Project description:Gene expression profile of LysMCre/Cre and KLF2∆/∆ primary peritoneal macrophages following 6 hours of LPS treatment. We used microarrays to detail the global program of gene expression following LPS stimulation of LysMCre/Cre and KLF2∆/∆ primary peritoneal macrophages. We identified distinct classes of genes that were altered following LPS stimulation.
Project description:Gene expression profile of LysMCre/Cre and KLF2?/? primary peritoneal macrophages following 6 hours of LPS treatment. We used microarrays to detail the global program of gene expression following LPS stimulation of LysMCre/Cre and KLF2?/? primary peritoneal macrophages. We identified distinct classes of genes that were altered following LPS stimulation. We injected 3 pairs of LysMCre/Cre (n=3) and KLF2?/? (n=3) mice with 2 ml of thioglycolate broth intraperitoneally. After 72 hours of thioglycolate injection, total cell suspension from peritoneal cavity were collected. These cells were seeded on 100mm tissue culture plates. Cells were allowed to attach for 4 hours and unattached cells were removed by washing with cell culture medium. These cells were placed in humidified incubator for additional 24 hours before LPS treatment. LPS was diluted to final concentration of 100ng/ml in culture medium and this medium was placed on peritoneal macrophages derived from LysM Cre/Cre and KLF2?/? mice for 6hours. Cells were lysed and total RNA was isolated and subjected to hybridization on Affymetrix microarrays.
Project description:Transcription profiling by array of LysMCre/Cre and KLF2∆/∆ (LysMCre/Cre: KLF2 FL/FL) primary peritoneal macrophages treated with lipopolysaccharide (LPS) for 6 hours
Project description:Akirin2 is an evolutionally conserved nuclear protein involved in the regulation of a set of inflammatory gene expression in various cell types. We used microarrays to examine the effect of Akirin2 deficiency in LPS-inducible gene expression in macrophages Peritoneal macrophages from wild-type and LysM-Cre+;Akirin2fl/fl mice were stimulated with LPS for 0, 2 and 4 hours, followed by RNA extraction and microarray analysis.
Project description:Krüppel-like factor 2 (KLF2) is a critical regulator of monocyte activation in response to inflammatory stimuli. Here, we report transcriptomic differences in peritoneal macrophages isolated from Lysozyme M Cre (control) and myeloid-specific KLF2 knockout mice.
Project description:PP2A regulates inflammatory cytokine/chemokine gene expression by dephosphorylating protein kinases at multiple signaling pathways from stimulated cells. In this dataset, Affymetrix mouse Gene ST 2.1 Array was used to assay total RNA extracted from LPS-treated PP2ACα knockout BMDM (PP2ACαfl/fl;lyM-Cre) and the control BMDM (PP2ACαfl/fl) In this dataset, we include the expression data obtained from LPS-stimulated PP2ACα conditional knockout BMDM (PP2ACαfl/fl;lyM-Cre) and control BMDM (PP2ACαfl/fl). The data are used to obtain 1080 genes that are differentially expressed in response to LPS stimulation
Project description:We have identified a number of miRNAs that are differentially expressed in LPS treated mouse peritoneal macrophages, as compared to untreated cells Peritoneal macrophages treated with LPS for 0, 4, 12, 24h. RNA was isolated and miRNA array assays were performed by Exiqon (miRCURY™ LNA Array version 10.0)
Project description:The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. With a single LPS stimulation, Ezh2 null(Ezh2flox/flox; LysM-Crecre/−) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre−/−), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.
Project description:Purpose: The goal of this study is to obtain the differential gene expression in MED1fl/fl and MED1ΔMac peritoneal macrophages (PMs) stimulated with or without LPS. Methods: Pooled PMs were collected from 8-week-old MED1fl/fl and MED1ΔMac mice (n=6 mice/group), and then treated without or with lipopolysaccharide (LPS; 50 ng/mL) for 6 hours. PMs mRNA profiles were generated by deep sequencing, in duplicate, using Illumina NextSeq500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: reads were mapped to the mm9 whole genome using tophat and RPKM were calculated using cufflink. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 12 million sequence reads per sample to the mouse genome (build mm9) and identified 24,130 transcripts in the PMs of MED1fl/fl and MED1ΔMac mice. Data analysis with TopHat and Cufflinks workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the detailed analysis of PMs transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results show that the expression of more than 20 genes involved in innate immune response and M1 polarization was significantly higher in MED1ΔMac than in MED1fl/fl macrophages after LPS treatment. Upregulated genes in MED1ΔMac macrophages are the proinflammatory genes.
Project description:TSA treatment blocks LPS-induction of several inflammatory target genes in primary macrophages. The microarray experiment was performed to identify LPS-inducible, LXR-sensitive target genes that retained LPS-induction in the presence of TSA. Thioglycollate-elicited macrophages were isolated from mice by peritoneal lavage 3 days following peritoneal injection of 2.5 ml 3% thioglycollate (DIFCO). Cells were plated in RPMI medium 1640 and 10% fetal bovine serum, washed after 5 h the medium was removed and cells were fed with fresh medium containing 0.5% fetal bovine serum. Cells were treated with TSA (Wako) at 100nM and/or with LPS (Sigma) at concentration of 100 ng/ml in the absence or presence of receptor-specific agonist for LXR (GW3965) at 1 µM. Keywords: TSA, LPS, LXR agonist