RNAseq analysis of the human neutrophil transcriptome, with and without in vitro cytokine stimulation
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ABSTRACT: We report cytokine specific changes in gene expression in the human neutrophil transcriptome using TNF-alpha and GM-CSF stimulation of healthy neutrophils Healthy human neutrophils were stimulated with TNF-alpha or GM-CSF for 1h in vitro. RNA was analysed by SOLiD and Illumina sequencing. RNA from one biological donor was sequenced on both platforms, and two different biological donors were sequenced by Illumina.
Project description:We report transcritomic characterization of changes in CTNNB1 mutation positive HCCs in comparison to paratumoral tissues 4 pair of HCC and paratumoral liver tissues are sequenced and compared
Project description:The objective of this study was to compare the transcriptional repertoire of mature human neutrophils before and after GM-CSF treatment by using oligonucleotide microarrays. Leukotriene B4 (LTB4) is an important pro-inflammatory lipid mediator generated by neutrophils upon activation. Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulation is known to enhance agonist-mediated LTB4 production of neutrophils within minutes, a process called “priming”. Here, we demonstrate that GM-CSF also limits the production of LTB4 by neutrophils via a transcriptional mechanism at later time points. We identified hematopoietic specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process. Neutrophils derived from RhoH/TTF-deficient (Rhoh-/-) mice demonstrated increased LTB4 production upon activation compared with normal mouse neutrophils. Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB4 upon activation compared with normal human neutrophils. Taken together, these data suggest that RhoH/TTF represents an inducible feedback inhibitor in neutrophils that is involved in the limitation of innate immune responses.
Project description:To check the dMyc function, RNA profiling was achieved by the RNA-seq assay comparing mRNA levels of lst81, dm0, lst81dm0 and rictorΔ1 in the adult heads of male mutant animals with wild-type controls Compare the mRNA profiles of 5-day old adult head materials of mutants (lst81, dmP0, lst81dmP0 and rictor1) to wild type W1118 by Illumina suquencing.
Project description:GM-CSF controls the development of granulocytes but little is known about the contribution of the downstream mediating transcription factor STAT5A/B. To elucidate this pathway, we generated mice lacking the Stat5a and 5b genes in blood cells. Peripheral neutrophils were decreased and administration of 5-FU and GM-CSF failed to induce granulopoiesis in Stat5a/b-mutant mice. GMPs were isolated and cultured with GM-CSF. Both the number and size of STAT5A/B-null colonies were reduced and GM-CSF-induced survival of mature STAT5A/B-null neutrophils was impaired. Time-lapse cinematography and single cell tracking of GMPs revealed that STAT5A/B-null cells were characterized by a longer generation time and an increased cell death. Gene expression profiling experiments suggested that STAT5A/B directs GM-CSF signaling through the regulation of cell survival genes.
Project description:Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA sequencing technology to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. We find that transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during the same postpartum time frame could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding M-NM-2-casein (CSN2) and a-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, PTPRF may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors to sub-optimal lactation in humans. Milk fat mRNA profiles were generated from Day 2 and mature milk samples obtained from lactating mothers
Project description:GM-CSF controls the development of granulocytes but little is known about the contribution of the downstream mediating transcription factor STAT5A/B. To elucidate this pathway, we generated mice lacking the Stat5a and 5b genes in blood cells. Peripheral neutrophils were decreased and administration of 5-FU and GM-CSF failed to induce granulopoiesis in Stat5a/b-mutant mice. GMPs were isolated and cultured with GM-CSF. Both the number and size of STAT5A/B-null colonies were reduced and GM-CSF-induced survival of mature STAT5A/B-null neutrophils was impaired. Time-lapse cinematography and single cell tracking of GMPs revealed that STAT5A/B-null cells were characterized by a longer generation time and an increased cell death. Gene expression profiling experiments suggested that STAT5A/B directs GM-CSF signaling through the regulation of cell survival genes. Experiment Overall Design: Mice lacking or with the Stat5a and 5b genes in blood cells, which were treated w/o GMP
Project description:GM-CSF controls the development of granulocytes but little is known about the contribution of the downstream mediating transcription factor STAT5A/B. To elucidate this pathway, we generated mice lacking the Stat5a and 5b genes in blood cells. Peripheral neutrophils were decreased and administration of 5-FU and GM-CSF failed to induce granulopoiesis in Stat5a/b-mutant mice. CMPs were isolated and cultured with GM-CSF.
Project description:Caspases, which are key effectors of apoptosis, have demonstrated non-apoptotic functions. One of these functions is the differentiation into macrophages of peripheral blood monocytes exposed to Colony-Stimulating Factor-1 (CSF1). Conversely, GM-CSF induces the differentiation of monocytes into macrophages in a caspase-independent manner. Macrophages generated by CSF1 and GM-CSF have distinct polarity. Macrophage polarization plays an important role in the pathogenesis of diverse human diseases as cancer, leading us to explore if caspase inhibition would affect macrophage polarization. To explore the role of caspases in CSF1 differentiation, we used human monocytes sorted from buffy coats treated by cytokines. We reported that caspase inhibition delays the ex vivo differentiation of peripheral blood monocytes exposed to CSF1 and modifies the phenotype of generated macrophages, e.g. cell shape, surface markers. Moreover, by RNAseq, we observed that the macrophages generated in presence of CSF1 and QVD are different from CSF1-treated monocytes and from GM-CSF-treated monocytes. Cell cycle and focal adhesion-related pathway genes were selectively down-regulated. This study confirms the importance of caspase activation in CSF1 differentiation.
Project description:We report gene expression in human neutrophils isolated by two methods: Polymorphprep (~95% purity) and negative selection (~99% purity) from two healthy donors - one donor with low eosinophil contamination of neutrophils and one donor with high eosinophil contamination of neutrophils. We report the effect of the presence of contaminating leukocytes in neutrophil preparations, and in reponse to inflammatory cytokines TNF-alpha and GM-CSF. Healthy human neutrophils were isolated using Polymorphprep or negative selection, and incubated for 1h in the absence or presence of TNF-alpha or GM-CSF. RNA was analysed by Illumina HiSeq 2000. The results from n=2 donors were analysed as biological replicates for differential expression analysis.
Project description:GM-CSF controls the development of granulocytes but little is known about the contribution of the downstream mediating transcription factor STAT5A/B. To elucidate this pathway, we generated mice lacking the Stat5a and 5b genes in blood cells. Peripheral neutrophils were decreased and administration of 5-FU and GM-CSF failed to induce granulopoiesis in Stat5a/b-mutant mice. CMPs were isolated and cultured with GM-CSF. Experiment Overall Design: Microarray Experiments for mice lacking or with the Stat5a and 5b genes in blood cells that were treated w/o CMP