Transcription profiling of T cells from healthy controls and patients with seasonal allergic rhinitis before and after one year of sublingual immunotherapy
ABSTRACT: We recruit 4 healthy controls (HC) and 4 patients (P) with seasonal allergic rhinitis before (b) sublingual immunotherapy at the same time and also the same patients after one year (a) of sublingual immunotherapy. Peripheral blood mononuclear cells (PBMCs) obtained from patients and controls were challenged with diluent (D) or allergen (A) extracts from birch pollen at a density of 106 cells/mL for 7 days in RPMI 1640 supplemented with 2 mM L-glutamine, 5% human AB serum, 5 μM β– mercaptoethanol and 50 μg/mL gentamicin. CD4+ T cells were isolated using flow cytometry and the quantity and quality of RNA was examined as described before. Gene expression microarrays (Illumina, San Diego, CA, USA) were performed (by using Agilent G4851B SurePrint G3 Hmn 8×60K V2 Microarray Kit).
Project description:Analysis of nasal epithelial cells from adult patients with seasonal allergic rhinitis and from non allergic controls. Results provide insight into the molecular mechanisms associated with inflammatory responses in nasal mucosa. Total RNA was obtained from nasal epithelial cells of 7 seasonal allergic rhinitis patients and 5 non-allergic control subjects
Project description:Seasonal allergic rhinitis (SAR) is a complex disease that is caused by many interacting genes and environmental factors. It is also an excellent model disease for clinical studies; it is common, it is seasonal, and since it takes place in the nasal cavity it can be studied in vivo non-invasively. Furthermore, the key disease cell, the Th2 cell is known. We study SAR using allergen-challenged CD4+ cells from allergic patients. Overall design: Samples were obtained from 3 patients with SAR; each separated into allergen-challenged samples and unchallenged controls.
Project description:Six patients with seasonal allergic rhinitis were challenged daily for 8 days with birch pollen extract. A mucosal biopsy was obtained from one nostril at basline (day 0) and from the other nostril after allergen challenge (day 9). The mucosal biopsies were digested into single cells, and then sorted into CD4 T cells and CD45+HLA-DR+ cells. Total RNA was extracted, amplified using whole transcriptome amplification, and gene expression was profiled on microarrays. The study design consisted of 6 subjects, 2 cell types (CD4 T cells, CD45+ HLA-DR+ cells), and 2 conditions (baseline, allergen challenge).
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:Epigenetic alterations may represent new therapeutic targets and/or biomarkers of allergic rhinitis (AR). Our aim was to examine genome-wide epigenetic changes induced by controlled pollen exposure in the Environmental Exposure Unit (EEU). 38 AR-sufferers and 8 non-allergic controls were exposed to grass pollen for 3h on two consecutive days. We interrogated DNA methylation at baseline and 3h in peripheral blood mononuclear cells (PBMCs) using the Infinium Methylation 450K array. We corrected for demographics, cell composition, and multiple testing (Benjamini-Hochberg), and verified hits using bisulfite PCR-pyrosequencing and qPCR. To extend these findings to a clinically relevant tissue, we investigated DNA methylation and gene expression of mucin 4 (MUC4), in nasal brushings from a separate validation cohort exposed to birch pollen. In PBMCs of allergic rhinitis participants, 42 sites showed significant DNA methylation changes of 2% or greater. DNA methylation changes in tryptase gamma 1 (TPSG1), schlafen 12 (SLFN12) and MUC4 in response to exposure were validated by pyrosequencing. SLFN12 DNA methylation significantly correlated with symptoms (p<0.05), and baseline DNA methylation pattern was found to be predictive of symptom severity upon grass allergen exposure (p<0.05). Changes in MUC4 DNA methylation in nasal brushings in the validation cohort correlated with drop in peak nasal inspiratory flow (Spearman r = 0.314, p = 0.034), and MUC4 gene expression was significantly increased (p<0.0001). This study revealed novel and rapid epigenetic changes upon exposure in a controlled allergen challenge facility, identified baseline epigenetic status as a predictor of symptom severity. Overall design: This cohort consist of genomic DNA extracted from lymphocyte-enriched blood samples from 15 Atopic and 8 non atopic participants. DNA was bisulphite converted and hybridized to the Illumina Infinium HumanMethylation450 Beadchip for genome wide DNA methylation profiling.
Project description:A Randomized, Placebo-Controlled Trial of Intradermal Allergen Immunotherapy for Grass Pollen Allergy Background: Repeated intradermal injection of grass pollen (nanograms of allergen) suppresses allergen-induced cutaneous late phase responses, in keeping with effects of conventional high dose subcutaneous and sublingual immunotherapy. We evaluated the efficacy and safety of grass pollen intradermal immunotherapy for treatment of allergic rhinitis.Methods: We randomly assigned 93 adults with grass pollen allergic rhinitis to receive 7 pre-seasonal Intradermal allergen immunotherapy injections (containing 7 ng of Phl p 5 major allergen) or histamine control. The primary end point was daily combined symptom-medication scores during the 2013 pollen season. Skin biopsies were taken after the pollen season following an intradermal allergen challenge. Cutaneous late phase responses were measured 4 and either 7, 10 or 13 months post-treatment. Results No difference in the primary endpoint was observed between treatment arms (median difference, 14; 95% confidence interval [CI], -172.5 to 215.1; P=0.80). Amongst secondary endpoints, nasal symptoms measured with daily scores (median difference, 35; 95% CI, 4.0 to 67.5; P=0.03) and visual-analogue scales (median difference, 53; 95% CI, -11.6 to 125.2; P=0.05) were higher in the intradermal treatment group. Intradermal immunotherapy increased serum Phl p-specific IgE (P=0.001) compared to the control arm and T cells cultured from biopsies showed higher and lower surface of surface markers for Type 2 (P=0.04) and Type 1 (P=0.01) T-helper cells, respectively, Interleukin-5 was differentially expressed by microarray (P=0.03). Late phase responses were still inhibited 7 months after treatment (P=0.03) but not at 10-13 months. Conclusions Grass pollen intradermal allergen immunotherapy was not clinically effective but resulted in immunological priming and worsening of allergic rhinitis symptoms. Overall design: T-cells were isolated from skin biopsy by explant culture from control subjects (8) and subjects undergoing IDIT (7). After 7 days T-cells were activated with PMA/Ionomycin. Control subjects were compared with IDIT subjects.
Project description:The link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium. We used micro array to profile gene expression of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles. 17 subjects were included in a cross-sectional study (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). RNA was extracted from isolated and cultured epithelial cells from bronchial brushes and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array).
Project description:Peripheral blood mononuclear cells (PBMC) were collected from ficoll gradients of whole blood and immediately processed for total RNA and stored at -80oC until use. Blood was from allergic patients at different timepoints pre or post immunotherapy (RIT). Gene expression was compared for the different timepoints. Blood samples were collected from allergic patients before or after rush immunotherapy (RIT), total RNA was prepared and gene expression assessed by Illumina. Samples were collected pre-rush or at 1 week, 7 weeks, 4 months (and for one patient 5 months) after beginning rush immunotherapy. Blood was taken at each timepoint prior to the patient receiving immunotherapy at each timepoint.
Project description:Allergic asthma and rhinitis are two common chronic allergic diseases that affect the lungs and nose, respectively. Both diseases share clinical and pathological features characteristic of excessive allergen-induced type 2 inflammation, orchestrated by memory CD4+ T cells that produce type 2 cytokines (TH2 cells). However, a large majority of subjects with allergic rhinitis do not develop asthma, suggesting divergence in disease mechanisms. Since TH2 cells play a pathogenic role in both these diseases and are also present in healthy non-allergic subjects, we performed global transcriptional profiling to determine whether there are qualitative differences in TH2 cells from subjects with allergic asthma, rhinitis and healthy controls. TH2 cells from asthmatic subjects expressed higher levels of several genes that promote their survival as well as alter their metabolic pathways to favor persistence at sites of allergic inflammation. In addition, genes that enhanced TH2 polarization and TH2 cytokine production were also upregulated in asthma. Several genes that oppose T cell activation were downregulated in asthma, suggesting enhanced activation potential of TH2 cells from asthmatic subjects. Many novel genes with poorly defined functions were also differentially expressed in asthma. Thus, our transcriptomic analysis of circulating TH2 cells has identified several molecules that are likely to confer pathogenic features to TH2 cells that are either unique or common to both asthma and rhinitis. RNA-sequencing of circulating TH2 cells isolated from a cohort of patients with allergic rhinitis (25), asthma (40) patients and healthy non allergic subjects (15). Cells were directly isolated from blood by flow cytometry. Total RNA was extracted, messenger RNA was selected and cDNA was amplified linearly with a PCR based method (Picelli et al. 2014). Libraries were prepared using the NexteraXT Illumina sequencing platform.
Project description:Background Atopic diseases, resulting from hypersensitivity to a wide variety of allergens, affect 10-20% of the population. Immunotherapy is an effective treatment for atopic diseases, but its mechanisms are not fully understood. Objective We studied gene expression profiles in the peripheral blood mononuclear cells and examined whether the individuals with allergic rhinitis have a unique gene expression profile and how the immunotherapy affect the gene expression profiles. Method We used cDNA microarray and “Expression Analysis Systemic Explorer” to examine the gene expression profiles in the peripheral blood mononuclear cells of atopic subjects and other groups. Results We identified a highly conserved gene expression profile in atopic subjects that permitted their accurate segregation from control or autoimmune subjects. A major feature of this profile was the under-expression of a variety of genes that encode proteins required for apoptosis and over-expression of genes that encode proteins critical for stress responses and signal transduction. We also identified 563 genes that can segregate individuals with allergic rhinitis based upon receipt of immunotherapy. Conclusion There is a highly conserved gene expression profile in the peripheral blood mononuclear cells of individuals with allergic rhinitis. This profile can be used to identify individuals with allergic rhinitis and to evaluate responses to immunotherapy. Quantitative endpoints, such as gene expression, may assist clinicians faced with clinical decisions in the diagnosis of patients and the evaluation of response to therapy. The knowledge of the possible genetic basis for immunotherapy efficacy may also lead to novel therapeutic approaches for atopic diseases.