Abstract
Inflammation associated oxidative stress leads to peroxidation of polyunsaturated fatty acids thereby generating volatile organic compounds (VOCs). The integrative analysis of the total amount of VOCs released by eosinophils and neutrophils in vitro enables the search for those compounds that discriminates between various inflammatory conditions.The approach comprises isolating eosinophils and neutrophils from 30 ml of blood of healthy non-smoking volunteers by gradient centrifugation, using lymphoprep. Eosinophils are separated from neutrophils by immunomagnetic cell separation using anti-CD16. Cells are activated with phorbol 12-myristate 13-acetate and VOCs from the headspace are collected at time 0′, 30′, 60′ and 90′ by introduction of ultra-pure nitrogen in the closed flasks at a flow rate of 200 ml min−1 during 10 min. The gases are trapped onto a sorption tube and analyzed by gas chromatography—time-of-flight—mass spectometry (GC-TOF-MS) in order to identify VOCs released in the headspace by activated neutrophils and eosinophils.Eosinophils and neutrophils were isolated from 26 healthy non-smoking volunteers. The average absolute number of eosinophils and neutrophils upon isolation was 3.5 × 106 and 19.4 × 106, respectively. The volatome in headspace consisted of 2116 compounds and those compounds present in at least 8% of the samples (1123 compounds) were used for further discriminant analysis. Discriminant analysis showed that two VOCs were able to distinguish between eosinophilic and neutrophilic cultures in the unactivated state with 100% correct classification of the entire data set and upon cross validation while five VOCs were able to discriminate between activated eosinophils and neutrophils with 96% correct classification in the original set and upon cross-validation.Analysis of VOCs seems to be a very promising approach in identifying eosinophilic and neutrophilic inflammation but it needs further development and in vivo confirmation.
Highlights
Asthma is a chronic inflammatory disease of the airways
Eosinophils and neutrophils were isolated from 26 healthy non-smoking volunteers
A combination of two volatile organic compounds (VOCs) was able to discriminate between unactivated eosinophils and neutrophils cell cultures
Summary
There is increasing evidence that phenotyping asthma according to airway inflammation allows the identification of subgroups of patients that are more likely to respond to targeted therapy The importance of these inflammatory phenotypes is that the underlying molecular mechanisms are different. While the eosinophilic phenotype is likely to reflect ongoing adaptive immunity in response to allergen with Th2 cytokine IL-4, IL-5 and IL-13 playing a key role, the neutrophilic is thought to reflect innate immune system activation in response to pollutants or infectious agents [1, 2]. There is, no evidence that inhaled corticosteroids may improve asthma control in the absence of uncontrolled eosinophilic inflammation as encountered in pauci-granulocytic asthma [9] and data suggest that severe neutrophilic asthma could be best targeted by using clarithromycin [10, 11]
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