Characterisation of the equine macrophage / monocyte

Abstract

Inflammatory airway disease (IAD) is a common performance limiting pulmonary disorder in young racehorses in training. Although the precise aetiopathogenesis is poorly understood, proposed mechanisms include opportunistic bacterial infections and/or suboptimal air-hygiene. Since alveolar macrophages (AMs) are the first line defence in the lungs of mammalian species, they may constitute an appropriate therapeutic target cell in the treatment and the prevention of opportunistic airway infections. This thesis aimed to investigate the basic biology of the equine AM. A series of experiments were conducted to investigate the function and phenotype of this cell and comparisons made with equine macrophages derived from other anatomical sites and macrophage datasets derived from other species. The lung environment is unique, and may direct a unique phenotype and function compared with macrophages derived from other sites. Macrophages were isolated from the lungs, peritoneal cavity and other regions of healthy horses. Excellent cell recovery was demonstrated and associated with good viability, RNA yield and a demonstrable response to several stimuli, both when fresh and following cryopreservation. AMs produced tumor necrosis factor alpha (TNFα) when stimulated with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly IC) and heat-killed Salmonella typhimurium and were actively phagocytic. By comparison, peritoneal macrophages (PMs) did not respond to these inducers and lacked phagocytic activity. In contrast to AMs, which showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and toll-like receptor 4 (TLR4), PMs lacked CD14. Moreover, gene expression analysis revealed an alternative macrophage activation for AMs, whereas PM showed a hybrid macrophage activation potentially attributed to the phenomenon of endotoxin tolerance. The response of equine AMs to LPS was analysed using microarrays. There was significant change in the expression of 240 genes. Those that were upregulated included well known inflammatory genes such as TNFα, IL1A and CXCL6. The pattern of response more closely resembled human and pig macrophages than mouse, including the LPS-induced expression of STAT4, IDO, IL7R genes and the failure to produce nitrite in response to LPS. These data suggest that the horse may represent a suitable animal model for human macrophage-associated lung inflammation, and conversely that data from humans may translate to horses. A final aim of this study was to investigate the effect of exercise on equine AM function. Therefore, AMs were isolated from bronchoalveolar lavage samples obtained from Standardbred racehorses at rest and during the training period and microarray analysis performed. Despite important limitations of the study, a few mechanisms at the molecular level were detected which may be involved in the development of either training-associated symptoms of, or susceptibility to IAD. Overall, this thesis aims to improve our understanding of equine macrophage biology and to provide useful information regarding the role of AMs in exercise-associated inflammation. Moreover, the findings presented here may help to inform future preventative pharmacological and/or managemental interventions for IAD.