Abstract
Airway inflammation is highly prevalent in horses, with the majority of non-infectious cases being defined as equine asthma. Currently, cytological analysis of airway derived samples is the principal method of assessing lower airway inflammation. Samples can be obtained by tracheal wash (TW) or by lavage of the lower respiratory tract (bronchoalveolar lavage (BAL) fluid; BALF). Although BALF cytology carries significant diagnostic advantages over TW cytology for the diagnosis of equine asthma, sample acquisition is invasive, making it prohibitive for routine and sequential screening of airway health. However, recent technological advances in sample collection and processing have made it possible to determine whether a wider range of analyses might be applied to TW samples. Considering that TW samples are relatively simple to collect, minimally invasive and readily available in the horse, it was considered appropriate to investigate whether, equine tracheal secretions represent a rich source of cells and both transcriptomic and proteomic data. Similar approaches have already been applied to a comparable sample set in humans; namely, induced sputum. Sputum represents a readily available source of airway biofluids enriched in proteins, changes in the expression of which may reveal novel mechanisms in the pathogenesis of respiratory diseases, such as asthma and chronic obstructive pulmonary disease. The aim of this study was to establish a robust protocol to isolate macrophages, protein and RNA for molecular characterization of TW samples and demonstrate the applicability of sample handling to rodent and human pediatric bronchoalveolar lavage fluid isolates. TW samples provided a good quality and yield of both RNA and protein for downstream transcriptomic/proteomic analyses. The sample handling methodologies were successfully applicable to BALF for rodent and human research. TW samples represent a rich source of airway cells, and molecular analysis to facilitate and study airway inflammation, based on both transcriptomic and proteomic analysis. This study provides a necessary methodological platform for future transcriptomic and/or proteomic studies on equine lower respiratory tract secretions and BALF samples from humans and mice.
Highlights
The European Thoroughbred horseracing sector has an annual economic impact of €12 billion, including approximately 155,000 employees in a variety of roles (British Horseracing Authority)
To the best of our knowledge, this study has shown for the first time that it is feasible to carry out both transcriptomic and proteomic analysis on a single tracheal wash (TW) collected from equines, in this case UK national hunt Thoroughbreds
Macrophages were successfully isolated from TW samples based on magnetic bead separation and isolated cells were deemed suitable for downstream transcriptomic analysis
Summary
The European Thoroughbred horseracing sector has an annual economic impact of €12 billion, including approximately 155,000 employees in a variety of roles (British Horseracing Authority). Recent technological advances in sample collection and processing procedures have made it possible to determine whether a wider range of analyses might be applied to TW samples This would be advantageous as equine tracheal secretions represent a rich source of: (i) cells, (ii) host-derived microbiome, (iii) transcriptome and (iv) local environment-derived proteins; and (v) are relatively simple to collect with minimal discomfort to the horse. The proteomic approach is novel and follows a recent upsurge of interest in proteomic analysis of human airway secretions as a highly informative noninvasive method of assessing the mechanisms associated with lung disease[15] Application of both a transcriptomic and proteomic approach to equine TW samples has the potential to reveal novel biomarkers of early disease development or disease associated tissue remodelling. We compared the relevance of our results to murine and paediatric samples, determining whether a similar approach might be adopted
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