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: 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
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:Comparison of gene expression in WT and MAL knockout (MALKO) mouse macrophages treated with 10ng/ml lipopolysaccharide (LPS) with that of mock-treated cells incubated for the same time (10 days). Cells from 4 mice of each genotype were used and each individual served as its own control. Hybridizations of treated and control samples were dye swapped. Two experiments: WT vs. LPS-treated WT, and MALKO vs. LPS-treated MALKO. 4 individual biological replicates for each genotype, and each mouse served as its own mock control.
Project description:Macrophages play a pivotal role in the immune system through recognition and elimination of microbial pathogens. Toll-like receptors (TLRs) on macrophages interact with microbial substances and initiate signal transduction through intracellular adapters. TLR4, which is important for the response to lipopolysaccharide (LPS), triggers downstream signaling mediators and eventually activates IkB kinase (IKK) complex and mitogen-activated protein kinases (MAPKs) such as p38. Previous reports revealed that, in addition to NF-kB, the induction of some LPS-inducible genes in macrophages required another transcription factor whose activity depends on p38. However, these transcription factors remained to be identified. Among these genes, NF-kB and C/EBPβ, a p38 downstream transcription factor, were predicted to co-regulate genes in LPS-stimulated BMDMs. Based on the subsequent results of a chromatin immunoprecipitation assay, we demonstrated that Tnfaip3 is regulated by both NF-kB and p38-dependent C/EBPβ. These results elucidate our understanding of the tight regulation of innate immunity. In order to identify p38-activated transcription factors that cooperate with NF-kB in response to LPS stimulation, microarrays were used to identify genes regulated by both NF-kB and p38 using wild-type, IKK-depleted, and p38 inhibitor-treated mouse bone marrow-derived macrophages (BMDMs). In silico analysis of transcription factor binding sites was used to predict the potential synergistic transcription factors from the co-expressed genes.
Project description:This SuperSeries is composed of the following subset Series: GSE19482: Transcriptional responses of human monocyte-derived macrophages (HMDM) to lipopolysaccharide (LPS) GSE19490: Transcriptional responses of mouse BMM and TEPM to lipopolysaccharide (LPS) GSE19765: Transcriptional responses of human monocyte-derived macrophages (HMDM) to lipopolysaccharide (LPS) - Illumina arrays GSE19766: Transcriptional responses of mouse bone marrow-derived macrophages (BMM) to lipopolysaccharide (LPS) - Illumina arrays Refer to individual Series
Project description:mRNA microarray analysis of bone marrow derived macrophages treated under four conditions, including Naïve (N). Bone marrow derived macrophages (BMDM) were derived from the bone marrow of mice and cultured in the presence of PAO, IFN-gamma, or lipopolysaccharide (LPS). Profiled groups include Naive, LPS, IFN, PAO. Compared each of the groups (PAO, LPS, IFN) with Naïve group.
Project description:The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. Results: We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Conclusions: Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. The domestic pig (Sus scrofa) provides a large animal model for human innate immune responses and inflammation that is also economically important in its own right. We demonstrate that macrophages can be harvested from 3 different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1. We have performed surface marker analysis and gene expression profiling on macrophages from these compartments, comparing 25 animals from 5 different breeds and their response to lipopolysaccharide. The results provide a clear distinction between alveolar macrophages (AM) and monocyte-derived (MDM) and bone-marrow-derived macrophages (BMDM). In particular, the lung macrophages express the growth factor, Flt1 and its ligand, VEGFA at high levels, suggesting a distinct pathway of growth regulation. We confirm that pig macrophages more closely resemble human, than mouse, in their set of LPS-inducible genes. Relatively few genes showed breed-specific differential expression, notably CXCR2 and CD302 in alveolar macrophages. In contrast, there was substantial inter-individual variation between pigs within breeds, mostly affecting genes annotated as being involved in immune responses. Future research will address whether such variation is heritable, and might form the basis for selective breeding for disease resistance or functional genomics. 140 Affymetrix Snowball microarray were analysed from 5 different breed (DR, LR, LW, PIE and HAM). 5 pig per breed were used and cells were harvested from Lungs, blood and bone-marrow (AM, MDM and BMDM). Cells were left untreated (0h) or stimulated with LPS Salmonella enterica serotype minnesota Re 595 - 100ng/ml (7h)
Project description:Interferon regulatory factor (IRF) 5 has a major role in defining inflammatory macrophage polarization, but the molecular mechanisms of its function as a transcriptional regulator of nflammatory genes are not fully understood. Here, we characterise the sites of IRF5 recruitment in inflammatory macrophages in response to lipopolysaccharide (LPS). Samples in this experiment have also been used in Illumina BeadChip expression profling assay (see ArrayExpress experiment E-MTAB-2032, https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2032).
Project description:Using GRO-Seq, we find extensive regulation of enhancer RNAs (eRNA) within super-enhancers in response to lipopolysaccharide treatment in macrophages. Both activation and repression of gene expression are associated with super-enhancers and eRNA transcription dynamics. Co-treatment of LPS and the anti-inflammatory drug dexamethasone targeted specific super-enhancers by attenuating their eRNA expression, leading to reduced expression of key inflammatory genes. We propose that super-enhancers function as molecular rheostats integrating the binding profiles of key regulators to produce dynamic profiles of gene expression. Nascent transcriptome (GRO-Seq) analysis over a time course (0, 20, 60, 180 min) of Lipopolisaccharide and Dexamethasone signaling in mouse bone marrow-derived macrophages.
Project description:Streptococcus Pneumoniae capsule is a major virulence factor. The capsule is known to inhibit complement fixation and phagocytes, but its impact on innate immune responses is not known. To address this question, we compared transcriptional responses by human monocyte derived macrophages in responses to wild type S. pneumoniae with isogenic mutant bacteria lacking their capsule. We conducted these experiments with and without macrophage pretreatment with cytochalasin D to evaluate the impact of phagocytosis, and we also made comparisons to mutant S.pneumoniae lacking bacterial lipoprotein which is known to be necessary for maximal innate immune responses.