Coughing,mucus accumulation,airway obstruction,and airway inflammation in control horses and horses affected with recurrent airway obstruction

OBJECTIVE: To investigate relationships between cough frequency and mucus accumulation, airway obstruction, and airway inflammation and to determine effects of dexamethasone on coughing and mucus score. ANIMALS: 13 horses with recurrent airway obstruction (RAO) and 6 control horses. PROCEDURE: 6 RAO-affected and 6 control horses were stabled for 3 days. Coughing was counted for 4 hours before and on each day horses were stabled. Before and on day 3 of stabling, tracheal mucus accumulation was scored, airway obstruction was assessed via maximal change in pleural pressure (deltaPpl(max)), and airway inflammation was evaluated by use of cytologic examination of bronchoalveolar lavage fluid (BALF). Effects of dexamethasone (0.1 mg/kg, IV, q 24 h for 7 days) were determined in 12 RAO-affected horses. RESULTS: To assess frequency, coughing had to be counted for 1 hour. In RAO-affected horses, stabling was associated with increases in cough frequency, mucus score, and deltaPpl(max). Control horses coughed transiently when first stabled. In RAO-affected horses, coughing was correlated with deltaPpl(max), mucus score, and airway inflammation and was a sensitive and specific indicator of deltaPpl(max) > 6 cm H2O, mucus score > 1.0, and > 100 neutrophils/microL and > 20% neutrophils in BALF Dexamethasone reduced cough frequency, mucus score, and deltaPpl(max), but BALF neutrophil count remained increased. CONCLUSIONS AND CLINICAL RELEVANCE: Because of its sporadic nature, coughing cannot be assessed accurately by counting during brief periods. In RAO-affected horses, coughing is an indicator of airway inflammation and obstruction. Corticosteroid treatment reduces cough frequency concurrently with reductions in deltaPpl(max) and mucus accumulation in RAO-affected horses.     

Mucin genes in horse airways: MUC5AC,but not MUC2, may play a role in recurrent airway obstruction

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.     

Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO‐affected and control horses

Reasons for performing study: Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. Objectives: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO‐affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. Methods: We performed quantitative RT‐PCR of eqMUC5AC, CLCA1 and EGFR; volume density measurements of intraepithelial mucosubstances; and cytological differentiation of intraluminal inflammatory cells in small cartilaginous airways from cranial left and right and caudal left and right lung lobes of 5 clinically healthy and 5 RAO‐affected horses that had been exposed to indoor stable environment for 5 days before euthanasia. Results: Neutrophils were increased in RAO‐affected horses compared to clinically healthy controls. EqMUC5AC mRNA levels were positively correlated with both CLCA1 and EGFR mRNA levels in RAO‐affected horses but only with CLCA1 in controls. The relationship between eqMUC5AC and CLCA1 differed in the 2 groups of horses with RAO‐affected animals overexpressing CLCA1 in relation to eqMUC5AC. Conclusions: These data implicate CLCA1 as a signalling molecule in the expression of eqMUC5AC in horses but also suggest differential regulation by CLCA1 and EGFR between horses with RAO and those with milder degrees of airway inflammation.     

The effect of adding oral dexamethasone to feed alterations on the airway cell inflammatory gene expression in stabled horses affected with recurrent airway obstruction

BACKGROUND: Chemokine expression in airway epithelium and bronchoalveolar lavage fluid (BALF) cells of horses with recurrent airway obstruction (RAO) is increased. HYPOTHESIS: For RAO-affected horses that are stabled and fed a pelleted ration, the addition of oral dexamethasone further improves pulmonary function and reduces inflammatory gene expression in pulmonary cells. ANIMALS: Twelve RAO-affected horses. METHODS: In a randomized cross-over experiment, the effect of feeding pellets in lieu of hay to stabled, RAO-affected horses was compared with the effect of feeding pellets and administering a 21-day decreasing dose regimen of oral dexamethasone on the expression (by kinetic polymerase chain reaction) of interleukin-8 (IL-8), chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1beta, IL-6, and beta-actin in the BALF cells and of IL-8, CXCL2, 2 IL-1 receptor (IL-1R2), Toll-like receptor 4 (TLR4), and glyceraldehyde 3-phosphate dehydrogenase in the bronchial epithelium 2 days after the final dose. RESULTS: Both treatments reduced airway neutrophilia and breathing efforts but the addition of dexamethasone was associated with fewer treatment failures. Compared with feed changes alone, dexamethasone administration further reduced the expression of IL-8, CXCL2, and IL-1beta in the BALF cells 3.3-, 2.5-, and 4.7-fold, respectively. In the airway epithelium, both treatments were equally efficacious in reducing the expression of IL-8 and CXCL2 expression relative to pretreatment values, but either treatment failed to alter the expression of IL-1R2 and TLR4. CONCLUSIONS AND CLINICAL IMPORTANCE: For a rapid and consistent improvement in pulmonary function and a reduction in inflammatory gene expression of the BALF cells, a decreasing dose of oral dexamethasone in combination with feed alterations is more efficacious for horses that must remain stabled.     

Neutrophil and platelet activation in equine recurrent airway obstruction is associated with increased neutrophil CD13 expression, but not platelet CD41/61 and CD62P or neutrophil–platelet aggregate formation

Recurrent airway obstruction (RAO) in mature horses is characterized by reversible airway obstruction and neutrophilic inflammation; there is also functional activation of circulating platelets and neutrophils. This study was undertaken to determine if changes in activation marker expression and heterotypic aggregate formation can be used as an indicator of this increased functional responsiveness.In vitro conditions for flow cytometric measurement of CD13, CD41/61 and CD62P expression on activated cells and heterotypic aggregate formation were established. Values were then compared before and after antigen challenge of RAO and healthy horses. Platelet adhesion to serum-coated plastic was measured as a functional marker of platelet activation.In vitro activation resulted in increased expression of neutrophil CD13 and platelet CD41/61 and CD62P. Activation of both cell types caused a significant increase in neutrophil–platelet aggregates.In horses with RAO, but not controls, there was a significant increase in the percentage of CD13 positive neutrophils at 10 h and 24 h and in the mean fluorescence intensity at 10 h. This was accompanied at 24 h by an increased mean platelet side scatter and thrombin-stimulated platelet adhesion.In conclusion, CD13 expression can be used as an indicator of equine neutrophil activation both in vitro and in vivo. Equine platelet activation in vitro can be detected by measuring CD41/61 or CD62P expression, and PAF-activated platelets and neutrophils form aggregates. However, despite evidence of circulating platelet activation, neither a change in expression of platelet activation markers, nor heterotypic aggregate aggregate formation could be detected.     

Effects of in vitro exposure to hay dust on expression of interleukin-17, -23, -8, and -1beta and chemokine (C-X-C motif) ligand 2 by pulmonary mononuclear cells isolated from horses chronically affected with recurrent airway disease

OBJECTIVE: To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or beta-glucan on cytokine expression in pulmonary mononuclear cells isolated from healthy horses and horses with recurrent airway obstruction (RAO). ANIMALS: 8 RAO-affected and 7 control horses (experiment 1) and 6 of the RAO-affected and 5 of the control horses (experiment 2). PROCEDURES: Bronchoalveolar lavage cells were isolated from horses that had been stabled and fed dusty hay for 14 days. Pulmonary mononuclear cells were incubated for 24 (experiment 1) or 6 (experiment 2) hours with PBS solution or solutions of hay dust, beta-glucan, or LPS. Gene expression of interleukin (IL)-17, IL-23(p19 and p40 subunits), IL-8, IL-1beta, and chemokine (C-X-C motif) ligand 2 (CXCL2) was measured with a kinetic PCR assay. RESULTS: Treatment with the highest concentration of hay dust solution for 6 or 24 hours increased expression of IL-23(p19 and p40), IL-8, and IL-1beta in cells from both groups of horses and increased early expression of IL-17 and CXCL2 in RAO-affected horses. Lipopolysaccharide upregulated early expression of IL-23(p40) and IL-8 in cells from both groups of horses but only late expression of these cytokines in cells from RAO-affected horses. Treatment with beta-glucan failed to increase cytokine expression at 6 or 24 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Cells from RAO-affected horses were not more responsive to the ligands tested than were cells from control horses, which suggests a minimal role of mononuclear cells in propagation of airway neutrophilia in horses with chronic RAO.