Allergen challenged CD4+ cells from allergic patients and healthy controls
ABSTRACT: Gene expression (Npatients = 21, Ncontrols = 21) of CD4+ T-cells failed to seperate patients with seasonal allergic rhinitis (SAR) and healthy controls in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen for 7 days. PBMCs from 21 patients (P) and 21 healthy controls (H) were challenged with grass pollen for 7 days. Diluent challenged control samples were obtained from all subjects. CD4+ cells were purified by MACS.
Project description:Medical research focuses on disease-specific genes. By contrast, here we systematically examined the roles of shared genes for disease susceptibility and as therapeutic and diagnostic targets. Meta-analysis of all published disease-related genome-wide association studies (GWAS) showed that T helper (Th) cell differentiation was the most shared pathway. Expression profiling data from highly diverse CD4+ T cell-associated diseases revealed shared disease-associated genes, which were enriched for Th cell differentiation, but also metabolic and proliferative pathways. This pleiotropy suggested that altered functions of shared genes could generally increase disease susceptibility. Indeed, compared to specific genes, the shared genes were enriched for disease-associated SNPs identified by all published disease-related GWAS. To examine if the shared genes induced disease-relevant pathways, we focused on transcription factors (TFs) that induced Th differentiation. Those TFs were enriched among the shared genes, as well as for disease-associated SNPs identified by GWAS, and disease-phenotypes in mice knock-out studies. Original GWAS and profiling data from patients with multiple sclerosis and allergy confirmed enrichment of disease-associated SNPs in the TFs, and that the TFs were differentially expressed at early disease stages, and their targets increased in parallel with disease development. From a clinical perspective, the shared genes were significantly enriched for known diagnostic and therapeutic targets. Prospective clinical studies of multiple sclerosis and allergy showed that shared or specific genes could be used to stratify patients for individualized medicine. Our findings show that shared disease genes generally increase disease susceptibility and are important therapeutic and diagnostic targets. Patients with seasonal allergic rhinitis (SAR) show considerable variations in response to glucocorticoids (GCs) treatment. Peripheral blood mononuclear cells (PBMCs) were collected from 8 high responders (HR) and 8 low responders (LR) to GC treatment. PBMCs were challenged with diluent, grass pollen extract (ALK-Abelló A/S; 100 μg/mL) as well as grass pollen extract plus glucocorticoids (100ug/mL) for one week. Total CD4+ T cells were enriched for the gene expression microarray analysis, which was performed using SurePrint G3 Human Gene Expression 8X60K microarrays.
Project description:Gene expression analysis in CD4+ T cells extracted from allergen-challenged PBMCs, isolated from discordant MZ twins with IAR MZ twins discordant for intermittent allergic rhinitis (IAR)
Project description:In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. Peripheral blood mononuclear cells (PBMCs) were prepared from fresh blood from 10 patients with seasonal allergic rhinitis and 10 healthy controls using Lymphoprep (Axis-Shield PoC, Oslo, Norway) according to the manufacturer’s protocol. PBMCs were stimulated with allergen extract (ALK-Abelló A/S; 100 μg/ml) or diluent (PBS) in RPMI 1640 supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 5% human AB serum (Lonza, Switzerland), 5 µM beta-mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 µg/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). After 17 hours of incubation, total CD4+ T cells were enriched from PBMCs by MACS negative sorting. Total RNA was extracted using a miRneasy Mini Kit (Qiagen, Valencia, CA, USA). The cRNA was prepared using a Low Input QuickAmp Labeling Kit. The expression microarray analyses were performed using Agilent SurePrint G3 Human Exon 4x180K Microarrays according to the manufacturer's instructions. Complementary microRNA data have been deposited in ArrayExpress under accession number E-MTAB-4900 ( http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4900/ ).
Project description:This SuperSeries is composed of the following subset Series: GSE37146: Gene expression analysis in MZ twins discordant for IAR [in vitro] GSE37155: Gene expression analysis in MZ twins discordant for IAR [in vivo] Refer to individual Series
Project description:DNA methylation has been shown to play a major role in determining cellular phenotype by regulating gene expression. Moreover, dysregulation of differentially methylated genes has been implicated in disease pathogenesis of various conditions including cancer development as well as autoimmune diseases such as systemic Lupus erythematosus and rheumatoid arthritis. Evidence is rapidly accumulating for a role of DNA methylation in regulating immune responses in health and disease. However, the exact mechanisms remain unknown. The overall aim of the project is to investigate the role of epigenetic mechanisms in regulating immunity and their impact on autoimmune disease pathogenesis.The aim of this pilot study is to perform whole genome methylation analysis in peripheral blood mononuclear cells (PBMCs) and cell subsets (CD4, CD8, CD14, CD19, CD16 and whole PBMCs) obtained from 6 healthy volunteers. Whole genome methylation analysis will be performed using two methodological approaches, the Infinium Methylation Bead Array K450 (Illumina) and MeDIP-seq. mRNA expression arrays will also be performed in order to correlate DNA methylation with gene expression as well as genotyping on the Illumina OmniExpress chip
Project description:To assess the nature of CD8+CD40L+ memory Tcells, we compared the gene expression to CD8+CD40L- and CD4+ counterparts, and found similarities in expression of genes encoding cytokines. PBMCs were isolated from five healthy donors. The cells were stimulated with PMA/Ionomycin for 6 h, and subsequently sorted into the CD4+CD45RA-CD40L+, CD8+CD45RA-CD40L-, CD8+CD45RA-CD40L+ populations by FACS-sort.
Project description:Expansion of circulating CD4+CD8+ double positive (DP) T-cells in a disease context is poorly understood. The study aims to identify mechanisms which may drive expansion of CD4+CD8+ double positive t-cells in GPA. PBMCs from 3 GPA patients and 3 healthy controls were used to generate mRNA profiles from CCD4+CD8+ double positive T-cells in a disease context
Project description:Background: Peripheral blood mononuclear cells (PBMCs) are relatively easily obtainable cells in humans. Gene expression profiles of PBMCs have been shown to reflect the pathological and physiological state of a person. Recently, we showed that the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) has a functional role in human PBMCs during fasting. However, the extent of the role of PPARα in human PBMCs remains unclear. In this study, we therefore performed gene expression profiling of PBMCs incubated with the specific PPARα ligand WY14,643. Results: Incubation of PBMCs with WY14,643 for 12 hours resulted in a differential expression of 1,373 of the 13,080 genes expressed in the PBMCs. Gene expression profiles showed a clear individual response to PPARα activation between six healthy human blood donors, which was not the result of the nutritional status of the donors. Pathway analysis showed that genes in fatty acid metabolism, primarily in β-oxidation were up-regulated upon activation of PPARα with WY14,643, and genes in several amino acid metabolism pathways were down-regulated. Conclusions: This study shows that PPARα in human PBMCs regulates fatty acid and amino acid metabolism. In addition, PBMC gene expression profiles show individual responses to WY14,643 activation. We show that PBMCs are a suitable model to study changes in PPARα activation in healthy humans. Keywords: metabolic state analysis PBMCs from six healthy Caucasian male blood donors, aged between 30 and 48 yr, were isolated directly after arrival of the buffy coat (max. 8 hours after donation) by Ficol-paque Plus density gradient centrifugation (Amersham Biosciences, Roosendaal, the Netherlands). PBMCs were incubated in RPMI1640 medium with 2 mmol/L L-glutamine, 10% fetal bovine serum and antibiotics (penicillin and streptomycin) in the presence of 5% CO2 at 37°C. at 1.0 x 106 cells per ml with either WY14,643 (50 μM) or vehicle (DMSO, 0.1%) for 12 hours. Total RNA from PBMCs was labeled using a one-cycle cDNA labeling kit (Affymetrix Inc, Santa Clara, CA) and hybridized to Affymetrix Human whole genome U133 plus 2.0 arrays (Affymetrix). Sample labeling, hybridization to chips and image scanning was performed according to the manufacturer’s GeneChip Expression Analysis Technical Manual (Affymetrix).