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: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. Keywords: time course
Project description:We report the genome-wide RNA sequencing analysis in Il10-/- bone marrow-derived macrophages (BMDMs) stimulated by lipopolysaccharide (LPS) where IL-10 effect in macrophage inflammatory response was examined in IL-10-deficient BMDMs upon LPS stimulation with addition of exogenous IL-10.
Project description:IL-6 induces IL4ralpha expression in macrophages. This mechanism is necessary to promote macrophage polarization towards an M2-phenotype and is crucial to limit the inflammatory response both upon obesity and LPS-endotoxemia. In this dataset, we include the expression data obtained from primary murine bone marrow-derived macrophages from control and IL6ralpha-deficient macrophages (n=4vs4) stimulated with interleukin-6 (IL-6) 8 samples were analyzed to compare control and IL6ralpha-deficient macrophages for their gene expression profiles upon stimulation with IL-6
Project description:In order to determine P2X7R secretome we analyzed the proteins present in cell-free supernatants from wild-type (P2rx7+/+) or P2rx7-/- bone marrow-derived macrophages (BMDMs) polarized either to M1 or M2 and subsequently treated with ATP. BMDMs were primed with LPS (M1) or IL-4 (M2) for 4 hours and the proteins secreted during this step were extensively washed with PBS before ATP was added in fresh buffer. The complex mixture of proteins obtained in the macrophages supernatants after ATP stimulation were fractionated using one dimension gel electrophoresis and 10 bands were selected for LC-MS/MS analysis based in their presence in higher intensity in P2rx7+/+ supernatant compared with P2rx7-/- supernatant.
Project description:Bone marrow-derived macrophages were produced from mice lacking IL-10 alone (IL10-def) or mice lacking both IL-10 and the p50/p105 subunit of NF-kB (p50/IL10), and left unstimulated, stimulated with LPS (1 ng/ml) or stimulated with LPS and IL-10 (0.3 ng/ml).
Project description:Mitochondrial electron transport chain (ETC) function modulates macrophage biology, however, mechanisms underlying mitochondrial ETC control of macrophage immune responses are not fully understood. Here we report that mutant mice with mitochondrial ETC complex III (CIII)-deficient macrophages exhibit increased susceptibility to influenza A virus and LPS-induced endotoxic shock. Cultured bone marrow-derived macrophages (BMDMs) isolated from these mitochondrial CIII-deficient mice released less IL-10 than controls following TLR3 or TLR4 stimulation. Surprisingly, restoring mitochondrial respiration without generating superoxide using alternative oxidase (AOX) was not sufficient to reverse LPS-induced endotoxic shock susceptibility or restore IL-10 release. However, activation of protein kinase A (PKA) rescued IL-10 release in mitochondrial CIII-deficient BMDMs following LPS stimulation. Additionally, mitochondrial CIII deficiency did not affect BMDM responses to interleukin-4 (IL-4) stimulation. Thus, our results highlight the essential role of mitochondrial CIII generated superoxide in the release of anti-inflammatory IL-10 in response to TLR stimulation
Project description:This study is focused to find new genes involved in Insulin signalling in bone marrow derived macrophages upon treatment with IL-4 (10 ng/ml) and LPS (100 nM). We will use primary macrophages differentiated from bone-marrow derived hematopoetic stem cells; our aim is to analyze the transcriptome upon anti-inflammatory (IL-4) and inflammatory (LPS) treatment.Total RNA was extracted with RNAEasy kit form Qiagen using manufacturer reccomendations and RIN values for all samples were between 9 and 10.
Project description:The nuclear orphan receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to exhibit an anti-inflammatory function in macrophages. To further elucidate the role of Nur77 in macrophage physiology, we compared the transcriptome of bone marrow-derived macrophages (BMM) from wild-type (WT) and Nur77-knockout (KO) mice both before and after stimulation with IL4 or LPS. Comparison of gene expression in bone marrow-derived macrophages, isolated from 3 wild-type (control) and 3 Nur77-/- mice (case), left untreated or stimulated in triplicate for 8 hours with LPS or IL-4
Project description:Bone marrow-derived macrophages, Unstimulated DMSO Bone marrow-derived macrophages, Unstimulated + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 1h DMSO Bone marrow-derived macrophages, LPS 1h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 2h DMSO Bone marrow-derived macrophages, LPS 2h + I-BET (GSK525762A) Bone marrow-derived macrophages, LPS 4h DMSO Bone marrow-derived macrophages, LPS 4h + I-BET (GSK525762A) LPS (100 ng/mL) was purchased from Sigma. Bone Marrow-derived macrophages (BMDMs) were differentiated from C57BL/6 bone marrow using 5 ng/mL each of recombinant M-CSF and IL-3 (Peprotech) for 7 days as described (Jeffrey et al, Nature Immunology, 2006). 2 x10^6 BMDMs were treated with DMSO or 1 μM of I-BET for 30 minutes before the addition of LPS (100 ng/mL) for 1, 2 or 4h. Unstimulated control samples were incubated with I-BET only for 1 hour. 500 ng of total RNA from 3 independent samples per group was used to prepare biotin-labeled RNA using Ambion Illumina TotalPrep RNA Amplification Kit (Applied Biosystems) and hybridized to Illumina MouseRef-8 v2.0 expression BeadChip kits. The chips were scanned using Illumina BeadArray Reader. 3 biological replicates and 4 timepoints
