Altered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy.

Polysaccharide storage myopathy (PSSM) is a widely described cause of exertional rhabdomyolysis in horses. Mitochondria play a central role in cellular energetics and are involved in human glycogen storage diseases but their role has been overlooked in equine PSSM. We hypothesized that the mitochondrial function is impaired in the myofibers of PSSM-affected horses. Nine horses with a history of recurrent exercise-associated rhabdomyolysis were tested for the glycogen synthase 1 gene (GYS1) mutation: 5 were tested positive (PSSM group) and 4 were tested negative (horses suffering from rhabdomyolysis of unknown origin, RUO group). Microbiopsies were collected from the gluteus medius (gm) and triceps brachii (tb) muscles of PSSM, RUO and healthy controls (HC) horses and used for histological analysis and for assessment of oxidative phosphorylation (OXPHOS) using high-resolution respirometry. The modification of mitochondrial respiration between HC, PSSM and RUO horses varied according to the muscle and to substrates feeding OXPHOS. In particular, compared to HC horses, the gm muscle of PSSM horses showed decreased OXPHOS- and electron transfer (ET)-capacities in presence of glutamate&malate&succinate. RUO horses showed a higher OXPHOS-capacity (with glutamate&malate) and ET-capacity (with glutamate&malate&succinate) in both muscles in comparison to the PSSM group. When expressed as ratios, our results highlighted a higher contribution of the NADH pathway (feeding electrons into Complex I) to maximal OXPHOS or ET-capacity in both rhabdomyolysis groups compared to the HC. Specific modifications in mitochondrial function might contribute to the pathogenesis of PSSM and of other types of exertional rhabdomyolyses.

The innate immune response of equine bronchial epithelial cells is altered by training.

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.

Training modifies innate immune responses in blood monocytes and in pulmonary alveolar macrophages.

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.

New Pathophysiological Insights from Serum Proteome Profiling in Equine Atypical Myopathy.

Equine atypical myopathy (AM) is a severe environmental intoxication linked to the ingestion of protoxins contained in seeds and seedlings of the sycamore maple (Acer pseudoplatanus) in Europe. The toxic metabolites cause a frequently fatal rhabdomyolysis syndrome in grazing horses. Since these toxic metabolites can also be present in cograzing horses, it is still unclear as to why, in a similar environmental context, some horses show signs of AM, whereas others remain clinically healthy. Label-free proteomic analyses on the serum of 26 diseased AM, 23 cograzers, and 11 control horses were performed to provide insights into biological processes and pathways. A total of 43 and 44 differentially abundant proteins between "AM vs cograzing horses" and "AM vs control horses" were found. Disease-linked changes in the proteome of different groups were found to correlate with detected amounts of toxins, and principal component analyses were performed to identify the 29 proteins representing a robust AM signature. Among the pathway-specific changes, the glycolysis/gluconeogenesis pathway, the coagulation/complement cascade, and the biosynthesis of amino acids were affected. Sycamore maple poisoning results in a combination of inflammation, oxidative stress, and impaired lipid metabolism, which is trying to be counteracted by enhanced glycolysis.

Airway Hyperresponsiveness, but Not Bronchoalveolar Inflammatory Cytokines Profiles, Is Modified at the Subclinical Onset of Severe Equine Asthma.

Airway hyperresponsiveness (AHR) and inflammation are both observed in human and equine asthma. The aim of this study was to assess the timeline and relationship of both features at the subclinical onset of severe equine asthma (SEA). First, the repeatability of the pulmonary function test (PFT) using impulse oscillometry system, and the methacholine bronchoprovocation test (BPT) were assessed at a 1-day interval on six SEA horses in clinical remission and six control horses. Then, clinical and ancillary tests were performed before and after a 1-week low-dust environmental challenge, including weighted clinical score, respiratory endoscopy, bronchoalveolar fluid cytology, PFT, and BPT. Both PFT and BPT showed acceptable repeatability. No test allowed SEA horses in clinical remission to be distinguished from control, unlike in human patients. Because of the low-dust environment, no significant difference was observed in the results of clinical and conventional ancillary examinations after the challenge. However, SEA horses showed increased AHR after the environmental challenge. At that stage, no signs of inflammation or changes in pro-inflammatory cytokines profiles (quantification and gene expression) were observed, suggesting AHR is present at an earlier stage of equine asthma than airway inflammation. This feature indicates SEA could present in a different disease pathway than neutrophilic human asthma.

In vitro virucidal activity of nebulized citrate-complexed silver nanoparticles against equine herpesvirus-1 and murine norovirus.

Viruses can be involved in respiratory disorders in horses, with limited therapeutic options. Citrate-complexed silver nanoparticles (C-AgNP) have shown bactericidal properties after in vitro nebulization. The aim of the present study was to assess the virucidal activity of C-AgNP after in vitro instillation or nebulization on equine herpesvirus-1 (EHV-1) and murine norovirus (MNV), the latter used as surrogate for small non-enveloped viruses. Both viruses were instilled or nebulized with C-AgNP of increasing concentrations, and titres were determined via TCID50 method. We demonstrated efficient inactivation of enveloped EHV-1 following instillation and nebulization of C-AgNP (infectivity losses of ≥ three orders of magnitude). While tenacious MNV was inactivated via 2000 ppm C-AgNP instillation, nebulized C-AgNP did not lead to reduction in MNV titres. Nebulization of C-AgNP may represent a novel virucidal therapeutic approach in horses. Further investigations are needed to assess its safety and effective concentrations for in vivo use.

Assessing fitness in endurance horses.

A field test and a standardized treadmill test were used to assess fitness in endurance horses. These tests discriminated horses of different race levels: horses participating in races of 120 km and more showed higher values of VLA4 (velocity at which blood lactate reached 4 mmol/L) and V200 (velocity at which heart rates reached 200 beats per min) than horses of lower race levels.

Accuracy of a heart rate monitor for calculating heart rate variability parameters in exercising horses.

Heart rate is evaluated in exercising horses to monitor the level of fitness to exercise, and it is usually acquired using heart rate monitors (HRM) or telemetric electrocardiograms (ECG). While HRM are commonly available and easy-to-use for horse's owners, ECG is a more expensive equipment requiring user's experience. Interest for heart rate variability (HRV) in horses is increasing for both research and clinical purposes. HRV is usually calculated from interbeat intervals (IBI) obtained by ECG. The aim of this study was to determine the accuracy of an HRM to detect IBI for the calculation of HRV in both resting and exercising horses. Simultaneous ECG and HRM recordings were performed on 13 horses and ponies under normal training conditions for at least 45 minutes. IBI from ECG were corrected using a dedicated software. IBI from HRM were exported without correction. Two HRV-parameters were calculated on both recordings for resting and exercising periods: the standard deviation of R-R intervals (SDRR) and the root mean square of successive differences (RMSSD). Agreement and concordance between the two systems were determined using Bland-Altman plot and Lin's correlation coefficient, respectively. Effects of variables were consequently assessed. For both HRV-parameters during resting and exercising periods, the means of the differences between the two systems were lower than 0.47 ms with a correlation coefficient higher than 0.999. Height, weight and body condition score had no effect on the results. The studied HRM could be of interest as easy-to-use device for obtaining HRV-parameters SDRR and RMSSD in resting and exercising horses.

Evaluation of the Bactericidal Effect of Nebulized Silver Nanoparticles on Common Respiratory Bacteria in Horses- In Vitro Studies.

Antimicrobial resistance is increasing in both human and veterinary medicine. Bacteria can be part of the etiology of respiratory disorders in horses. Bactericidal activity of silver has been largely described and silver is currently used in veterinary therapeutic applications such as wound dressings. The aim of this study was to assess the in vitro bactericidal effects of nebulized silver nanoparticles (AgNP) on 2 common equine respiratory bacteria, Streptococcus equi subsp. zooepidemicus and Actinobacillus equuli subsp. equuli. Firstly, antimicrobial susceptibility of AgNP was determined over time by turbidity assessment in liquid broth. Secondly, bacterial growth inhibition was tested after instillation or after nebulization of low (100 ppm) and high (500, 1,000 and 2,000 ppm) concentrations of AgNP on agar plate. Both bacteria were susceptible to AgNP, even at dilution 1:4 for A. equuli and 1:8 for S. zooepidemicus after 8 hours of incubation, and 1:256 for both bacteria after 24 hours of incubation. The bacterial growth was partially inhibited at low concentration and completely inhibited at high concentrations of instilled AgNP. The bacterial growth was completely inhibited after nebulization of low concentrations of AgNP for A. equuli and high concentrations of AgNP for S. zooepidemicus. We concluded nebulized AgNP could be a candidate for innovative therapeutic way against bacterial respiratory disorders in horses. Nevertheless, further investigations are required to assess the in vivo potential and toxicity of nebulized AgNP.

Acylcarnitine profile in Alaskan sled dogs during submaximal multiday exercise points out metabolic flexibility and liver role in energy metabolism.

Alaskan sled dogs develop a particular metabolic strategy during multiday submaximal exercise, allowing them to switch from intra-muscular to extra-muscular energy substrates thus postponing fatigue. Specifically, a progressively increasing stimulus for hepatic glycogenolysis and gluconeogenesis provides glucose for both fueling exercise and replenishing the depleted muscle glycogen. Moreover, recent studies have shown that with continuation of exercise sled dogs increase their insulin-sensitivity and their capacity to transport and oxidize glucose and carbohydrates rather than oxidizing fatty acids. Carnitine and acylcarnitines (AC) play an essential role as metabolic regulators in both fat and glucose metabolism; they serve as biomarkers in different species in both physiologic and pathologic conditions. We assessed the effect of multiday exercise in conditioned sled dogs on plasma short (SC), medium (MC) and long (LC) chain AC by tandem mass spectrometry (MS/MS). Our results show chain-specific modification of AC profiles during the exercise challenge: LCACs maintained a steady increase throughout exercise, some SCACs increased during the last phase of exercise and acetylcarnitine (C2) initially increased before decreasing during the later phase of exercise. We speculated that SCACs kinetics could reflect an increased protein catabolism and C2 pattern could reflect its hepatic uptake for energy-generating purposes to sustain gluconeogenesis. LCACs may be exported by muscle to avoid their accumulation to preserve glucose oxidation and insulin-sensitivity or they could be distributed by liver as energy substrates. These findings, although representing a "snapshot" of blood as a crossing point between different organs, shed further light on sled dogs metabolism that is liver-centric and more carbohydrate-dependent than fat-dependent and during prolonged submaximal exercise.

In Vitro Assays for the Assessment of Impaired Mitochondrial Bioenergetics in Equine Atypical Myopathy.

Equine atypical myopathy is a seasonal intoxication of grazing equids. In Europe, this poisoning is associated with the ingestion of toxins contained in the seeds and seedlings of the sycamore maple (Acer pseudoplatanus). The toxins involved in atypical myopathy are known to inhibit ß-oxidation of fatty acids and induce a general decrease in mitochondrial respiration, as determined by high-resolution respirometry applied to muscle samples taken from cases of atypical myopathy. The severe impairment of mitochondrial bioenergetics induced by the toxins may explain the high rate of mortality observed: about 74% of horses with atypical myopathy die, most within the first two days of signs of poisoning. The mechanism of toxicity is not completely elucidated yet. To improve our understanding of the pathological process and to assess therapeutic candidates, we designed in vitro assays using equine skeletal myoblasts cultured from muscle biopsies and subjected to toxins involved in atypical myopathy. We established that equine primary myoblasts do respond to one of the toxins incriminated in the disease.

Epithelial RABGEF1 deficiency promotes intestinal inflammation by dysregulating intrinsic MYD88-dependent innate signaling.

Intestinal epithelial cells (IECs) contribute to the regulation of intestinal homeostasis and inflammation through their interactions with the environment and host immune responses. Yet our understanding of IEC-intrinsic regulatory pathways remains incomplete. Here, we identify the guanine nucleotide exchange factor RABGEF1 as a regulator of intestinal homeostasis and innate pathways dependent on IECs. Mice with IEC-specific Rabgef1 deletion (called Rabgef1IEC-KO mice) developed a delayed spontaneous colitis associated with the local upregulation of IEC chemokine expression. In mouse models of colitis based on Interleukin-10 deficiency or dextran sodium sulfate (DSS) exposure, we found that IEC-intrinsic RABGEF1 deficiency exacerbated development of intestinal pathology and dysregulated IEC innate pathways and chemokine expression. Mechanistically, we showed that RABGEF1 deficiency in mouse IECs in vitro was associated with an impairment of early endocytic events, an increased activation of the p38 mitogen-activated protein kinase (MAPK)-dependent pathway, and increased chemokine secretion. Moreover, we provided evidence that the development of spontaneous colitis was dependent on microbiota-derived signals and intrinsic MYD88-dependent pathways in vivo. Our study identifies mouse RABGEF1 as an important regulator of intestinal inflammation, MYD88-dependent IEC-intrinsic signaling, and chemokine production. This suggests that RABGEF1-dependent pathways represent interesting therapeutic targets for inflammatory conditions in the gut.

Expression microarrays in equine sciences.

Microarrays have become an important research tool for life science researchers. Expression microarrays are capable of profiling the gene expression pattern of tens of thousands of genes in a single experiment. It appears to be the platform of choice for parallel gene expression profiling. Various equine-specific gene expression microarrays have been generated and used. However, homologous microarrays are not yet commercially available for the horse. An alternative is the use of heterologous microarrays, mainly microarrays specific for mice or humans. Although the use of microarrays in equine research is still in its infancy, gene expression microarrays have shown their potential in equine research. This review presents the previous, current and potential use of expression microarrays in equine research.

Epithelial expression of mRNA and protein for IL-6, IL-10 and TNF-alpha in endobronchial biopsies in horses with recurrent airway obstruction.

BACKGROUND: The aim of this study was to evaluate the contribution of bronchial epithelium to airway inflammation, with focus on mRNA and protein expression of cytokines of innate immunity IL-6, IL-10 and TNF-alpha, in horses with Recurrent Airway Obstruction (RAO) during exacerbation and in remission. RESULTS: Despite marked clinical and physiologic alterations between exacerbation and after remission in the RAO horses no differences were detected in either cytokine mRNA or protein levels. Moreover, the expression of investigated cytokines in RAO horses on pasture did not differ from controls. In comparing real-time PCR analysis to results of immunohistochemistry only IL-10 mRNA and protein levels in RAO horses on pasture were significantly correlated (rs = 0.893, p = 0.007). Curiously, in controls examined on pasture the TNF-alpha protein level was positively correlated to IL-10 mRNA expression (rs = 0.967, p = 0.007) and negatively correlated to IL-6 mRNA expression (rs = -0.971, p = 0.001). CONCLUSION: Given the complementary relationship of assessing cytokines directly by immunohistochemistry, or indirectly by PCR to mRNA, the lack of significant changes in either mRNA or protein levels of IL-6, IL-10 or TNF-alpha mRNA in RAO horses in exacerbation suggests that these particular cytokines in bronchial tissue may not play a substantive role in the active inflammation of this disease. To support this contention further studies examining time dependency of expression of IL-6, IL-10 or TNF-alpha are needed, as is expansion of the range of cytokines to include other key regulators of airway inflammation.

Relevance of using a human microarray to study gene expression in heaves-affected horses.

Environmental causes of heaves are well described, but the molecular mechanisms of the disease remain unclear. Previous studies have highlighted the implications of variations in gene expression, most using reverse transcription polymerase chain reaction (RT-PCR). This well-known technique limits the number of genes that can be studied in a single assay. Microarray appears to be a valuable tool to by-pass this limitation, but so far there has been no equine-specific microarray available on the market. The present study was performed to determine whether a human microarray could be used to study gene expression in nucleated cells originating from peripheral blood and bronchoalveolar lavage fluid (BALF) in heaves-affected horses. With a four-fold cut-off, a total of 46 candidates were identified with differentially regulated genes between heaves-affected horses and controls. A real-time quantitative RT-PCR (RT-QPCR) conducted on a selection of genes, determined on the basis of previous publications, was used to validate the microarray results. The microarray failed to detect the presence of interleukin (IL)-1beta and IL-8 mRNA in the nucleated cells from BALF otherwise confirmed by real-time RT-QPCR. Although some candidate genes have been identified using this method, a complete expression profile of genes related to heaves could not be obtained with the use of the human microarray.

Change in blood antioxidant status of horses moved from a stable following diagnosis of equine motor neuron disease.

The antioxidant status of 10 horses living in stable 1 where 2 cases of equine motor neuron disease had previously been diagnosed was assessed before and 9 weeks after moving to another stable. Duration of residence in stable 1, subsequent moving, or both, significantly affected several parameters of the antioxidant status.

DNA binding activity of transcription factors in bronchial cells of horses with recurrent airway obstruction.

Horses with recurrent airway obstruction (RAO) present many similarities with human asthmatics including airway inflammation, hyperresponsiveness, reversible obstruction, and increased NF-kappaB expression. Studies in experimental asthma models have shown that transcriptions factors such as activator protein-1 (AP-1), GATA-3, cyclic AMP response element binding protein (CREB) and CAAT/enhancer binding protein (C/EBP) may also play an important role in airway inflammation. The purpose of this study was to measure DNA binding activity of these transcription factors in the airways of horses with RAO and to compare it to pulmonary function and bronchoalveolar lavage fluid (BALF) cytology. Seven horses with RAO and six control animals were studied during a moldy hay challenge and after 2 months at pasture. Pulmonary function, BALF cytology and transcription factors' activities in bronchial brushings were measured during hay and pasture exposures. During moldy hay challenge, RAO-affected horses developed severe airway obstruction and inflammation and a significantly higher airway AP-1 binding activity than in controls. After 2 months on pasture, pulmonary function and airway AP-1 binding activity were not different between RAO and control horses. The DNA binding activity of CREB in airways of RAO-affected horses increased significantly after 2 months at pasture and became higher than in controls. A significant positive correlation was detected between AP-1 binding activity and indicators of airway obstruction and inflammation. Airway GATA-3, CEBP and CREB binding activities were negatively correlated with indices of airway obstruction. However, contrarily to CREB binding activity, GATA-3 and CEBP binding activities were not different between RAO and control horses and were unaffected by changes in environment. These data support the view that AP-1 and CREB play a role in modulating airway inflammation in horses with RAO.

Effect of beclomethasone dipropionate and dexamethasone isonicotinate on lung function, bronchoalveolar lavage fluid cytology, and transcription factor expression in airways of horses with recurrent airway obstruction.

Glucocorticoid (GC) therapy is recognized to be effective for the treatment of recurrent airway obstruction (RAO) in horses. Anti-inflammatory properties of GC are thought to be mediated by suppression of inflammatory gene expression via inhibition of transcription factors such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). The purpose of this study was to evaluate the effect of low-dose inhaled beclomethasone dipropionate and injectable dexamethasone 21-isonicotinate on clinical signs, pulmonary function, airway cytology, and activity of NF-kappaB and AP-1 in bronchial cells of RAO-affected horses. Seven horses with RAO were exposed to moldy hay until they developed airway obstruction on 3 separate occasions. In a crossover design, they were then treated with a placebo (injection on day 1), inhaled beclomethasone (500 microg q12h for 10 days), or dexamethasone (0.06 mg/kg, IM on day 1) and monitored for 10 days. Pulmonary function, bronchoalveolar lavage fluid cytology, and NF-kappaB and AP-1 activity in bronchial brushing cells were measured before (day 1) and after treatment (day 10). Treatment with beclomethasone resulted in significantly improved pulmonary function of RAO-affected horses compared with placebo and dexamethasone treatments. However, none of the treatments had an effect on bronchoalveolar lavage fluid cytology or NF-kappaB and AP-1 activity. These findings reveal that, in a model of severe RAO, the benefits of low-dose inhaled beclomethasone on pulmonary function are not accompanied by a decrease in airway inflammatory cells or a suppression of transcription factors NF-kappaB and AP-1 DNA-binding activity.

Myeloperoxidase concentration in bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction.

The aim of this work was to measure the myeloperoxidase (MPO) concentration in bronchoalveolar lavage (BAL) fluid collected from horses with recurrent airway obstruction (RAO), both in crisis and in remission, as well as from healthy horses. Seven horses with RAO were exposed to moldy hay until the maximum change in pleural pressure was greater than 1.5 kPa. At that point, BAL was performed, and the total cell counts and percentages in the fluid were immediately determined. To measure the MPO concentration in BAL-fluid supernatant, we used a specific enzyme-linked immunosorbent assay with polyclonal antibodies against equine MPO. The tests were repeated on the horses with RAO after they had spent 2 mo on pasture. Six healthy horses serving as controls underwent the same tests. The absolute and relative neutrophil counts and the MPO concentration in the BAL fluid were significantly greater in the horses with an RAO crisis than in the control horses. After 2 mo on pasture, the horses that had been in RAO crisis were clinically normal, and their neutrophil counts and MPO levels in BAL fluid had significantly decreased; during remission their neutrophil counts were not significantly different from those in the healthy horses, but their MPO concentration remained significantly higher. This study showed that determining the MPO concentration in a horse's BAL fluid is technically possible and that during remission from RAO the concentration remains higher than normal. Thus, MPO may be a marker of neutrophil presence and activation in the lower airways.

Expression microarray as a tool to identify differentially expressed genes in horses suffering from inflammatory airway disease.

BACKGROUND: Inflammatory airway disease (IAD) affects performance and well-being of horses. Diagnosis is primarily reached by bronchoalveolar lavage (BAL) cytology which is invasive and requires sedation. OBJECTIVES: The purpose of this study was to identify differential gene expression in peripheral blood of horses with IAD using species-specific expression microarrays. METHODS: Equine gene expression microarrays were used to investigate global mRNA expression in circulating leukocytes from healthy, IAD-affected, and low-performing Standardbred and endurance horses. RESULTS: Nine genes in Standardbred and 61 genes in endurance horses were significantly differentially regulated (P < .001). These genes were related to inflammation (eg, ALOX15B, PLA2G12B, and PENK), oxidant/antioxidant balance (eg, DUOXA2 and GSTO1-1), and stress (eg, V1aR, GRLF1, Homer-2, and MAOB). All these genes were up-regulated, except down-regulated Homer-2 and MAOB. DUOXA2, ALOX15B, PLA2G12B, MAOB, and GRLF1 expression was further validated by RT-qPCR. An increase in glutathione peroxidase (GPx) activity in heparinized whole blood of IAD-affected Standardbred (P = .0025) and endurance horses (P = .0028) also suggests a deregulation of the oxidant/antioxidant balance. There was good correlation (r = .7354) between BAL neutrophil percentage and whole blood GPx activity in all horses. CONCLUSIONS: This study showed that circulating blood cell gene expression reflects inflammatory responses in tissues. Whether any of the genes have potential for diagnostic applications in the future remains to be investigated. Although not specific for IAD, whole blood GPx activity appears to be correlated with BAL neutrophil percentage. This finding should be further assessed by testing a larger number of horses.