Application across species of a one health approach to liquid sample handling for respiratory based -omics analysis

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.

Training associated alterations in equine respiratory immunity using a multiomics comparative approach

“…The present study used a series of 32 horse-derived TW samples from 16 Thoroughbred racehorses at two time points (T0-resting period, T1-training period) and an average of 10.8 × 10 6 (± 1.9) cells per sample were isolated, consistent with previously reported data 16 , 17 . None of the animals had clinical signs of respiratory disease or a history of poor performance.…”

Training Associated Alterations in Equine Respiratory Immunity Using a Multiomics Comparative Approach

“…These protein's functions were related to immune response and lung biology and included pulmonary surfactant-associated proteins (SFTPA1, SFTPB), complement factors (CA6,C1R), heat shock proteins (HSPE1), antigen presentation molecules (DRA, PIP), antioxidants (PRDX3, SOD2), haemopoietic factors (HBE1, HBA, FGA, TFRC), protein synthesis (PSMA5, HSPE1, PHB) and apoptosis related factors (GSN). Thirty percent of the proteins detected here were also previously detected from TW samples derived from racehorses 16 .…”

“…The present study used a series of 32 horse-derived TW samples from 16 Thoroughbred racehorses at two time points (T0-resting period, T1-training period) and an average of 10.8x10 6 (+1.9) cells per sample were isolated, consistent with previously reported data 15,16 . None of the animals had clinical signs of respiratory disease or a history of poor performance.…”

“…Samples containing haemosiderophages were considered consistent with a diagnosis of EIPH 13. Total cell Isolation from Tracheal Wash A 1ml aliquot of sample was stored at -80°C for future proteomic analyses and the remaining sample incubated for 15 minutes at room temperature in 0.1% DTT to depolymerize secreted mucin, as previously described 16. Dithiothreitol has been demonstrated to cause no deleterious effects on human sputum derived cells or interfere with surface marker measurements using ow cytometry 74,75.…”

“…Twenty micrograms per sample were pooled for each time point and samples were processed as described above. In order to visualise the total protein load of all the samples, samples were run on gradient gels and stained with instant blue protein stain, as previously described 16 . Subsequently, protein samples were submitted for proteomic analysis.…”