| Introduction. Recurrent airway obstruction (RAO) is characterized by neutrophilic airway inflammation and obstruction, and stabling of susceptible horses triggers acute disease exacerbations. Stable dust is rich in endotoxin and fungi, such as Faenia rectivirgula, Thermoactinomyces vulgaris and Aspergillus fumigatus. Toll-like receptors (TLRs) are pathogen recognition receptors that are transmembrane proteins on a variety of airway cells (e.g. epithelial cells). Toll-like receptor 4 is the principal recognition receptor for lipopolysaccharide (LPS), and TLR2 is the principal recognition receptor for fungal products. In human bronchial epithelium, TLR4 and TLR2 stimulation leads to increased production of interleukin (IL)-8, a potent neutrophil attractant. The zinc finger protein A20 negatively regulates these pathways. Hypotheses. We hypothesized (i) that horses in stables are exposed to higher endotoxin concentrations than on pasture; (ii) that during stabling TLR4, TLR2, and IL-8 mRNA expression are increased in RAO-affected horses compared to controls, and that this would be paralleled by elevated neutrophil counts in bronchoalveolar lavage fluid (BAL); and (iii) that A20 mRNA expression is not increased in RAO-affected horses during stabling compared to controls. Materials and methods. We determined endotoxin concentrations in the breathing zone of six horses. Furthermore, we measured the maximal change in pleural pressure (DeltaPplmax), determined inflammatory cell counts in BAL, and quantified TLR4, TLR2, IL-8, and A20 mRNA in bronchial epithelium by quantitative real-time polymerase chain reaction (qRT-PCR). For these experiments, we studied six age-matched horse pairs, each pair consisting of one RAO-affected horse and one control horse. Each pair was studied when the RAO-affected horse had airway obstruction induced by stabling, and after 7, 14 and 28 days on pasture. Results . Endotoxin concentrations in the breathing zone of stabled horses were significantly higher than in the breathing zone of these horses on pasture. While stabling increased BAL neutophils and DeltaPplmax as well as TLR4 and IL-8 mRNA expression significantly, TLR2 and A20 were unaffected. TLR4, but not TLR2, correlated with IL-8 and IL-8 was significantly correlated with BAL neutrophils and A20. When data were pooled, TLR2 and A20 were significantly correlated and A20 was negatively associated with inflammatory cells. Conclusions. In our stables, horses are exposed to an at least 8-fold higher concentration of endotoxin than on pastures. Elevated TLR4 expression and lack of A20 upregulation in bronchial epithelial cells from RAO-affected horses may contribute to elevated IL-8 production, leading to exaggerated neutrophilic airway inflammation in response to inhalation of stable dust. Stable dust exposure does not lead to an increase in TLR2 mRNA expression. The localization or the amount of the TLR2 receptor complex rather than the amount of TLR2 mRNA may be important in fungal-induced airway inflammation. Equine epithelial-derived A20 may protect against airway inflammation by decreasing the number of bronchoalveolar lavage inflammatory cells, and is involved in modulation of airway inflammation. |
