Chicken CSF2 and IL-4-, and CSF2-Dependent Bone Marrow Cultures
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ABSTRACT:
PROVIDER: PRJEB56177 | ENA |
REPOSITORIES: ENA
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Project description:Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.
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Project description:Femur and tibia bones were harvested from 8-week-old wild-type (WT) C57BL/6 mice. Bone marrow was flushed out into cold PBS (Life Technologies) plus 2% heat-inactivated FBS, passed through a needle five times to dissociate the cells, and then passed through a 70-μm cell strainer (Becton Dickinson) to remove cell clumps, bone, hair, and other cells/tissues. After addition of three volumes of NH4Cl solution (0.8% NH4Cl solution; Beyotime Institute of Biotechnology, Jiangsu, China), the mixture was incubated on ice for 10 min to remove red blood cells; the cells were then spun down and resuspended in cold PBS with 2% FBS. The harvested cells were cultured in DMEM containing 10% FBS and supplemented with 10 ng/ml recombinant mouse CSF1 (R&D Systems) or 10 ng/ml recombinant mouse CSF2 (R&D Systems) for 7 days to obtain CSF1- or CSF2-induced BMDMs: M(CSF1) or M(CSF2) cells, respectively. To test the difference genes expression of TAMs obtained from M(CSF1) and M(CSF2) cells in C57BL/6 mice. We used microarrays to detail the global programme of gene expression and identified M(CSF1) and M(CSF2) cells during this process.
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Project description:Mast cells are tissue resident granulocytes which are most abundant at the interface between tissues and the external environment, such as around blood vessels, in the skin or mucosal surfaces in the lungs and gut. Pathologically they are involved in allergic reactions and anaphylaxis, however they may also play protective roles in responses to some infections, particularly to pathogenic helminths. Mast cells also express high levels of the IL-33 receptor, which like TLRs, activates Myd88 dependent signalling pathways to drive de novo cytokine production in mast cells.IL-33 is a member of the IL-1 family known to stimulate a number of immune cell types including mast cells. IL-33 is a strong activator of de novo cytokine production in mast cells without inducing degranulation, although it has also been shown to synergise with other signals to promote degranulation. Bone Marrow-Derived Mast cells (BMMCs) were cultured as described previously [27]. Briefly, bone marrow was flushed in PBS and the cells pelleted by centrifugation. Cells were cultured at 1 million cells per ml in RPMI 1640 supplemented with 10% FBS (Biosera/Labtech), 5 mM l‐Glutamine (GIBCO Life Technologies), 100 U/ml Penicillin (GIBCO Life Technologies), 100 μg/ml Streptomycin (GIBCO Life Technologies), 25 mM HEPES (Lonza), 1 mM sodium pyruvate (Lonza), 1X nonessential amino acids (Lonza), 50 μM 2‐mercaptoethanol and 30 ng/ml IL‐3 (PeproTech). Cells were passaged twice per week and used between passage 12 and 16. 4 independent BMMC cultures were either stimulated with 10 ng/ml IL-33 for 48 hours or left unstimulated, followed by single shot LC-MS analysis.
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Project description:We aimed to study the effects of CSF2 treatment on the transcriptome of the Day 15 female and male embryo. Within the raw files, channel 1= Cy3 and channel 2= Cy5 Transcriptional profiling of bovine extra-embryonic membrane at Day 15 post-insemination produced by IVF. Oocytes were matured and fertilized in vitro using a single Holstein bull. After 5 days in vitro culture, the embryos were either treated with 10 ng/ml bovine recombinant CSF2 or the Control (DPBS/BSA) until Day 7. At Day 7, the embryos were transferred into recipients. At day 15 following insemination, embryos were flushed and collected for total RNA and gDNA extraction. The gender of the embryos were determined by PCR prior to microarray analysis. CSF2 males were compared to Control males while CSF2 females were compared to Control females. Control females were also compared to control males.
2014-10-01 | E-GEOD-55364 | biostudies-arrayexpress
Project description:We aimed to study the effects of CSF2 treatment on the methylome of the Day 15 female and male embryo. Methylomic profiling of bovine extra-embryonic membrane at Day 15 post-insemination produced by IVF. Oocytes were matured and fertilized in vitro using a single Holstein bull. After 5 days in vitro culture, the embryos were either treated with 10 ng/ml bovine recombinant CSF2 or the Control (DPBS/BSA) until Day 7. At Day 7, the embryos were transferred into recipients. At day 15 following insemination, embryos were flushed and collected for total RNA and gDNA extraction. The gender of the embryos were determined by PCR prior to microarray analysis. CSF2 males were compared to Control males while CSF2 females were compared to Control females
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Project description:Analysis of stratified epidermal cultures treated with IL-1a, IL-1F5, IL-1F6, IL-1F8 and IL-1F9 to determine the effects of these cytokines at 24h. Results provide insight into the role of IL-1 family cytokines in the pathogenesis of psoriasis. 16 samples. 2 untreated controls, 14 cytokine treated
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