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NIH/NIAID Neutrophil Response to A. phagocytophilum

ABSTRACT: Polymorphonuclear leukocytes (PMNs) were obtained from healthy individuals in accordance with protocols approved by the Institutional Review Board for Human Subjects at the University of Minnesota and the National Institute of Allergy and Infectious Diseases. PMNs (107) were combined on ice with live S. aureus (108) or with live or heat-killed A. phagocytophilum (bacteria isolated from 5x106 infected HL60 cells for a ratio of 1 infected HL60 cell: 2 PMNs, ~ 5-20 A. phagocytophilum: PMN) in wells of a 12-well tissue culture plate (pre-coated with 20% autologous normal human serum). Unstimulated control assays received either buffer (for S. aureus comparisons) or clarified HL60 lysate (for A. phagocytophilum comparisons). Plates were centrifuged at 350 x g for 8 min at 4oC to synchronize phagocytosis and incubated at 37 deg. C in a CO2 incubator for the indicated times. At the indicated times, tissue culture medium was aspirated from the plate and PMNs were lysed directly with RLT buffer (Qiagen, Valencia, CA). Purification of PMN RNA and subsequent preparation of labeled cRNA target was performed as described in Methods. Labeling of samples, hybridization of cRNA with HU133A oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols ( http://www.affymetrix.com/pdf/expression_manual.pdf ). Experiments were performed in triplicate, using PMNs from three healthy individuals for each treatment. Keywords = HUMAN NEUTROPHILS BACTERIAL APOPTOSIS GENE REGULATION Keywords: ordered

ORGANISM(S): Homo sapiens

PROVIDER: GSE2405 | GEO | 2005/05/15

SECONDARY ACCESSION(S): PRJNA91613

REPOSITORIES: GEO

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Transcription profiling time series of the human polymorphonuclear leukocyte response to exposure to Anaplasma phagocytophilum or Staphylococcus aureus

Project description:Polymorphonuclear leukocytes (PMNs) were obtained from healthy individuals in accordance with protocols approved by the Institutional Review Board for Human Subjects at the University of Minnesota and the National Institute of Allergy and Infectious Diseases. PMNs (107) were combined on ice with live S. aureus (108) or with live or heat-killed A. phagocytophilum (bacteria isolated from 5x106 infected HL60 cells for a ratio of 1 infected HL60 cell: 2 PMNs, ~ 5-20 A. phagocytophilum: PMN) in wells of a 12-well tissue culture plate (pre-coated with 20% autologous normal human serum). Unstimulated control assays received either buffer (for S. aureus comparisons) or clarified HL60 lysate (for A. phagocytophilum comparisons). Plates were centrifuged at 350 x g for 8 min at 4oC to synchronize phagocytosis and incubated at 37 deg. C in a CO2 incubator for the indicated times. At the indicated times, tissue culture medium was aspirated from the plate and PMNs were lysed directly with RLT buffer (Qiagen, Valencia, CA). Purification of PMN RNA and subsequent preparation of labeled cRNA target was performed as described in Methods. Labeling of samples, hybridization of cRNA with HU133A oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols ( http://www.affymetrix.com/pdf/expression_manual.pdf ). Experiments were performed in triplicate, using PMNs from three healthy individuals for each treatment.

2007-08-25 | E-GEOD-2405 | biostudies-arrayexpress

NIH/NIAID Chronic Granulomatous Disease Neutrophils

Project description:Polymorphonuclear leukocytes (PMNs) were obtained from healthy individuals (Healthy1, Healthy2, Healthy3, and Healthy4) or patients with X-linked chronic granulomatous disease (XCGD1, XCGD2, XCGD3, XCGD4, XCGD5, and XCGD6). The studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases, and the Institutional Review Board for Human Subjects at the University of Iowa. All studies were conducted according to Declaration of Helsinki principles. PMNs (107) were combined with or without IgG and C3bi-coated latex beads (8 x 107) in wells of a 12-well tissue culture plate (pre-coated with 20% normal human serum) and centrifuged at 350 x g for 8 min at 4oC to synchronize phagocytosis. For the purpose of these studies, activated or "Stimulated" PMNs are defined as those that have been stimulated by phagocytosis of IgG- and C3bi-coated latex beads. "Control" PMNs are defined as resting cells or those left unstimulated throughout the time-course. Following centrifugation, plates were incubated at 37 deg. C in a CO2 for the indicated times. At the indicated times, tissue culture medium was aspirated from the plate and PMNs were lysed directly with RLT buffer (Qiagen, Valencia, CA). Purification of PMN RNA and subsequent preparation of labeled cRNA target (12 g) was performed as described in Methods. Labeling of samples, hybridization of cRNA with Hu95Av2 oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols. Gene expression in healthy control and XCGD individuals was always compared at the same time points after phagocytosis Keywords = HUMAN PMN CHRONIC GRANULOMATOUS DISEASE NEUTROPHILS Keywords: parallel sample

2003-12-31 | GSE935 | GEO

Transcription profiling of human polymorphonuclear leukocytes (PMNs) from healthy individuals vs patients with X-linked chronic granulomatous disease

Project description:Polymorphonuclear leukocytes (PMNs) were obtained from healthy individuals (Healthy1, Healthy2, Healthy3, and Healthy4) or patients with X-linked chronic granulomatous disease (XCGD1, XCGD2, XCGD3, XCGD4, XCGD5, and XCGD6). The studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases, and the Institutional Review Board for Human Subjects at the University of Iowa. All studies were conducted according to Declaration of Helsinki principles. PMNs (107) were combined with or without IgG and C3bi-coated latex beads (8 x 107) in wells of a 12-well tissue culture plate (pre-coated with 20% normal human serum) and centrifuged at 350 x g for 8 min at 4°C to synchronize phagocytosis. For the purpose of these studies, activated or Stimulated PMNs are defined as those that have been stimulated by phagocytosis of IgG- and C3bi-coated latex beads. Control PMNs are defined as resting cells or those left unstimulated throughout the time-course. Following centrifugation, plates were incubated at 37 °C in a CO2 for the indicated times. At the indicated times, tissue culture medium was aspirated from the plate and PMNs were lysed directly with RLT buffer (Qiagen, Valencia, CA). Purification of PMN RNA and subsequent preparation of labeled cRNA target (12 g) was performed as described in Methods. Labeling of samples, hybridization of cRNA with Hu95Av2 oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols. Gene expression in healthy control and XCGD individuals was always compared at the same time points after phagocytosis

2007-09-29 | E-GEOD-935 | biostudies-arrayexpress

Transcription profiling of neutrophils exposed to S. aureus infection

Project description:Neutrophil lysis after phagocytosis is a process potentially important in the pathogenesis of community-associated methicillin-resistant S. aureus (CA-MRSA) infection. The mechanism for this process is not currently known. Therefore, to better understand CA-MRSA virulence we used human oligonucleotide microarrays to investigate the mechanism underlying enhanced PMN lysis that occurs after phagocytosis of CA-MRSA. In order to examine the effect of S. aureus on the neutrophil transcriptome and to elucidate any possible differences in this effect between hospital- and community-associated S. aureus, we performed microarray expression analysis on human neutrophils treated with hospital- and community-associated S. aureus. Polymorphonuclear leukocytes (PMNs) were isolated from the blood of healthy donors. Control and S. aureus-exposed PMNs were incubated at 37C for 1, 2, 3 or 6 hours.

2010-06-09 | E-GEOD-16837 | biostudies-arrayexpress

Sublytic concentrations of Staphylococcus aureus Panton-Valentine leukocidin alter human polymorphonuclear leukocyte gene expression and enhance bactericidal capacity.

Project description:Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are often caused by strains encoding Panton-Valentine leukocidin (PVL). PVL can cause lysis of polymorphonuclear leukocytes (PMNs) and other myeloid cells in vitro, a function considered widely as the primary means by which PVL might contribute to disease. However, at sublytic concentrations PVL can function as a PMN agonist. To better understand this phenomenon, we investigated the ability of PVL to alter human PMN function. PMNs exposed to PVL had enhanced capacity to produce superoxide in response to N-formyl-methionyl-leucyl-phenylalanine (fMLF), but unlike priming by lipopolysaccharide, this response did not require Toll-like receptor signal transduction. On the other hand, there was subcellular redistribution of NADPH oxidase components in PMNs following exposure of these cells to PVL - a finding consistent with priming. Priming of PMNs with other agonists such as IL-8 or GM-CSF altered the ability PVL to cause formation of pores in the plasma membranes of these cells. Microarray analysis revealed significant changes in the human PMN transcriptome following exposure to PVL, including up-regulation of molecules that regulate the inflammatory response. Consistent with the microarray data, mediators of the inflammatory response were released from PMNs after stimulation with PVL. We conclude that exposure of human PMNs to sublytic concentrations of PVL elicits a proinflammatory response that is regulated in part at the level of gene expression. We propose that PVL-mediated priming of PMNs enhances the host innate immune response.

2012-09-06 | GSE33939 | GEO

NIH/NIAID GM-CSF Modulates Global Changes in Neutrophil Gene Expression

Project description:PMNs were isolated from venous blood of healthy individuals in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. Human PMNs (107) were cultured in RPMI/H -/+ 100 ng/ml GM-CSF at 37oC and 5% CO2 for up to 24 h as indicated. At the indicated time points, tissue culture medium was aspirated from each well and PMNs were lysed with RLT buffer (Qiagen). Purification of PMN RNA and subsequent preparation of labeled cRNA target was performed as described in Methods. Labeling of samples, GeneChip hybridization (Hu95Av2 oligonuceotide microarrays) and scanning were performed according to standard Affymetrix protocols (see http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Experiments were performed in triplicate, using PMNs from three healthy individuals for each treatment. These analyses allowed comparison of global gene expression in human neutrophils during spontaneous apoptosis with that in cells cultured with human GM-CSF. Keywords: time course

2005-10-07 | GSE2803 | GEO

Sublytic concentrations of Staphylococcus aureus Panton-Valentine leukocidin alter human polymorphonuclear leukocyte gene expression and enhance bactericidal capacity.

2012-09-06 | E-GEOD-33939 | biostudies-arrayexpress

Transcription profiling of human PMNs to compare global gene expression in human neutrophils during spontaneous apoptosis with that in cells cultured with human GM-CSF

Project description:PMNs were isolated from venous blood of healthy individuals in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. Human PMNs (107) were cultured in RPMI/H -/+ 100 ng/ml GM-CSF at 37°C and 5% CO2 for up to 24 h as indicated. At the indicated time points, tissue culture medium was aspirated from each well and PMNs were lysed with RLT buffer (Qiagen). Purification of PMN RNA and subsequent preparation of labeled cRNA target was performed as described in Methods. Labeling of samples, GeneChip hybridization (Hu95Av2 oligonuceotide microarrays) and scanning were performed according to standard Affymetrix protocols (see http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Experiments were performed in triplicate, using PMNs from three healthy individuals for each treatment. These analyses allowed comparison of global gene expression in human neutrophils during spontaneous apoptosis with that in cells cultured with human GM-CSF. Experiment Overall Design: 39 samples total: 9 time zero controls, -/+ GM-CSF at 5 time points in triplicate

2007-09-20 | E-GEOD-2803 | biostudies-arrayexpress

Gene expression data from S. aureus-exposed neutrophils

2010-06-01 | GSE16837 | GEO

FOXO3 Deficiency in Neutrophils Drives Colonic Inflammation and Tumorigenesis

Project description:Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to accumulation of their intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor FOXO3, our aim is to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs’ coat protein, PLIN2. Mouse peritoneal PMNs with increased LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3389) separated transcriptomes of affected tissue in IBD (p=0.00018) and colon cancer (p=0.0037) from control. PMN-FOXO3389 predicted colon cancer invasion (lymphovascular p= 0.015; vascular p= 0.046; perineural p=0.03), and poor survival. Validated DEGs from PMN-FOXO3389 (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p<0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.

2023-06-05 | GSE234072 | GEO

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