Abstract
BackgroundThe mechanism for the contribution of eosinophils (EOS) to asthma pathophysiology is not fully understood. Genome-wide expression analysis of airway EOS by microarrays has been limited by the ability to generate high quality RNA from sufficient numbers of airway EOS.ObjectiveTo identify, by genome-wide expression analyses, a compendium of expressed genes characteristic of airway EOS following an in vivo allergen challenge.MethodsAtopic, mild asthmatic subjects were recruited for these studies. Induced sputum was obtained before and 48h after a whole lung allergen challenge (WLAC). Individuals also received a segmental bronchoprovocation with allergen (SBP-Ag) 1 month before and after administering a single dose of mepolizumab (anti-IL-5 monoclonal antibody) to reduce airway EOS. Bronchoalveolar lavage (BAL) was performed before and 48 h after SBP-Ag. Gene expression of sputum and BAL cells was analyzed by microarrays. The results were validated by qPCR in BAL cells and purified BAL EOS.ResultsA total of 299 transcripts were up-regulated by more than 2-fold in total BAL cells following SBP-Ag. Mepolizumab treatment resulted in a reduction of airway EOS by 54.5% and decreased expression of 99 of the 299 transcripts. 3 of 6 post-WLAC sputum samples showed increased expression of EOS-specific genes, along with the expression of 361 other genes. Finally, the intersection of the 3 groups of transcripts (increased in BAL post SBP-Ag (299), decreased after mepolizumab (99), and increased in sputum after WLAC (365)) was composed of 57 genes characterizing airway EOS gene expression.ConclusionWe identified 57 genes that were highly expressed by BAL EOS compared to unseparated BAL cells after in vivo allergen challenge. 41 of these genes had not been previously described in EOS and are thus potential new candidates to elucidate EOS contribution to airway biology.
Highlights
Recruitment of EOS to the lung has been reproducibly reported in allergic asthma [1]
We identified 57 genes that were highly expressed by Bronchoalveolar lavage (BAL) EOS compared to unseparated BAL cells after in vivo allergen challenge. 41 of these genes had not been previously described in EOS and are potential new candidates to elucidate EOS contribution to airway biology
Subjects had a history of mild atopic asthma as defined by at least one positive skin prick test and a history of allergen-induced asthma exacerbation with reversibility to albuterol .12% and/or a provocative concentration of methacholine producing a 20% fall in FEV1 (PC20) of,8 mg/ml
Summary
Recruitment of EOS to the lung has been reproducibly reported in allergic asthma [1]. One approach to understanding the biology of EOS in asthma is gene expression analysis by microarrays. A subsequent study performed by our group showed that the expression of more than 200 genes was increased in vitro in IL-5- and GM-CSF-activated EOS, including the anti-apoptotic serine/threonine protein kinase Pim-1 [14,15]. During their egress to the airway, in response to allergen, the phenotype of peripheral blood EOS changes dramatically [16,17,18]; gene analysis with microarrays of airway EOS has not been explored.
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