Equine Recurrent Airway Obstruction Research Articles

Effect of lipopolysaccharide on the responsiveness of equine bronchial tissue

Recurrent airway obstruction (RAO) is a main characteristic of horses with severe equine asthma syndrome. The presence of bacterial lipopolysaccharide (LPS) in the airways of horses is thought to play a crucial role in the clinical expression of this disorder. This study pharmacologically characterized the effect of LPS on the responsiveness of equine bronchial tissue. Equine isolated bronchi were incubated overnight with LPS (0.1–100 ng/ml) and then stimulated by electrical field stimulation (EFS). The role of capsaicin sensitive-sensory nerves (capsaicin desensitization treatment), neurokinin-2 (NK2) receptors (blocked by GR159897), transient receptor potential vanilloid type 1 receptors (TRPV1; blocked by SB366791), and neurokinin A (NKA) were investigated. Untreated bronchi were used as control tissues. LPS (1 ng/ml) significantly increased the EFS-evoked contractility of equine bronchi compared with control tissues (+742 ± 123 mg; P < 0.001). At higher concentrations LPS induced desensitization to airways hyperresponsiveness (AHR; EC50: 5.9 ± 2.6 ng/ml). Capsaicin desensitization and GR159897 significantly prevented AHR induced by LPS at EFS1–50Hz (−197 ± 25%; P < 0.01). SB366791 inhibited AHR at very low EFS frequency (EFS1Hz −193 ± 29%; P < 0.01 vs. LPS-treated bronchi). LPS (1 ng/ml) significantly (P < 0.01) increased 3.7 ± 0.7 fold the release of NKA compared with control bronchi. LPS induces biphasic dysfunctional bronchial contractility due to the stimulation of capsaicin sensitive-sensory nerves, increased release of NKA, and activation of NK2 receptors, whereas TRPV1 receptors appear to play a marginal role in this response. The overnight challenge with low concentrations of LPS represents a suitable model to investigate pharmacological options that may be of value in the treatment of equine RAO.

A genome-wide association study for equine recurrent airway obstruction in European Warmblood horses reveals a suggestive new quantitative trait locus on chromosome 13.

Recurrent airway obstruction (RAO), also known as heaves, is an asthma-like respiratory disease. Its development is strongly influenced by environmental risk factors such as sensitization and exposure to moldy hay, straw bedding and stabling indoors. A hereditary component has been documented in previous studies; however, so far no causative genetic variant that influences the risk of developing RAO has been identified. In this study, we revised an existing dataset and selected 384 horses for genotyping on the Affymetrix high-density equine SNP array. We performed an allelic case-control genome-wide association study, which revealed a suggestively significant association on equine chromosome 13 at 32843309bp. This SNP is located in the protein-coding gene TXNDC11, which is possibly involved in the folding process of the multiprotein complexes DUOX1 and DUOX2. In humans, these proteins are known to take part in regulating the production of H2 O2 in the respiratory tract epithelium as well as in MUC5AC mucin expression. Therefore, TXNDC11 may be considered a functional candidate gene, and further research is needed to explore its potential role in RAO-affected horses.

Mechanisms of equine recurrent airway obstruction: changes in understanding and management

Recurrent airway obstruction (RAO) is an inflammatory and obstructive disease that is more prevalent in horses stabled for long periods. The most common clinical signs include cough, exercise intolerance, dyspnea, nasal secretion and absence of fever. The pathophysiological effects of RAO in the lungs include goblet cell metaplasia, alveolar fibrosis, neovascularization, airway wall thickening affecting all tissue layers, and bronchial smooth muscle hypertrophy. The alveolar macrophage is the main effector of particle elimination in the inflammatory process of RAO, with the ability to increase or suppress inflammatory responses. Activation of macrophages increases the release of cytokines that enhance the inflammatory reaction, such as interleukin-1, tumor necrosis factor alpha, and interleukin-8, resulting in the attraction of neutrophils to the alveolar environment, which becomes the predominant cell type during periods of crisis. Although the clinical signs of RAO are well-defined, the underlying immunologic mechanisms are still being investigated. This review provides information about this disease, the understanding of which has changedover time.

Metalloproteinases and their inhibitors are influenced by inhalative glucocorticoid therapy in combination with environmental dust reduction in equine recurrent airway obstruction

BackgroundOverexpression of matrix-metalloproteinases (MMPs) has been shown to lead to tissue damage in equine recurrent airway obstruction (RAO), as a misbalance with their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), occurs. This favors irreversible pulmonary fibrosis formation. Increased levels of MMPs, TIMPs or altered ratios between them can be used as biomarkers of respiratory disease. We hypothesized that levels of MMPs, TIMPs and their ratios correlate with improvement in clinical findings and bronchoalveolar lavage fluid (BALF) cytology after 10 days of inhalative glucocorticoid therapy and environmental dust reduction (EDR) and may be used to monitor treatment success.Ten horses with a history of RAO participated in a prospective clinical study. Clinical and cytological scoring was performed before and after inhalative therapy using budesonide (1500 μg BID over 10 days) and EDR (bedding of wood shavings and wet hay as roughage). Gelatin zymography was performed for qualitative and semi-quantitative evaluation of MMP-2 and MMP-9 in BALF supernatant, while fluorimetry was used to evaluate MMP-8 activity. Additionally, specific equine ELISA assays were used for quantitative assessment of MMP-2, MMP-9, TIMP-1 and TIMP-2.ResultsA significant reduction in the total and several single parameters of the clinical score were found after 10 days of inhalative therapy and EDR. The concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 (ELISA) as well as their activities (MMP-2 and MMP-9 zymography and MMP-8 fluorimetry) were significantly decreased after therapy. Significant improvements in MMP-8/TIMP-1 and MMP-8/TIMP-2 ratios were also found, differences between other ratios before and after therapy were insignificant.ConclusionsMetalloproteinases and their inhibitors, in particular MMP-9 and TIMP-2, are valuable markers for clinical improvement in RAO.

Intratracheal therapy with autologous bone marrow-derived mononuclear cells reduces airway inflammation in horses with recurrent airway obstruction

This research evaluated the effects of bone marrow-derived mononuclear cells (BMMCs) on the inflammatory process in the equine recurrent airway obstruction (RAO). Eight horses in RAO clinical score were divided into cell therapy group (Gcel) treated with a single intratracheal dose of BMMCs, and dexamethasone group (Gdex) treated with 21days of oral dexamethasone. The horses were clinically revaluated on days 7 and 21, together with cytological evaluation of the BALF, and detection of inflammatory markers (interleukins [IL]-10, -4, and -17, and interferon γ and α). There were decreases in respiratory effort and clinical score on days 7 and 21(p<0.05) for both groups. The percentage of neutrophils decreased and macrophages increased on days 7 and 21 (p<0.005) in both groups. IL-10 levels increased in the Gcel group on day 21 compared to days 0 and 7 (p<0.05), but this was not observed in the Gdex group. The quantification of IL-4, IL-17, IFN-γ, and IFN-α did not change between evaluations in both groups. These preliminary results suggest that BMMCs may ameliorate the inflammatory response of RAO.

Procalcitonin Under the Course of Budesonide Inhalation Therapy in Recurrent Airway Obstruction

Procalcitonin (PCT), a precursor protein of the hormone calcitonin, is a sensitive inflammatory marker useful in diagnosis of exacerbation of asthma and chronic obstructive pulmonary disease. In this study, PCT was evaluated as a potential biomarker for the success of budesonide inhalation therapy in equine recurrent airway obstruction (RAO). Twelve horses suffering from RAO were included in a prospective clinical study. Clinical examinations, exercise test, blood gas analysis, endoscopy, bronchoalveolar lavage fluid cytology, and thoracic radiography were performed before and after therapy and results included in a scoring system. Inhalative therapy using budesonide at a dosage of 1,500 μg twice daily was performed over 10 days. Equine-specific enzyme-linked immunosorbent assays were used to evaluate concentrations of PCT as well as interleukins-1ß and 6 in bronchoalveolar lavage fluid. A significant reduction in clinical score, in particular in dyspnea, amount, and viscosity of tracheal secretion, was found after 10 days of inhalation (P = .005). For PCT, no difference was found before and after therapy. The median PCT concentration increased insignificantly from 13.85 (6.8–42.09) ng/mL to 16.47 (2.04–151.01) ng/mL after therapy (P = .158). In conclusion, PCT does not seem to be a useful marker to monitor treatment success of glucocorticoid inhalation in RAO.

Analysis of genomic copy number variation in equine recurrent airway obstruction (heaves).

We explored the involvement of genomic copy number variants (CNVs) in susceptibility to recurrent airway obstruction (RAO), or heaves-an asthmalike inflammatory disease in horses. Analysis of 16 RAO-susceptible (cases) and six RAO-resistant (control) horses on a custom-made whole-genome 400K equine tiling array identified 245 CNV regions (CNVRs), 197 previously known and 48 new, distributed on all horse autosomes and the X chromosome. Among the new CNVRs, 30 were exclusively found in RAO cases and were further analyzed by quantitative PCR, including additional cases and controls. Suggestive association (P=0.03; corrected P=0.06) was found between RAO and a loss on chromosome 5 involving NME7, a gene necessary for ciliary functions in lungs and involved in primary ciliary dyskinesia in humans. The CNVR could be a potential marker for RAO susceptibility but needs further study in additional RAO cohorts. Other CNVRs were not associated with RAO, although several involved genes of interest, such as SPI2/SERPINA1 from the serpin gene family, which are associated with chronic obstructive pulmonary disease and asthma in humans. The SPI2/SERPINA1 CNVR showed striking variation among horses, but it was not significantly different between RAO cases and controls. The findings provide baseline information on the relationship between CNVs and RAO susceptibility. Discovery of new CNVs and the use of a larger population of RAO-affected and control horses are needed to shed more light on their significance in modulating this complex and heterogeneous disease.

Pulmonary Remodeling in Equine Asthma: What Do We Know about Mediators of Inflammation in the Horse?

Equine inflammatory airway disease (IAD) and recurrent airway obstruction (RAO) represent a spectrum of chronic inflammatory disease of the airways in horses resembling human asthma in many aspects. Therefore, both are now described as severity grades of equine asthma. Increasing evidence in horses and humans suggests that local pulmonary inflammation is influenced by systemic inflammatory processes and the other way around. Inflammation, coagulation, and fibrinolysis as well as extracellular remodeling show close interactions. Cytology of bronchoalveolar lavage fluid and tracheal wash is commonly used to evaluate the severity of local inflammation in the lung. Other mediators of inflammation, like interleukins involved in the chemotaxis of neutrophils, have been studied. Chronic obstructive pneumopathies lead to remodeling of bronchial walls and lung parenchyma, ultimately causing fibrosis. Matrix metalloproteinases (MMPs) are discussed as the most important proteolytic enzymes during remodeling in human medicine and increasing evidence exists for the horse as well. A systemic involvement has been shown for severe equine asthma by increased acute phase proteins like serum amyloid A and haptoglobin in peripheral blood during exacerbation. Studies focusing on these and further possible inflammatory markers for chronic respiratory disease in the horse are discussed in this review of the literature.

Serum 8-hydroxy-2-deoxyguanosine as a marker of DNA oxidative damage in horses with recurrent airway obstruction.

BackgroundIt has been reported that equine recurrent airway obstruction (RAO) is a state of oxidative stress. Oxidant-antioxidant imbalance is known to increase the conversion of deoxyguanosine to 8-hydroxy-2-deoxyguanosine (8-OHdG) in DNA. 8-OHdG can easily be measured using ELISA tests in serum or urine samples. In this study, we analysed serum 8-OHdG levels in horses with recurrent airway obstruction and in healthy controls.ResultsThe study material consisted of seven healthy horses and seven horses with symptomatic RAO. All horses were exposed to moldy hay and straw for 48 h to induce clinical exacerbation of RAO. The serum 8-OHdG levels were determined using the ELISA Highly Sensitive 8-OHdG kit. The difference between the levels of 8-OHdG in healthy and RAO-affected horses was significant. The median level of 8-OHdG was 0.044 ng/ml in the healthy controls versus 0.498 ng/ml in RAO horses (P = 0.0021).ConclusionsThe results of the study strongly suggest that DNA damage coexists in the course of equine RAO. We therefore propose that future research should aim at the development of new drugs that target pro-inflammatory molecules, since DNA damage appears to be the result of chronic inflammation.

Tamoxifen as a new therapeutic tool for neutrophilic lung inflammation.

Neutrophilic asthma is an important disease subgroup, including patients with severe phenotypes and erratic responses to standard treatments. Tamoxifen (TX), a selective estrogen receptor modulator (SERM) used as treatment of human breast cancer, has been shown to induce early apoptosis of equine blood and bronchoalveolar lavage fluid (BALF) neutrophils in vitro. Equine recurrent airway obstruction (RAO) is a naturally occurring neutrophilic condition, closely related with human asthma. Our purpose was to investigate the therapeutic potential of tamoxifen in horses with neutrophilic lung inflammation. Twelve horses underwent acute lung inflammation through exposure to allergens known to cause RAO, after which they received treatment with either tamoxifen or dexamethasone. Outcome measures included evaluation of clinical signs, BALF cytology, and early apoptosis of blood and BALF neutrophils. Tamoxifen treatment decreased BALF neutrophil counts (65.3 ± 19.38% before treatment; 7.6 ± 4.5% 2 days post-treatment,; and 13.6 ± 9.3% 5 days post-treatment). A similar decrease was observed with dexamethasone treatment (48.6 ± 5.88% before treatment; 11.5 ± 8.1% 2 days post-treatment; 14.6 ± 10.3% 5 days post-treatment). Clinical and endoscopic scores improved in both treatment groups. Tamoxifen treatment significantly increased early apoptosis of peripheral blood neutrophils at 5 days post-treatment (27.04 ± 15.2%), and in BALF neutrophils at 2 and 5 days post-treatment (42.11 ± 11.67% and 48.98 ± 2.6%, respectively). Tamoxifen treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction in BALF neutrophils and improvement in the animals' clinical status.

Serum concentrations of allergen-specific IgE in horses with equine recurrent airway obstruction and healthy controls assessed by ELISA.

Equine recurrent airway obstruction (RAO), also known as heaves, is one of the most common respiratory problems in older horses. When RAO-affected horses stay pastured or in a dust-free environment for a prolonged time, clinical signs as well as airway inflammation wane. A number of environmental, immunologic, infectious, and genetic factors play an important role in the pathogenesis of RAO, and the immunologic basis of this disease is still poorly understood. The aim of this study was to investigate the concentrations of allergen-specific IgE in the serum of horses suffering from RAO and healthy controls. The study included a group of 14 adult Polish Konik horses, kept in a standardized environment, and divided into 2 groups: 7 horses which did not have any respiratory problems comprised the control group and 7 horses with a history of RAO constituted the study group. A clinical and laboratory evaluation, endoscopic examination, and bronchoalveolar lavage (BAL) were performed in all horses. Sera of all horses were tested against allergens from 9 molds and 3 mites using the Heska Allercept assay. In the serologic tests, a statistically significant difference between both groups was found for specific IgE against mites, wherein Tyrophagus putrescentia correlated most clearly with RAO. There was no difference between groups for IgE specific against molds. On the basis of our observations and results, we conclude that RAO is associated with increased serum concentrations of specific serum IgE against mites, in particular Tputrescentia.

Equine Recurrent Airway Obstruction

Abstract Equine Recurrent Airway Obstruction (RAO), also known as heaves or broken wind, is one of the most common disease in middle-aged horses. Inflammation of the airway is inducted by organic dust exposure. This disease is characterized by neutrophilic inflammation, bronchospasm, excessive mucus production and pathologic changes in the bronchiolar walls. Clinical signs are resolved in 3-4 weeks after environmental changes. Horses suffering from RAO are susceptible to allergens throughout their lives, therefore they should be properly managed. In therapy the most importanthing is to eliminate dust exposure, administration of corticosteroids and use bronchodilators to improve pulmonary function.

Oxidant‐Antioxidant Status in the Blood of Horses with Symptomatic Recurrent Airway Obstruction (RAO)

BackgroundSystemic oxidative stress in horses with recurrent airway obstruction (RAO) is poorly characterized.ObjectivesThe goal of this study was to investigate whether equine RAO is associated with systemic disturbances in the oxidant‐antioxidant equilibrium.AnimalsSeven healthy horses and 7 horses with symptomatic RAO.MethodsA prospective study. Healthy and RAO‐affected horses were exposed to a 48‐hour challenge with moldy hay and straw to induce clinical exacerbation of RAO. Venous blood was collected and the activities of the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in equine erythrocyte lysates were measured. The concentration of thiobarbituric acid‐reactive substances (TBARSs) was assessed both in erythrocyte lysates and in plasma.ResultsA significant increase in the activities of GPx and SOD was detected in RAO‐affected horses compared with the control animals. There was no significant difference between groups in terms of the erythrocyte lysate activities of CAT, GR, or TBARs or the plasma concentration of TBARs.Conclusion and Clinical ImportanceOur results support the hypothesis that RAO in horses is associated with systemic oxidative stress. Future studies are needed to assess whether horses suffering from RAO can benefit from antioxidant supplementation.

Concomitant inhibition of primary equine bronchial fibroblast proliferation and differentiation by selective β2-adrenoceptor agonists and dexamethasone

Altered airway cell proliferation plays an important role in the pathogenesis of human bronchial asthma and chronic obstructive pulmonary disease (COPD) as well as the equine recurrent airway obstruction (RAO) with consistent changes, i.e. narrowing the airway wall, explained by proliferation and differentiation of fibroblasts. In permanent cell lines, it has been suggested that β2-adrenoceptor agonists and glucocorticoids regulate cell proliferation via the β2-adrenoceptor pathway; indeed, no study was carried out in fresh isolated primary equine bronchial fibroblasts (EBF). We characterized the β-adrenoceptors in EBF, and compared effects of long-acting (clenbuterol) and short-acting (salbutamol and isoproterenol) β2-agonists and dexamethasone on proliferation, differentiation and collagen synthesis. High density (Bmax; 5037±494 sites/cell) of β2-adrenoceptor subtype was expressed in EBF. β2-agonists inhibited concentration-dependently EBF proliferation with potency of clenbuterol>salbutamol »isoproterenol which was inhibited by ICI 118.551 and propranolol but not by CGP 20712A. In contrast, dexamethasone alone inhibited less EBF proliferation, but the effect was high when dexamethasone was combined with β2-agonists. Transforming growth factor-β1 (TGF-β1) increased transformation of fibroblasts into myofibroblasts, which was inhibited by clenbuterol and dexamethasone alone and drug combination resulted in high inhibition rate. Collagen synthesis in EBF was rather hampered by dexamethasone than by β-agonists. Collectively, the expression of β2-adrenoceptor subtype in EBF and the anti-proliferative effect of clenbuterol suggest that β2-adrenoceptors are growth inhibitory and anti-fibrotic in EBF. These β2-agonist effects in EBF were synergistically enhanced by dexamethasone, providing the additive effects of glucocorticoids to counteract airway remodelling and morbidity of asthma and RAO.

Secretoglobin 1A1 and 1A1A differentially regulate neutrophil reactive oxygen species production, phagocytosis and extracellular trap formation.

Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.

Effect of the ROCK inhibitor Y-27632 on Proliferation and Differentiation of Equine Bronchial Epithelial Cells

The airway epithelium plays an important role in physiological and pathological processes including inflammation, innate immunity and regenerative responses in the respiratory tract with regard to airway diseases such as human asthma, COPD and Equine Recurrent Airway Obstruction (RAO). Airway epithelial cell lines which would serve as an in vitro cell model are, indeed, missing for the horse. The objective of the study is to establish long-term equine bronchial epithelial cell cultures using the ROCK inhibitor Y-27632 and characterize them. Fresh isolated EBEC were cultured in the presence and absence of 10µM Y-27632 under conventional and air-liquid-interface culture conditions. Cell viability, morphology, proliferation and differentiation states were examined. Under conventional culture, Y-27632 induced higher growth rate of primary EBEC over a short period of time when compared to non-treated cells and increased the passage number. Epithelial cells grew faster and retain their epithelial cell morphology for a number of passages, with less contamination with fibroblasts. EBEC cultured on membrane inserts revealed high TEER values in the presence of Y-27632 and switching to Air-Liquid-Interface (ALI) did not cause decrement of TEER values. Inmunostaing of EBEC showed a different pattern of CK, TJP-1 and Vim expression indicating rapid differentiation of Y-27632-treated cells. Also, light and scanning electron microscopic imaging showed high amount of cilia, microvilli and ridges as well as more defined polygonal-shaped epithelial cells in the presence of Y-27632. Taken together, these results suggest that the ROCK inhibitor Y-27632 permits establishing conditional long-term in vitro airway epithelial cell models to investigate their role in equine airway diseases and as pharmacological targets.

Evaluation of Coughing and Nasal Discharge as Early Indicators for An Increased Risk to Develop Equine Recurrent Airway Obstruction (RAO)

BackgroundIt is often assumed that horses with mild respiratory clinical signs, such as mucous nasal discharge and occasional coughing, have an increased risk of developing recurrent airway obstruction (RAO).HypothesisCompared to horses without any clinical signs of respiratory disease, those with occasional coughing, mucous nasal discharge, or both have an increased risk of developing signs of RAO (frequent coughing, increased breathing effort, exercise intolerance, or a combination of these) as characterized by the Horse Owner Assessed Respiratory Signs Index (HOARSI 1–4).AnimalsTwo half‐sibling families descending from 2 RAO‐affected stallions (n = 65 and n = 47) and an independent replication population of unrelated horses (n = 88).MethodsIn a retrospective cohort study, standardized information on occurrence and frequency of coughing, mucous nasal discharge, poor performance, and abnormal breathing effort—and these factors combined in the HOARSI—as well as management factors were collected at intervals of 1.3–5 years.ResultsCompared to horses without clinical signs of respiratory disease (half‐siblings 7%; unrelated horses 3%), those with mild respiratory signs developed clinical signs of RAO more frequently: half‐siblings with mucous nasal discharge 35% (P < .001, OR: 7.0, sensitivity: 62%, specificity: 81%), with mucous nasal discharge and occasional coughing 43% (P < .001, OR: 9.9, sensitivity: 55%, specificity: 89%); unrelated horses with occasional coughing: 25% (P = .006, OR = 9.7, sensitivity: 75%, specificity: 76%).Conclusions and Clinical importanceOccasional coughing and mucous nasal discharge might represent an increased risk of developing RAO.

Participation of T regulatory cells in equine recurrent airway obstruction

Recurrent airway obstruction (RAO) is an equine immune-mediated disease with a high incidence worldwide. The aim of this work was to contribute to the understanding of RAO pathogenesis by studying T cells bearing regulatory markers in peripheral blood (PB) and in bronchoalveolar lavage fluid (BALF) recovered from the same group of susceptible horses before and after exposure to moldy hay, which has been shown to induce RAO signology in our horse herd. With this purpose, mononuclear cells were obtained from the BALF and PB from horses before and after antigenic challenge and were stained with fluorochrome-conjugated antibodies against CD4, CD25 and Foxp3 and subsequently analyzed by flow cytometry. The results indicated that the percentage of CD4+, Foxp3+ cells clearly increased in PB and BALF obtained from horses with RAO. In addition, the percentage of CD4+, CD25high cells was greatly augmented in BALF of RAO positive horses compared with a baseline. No changes were observed in the PB compartment. The percentage of CD4+, CD25high, Foxp3+ cells in BALF increased in horses with active disease compared to horses in remission; this cell population also does not show changes in the PB compartment when RAO positive and RAO negative horses were compared. On the other hand, when the percentage of CD4, Foxp3 positive cells were compared with the percentage of CD4+, CD25high cells, the numbers were very similar. This observation was true for PB and BALF cells from non exposed horses as well as horses exposed to antigen. In all the experimental situations studied, the population expressing all of the markers CD4+, CD25high, Foxp3+ represent only a minor percentage of CD4+, CD25high or CD4+, Foxp3 subpopulations; therefore, an significant number of CD4+, CD25high, Foxp3- and CD4+, CD25null, Foxp3+ cells must exist. Finally, we conclude that horses with RAO show an airway accumulation of T cells bearing regulatory markers that probably are modulating the course of this disease, and that these T cells may be involved in the resolution of immune-mediated bronchial inflammation.

Neutrophil and macrophage apoptosis in bronchoalveolar lavage fluid from healthy horses and horses with recurrent airway obstruction (RAO)

BackgroundDysregulation of apoptosis has been implicated in a range of diseases including tumors, neurodegenerative and autoimmine diseases, as well as allergic asthma and chronic obstructive pulmonary disease (COPD) in humans. Although it has a different pathophysiology, delayed apoptosis of various inflammatory cells may play a pivotal role in the development of recurrent airway obstruction (RAO) in horses. Reduction of inflammatory cell apoptosis or a dysregulation of this process could lead to chronic inflammation and tissue injury. Therefore, the aim of this study was to investigate the rate of apoptosis and necrosis of neutrophils and macrophages in bronchoalveolar lavage fluid obtained from seven horses suffering from RAO (study group) and seven control horses.ResultsWe demonstrated that neutrophil/macrophage apoptosis is altered in RAO-affected horses compared with the control group in the BAL fluid. We found a significant difference between the median percentage of early and late apoptosis of neutrophils between the study and control group of horses. Moreover, we found a positive correlation between the rate of apoptosis and the median percentage of macrophages in RAO-affected horses.ConclusionThe findings suggest that apoptosis dysregulation may play a significant role in the pathogenesis of RAO. However, further studies are needed to clarify the role of altered apoptosis in the course of equine recurrent airway obstruction.

Effect of hay dust extract and cyathostomin antigen stimulation on cytokine expression by PBMC in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is an inflammatory, obstructive airway disease induced by exposure of susceptible horses to inhaled organic dust particles. The immunological process underlying RAO is still unclear. Previous studies have shown that RAO is linked to the Interleukin-4 receptor (IL-4R) gene in one Warmblood family (F1), but not in another (F2). It has also been shown that in F1, but not in F2, RAO is associated with resistance against parasites, suggesting that this association may have an immuno-genetic basis. Therefore, we hypothesized that the T helper (h)1/Th2/regulatory (Treg) cytokine profiles of RAO-associated antigen- and parasite-antigen-stimulated peripheral blood mononuclear cells (PBMC) differ between RAO-affected and healthy horses depending on their genetic background. In our study, PBMC from 17 RAO-affected and 14 healthy control horses of F1 and F2 were stimulated for 24h with antigens relevant to RAO [hay dust extract (HDE), Aspergillus fumigatus extract (AFE) and lipopolysaccharids (LPS)]; cyathostomin extract (CE) and recombinant cyathostomin antigen (RCA) or with concanavalin A (ConA). Total mRNA levels of IL-4, IL-4R, IL-13, interferon (INF)-γ and IL-10 were examined by qRT-PCR. Stimulation with either HDE or RCA resulted in significant differences in IL-4R mRNA levels between RAO-affected and control horses in F1, but not in F2. For IL-10 mRNA expression, a significant difference between RAO-affected and control horses in F1 but not in F2 was observed only following stimulation with HDE. In contrast to HDE, stimulation with CE resulted in a significant difference of IL-10 mRNA expression level between RAO-affected horses of F2 and healthy horses of F1. No significant differences were detected upon stimulation with any of the other challenge agents. These findings indicate that the immunological response, specifically IL-4R expression, in response to hay dust and cyathostomin antigens, differs between RAO-affected and healthy horses depending on their genetic background. This study shows that analysis of PBMC reveals systemic changes associated with RAO and helps to elucidate immunological pathways involved in this disease.