuvants induced antibody responses to a similar level and with balanced Ig isotypes, in two separate mouse immunization models. However, E6020 induced superior non-specific protection in a model of Streptococcus pneumoniae infection, compared to sMPL, which correlates with the activation of reactive nitrogen and oxygen species. These data provide evidence that a TRIF bias is not necessarily predictive of superior adjuvanticity, but may activate stronger innate defenses from infection. TLR4 is unique among TLRs that it stimulates both the MyD88- and TRIF-dependent pathways (reviewed in Takeda K and Akira S, 2004, Semin Immunol). To compare the relative stimulatory capacities of two synthetic TLR4 agonist adjuvants, PHAD® (sMPL; Avanti Polar Lipids LLC) and E6020 (Eisai, Inc), we performed microarray analysis of primary thioglycollate-elicited mouse peritoneal macrophages that were either cultured in media alone (control) or stimulated with 100 ng/mL (57.3 uM sMPL, 61.6 uM E6020) of the TLR4 agonists for two hours. Total RNA was extracted from three biological repicates (numbered 1-3) per treatment, and the transcriptomes analyzed individually by whole transcriptome microarray (Affymetrix Clariom D). Of the differentially expressed genes (DE genes; average of three replicates greater or equal to 2-fold difference in any comparison of treatments and false discovery rate below 0.05) almost 94% were protein-coding genes (includes those listed as either protein-coding or complex in the database), on which we focused for further analysis. sMPL induced expression of 155 protein-coding genes, including those that encode IL-1beta, TNF-alpha, Cxcl1 and Cxcl2--proteins that are associated with classic macrophage activation and are essential for inducing fever and attracting neutrophils. sMPL suppressed 15 protein-coding genes. In contrast, E6020 induced expression of 413 protein-coding genes and suppressed 88 protein-coding genes, which included all but one of the sMPL-regulated protein-coding genes. The majority of the genes induced by E6020, but not sMPL, are associated with Type I IFN expression (reference: Interferome.org database). This interferon signature points to stronger activation of the TRIF/IRF3 pathway in E6020-treated macrophages and includes genes that are secondarily induced by Type I IFNs. Similar expression of MyD88 dependent genes Il1b, Il10, and Tnf, and differential expression of TRIF-dependent genes Ifnb1, Cxcl10, and Ccl5 between the two synthetic agonists was confirmed by qRT-PCR.

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