Project description:Foxp3+ regulatory T cells (Treg) play a central role for tolerance against self and innocuous environmental antigens. However, the role of antigen-specificity for Treg-mediated tolerance is only incompletely understood. Here we show by direct ex vivo characterization of human CD4+ T cells, that the response against innocuous airborne antigens, such as plant pollen or fungal spores, is dominated by memory-like antigen-specific Treg. Surprisingly, breakdown of tolerance in atopic donors was not accompanied by a quantitatively or qualitatively altered Treg response, but instead correlated with a striking dichotomy of Treg versus Th2 target specificity. Allergenic proteins, are selectively targeted by Th2 cells, but not Treg. Thus human Treg specific for airborne antigens maintain tolerance at mucosal sites and the failure to generate specific Treg against a subgroup of antigens provides a window of opportunity for allergy development. PBMCs from sex and age matched birch pollen allergic patients and healthy controls, were stimulated (7h) with airborne fungal (A. fumigatus) or birch pollen antigen (birch) and sorted into antigen specific conventional and regulatory T cells according to their expression of CD154+ and CD137+ on CD4+ T cells, respectively. Number of samples per group in parentheses: Healthy controls stimulated with A. fumigatus (n=5), allergic patients stimulated with A. fumigatus (n=6), healthy controls stimulated with birch (n=6), allergic patients stimulated with birch (n=4).
Project description:Analysis of gene-expression profiles with microarrays can be very useful to dissect specific responses and to characterize with a global view, new elements for improving the diagnosis, treatment and understanding of allergic diseases. We have used this approach for studying the olive pollen response, taking advantage our previous results of T-cell epitope mapping on Ole e 1 molecule (the major allergen from olive pollen) in order to analyze the stimuli influence on the gene-expression of olive pollen allergic patients. Peripheral blood mononuclear cells (PBMCs) from 6 healthy controls and 6 allergic subjects were stimulated 24 hours with olive pollen stimuli: Ole e 1 molecule and two Ole e 1 peptides previously defined as P2+3 (aa10-31), mainly recognized by non-allergic subjects (possible immunoregulatory epitope) and P10+12+13 (aa90-130), immunodominant T-cell epitope. RNA extracted from basal and stimulated PBMCs was analyzed by HuGeU133 plus 2.0 GeneChip, Affymetrix (38.500genes). After assessment of data quality by standard quality checks and principal components analysis (PCA), differential gene-expression by experimental conditions was performed by multiple testing, using microarrays specific software. Differences in functional analysis were performed by KEGG, for pathways and Gene-Ontology for biological process. The results of gene-expression by PCA showed differential clusters that correlated with the experimental conditions from samples of allergic patients. Analysis of differential gene-expression by multiple testing, and functional analysis by KEGG and Gene-Ontology revealed differential genes and pathways among the 4 experimental conditions. Overall design: The study population comprised a total of 12 subjects (6 healthy controls and 6 Olive pollen–allergic patients), selected from a previous immunological study (Aguerri et al. Eur. J. Inflammation 2012, ), from Andalusia, who were recruited in 2 olive pollen exposure situations: during (April-June) and outside the pollen season (October-December). The subjects were unrelated and recruited at the Allergy Service of 4 hospitals in Andalusia (Granada, Jaén, Sevilla, and Málaga). These subjects fulfilled the following criteria: seasonal rhinitis and/or asthma from April to June, a positive skin prick test result for O. europaea pollen extract (ALK Abelló, Madrid, Spain), and no previous immunotherapy. Informed consent was obtained from each subject. Ethical approval for the study was obtained from the Ethical and Research Committee of the participating hospitals. PBMCs were isolated from heparin-containing peripheral blood samples taken during and outside pollen season, by gradient centrifugation on Lymphoprep (Comercial Rafer, Zaragoza, Spain) following the manufacturer’s instructions. These PBMCs (1,000,000 cls) were cultured with and without olive pollen stimuli [complete olive pollen extract (Olea, 25 µg/ ml)] peptide 2+3 (5 µg/ ml) and peptide 10+12+13 (5 µg/ ml) during 24 hours. After cell culture, total RNA was extracted using Trizol method (Invitrogen, Carlsbad, CA, USA). A total of 96 RNAs samples were studied (12 subjects x 2 exposure moments x 4 experimental conditions).
Project description:Analysis of gene-expression profiles by microarrays can be very useful to characterize new potential candidate genes, key regulatory networks, and to define phenotypes or molecular signatures to improve the diagnosis or classification of the disease. We have used this approach in the study of one of the major causes of allergic diseases in Mediterranean countries, the olive pollen response, in order to find differential molecular markers among five clinical groups, Non-allergic, Asymptomatic, Allergic but not to olive pollen, Non-treated, olive pollen allergic patients and Olive pollen allergic patients (under specific-immunotherapy). The results of gene-expression by principal components analysis (PCA) clearly showed five clusters of samples that correlated with the five clinical groups. Analysis of differential gene-expression by multiple testing, and functional analysis by KEGG and Gene-Ontology revealed differential genes and pathways among the 5 clinical groups. The study population comprised 28 subjects, selected from a previous immunological study (Aguerri et al. Eur. J. Inflammation 2012, in press), from Andalusia, who were recruited in 2 olive pollen exposure situations: during (April-June) and outside the pollen season (October-December). We established 5 groups, and 6 subjects from each group were selected for gene-expression analysis: Group 1, non-allergic subjects; Group 2, asymptomatic subjects (diagnosed with olive pollen allergy by skin testing, with no seasonal respiratory symptoms [rhinitis and/or asthma], and who consulted for adverse reaction to drugs); Group 3, patients who were allergic, but not to olive pollen; Group 4, non-treated olive pollen–allergic; and Group 5, olive pollen–allergic patients (receiving olive pollen–specific immunotherapy).The subjects were unrelated and recruited at the Allergy Service of 4 hospitals in Andalusia (Granada, Jaén, Sevilla, and Málaga). Olive pollen–allergic patients fulfilled the following criteria: seasonal rhinitis and/or asthma from April to June, a positive skin prick test result for O. europaea pollen extract (ALK Abelló, Madrid, Spain), and no previous immunotherapy. Informed consent was obtained from each subject. Ethical approval for the study was obtained from the Ethical and Research Committee of the participating hospitals. PBMCs were isolated from heparin-containing peripheral blood samples taken during and outside pollen season, by gradient centrifugation on Lymphoprep (Comercial Rafer, Zaragoza, Spain) following the manufacturer’s instructions.
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: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: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:Timothy grass (TG) pollen is a common seasonal airborne allergen associated with symptoms ranging from mild rhinitis to severe asthma. The aim of this study was to characterize changes in TG-specific T cell responses as a function of seasonality. Peripheral blood mononuclear cells (PBMC) obtained either during the pollen season or out of season, from allergic individuals and non-allergic controls were stimulated either with TG extract or a pool of previously identified immunodominant antigenic regions. PBMC from in season allergic subjects exhibit higher IL-5 and IL-10 responses compared to out of season donors. In the case of non-allergic subjects, as expected we observed lower IL-5 responses and robust production of IFNγ compared to allergic individuals. Strikingly, non-atopic donors exhibited an opposing pattern with decreased immune reactivity in-season. The broad downregulation in non-allergic donors indicates that healthy individuals are not oblivious to allergen exposure but rather react with an active modulation of the responses following the antigenic stimulus provided during the pollen season. Transcriptomic analysis of allergen-specific T cells defined genes modulated in concomitance with allergen exposure and inhibition of responses in non-allergic donors. Magnitude and functionality of T-helper cell responses differ substantially for in season versus out of season in allergic and non-allergic subjects. The results indicate specific and opposing modulation of immune responses following the antigenic stimulation during the pollen season. This seasonal modulation reflects the enactment of specific molecular programs associated with health and allergic disease. Overall design: 11 allergen-specific T cell RNA samples were analyzed: 5 isolated from PBMC of allergic individuals and 6 from non-allergic individuals (considered as the control group).
Project description:This study investigated temporal transcriptomic changes in response to nasal allergen challenge of titrated timothy grass pollen. This is an open single-center observational study conducted outside the pollen season. Twelve participants with seasonal allergic rhinitis underwent a control (diluent) challenge followed by nasal allergen challenge after an interval of 14 days. On each challenge day, nasal challenge with control or titrated timothy grass pollen (Aquagen, phleum pratense; ALK) was administered. Peripheral blood was collected before nasal challenge (baseline) and at 3, 6 and 24 hours following challenge. RNA was extracted from whole blood and CD4 cells for microarray experiment using Affymetrix Human Gene 1.0 ST arrays.
Project description:To investigate the allergenicity of the major birch pollen allergen Bet v 1 and the impact of known adjuvants coming from pollen, such as lipopolysaccharide (LPS), we performed quantitative proteome analysis of stimulated monocyte-derived dendritic cells (moDCs). Thus, we treated cells with birch pollen extract (BPE), a recombinant variant of Bet v 1 or LPS followed by proteomic profiling by means of high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) using isobaric labelling.
Project description: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.