Project description:To compare the expression profile of differentiated mouse bone marrow macrophages (BMM) in response to IL-4, we have employed whole genome microarray expression profiling. For this purpose, bone marrow cells were isolated from 8 to 12 weeks old C57BL6/J and Balb/cAnNCrl mice and cultured in the presence of the macrophage colony-stimulating factor (M-CSF). After seven days of culture, IL-4 was added for 4 and 18 hours. Keywords: Mice strain comparison; Gene expression profiling IL-4 induced gene expression was investigated in mouse bone marrow macrophages (BMM) of C57BL6/J and Balb/cAnNCrl mice. Differentiated BMM were incubated with mouse recombinant IL-4 for 4 or 18 hours or without for 18 hours. Two independent experiments were performed at each time (mock, 4 and 18 hours) using different mice littermates for each experiment.
Project description:To compare the expression profile of differentiated mouse bone marrow macrophages (BMM) in response to IL-4, we have employed whole genome microarray expression profiling. For this purpose, bone marrow cells were isolated from 8 to 12 weeks old C57BL6/J and Balb/cAnNCrl mice and cultured in the presence of the macrophage colony-stimulating factor (M-CSF). After seven days of culture, IL-4 was added for 4 and 18 hours. Keywords: Mice strain comparison; Gene expression profiling
Project description:Bone marrow cells were isolated, primed with M-CSF (M-BMDM) or GM-CSF (GM-BMDM) and cultured for 7 days. The proteomic difference between GM-BMDM and M-BMDM were analyzed to describe the phenotye and function of two types of macrophages.
Project description:We use tolDCs from syngeneic (C57BL6) and allogeneic (BALB/c) mice to show potent renoprotective abilities in a ischemia reperfusion AKI model. Dendritic cells were derived from mice bone marrow and cultured for 7 days with GM-CSF and IL-4. Tolerogenic DCs were cultured in the same DC media but with the addition of VitD3 and IL10. LPS was added on day 6 and cells harvested for use or analysis on day 7.
Project description:Pattern recognition receptors (PRR) detect microbial products and induce cytokines which shape the immunological response. Interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α) and IL-1β are proinflammatory cytokines which can be essential for resistance against infection, but if produced at high levels, may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine which dampens proinflammatory responses, but can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response. Here, we show that macrophages derived from C57BL/6 mice produce low levels of IL-12, TNF-α and IL-1β, but high levels of IL-10 in response to TLR4 and TLR2 ligands LPS and PamCSK4, and Burkholderia pseudomallei a Gram-negative bacterium which activates TLR 2/4. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in B. pseudomallei and LPS stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN dependent, but IL-27 independent mechanism. Further, type I IFN contributed to differential IL-1β and IL-12 production in B. pseudomallei and LPS stimulated C57BL/6 and BALB/c macrophages, via both IL-10-dependent and independent mechanisms. These findings highlight key pathways responsible for the regulation of pro- and anti-inflammatory cytokines in macrophages and reveal how they may differ according to the genetic background of the host. Total RNA obtained from bone-marrow derived macrophages of C57BL/6 WT, C57BL/6 Ifnar1-/- and BALB/c mice stimulated with heat-killed Burkholderia pseudomallei or media as controls.
Project description:Macrophages were derived from the bone-marrow of 3 x fl/+ Dicer LysCre +/- (wild-type) and 3 x fl/fl Dicer LysCre +/- mice and stimulated with IL-4 (50ng/mL) for 72h. Total RNA was isolated and analyzed by gene array. In this experiment, we derived Dicer deficient bone-marrow macrophages using Dicer fl/+ LysM-Cre by Dicer fl/+ crossed mice to obtain Dicer fl/fl LysM-cre progeny (and Dicer deficient macrophages). Next, we studied the effects of IL-4 stimulation in macrophage with a deficiency in Dicer/microRNAs.
Project description:Effects of SOCS3 on the transcriptional response of bone marrow-derived macrophages to IL-6. Fetal liver cells from SOCS3+/+ or SOCS3-/- embryos were used to reconstitute recipient mice. Donor derived bone marrow from these mice was differentiated to macrophages. Macrophages were either unstimulated, or stimulated for 100 or 400 minutes with 10 ng/ml IL-6. Keywords = macrophage, SOCS3, IL-6, interferon
Project description:Conditional macrophage-specific PPARg knockout mice were generated on C57Bl/6 background by breeding PPARg fl/- (one allele is floxed, the other is null) and lysozyme Cre transgenic mice. PPARg and IL-4 signaling was analyzed on bone marrow-derived macrophages. Bone marrow of 3 mice per group was isolated and differentiated to macrophages with M-CSF (20 ng/ml). 20 ng/ml IL-4 was used to induce alternative macrophage activation and 1 uM Rosiglitazone (RSG) was used to activate PPARg. From each mouse 4 samples were generated: 1. M-CSF, 2. M-CSF+RSG, 3. IL-4 and 4. IL-4+RSG. All compounds were added throughout the whole differentiation process, and fresh media was added every other day. Control cells were treated with vehicle (DMSO:ethanol). After 10 days, RNA was isolated and gene expression profiles were analyzed using Mouse Genome 430 2.0 microarrays from Affymetrix. 3 PPARg +/- LysCre and 3 PPARg fl/- LysCre mice were used to isolate bone marrow and from each macrophages were differentiated with or without IL-4 and simultaneously treated with vehicle or RSG. Altogether we analyzed 24 samples with 3 biological replicates as below.
Project description:Interferon (IFN)γ and interleukin (IL)-4 are central regulators of T helper 1 (Th1) and T helper 2 (Th2) immune responses, respectively. Both cytokines have a major impact on macrophage phenotypes: IFNγ–priming and subsequent TLR4 activation induces so called classically activated macrophages that are characterized by pronounced pro-inflammatory responses, whereas IL-4–treated macrophages, commonly called alternatively activated, are known to develop enhanced capacity for endocytosis, antigen presentation, and tissue repair and are generally considered anti-inflammatory. Considering IL-4 as priming rather than activating stimulus, we now compared the TLR4–dependent global gene activation program in IFNγ– versus IL-4–pretreated mouse macrophages, which has rarely been studied so far. Although both cytokines frequently induced opposing effects on gene transcription, the subsequent activation of bone marrow-derived macrophages by lipopolysaccharide (LPS) produced a strong, priming dependent pro-inflammatory response in both macrophage types. For example, the production of key pro-inflammatory cytokines IL-6 and IL-12 was significantly higher in IL-4– versus IFNγ–primed macrophages and several cytokine genes, including Il19, Ccl17, Ccl22, Ccl24 and Cxcl5, were preferentially induced in alternatively primed and LPS activated mouse macrophages. In a subset of genes, including IL12a, IFNγ priming was actually found to suppress LPS–induced gene expression in a Stat1–dependent manner. Our data suggest that IL-4–priming is not per se anti-inflammatory but generates a macrophage that is “tissue protective” but still capable of mounting a strong inflammatory response after TLR4–dependent activation. Keywords: Gene expression profiling Gene expression was investigated in mouse bone marrow-derived macrophages (BMM). On day 7, BMM were stimulated with either IL-4 or IFNγ overnight (18h in total). LPS treatment was performed in primed and unprimed macrophages 4 h prior to harvesting. At least three independent experiments were performed for each condition.