Genome wide Affymetric Microarray analysis for RIPK1 dependent inflammatory changes in Bone Marrow Derived Macrophages (BMDMs)
Ontology highlight
ABSTRACT: Gene level expression estimate using the Whole Transcript (WT) Assay approach of the Gene 1.0 ST Array System for Mouse. This assay was done to identify the RIPK1-dependent gene expression changes in mouse BMDMs. Cost-effective gene-level analysis based on whole-transcript coverage. We analyzed Bone Marrow Derived Macrophages (BMDMs) under 4 different conditions (Control, LPS, LPS/zVAD, LPS/zVAD/Nec-1) to assess inflammatory changes in RIPK1 kinase dependent manner compared to LPS, LPS/zVAD plus RIPK1 inhibitor Nec-1 and control.
Project description:We characterized the CDK9 and Hes1 occupancy on gene loci in conditions of unstimuated and LPS stimualtion in BMDMs BMDMs were left untreated or stimulated with LPS for 1 hour. CDK9 or Hes1 ChIP was performed and the DNA products were subject to ChIPseq
Project description:We characterized the RNA polymerase II occupancy on gene loci in WT and Hes1 KO BMDMs under untreated and LPS-stimulated conditions WT and Hes1 KO BMDMs were left untreated or stimulated with LPS for 1 hour. Pol II ChIP was performed and the DNA products were subject to ChIPseq
Project description:Differential macrophage activation mediate genetic differences to a variety of inflammatory pathologies. We wanted to elucidate the transcriptional and regulatory programs regulating differential macrophage activation in genetically diverse mouse strains. Bone marrow-derived macrophages (BMDMs) from AJ and C57BL/6j mice were left unstimulated, stimulated with IFN/TNF, or IL-4, or CpG, or LPS or IFN/TNF and infected with a type II (Pru A7) strain of Toxoplasma gondii, or infected with Pru A7 and gene expression analyzed 18 hrs later.
Project description:TLR ligands consistently induce expression of two Hes family members Hes1 and Hey1 in macrophages.To evaluate the effects of these two factors on inflammatory responses, we generated mice lacking both Hes1 and Hey1 (DKO). WT and DKO BMDMs were then untreated or exposed to LPS for 3 hours, and microarray was performed to examine global gene expression profiles to identify Hes1 and Hey1-regulated inflammatory genes Examination of Hes1 and Hey1-regulated inflammatory genes in macrophages
Project description:The specific binding of transcription factors to cognate sequence elements is thought to be critical for the generation of specific gene expression programs. The transcription factors nuclear factor kB (NF-kB) and the interferon (IFN) regulatory factors (IRFs) bind to the kB site and the interferon response element (IRE), respectively, of target genes, and they are activated in macrophages after exposure to pathogens. However, how these factors produce pathogen-specific inflammatory and immune responses remains poorly understood. Combining top-down and bottom-up systems biology approaches, we have identified the NF-kB p50 homodimer (p50:p50) as a regulator of IRF responses. First, unbiased genome-wide expression analysis revealed that p50 repressed a subset of IFN-inducible genes through a previously uncharacterized subclass of guanine-rich IRE (G-IRE) sequences, which was substantiated by biochemical and structural analyses. Second, mathematical modeling predicted that p50:p50 might enforce the stimulus-specificity of composite promoters. Indeed, the production of the antiviral regulator IFN-b was rendered stimulus-specific by the binding of p50:p50 to the G-IRE–containing IFNb enhancer to suppress cytotoxic IFN signaling. Specifically, a deficiency in p50 resulted in the inappropriate production of IFN-b in response to bacterial DNA sensed by Toll-like receptor 9. This role for NF-kB p50 in enforcing the specificity of the cellular response to pathogens by binding to a previously uncharacterized subset of IRE sequences alters our understanding of how the NF-kB and IRF signaling systems cooperate to regulate antimicrobial immunity. [BMDM]: Total RNA extracted from wt, p50-/- or ifnar-/- bone marrow derived macrophages (BMDMs) were subjected to stimulation with LPS, CpG or IFNb [MEF]: Total RNA extracted from wt or p50KO primary mouse embryonic fibroblasts were subjected to stimulation with LPS or IFNb
Project description:To investigate the effects of the differences seen in the JUND binding patterns between LEW, WKY and WKY.LCrgn2 bone marrow derived macrophages (BMDMs), we have 1) analysed gene expression changes over a time course (4 time points: 0,2,4 and 8 hrs) of stimulation with lipopolysaccharide (LPS) using whole transcript expression microarrays, 2) knocked down JunD in WKY BMDMs and analysed gene expression by microarrays.
Project description:IL-10 or IL-6 stimulation of control 129xC57BL/6 murine bone marrow derived macrophages in the presence of LPS. We used microarrays to detail the global programme of gene expression changes in response to IL-6 or IL-10 stimulation in the presence of lipopolysaccharide. BMDMs were isolated from control, IL-6-/-, and IL-10-/- mice on a 129XBL/6 mixed background mice and differentiated in the presence of CSF-1 for 6-7 days. Cells were scraped and plated in 6 well plates at 2x10e6/well. Cells were washed with complete DMEM and rested for 1-2 hr before stimulation with combinations of IL-10 (10 ng/ml), IL-6 (2 ng/ml) or LPS (100 ng/ml) for 45 min or 180 mins. Complete biological replicates were performed. Experiment Overall Design: Data sets from wild-type, IL-10-/- and IL-6-/- BMDMs treated with IL-6 or IL-10 in the presence of LPS over time
Project description:BMDMs were generated via differentiation of bone marrow cells in culture media containing m-CSF for 7 day. Upon differentiation, BMDMs were left untreated or stimulated with LPS, IFNa, PGE2, LPS+PGE2, or IFNa+PGE2.
Project description:To investigate the plasticity of Lipolysaccharide (LPS) tolerance, we employed microarray profiling to analyse the gene expression profile in macrophage. Four macrophage populations were induced; Untreated macrophages (Control group), Acute response to LPS (LPS activation group), LPS tolerance (T – Tolerant group) and recovered (R = recovered macrophage group) Using transcriptional analysis we demonstrate that recovery from LPS tolerance (R – Recovery), as defined by cytokine gene expression, is associated with a global change in the transcriptional profile of macrophage. This data confirms that LPS tolerance is a transient state which results in induction of novel hybrid macrophage activation state with a unique transcriptional signature. Bone marrow derived macrophages were polarised into three activation states; Acute response to LPS (A), LPS tolerant (T) and recovered (R). Gene expression was measured at 4 hours post stimulation with LPS. Three independent experiments were performed to measure gene expression changes between each macrophage group.
Project description:We investigated the molecular mechanisms by which ERRM-NM-1 negatively regulates TLR-signaling pathways. To examine this, we performed global gene expression analysis of ERRM-NM-1+/+ and ERRM-NM-1-/- BMDMs after LPS stimulation. Microarray analysis revealed that several genes encoding various TLR-negative regulators were downregulated in LPS-stimulated ERRM-NM-1-/- BMDMs, when compared with ERRM-NM-1+/+ BMDMs Bone marrow-derived macrophages were isolated in 6~8 week old male C57BL6 mice and then divided into 4 groups 1) Solvent control-treated ERRM-NM-1+/+ BMDM 2) LPS-treated ERRM-NM-1+/+ BMDM 3) Solvent control-treated ERRM-NM-1-/- BMDM 4) LPS-treated ERRM-NM-1-/- BMDM