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Clinical examination, bronchoalveolar lavage fluid (BALF) cytology, acute-phase protein, and pulmonary hemostasis and fibrinolysis marker (fibrinogen, serum amyloid A [SAA], and D-dimer) results were compared between control and respiratory disease-affected horses. Using a clinical scoring system, horses (n = 58) were classified as respiratory disease-free (Controls, n = 15) or with recurrent airway obstruction (RAO; n = 18), inflammatory airway disease (n = 14) or chronic interstitial pneumopathy (n = 11). There were no significant differences in fibrinogen concentrations among groups, but there was a trend toward a lower value in controls (median 0.0024 g/L) than in horses with chronic pneumopathies (median 0.0052 g/L), in particular, those with RAO (median 0.0062 g/L). Fibrinogen concentration was positively correlated with percentage of neutrophils in BALF ( r s = 0.377, p = 0.004). SAA concentrations were low; 65.5% of samples were below the detection limit. D-dimer concentrations were also low and quantifiable concentrations were only obtained after ultrafiltration and only in RAO (median 0.1 mg/L). In conclusion, there was limited evidence of increased coagulatory activity in chronic pneumopathies, apart from RAO. It is uncertain whether fibrinogen and D-dimer concentrations increased due to their role as acute-phase proteins or as a misbalance of coagulation and fibrinolysis.

Recurrent airway obstruction (RAO) represents a serious health problem and is traditionally classified as an allergic disease, where contact with an antigen can induce clinical airway inflammation, bronchial hyper-responsiveness and reversible airway obstruction. Previous studies have demonstrated the presence of the Th2 response in the lungs of human patients with asthma and horses with heaves. These cells are involved in the production of cytokines which regulate the synthesis of immunoglobulins. 40 horses were evaluated: 30 horses with RAO and 10 healthy animals. The expression levels of interferon-alpha 1 (IFN-α1), interferon-gamma (IFN-γ), interleukin-1β, (IL-1β), IL-2, IL-4, IL-13 and tumor necrosis factor alpha (TNF-α) were measured in the serum obtained from control and RAO-susceptible horses during crisis. In all the patients, serum cytokine levels were detected. Serum median IL-13 and IFN-γ levels were significantly higher in RAO-affected horses than in the healthy group (p < 0.001). The serum median IFN-α1, IL-1β, IL-2, IL-4, and TNF-α levels were similar in both groups. These results indicate a low variability of the levels of cytokines and a high frequency of their detection in serum samples from horses with RAO. Immune mechanisms involved in equine RAO are more complex than those defined by a simple Th1/Th2 dichotomy.

Recurrent airway obstruction (RAO) is a pulmonary inflammatory condition that afflicts certain mature horses exposed to organic dust particulates in hay. Its clinical and pathological features, manifested by reversible bronchoconstriction, excessive mucus production and airway neutrophilia, resemble the pulmonary alterations that occur in agricultural workers with occupational asthma. The immunological basis of RAO remains uncertain although its chronicity, its localization to a mucosal surface and its domination by a neutrophilic, non-septic inflammatory response, suggest involvement of Interleukin-17 (IL-17). We examined global gene expression profiles in mediastinal (pulmonary-draining) lymph nodes isolated from RAO-affected and control horses. Differential expression of > 200 genes, coupled with network analysis, supports an IL-17 response centered about NF-κB. Immunohistochemical analysis of mediastinal lymph node sections demonstrated increased IL-17 staining intensity in diseased horses. This result, along with the finding of increased IL-17 concentrations in lymph node homogenates from RAO-affected horses (P = 0.1) and a down-regulation of IL-4 gene and protein expression, provides additional evidence of the involvement of IL-17 in the chronic stages of RAO. Additional investigations are needed to ascertain the cellular source of IL-17 in this equine model of occupational asthma. Understanding the immunopathogenesis of this disorder likely will enhance the development of therapeutic interventions beneficial to human and animal pulmonary health.

Background: Systemic oxidative stress in horses with recurrent airway obstruction (RAO) is poorly characterized., Objectives: The goal of this study was to investigate whether equine RAO is associated with systemic disturbances in the oxidant-antioxidant equilibrium., Animals: Seven healthy horses and 7 horses with symptomatic RAO., Methods: A 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., Results: A 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 Importance: Our 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., (Copyright © 2014 by the American College of Veterinary Internal Medicine.)

Heaves, an obstructive neutrophilic airway inflammation of horses, is triggered by dust components such as endotoxin and has similarities to human asthma. Pulmonary intravascular macrophages (PIMs) increase horses' sensitivity to endotoxin-induced lung inflammation; however, their role in an airborne pathology remains unknown. Therefore, we investigated the role of PIMs in the development of heaves in horses. Clinical and inflammatory responses were evaluated following induction of heaves by moldy hay exposure and PIM depletion with gadolinium chloride (GC). Mares (N = 9) were exposed to four treatments: alfalfa cubes (Cb), alfalfa cubes + GC (Cb-GC), moldy hay (MH), and moldy hay + GC (MH-GC). Clinical scores and neutrophil concentrations in bronchoalveolar lavage (BAL) fluid were higher when mares received MH compared with MH-GC. BAL cells from MH-GC-treated mares had significantly lower IL-8 and TLR4 mRNA expression compared with MH-treated mares. In vitro LPS challenge significantly increased IL-8 but not TLR4 mRNA expression in BAL cells recovered from horses fed with MH, but not from the MH-GC treatment. In summary, PIM depletion attenuated clinical scores, reduced the alveolar migration of neutrophils, and decreased the expression of proinflammatory molecules in BAL cells of heaves horses, suggesting a proinflammatory role of PIMs in the development of airborne pathology.

Recurrent airway obstruction (RAO) in the horse is a disease characterized by reversible bronchoconstriction and by mucus and neutrophil accumulation in the airways. It has been hypothesized that in horses with RAO, remodeling changes occur that are similar to those described in humans with asthma. Although collagen fibrils are present surrounding normal airways, they are a prominent feature of airway remodeling in human asthma with evidence of enhanced collagen III and I fibril deposition. An immunolabeling method was developed to identify collagen I and III in equine lung and to describe the collagen fiber type and distribution within the walls of the noncartilagenous bronchioles. The health status of 14 horses was characterized by clinical respiratory exam, bronchoalveolar lavage cytology, and pulmonary function tests. Following postmortem examination and histological assessment, horses were divided into RAO-affected (n = 4) and nonaffected (n = 10) groups. Eight sections per horse from all lung regions were evaluated histologically. Results of the study showed that collagens I and III were present in the lamina propria and adventitial area of the noncartilaginous bronchioles. There was clear staining differentiation between collagen I or III, airway smooth muscle, and the airway epithelium. Collagen I and III were present in the lamina propria and adventitial areas of the noncartilaginous bronchioles of horses, and there was no significant difference in the relative amount of collagen I and III between this group of RAO-affected and nonaffected horses.

The long pentraxin 3 (PTX3) plays an important role in host defence and its over-expression may contribute to airway injury. The aim of the present study was therefore to characterize in more detail PTX3 and its expression in the horses' airway. Six healthy horses and six horses affected by recurrent airway obstruction (R.A.O.) were submitted to a dusty environment challenge. PTX3 DNA and cDNA were cloned and sequenced. PTX3 expression was evaluated by RT-qPCR, Western blotting and immunohistochemistry in bronchoalveolar lavage fluid (BALF) cells, BALF supernatant and bronchial epithelial cells. An alternative splicing of the second exon of PTX3 occurred, resulting in two forms of the protein: "spliced" (32 kDa) and "full length" (42 kDa). PTX3 was detected in BALF macrophages, neutrophils and bronchial epithelial cells. It was over-expressed in the BALF supernatant from R.A.O.-affected horses in crisis. However, dust was unable to induce PTX3 in BALF cells ex vivo, indicating that dust is an indirect inducer of PTX3. Dust exposure in-vivo induced PTX3 in BALF macrophages but there was no significant difference between healthy and R.A.O.-affected horses. Conversely, PTX3 was over-expressed in the bronchial epithelial cells from R.A.O.-affected horses in crisis. These data indicate a differential regulatory mechanism in inflammatory and bronchial epithelial cells and offer therapeutically interesting perspectives., (INRA, EDP Sciences, 2009)

Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horses. Recombinant equine CCSP and specific antiserum were produced, and percutaneous lung biopsies were obtained from 3 healthy horses and from 3 RAO-affected horses before and after induction of RAO. CCSP relative gene expression in tissue, as well as protein concentration in lung lavage fluid, was determined. Immunocytochemical analysis, using both light and immunogold ultrastructural methods, demonstrated reduced CCSP staining in lung tissue of animals with RAO. Immunogold label in Clara cell granules was less in animals with chronic RAO than in normal animals, and absent in animals that had active disease. Median lung lavage CCSP concentration was 132 and 129 ng/ml in healthy horses, and 62 and 24 ng/ml in RAO horses before and after challenge, respectively. CCSP lung gene expression was significantly higher in healthy animals than in animals with chronic RAO. Together, these preliminary findings suggest that reduced production of CCSP and subcellular changes in Clara cells are features of chronic environmentally induced lung inflammation in horses.

In recurrent airway obstruction (RAO) or heaves, bronchospasm has been attributed to enhanced cholinergic activity. However, the expression and function of muscarinic acetylcholine receptors (mAChR) and their signaling components are not yet known. Thus, we examined the expression, subtype distribution and postreceptor signaling pathways of mAChR in the peripheral lung, bronchial and tracheal epithelia with the underlying smooth muscle from nine horses with RAO and 11 healthy control horses. In RAO horses, no significant segment-dependent alteration in mAChR density and subtype distribution (assessed by [N-methyl-3H]-scopolamine binding; ([3H]-NMS)), was found, except a trend in receptor down-regulation in some peripheral parts of the lung. The total number of high mAChR binding sites (assessed by carbachol-displacement experiments in the presence or absence of guanosine 5'-triphosphate) was not changed in RAO, suggesting that the functional coupling of mAChR to the corresponding G-proteins is intact. The M2-mediated inhibition of adenylate cyclase (AC) as well as the M3-receptor-G(q/11)-phospholipase C (PLC) activity was not different between RAO and control airway tissues. In conclusion, in equine RAO airways, mAChR expression and function were not altered, and thus appear not to account for the enhanced cholinergic activity in RAO.

The aim of this study was to assess the effects of changes to the stable environment on exhaled markers of respiratory inflammation in six horses with clinical histories of recurrent airway obstruction. The horses were maintained for two weeks under conventional stable management (straw bedding and hay) and for two weeks on a reduced-dust regimen (paper bedding and ensiled grass), in a crossover study design. Exhaled ethane and carbon monoxide (CO) and exhaled breath condensate hydrogen peroxide (H(2)O(2)) were measured every three days under each regimen. The presence of clinical signs of airway inflammation (nasal discharge and cough) was monitored daily. The reduced-dust regimen was associated with fewer clinical signs of airway inflammation than the conventional regimen. Exhaled ethane and CO were significantly lower on the reduced-dust regimen and these markers were correlated with clinical signs of respiratory inflammation, but exhaled H(2)O(2) was not affected by the management regimen.

Reasons for Performing Study: Increased mucin gene expression may be an important cause of mucus accumulation observed in recurrent airway obstruction (RAO)-affected horses. To date, however, no mucin gene sequences are available for the horse., Objectives: To identify equine homologues of gel-forming mucins and investigate their expression at different airway generations of healthy and RAO-affected horses., Methods: Two equine homologues were identified by cloning and sequencing fragments of equine (eq)MUC5AC and eqMUC2., Results: Semiquantitative RT-PCR on RNA from airways (generations 1, 5, 10, 15; small airways and parenchyma), stomach (glandular), and colon revealed that eqMUC5AC is expressed in equine stomach and in all of the airway samples. In contrast, eqMUC2 steady-state mRNA levels were detected in colon and very faintly in stomach, but not in airway tissue. EqMUC5AC expression was also compared to that of ZO-1, a tight junction protein, and eqMUC5AC/ZO-1 ratios were higher in RAO-affected compared to control horses at all airway generations., Conclusions: That eqMUC5AC is expressed in horse airways, but any expression of MUC2 is undetectable and unlikely to be of physiological consequence., Potential Relevance: EqMUC5AC up-regulation may be a primary mechanism responsible for mucus hypersecretion and accumulation in RAO.

Objective: To evaluate leukotriene (LT) biosynthetic capacity in lung tissue from healthy horses and horses with recurrent airway obstruction (RAO)., Sample Population: Lung parenchyma and airway specimens from 8 RAO-affected and 5 healthy horses., Procedure: Horses were stabled for > or = 72 hours. Blood was drawn before euthanasia, after which lung specimens were collected. Tissue strips from small airways and parenchyma were incubated in organ baths with the precursor LTA4 or stimulated with calcium ionophore A23187 or the tripeptide N-formyl-Met-Leu-Phe (fMLP), with or without exogenous arachidonic acid, in the presence of isolated blood neutrophils., Results: Stabling induced typical clinical signs of airway obstruction in RAO-affected horses but not control horses. When lung parenchyma or airway specimens from both groups of horses were incubated with calcium ionophore, with or without arachidonic acid, they did not form LT. In contrast, addition of LTA4 to both tissues resulted in conversion to LTB4, although concentrations of LTC4 were negligible in airways and parenchymal strips from healthy and RAO-affected horses. Incubation of airway and parenchymal strips with suspensions of autologous neutrophils did not influence formation of LT stimulated by calcium ionophore or fMLP, with or without exogenous arachidonic acid., Conclusions and Clinical Relevance: Results suggest that lung parenchyma and airway tissues themselves are not of substantial importance for LT formation in the lungs, although these tissues possessed some LTA4 hydrolase activity, enabling LTB4 formation. It may be speculated that LTB4 originates primarily from neutrophils and may play a role in the inflammatory events of RAO.

In most cells trans-activating NF-kappaB induces many inflammatory proteins as well as its own inhibitor, IkappaB-alpha, thus assuring a transient response upon stimulation. However, NF-kappaB-dependent inflammatory gene expression is persistent in asthmatic bronchi, even after allergen eviction. In the present report we used bronchial brushing samples (BBSs) from heaves-affected horses (a spontaneous model of asthma) to elucidate the mechanisms by which NF-kappaB activity is maintained in asthmatic airways. NF-kappaB activity was high in granulocytic and nongranulocytic BBS cells. However, NF-kappaB activity highly correlated to granulocyte percentage and was only abrogated after granulocytic death in cultured BBSs. Before granulocytic death, NF-kappaB activity was suppressed by simultaneous addition of neutralizing anti-IL-1beta and anti-TNF-alpha Abs to the medium of cultured BBSs. Surprisingly, IkappaB-beta, whose expression is not regulated by NF-kappaB, unlike IkappaB-alpha, was the most prominent NF-kappaB inhibitor found in BBSs. The amounts of IkappaB-beta were low in BBSs obtained from diseased horses, but drastically increased after addition of the neutralizing anti-IL-1beta and anti-TNF-alpha Abs. These results indicate that sustained NF-kappaB activation in asthmatic bronchi is driven by granulocytes and is mediated by IL-1beta and TNF-alpha. Moreover, an imbalance between high levels of IL-1beta- and TNF-alpha-mediated IkappaB-beta degradation and low levels of IkappaB-beta synthesis is likely to be the mechanism preventing NF-kappaB deactivation in asthmatic airways before granulocytic death.

To test the hypothesis that reactive oxygen species could be associated to the lower airway disorders occurring in horses suffering from recurrent airway obstruction (RAO), indices of oxidative stress were studied in blood and pulmonary epithelium lining fluid in 5 RAO horses either in clinical remission or 24 h after the onset of a crisis of bronchospasm and in 5 healthy horses. Venous blood and bronchoalveolar lavage fluid (BALF) samples were collected and analysed for reduced glutathione (GSH), oxidised glutathione (GSSG), total glutathione (TGSH), glutathione redox ratio (GRR) in blood haemolysate and pulmonary epithelium lining fluid (PELF). The haemolysate concentrations of GSH, GSSG, TGSH and GRR were similar in the 3 groups. The PELF glutathione status was significantly different in the RAO horses in acute crisis compared to healthy horses, indicating the occurrence of an oxidative stress. When RAO horses were in crisis their GSH and TGSH remained unchanged but their GSSG and GRR were significantly increased compared to the remission. These results support the hypothesis that oxidative stress is associated with lower airway disorders occurring in horses suffering from RAO.

Heaves is a respiratory disorder of horses and ponies characterized by bouts of acute airway obstruction and airway hyperresponsiveness. We measured prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (15-HETE) production in vitro in tracheal epithelium obtained from six affected horses at the time of acute airway obstruction as compared with six matched control horses. Strips of epithelium and subepithelial tissue were prepared and stimulated with A23187, histamine, and bradykinin. The PGE2 and 15-HETE in media from strips was quantitated by radioimmunoassay. 15-HETE above the limits of accurate detection was found in epithelial strips of only two principal animals and in none of the control horses, and the amount of 15-HETE was not increased when strips were stimulated. Epithelial strips from affected horses tended to produce less PGE2 than did strips from control horses, and there was a significant correlation between epithelial PGE2 production and the time taken for affected animals to develop airway obstruction. Subepithelial tissue strips from control horses produced significantly more PGE2 in response to A23187 and bradykinin than did strips from affected horses. We conclude that equine tracheal epithelium is not a significant source of 15-HETE. Airway mucosal PGE2 production is reduced in horses with heaves, which suggests that a relative decrease in this bronchorelaxant substance may be a factor in the pathogenesis of this model of asthma.