Abstract
BackgroundThe airway epithelium is thought to play an important role in the pathogenesis of asthmatic disease. However, much of our understanding of airway epithelial cell function in asthma has been derived from in vitro studies that may not accurately reflect the interactive cellular and molecular pathways active between different tissue constituents in vivo.MethodsUsing a sheep model of allergic asthma, tracheal explants from normal sheep and allergic sheep exposed to house dust mite (HDM) allergen were established to investigate airway mucosal responses ex vivo. Explants were cultured for up to 48 h and tissues were stained to identify apoptotic cells, goblet cells, mast cells and eosinophils. The release of cytokines (IL-1α, IL-6 and TNF-α) by cultured tracheal explants, was assessed by ELISA.ResultsThe general morphology and epithelial structure of the tracheal explants was well maintained in culture although evidence of advanced apoptosis within the mucosal layer was noted after culture for 48 h. The number of alcian blue/PAS positive mucus-secreting cells within the epithelial layer was reduced in all cultured explants compared with pre-cultured (0 h) explants, but the loss of staining was most evident in allergic tissues. Mast cell and eosinophil numbers were elevated in the allergic tracheal tissues compared to naïve controls, and in the allergic tissues there was a significant decline in mast cells after 24 h culture in the presence or absence of HDM allergen. IL-6 was released by allergic tracheal explants in culture but was undetected in cultured control explants.ConclusionsSheep tracheal explants maintain characteristics of the airway mucosa that may not be replicated when studying isolated cell populations in vitro. There were key differences identified in explants from allergic compared to control airways and in their responses in culture for 24 h. Importantly, this study establishes the potential for the application of tracheal explant cultures in relevant ex vivo investigations on the therapeutic and mechanistic modalities of asthmatic disease.
Highlights
The airway epithelium is thought to play an important role in the pathogenesis of asthmatic disease
Priming of the airways with allergen induced a marked recruitment of eosinophils (15-32% of bronchoalveolar lavage (BAL) leukocytes) into the BAL fluid when assessed at 48 h post-challenge, similar to that observed in our previous studies [14,16]
Histological and functional changes in tracheal explant cultures Tracheal tissues were collected from control animals to assess the effects of maintaining tracheal explants in culture for 0 h, 5 h, 24 h, and 48 h
Summary
The airway epithelium is thought to play an important role in the pathogenesis of asthmatic disease. In vitro cell-based studies have allowed detailed investigations of the molecular mechanisms underlying the pathology of asthmatic disease This has included studies in humans and appropriate animal models using primary airway epithelial, fibroblast and smooth muscle cells cultured from biopsy samples collected by fibreoptic bronchoscopy or post-mortem tissues [5,6]. Tracheal explants established in sheep display many key features of the in vivo airways such as mucus coverage, mucociliary clearance and cell structure [9] While such studies have been used to examine approaches for gene delivery [9,10,11] and mechanisms of epithelial cell mucus secretion [12], none to date have used tracheal explants to investigate the allergic basis of bronchial asthma. In this study we use a validated model of human allergic asthma [13,14,15] to investigate airway mucosal responses ex vivo in tracheal explants derived from normal sheep and allergic sheep exposed to house dust mite (HDM) allergen
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