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RNA-Seq analysis of human neutrophils isolated by different protocols (Polymorphprep and negative selection) and incubated with and without in vitro cytokine stimulation

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Helen Wright

PROVIDER: E-GEOD-70068 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA-Seq analysis of human neutrophils isolated by different protocols (Polymorphprep and negative selection) and incubated with and without in vitro cytokine stimulation

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.

2015-09-24 | GSE70068 | GEO

RNAseq analysis of the human neutrophil transcriptome, with and without in vitro cytokine stimulation

Project description: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.

2013-03-06 | E-GEOD-40548 | biostudies-arrayexpress

RNAseq analysis of the human neutrophil transcriptome, with and without in vitro cytokine stimulation

Project description:We report cytokine specific changes in gene expression in the human neutrophil transcriptome using TNF-alpha and GM-CSF stimulation of healthy neutrophils

2013-03-06 | GSE40548 | GEO

Expression data from control, aged and activated neutrophils in mouse blood

Project description:Neutrophils provide immune protection against pathogens but also may promote tissue injury in inflammatory diseases. Although neutrophils are generally considered as a relatively homogeneous population, evidence for heterogeneity is emerging. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and the replenishment by newly released neutrophils from the bone marrow. We used microarray to globally analyze gene expression in aged neutrophils and characterize the inflammatory programs that are activated during the aging process in the circulation. Control, aged and activated neutrophils were sorted directly from mouse blood for RNA extraction and hybridization on Affymetrix microarrays. We transfused whole blood and harvested donor neutrophils marked by the CD45.1 allele 6h later to derive neutrophils that had truly aged in vivo. Sorted neutrophils were compared to control donor neutrophils that had been transferred for only 10 min. Additionally, we harvested circulating neutrophils from TNF-? treated mice for comparison with neutrophils activated by systemic inflammation.

2015-09-24 | E-GEOD-69886 | biostudies-arrayexpress

Project description:Host defense against bacterial and fungal infections diminishes with age. In humans, this is thought to be caused in part by a decline in neutrophil responses. However, it remains unclear whether a similar decline in neutrophil function occurs in mice. Here, we show that old mice have a reduced capacity to clear pathogenic E. coli during septic peritonitis. Neutrophil recruitment to the peritoneum was elevated during lipopolysaccharide (LPS)-induced septic peritonitis but not aseptic peritonitis. Neutrophils from old mice showed reduced chemoattractant-induced reactive oxygen species (ROS) production upon priming with LPS but not GM-CSF/TNF. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas NETosis was impaired independently of LPS. The chemoattractant-stimulated production of PIP3 was reduced upon priming with LPS but not GM-CSF/TNF, whereas PI(4,5)P2 levels were constitutively low. Unexpectedly, chemotaxis was normal regardless of priming pathway, as were the chemoattractant-stimulated activities of Rac1 and Rac2. The expression of 5% of neutrophil proteins was deregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as toll-like receptor (TLR) pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. Upregulation of Cd180 and downregulation of MyD88 may contribute to the impaired LPS priming and LPS-dependent PIP3 production. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS-TLR4 pathway dependence resolves some of the controversy regarding the effects of age on murine neutrophils and confirms mice are an appropriate model for the declining human neutrophil function.

2022-08-20 | PXD035577 | Pride

RNA-seq analysis of lung-infiltrating neutrophils from WT and Atgl-cKO mice

Project description:Atglflox/flox (B6N.129S-Pnpla2tm1Eek/J), S100A8-cre+/- (B6.Cg-Tg(S100A8-cre,-EGFP)1Ilw/J) mice were obtained from The Jackson Laboratory. Atglflox/flox mice were bred to S100A8-cre+/- mice to generate Atglflox/WTS100A8-cre+/- mice, which were backcrossed onto Atglflox/flox mice to generate Atglflox/floxS100A8-cre+/- mice (Atgl neutrophils-specific knock out, Atgl-cKO). Age-matched littermate Atglflox/flox mice were used as wild-type (WT) controls. To compare of the gene expression of the lung-infiltrating neutrophils isolated from Atgl-cKO mice and their WT littermates, AT3-g-csf cells were injected into the fourth mammary fat pads of female WT and Atgl-cKO mice (10-week-old, n = 4/group). The AT3-g-csf cell line is based on a murine breast cancer cell line (AT3) derived from MMTV-PyMT tumors in the C57BL/6 background, and further constructed to overexpress granulocyte-colony stimulating factor (G-CSF) for induction of the host inflammatory condition. At day 10 (pre-metastatic stage), the mice were euthanized and then Ly6G+ neutrophils were isolated from lung by using anti-Ly6G MicroBead Kit (Miltenyi Biotec) following manufacturer’s instructions. The isolated neutrophils were analyzed by flow cytometry and the cells with a > 95% purity were used for the next procedure. Total RNA was isolated from neutrophils using the miRNeasy Mini kit (Qiagen) and the transcriptional profiles of neutrophils were analyzed by RNA sequencing.

2020-06-28 | E-MTAB-9235 | biostudies-arrayexpress

Human Eosinophils Express a Distinct Gene Expression Program in Response to IL-3 Compared to Common Beta-Chain Cytokines IL-5 and GM-CSF

Project description:Background: Despite recent advances in asthma management with anti-IL-5 therapies, many patients with eosinophilic asthma remain poorly controlled. IL-3 shares a common beta subunit receptor with both IL-5 and GM-CSF, but through alpha subunit-specific properties, uniquely influences eosinophil biology and may serve as a potential therapeutic target. Objective: We aimed to globally characterize the transcriptomic profiles of GM-CSF, IL-3, and IL-5 stimulation and identify differences in gene expression using advanced statistical modeling. Methods: Human eosinophils were isolated from the peripheral blood of healthy volunteers and stimulated with either GM-CSF, IL-3 or IL-5 for 48 hours. RNA was then extracted and bulk sequencing performed. DESeq analysis identified differentially expressed genes and weighted gene co-expression network analysis independently defined modules of genes that are highly co-expressed. Results: IL-3 stimulation yielded the most numbers of differentially expressed genes that were also highly co-expressed. GM-CSF and IL-5 stimulation demonstrated redundancy in eosinophil gene expression. Conclusion: IL-3 produces a distinct eosinophil gene expression program among the beta-chain receptor cytokines.

2019-09-06 | GSE128027 | GEO

NK cell-derived GM-CSF is critical for enhancing pathological arthritis

Project description:GM-CSF is involved in immune complex (IC)-mediated arthritis. However, little is known about what is the cellular source of GM-CSF and how it is regulated during IC-mediated inflammation. Using novel GM-CSF reporter mice, we show that NK cells produce GM-CSF during an IC-mediated model of inflammatory arthritis. NK cells promoted STIA in a GM-CSF-dependent manner, as deletion of NK cells and selective removal of GM-CSF production by NK cells abrogated disease. Furthermore, we show that myeloid cell activation by GM-CSF is restrained by induction of JAK/STAT checkpoint inhibitor cytokine-inducible SH2-containing protein, CIS. Myeloid cells from CIS-deficient mice had exaggerated responses to GM-CSF, and these mice develop exacerbated STIA. Our data suggest that tissue NK cells may amplify joint inflammation in arthritis via GM-CSF production and thus represent a novel target in IC-mediated pathology. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor and strategies that boost its function may provide an alternative anti-inflammatory approach.

2020-02-19 | PXD012794 | Pride

G-CSFR expression is not essential for neutrophil differentiation but for progenitor formation and emergency neutrophil responses

Project description:Severe congenital neutropenia (SCN) is a rare disorder characterized by a maturation arrest of myeloid progenitor cells in the bone marrow and severe reduction in the amount of circulating neutrophils. Loss-of-function mutations in the CSF3R (the gene encoding the granulocyte colony-stimulating factor (G-CSF) receptor) have been reported in a handful of cases. We describe two novel pedigrees with moderate neutropenia. G-CSFR immunostaining was greatly reduced on patient neutrophils. G-CSF did not prolong neutrophil survival or enhanced reactive oxygen species generation, and STAT-3 phosphorylation was absent, while neutrophils did respond to granulocyte-macrophage colony-stimulating factor (GM-CSF). Despite a lack of G-CSF signaling, morphology and cellular proteomics were normal. We suggest the major role of G-CSF is not in myeloid differentiation, but in generation of sufficient number of committed progenitor cells for neutrophil release and their survival during inflammation, which corresponds with G-CSFR expression in myeloid cell fractions from bone marrow of healthy individuals.

2020-09-25 | PXD018178 | Pride

Identification of genes effected by GM-CSF treatment in mature human neutrophils

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.

2009-03-31 | GSE15139 | GEO

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