ABSTRACT
Respiratory diseases, including inflammatory airway disease (IAD), viral and bacterial infections, are common problems in exercising horses. The airway epithelium constitutes a major physical barrier against airborne infections and plays an essential role in the lung innate immune response mainly through toll-like receptor (TLR) activation. The aim of this study was to develop a model for the culture of equine bronchial epithelial cells (EBEC) in vitro and to explore EBEC innate immune responses in trained horses. Bronchial epithelial biopsies were taken from 6 adult horses during lower airway endoscopy. EBEC were grown in vitro by an explant method. The innate immune response of EBEC was evaluated in vitro by treatment with TLR ligands. TLR3 is the most strongly expressed TLR at the mRNA level in EBEC and stimulation of EBEC with Poly(I:C), an analog of viral dsRNA, triggers a strong secretion of IFN-ß, TNF-α, IL-6 and CXCL8. We further evaluated the EBEC innate immune response in horses that underwent a 4-month-training program. While training had no effect on TLR mRNA expression in EBEC as well as in bronchial biopsies, it increased the production of IFN-ß after stimulation with a TLR3 ligand and decreased the secretion of TNF-α and IL-6 after stimulation with a TLR2 and TLR3 ligand. These findings may be implicated in the increased risk for viral and bacterial infections observed in sport horses. Altogether, we report a successful model for the culture of EBEC that can be applied to the investigation of pathophysiologic conditions in longitudinal studies.
Subject(s)
Horses/physiology , Immunity, Innate , Physical Conditioning, Animal , Respiratory Mucosa/immunology , Toll-Like Receptors/genetics , Animals , Bronchi/cytology , Bronchi/immunology , Cell Culture Techniques/methods , Cells, Cultured , Cytokines/metabolism , Female , Horses/genetics , Horses/immunology , Male , Poly I-C/pharmacology , Respiratory Mucosa/cytology , Toll-Like Receptors/metabolismABSTRACT
In humans, strenuous exercise causes increased susceptibility to respiratory infections associated with down-regulated expression of Toll-like receptors (TLRs) and costimulatory and antigen-presenting molecules. Lower airway diseases are also a common problem in sport and racing horses. Because innate immunity plays an essential role in lung defense mechanisms, we assessed the effect of acute exercise and training on innate immune responses in two different compartments. Blood monocytes and pulmonary alveolar macrophages (PAMs) were collected from horses in untrained, moderately trained, intensively trained, and deconditioned states before and after a strenuous exercise test. The cells were analyzed for TLR messenger ribonucleic acid (mRNA) expression by real-time PCR in vitro, and cytokine production after in vitro stimulation with TLR ligands was measured by ELISA. Our results showed that training, but not acute exercise, modified the innate immune responses in both compartments. The mRNA expression of TLR3 was down-regulated by training in both cell types, whereas the expression of TLR4 was up-regulated in monocytes. Monocytes treated with LPS and a synthetic diacylated lipoprotein showed increased cytokine secretion in trained and deconditioned subjects, indicating the activation of cells at the systemic level. The production of TNF-α and IFN-ß in nonstimulated and stimulated PAMs was decreased in trained and deconditioned horses and might therefore explain the increased susceptibility to respiratory infections. Our study reports a dissociation between the systemic and the lung response to training that is probably implicated in the systemic inflammation and in the pulmonary susceptibility to infection.
Subject(s)
Immunity, Innate/immunology , Inflammation/immunology , Lung/immunology , Macrophages, Alveolar/immunology , Monocytes/immunology , Physical Conditioning, Animal , Toll-Like Receptors/metabolism , Animals , Blotting, Western , Female , Horses , Hydrocortisone/metabolism , Inflammation/metabolism , Inflammation/pathology , Interferon-beta/genetics , Interferon-beta/metabolism , Longitudinal Studies , Lung/metabolism , Macrophages, Alveolar/metabolism , Monocytes/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Endotoxemia is responsible for severe illness in horses. Individuals can become clinically unresponsive to the endotoxin molecule after an initial exposure; a phenomenon referred to as 'endotoxin tolerance' (ET). ET has been observed clinically in horses in vivo; however, cytokine expression associated with ET has not been investigated. The purpose of this study was to develop and validate a method for inducing ET in equine peripheral blood mononuclear cells (PBMC) in vitro, and to describe selected cytokine responses which are associated with ET. ET was induced by culturing cells with three concentrations of endotoxin, and evaluated after a second dose of endotoxin given to challenge the cells. The relative mRNA expression of IL-10 and IL-12 was measured by use of quantitative PCR. ET was induced in all cells exposed to the 2-step endotoxin challenge. The relative mRNA expression of IL-10 in tolerized cells was not different from positive control cells. In contrast, the relative mRNA expression of IL-12 in tolerized cells was decreased by 15-fold after the second endotoxin challenge. This experiment demonstrated a reliable method for the ex vivo induction of ET in equine PBMC. A marked suppression of IL-12 production is associated with ET.
