Project description:The role of microRNAs in the inflammatory response of macrophages to LPS was adressed by expression analysis in DicerKO (LysMcre/+;Dicerfl/fl;ApoE-/-) vs wildtype (LysMcre/+;Dicer+/+ApoE-/-) macrophages when challenged either with vehicle (PBS) or LPS.
Project description:Analysis of gene expression in explanted peritoneal macrophages from Aoah -/- and Aoah +/+ mice treated with LPS 21 days prior to harvest. Explanted peritoneal machrophages were challenged with LPS or control (PBS). The study seeks to characterize global gene expression in the state of prolonged LPS tolerance induced in mice lacking the LPS-inactivating enzyme Aoah. Groups of 9 C57BL/6 Aoah+/+ or Aoah-/- mice were given i.p. injections of 10 µg E. coli LPS/mouse. Twenty-one days later (when Aoah-/- mice remain tolerant and Aoah+/+ mice have recovered), peritoneal macrophages were harvested and the yields from three mice were pooled to form 3 samples per group (i.e., three samples of Aoah+/+ and Aoah-/- mice, with each sample comprised of peritoneal macrophages from 3 mice). Next day, cells were challenged with LPS or PBS and whole RNA was isolated 2 hours later and used for microarray experiments.
Project description:Analysis of gene expression in explanted peritoneal macrophages from Aoah -/- and Aoah +/+ mice treated with LPS 21 days prior to harvest. Explanted peritoneal machrophages were challenged with LPS or control (PBS). The study seeks to characterize global gene expression in the state of prolonged LPS tolerance induced in mice lacking the LPS-inactivating enzyme Aoah.
Project description:The activation profiles of macrophages under different immune and inflammatory conditions have generated great interest. LPS, in particular, is a commonly used in vitro model of infection and inflammation studies in macrophages. We have used gene expression microarrays to define the effects of each of three variables; LPS dose, LPS vs. interferons beta and gamma, and genetic background on the transcriptional response of mouse bone marrow-derived macrophages Macrophages derived from the C57BL/6 strain of mouse were challenged with increasing doses of LPS over a 24 hour time-course (0.5ng/ml, 5ng/ml, or 50ng/ml LPS). BALB/c derived macrophages were treated with any of 5ng/ml LPS, 10U/ml of recombinant mouse interferon-beta, or 10U/ml interferon-gamma, over a 24 hour timecourse. Sampling times were 0 hours (pre-treatment) and 1, 2, 4, 8, and 24 hours post-treatment in each case.
Project description:To investigate the mechanism of TRPC1 or TRPC6 on the regulation of endotoxemic cardiac dysfunction, we established Trpc1-/- and Trpc6-/- mice. LPS-challenged endotoxemic mouse model was built, and the gene expression profile was analyzed using data obtained from RNA-seq of WT, LPS-challenged WT, LPS-challenged Trpc1-/-, and LPS-challenged Trpc6-/- mice hearts.
Project description:The activation profiles of macrophages under different immune and inflammatory conditions have generated great interest. LPS, in particular, is a commonly used in vitro model of infection and inflammation studies in macrophages. We have used gene expression microarrays to define the effects of each of three variables; LPS dose, LPS vs. interferons beta and gamma, and genetic background on the transcriptional response of mouse bone marrow-derived macrophages Macrophages derived from the C57BL/6 strain of mouse were challenged with increasing doses of LPS over a 24 hour time-course (0.5ng/ml, 5ng/ml, or 50ng/ml LPS). BALB/c derived macrophages were treated with any of 5ng/ml LPS, 10U/ml of recombinant mouse interferon-beta, or 10U/ml interferon-gamma, over a 24 hour timecourse. Sampling times were 0 hours (pre-treatment) and 1, 2, 4, 8, and 24 hours post-treatment in each case. 64 samples in total were analysed. These incorporated 6 different timecourse studies. A combination of statistical filtering using the Empirical Bayes function in Bioconductor package (R statistical software), and co-expression analysis using the network analysis tool BioLayout Express 3D, was used to compare the timecourse studies.
Project description:We report the transcriptome of murine macrophages (cell line: RAW264.7) after supplementation with polyunsaturated fatty acids (PUFA) and stimulation with LPS. The fatty acids docosahexaenoic acid (DHA, C22:6n3) or arachidonic acid (AA, C20:4n6) were included in the culture medium in concentrations of 15 µmol/L using ethanol as a vehicle (0.2 % v/v final ethanol concentration). Cells were cultured in the enriched media totaling 72 h. Stimulation of cells was performed in the last 24 h of fatty acid supplementation by addition of LPS (1 µg/mL; from E. coli serotype 0111:B4).
Project description:Neutrophils play important roles in inflammatory airway diseases. Here, we assessed whether apolipoprotein A-I (apoA-I) modifies neutrophil heterogeneity as part of the mechanism by which it attenuates acute airway inflammation. Neutrophilic airway inflammation was induced by daily intranasal administration of LPS plus house dust mite (LPS+HDM) to Apoa1-/- and Apoa1+/+ mice for 3 days. Single cell RNA sequencing was performed on cells recovered in bronchoalveolar lavage fluid (BALF) on day 4. Unsupervised profiling identified 10 clusters of neutrophils in BALF from Apoa1-/- and Apoa1+/+ mice. LPS+HDM-challenged Apoa1-/- mice had an increased proportion of the Neu4 neutrophil cluster that expressed S100a8, S100a9, and Mmp8, and had high maturation, aggregation, and TLR4 binding scores. There was also an increase in the Neu6 cluster of immature neutrophils, whereas neutrophil clusters expressing interferon-stimulated genes were decreased. An unsupervised trajectory analysis showed that Neu4 represented a distinct lineage in Apoa1-/- mice. LPS+HDM-challenged Apoa1-/- mice also had an increased proportion of recruited airspace macrophages, which was associated with a reciprocal reduction in resident airspace macrophages. Increased expression of a common set of pro-inflammatory genes, S100a8, S100a9, and Lcn2, was present in all neutrophils and airspace macrophages from LPS+HDM-challenged Apoa1-/- mice. Apoa1-/- mice have increases in specific neutrophil and macrophage clusters in the lung during acute inflammation mediated by LPS+HDM, as well as enhanced expression of a common set of pro-inflammatory genes. This suggests that modifications in neutrophil and macrophage heterogeneity contribute to the mechanism by which apoA-I attenuates acute airway inflammation.
Project description:To investigate how the phenotype of macrophages that have engulfed engineered nanoparticles (ENPs) differs from normal macrophages, we conducted Affymetrix microarray studies to identify the gene regulatory pathways affected by the ENPs. To mimic potential occupational exposure scenarios, the experimental design involved pretreatment of mouse primary bone marrow macrophages with the ENPs (25 mg/ml) for 24 hr, followed by removal of residual ENPs and challenging the macrophages with the TLR4 ligand and surrogate bacterial stimulus, lipopolysachharide (LPS) for 4 hr. The 4 hr challenge time was chosen based on preliminary studies which showed many of the proinflammatory gene expression responses peak between 2-6 hr after LPS treatment. Transcriptional responses were measured by global microarray analysis of mouse primary bone marrow macrophage cells. Groups (N=3 replicates) of primary mouse bone marrow macrophages were pretreated with selected engineered nanoparticless (33 nm iron oxide(SPIO) or 50 nm amorphous silica) at concentrations of 25 mg/ml for 24 hours. After removing the nanoparticles, macrophages were challenged with fresh media containing 10ng/ml lipopolysaccharide (LPS).
Project description:Setd1bKO primary murine bone marrow-derived macrophages (BMDMs) were treated with lipopolysaccharide (LPS) or dexamethasone and LPS (Dex+LPS) and gene expression differences in response to treatment analysed by PolyA RNA-Sequencing. No Dex-treatment dependent gene expression differences were identified. Setd1aDel/+ Raw264.7 cells with reduced Setd1a expression were analyzed with regards to their reponse to Dex when inflammatorily challenged with LPS by mRNA-Seq. We observed reduced GR-dependent gene acivation in Setd1a hypermorphic Raw264.7 cells. Wild type and Setd1aDel/+ Raw264.7 cells were treated with LPS or LPS and interferon beta (IFNB1) to show the IFNB1-dependent loss of gene expression in LPS-stimulated Setd1aDel/+ cells.