Alveolar macrophages (AMs) constitute the first line of defence in the lung of all species, playing a crucial role in the regulation of immune responses to inhaled pathogens. A detailed understanding of the function and phenotype of AMs is a necessary pre-requisite to both elucidating their role in preventing opportunistic bacterial colonisation of the lower respiratory tract and developing appropriate preventative strategies. The purpose of the study was to characterise this important innate immune cell at the tissue level by making functional and phenotypic comparisons with peritoneal macrophages (PMs). We hypothesised that the tissue of origin determines a unique phenotype of AMs, which may constitute an appropriate therapeutic target for certain equine respiratory diseases. Macrophages isolated from the lung and the peritoneal cavity of 9 horses were stimulated with various toll like receptor (TLR) ligands and the production of nitrite, tumour necrosis factor alpha (TNFα), interleukin (IL) 10 and indoleamine 2,3-dioxygenase (IDO) were measured by the Griess reaction and enzyme linked immunosorbent assay (ELISA) and/or quantitative polymerase chain reaction, respectively. Cells were also compared on the basis of phagocytic-capacity and the expression of several cell surface markers. AMs, but not PMs, demonstrated increased TNFα release following stimulation with LPS, polyinosinic polycytidylic acid (Poly IC) and heat-killed Salmonella typhinurium and increased TNFα and IDO mRNA expression when stimulated with LPS. AMs showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and TLR4, whereas PMs showed high expression of TLR4 only. AMs, but not PMs, demonstrated efficient phagocytic activity. Our results demonstrate that AMs are more active than PMs when stimulated with various pro-inflammatory ligands, thus supporting the importance of the local microenvironment in the activation status of the macrophage. This information provides a valuable knowledge base on which to improve our understanding of the role of macrophages and their microenvironment in equine innate immunity.

SummaryReasons for performing studyAlveolar macrophages (AMs) are the first line of defence against pathogens in the lungs of all mammalian species and thus may constitute appropriate therapeutic target cells in the treatment and prevention of opportunistic airway infections. Therefore, acquiring a better understanding of equine macrophage biology is of paramount importance in addressing this issue in relation to the horse.ObjectivesTo compare the transcriptome of equine AMs with that of equine peritoneal macrophages (PMs) and to investigate the effect of lipopolysaccharide (LPS) on equine AM.Study designGene expression study of equine AMs.MethodsCells from both bronchoalveolar and peritoneal lavage fluid were isolated from systemically healthy horses that had been submitted to euthanasia. Cells were cryopreserved. RNA was extracted and comparative microarray analyses were performed in AMs and PMs, and in AMs treated and untreated with LPS. Comparisons with published data derived from human AM studies were made, with particular focus on LPS‐induced inflammatory status.ResultsThe comparison between AMs and PMs revealed the differential basal expression of 451 genes. Gene expression analysis revealed an alternative (M2) macrophage polarisation profile in AMs and a hybrid macrophage activation profile in PMs, a phenomenon potentially attributable to a degree of induced endotoxin tolerance. The gene expression profile of equine AMs following LPS stimulation revealed significant changes in the expression of 240 genes, including well‐known upregulated inflammatory genes. This LPS‐induced gene expression profile of equine AMs more closely resembles that of human rather than murine macrophages.ConclusionsThis study improves current understanding of equine macrophage biology. These data suggest that the horse may represent a suitable animal model for the study of human macrophage‐associated lung inflammation and data derived from human macrophage studies may have significant relevance to the horse.

Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined the changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer.

pANCA were detected in a high percentage of dogs with IMHA and vector-borne infectious diseases. Therefore, pANCA may be a relatively nonspecific marker for dogs with inflammatory bowel disease, although they could represent a biomarker for immune-mediated diseases and infections.

Mild to moderate equine asthma is prevalent in young racehorses, particularly early in their training period. Although the precise aetiopathogenesis remains undetermined, it is possible that the susceptibility of this population might partly reflect an exercise-associated immune derangement at the level of the airway. We performed a genome-wide basal gene expression scan on alveolar macrophages (AMs) isolated from Standardbred racehorses prior to and after commencement of competition race training with a view to identifying any exerciseassociated gene expression modulation consistent with functional alterations which might reflect training-associated immunological derangement. Microarray technology was used to analyse the basal gene expression profiles of bronchoalveolar fluid-derived AMs, harvested from six systemically healthy Standardbred racehorses prior to (T0) and following (T1) entry into training. Additionally, AM LPS-induced TNF-α and IL-10 release at T0 and T1 was assessed. Although the data revealed significant inter-horse heterogeneity in relation to the magnitude of individual gene expression at each time-point, within each horse, several inflammatory related genes (e.g. chemokine ligands, interferons and NFKB) declined in expression from T0 to T1. Entry into training did not significantly alter AM LPS-induced TNF-α or IL-10 release. The data support a direct effect of training on AM basal gene expression, particularly with respect to immune-related genes. The pattern of trainingassociated differential gene expression may indicate relative downregulation of inflammatory-related genes, consistent with an immunosuppressive effect of training and an increased susceptibility to opportunistic pathogens.

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.

Horses are already recognised as models for several human diseases, including metabolic syndrome, asthma, musculoskeletal diseases, melanoma and autoimmune uveitis. 1-6 More than 100 equine hereditable conditions may serve as models for human disorders, including inflammation, muscular or fertility disorders, osteoarthritis and even depression. 7-10 The horse has the potential to represent an appropriate

Neutrophilic airway inflammation is highly prevalent in racehorses in training, with the term mild to moderate equine asthma (MMEA) being applied to the majority of such cases. Our proposed study is largely derived from the strong association between MMEA in racehorses and their entry into a race training program. The objectives of this study are to characterise the effect of training on the local pulmonary immune system by defining the gene and protein expression of tracheal wash (TW) derived samples from Thoroughbred racehorses prior to and following commencement of race training. Multiomics analysis detected 2138 differentially expressed genes and 260 proteins during the training period. Gene and protein sets were enriched for biological processes related to acute phase response, oxidative stress, haemopoietic processes, as well as to immune response and inflammation. This study demonstrated TW samples to represent a rich source of airway cells, protein and RNA to study airway immunity in the horse and highlighted the benefits of a multiomics methodological approach to studying the dynamics of equine airway immunity. Findings likely reflect the known associations between race-training and both airway inflammation and bleeding, offering further insight into the potential mechanisms which underpin training associated airway inflammation.