Influence of olive pollen stimuli on the gene- expression profile in healthy controls and allergic patients

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. 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).