Airway inflammation and mucus in two age groups of asymptomatic well-performing sport horses

REASONS FOR PERFORMING STUDY: Horses housed in conventional stable environments are exposed to high dust loads. Long-term exposure to inhaled dust may lead to subclinical airway disease. HYPOTHESES: Airway inflammation and mucus accumulation are 1) common in asymptomatic well-performing sport horses, 2) increased in older compared to younger individuals and 3) correlated. METHODS: Mucus quantity and quality (endoscopic scoring) and BALF differential cytology were assessed in 13 younger horses (mean age 5 years) and 13 older horses (mean age 15 years), which had no historical or clinical evidence of lower airway disease, were performing well and were housed permanently in a conventional stable environment. RESULTS: 1) Airway inflammation and/or mucus accumulation were very common. All sport horses showed evidence of subclinical inflammatory airway disease (IAD). 2) Older horses, having been exposed for 10 more years to a conventional stable environment, did not show increased subclinical airway inflammation or mucus accumulation. The only differences between the age groups were increased BALF total and lymphocyte cell counts in the younger horses. 3) Mucus quantity and quality scores were not significantly correlated with each other or with BALF neutrophil percentages and absolute numbers. CONCLUSIONS: Although clinically healthy and performing well, all of the examined horses housed in a conventional stable environment showed evidence of IAD. The degree of IAD was not increased in older horses that had not developed clinical signs. POTENTIAL RELEVANCE: The effect of subclinical degrees of IAD on performance in dressage and show jumping should be investigated further to better understand the clinical significance of IAD in sport horses.     

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-β, 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-β 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.     

Histamine bronchoprovocation does not affect bronchoalveolar lavage fluid cytology, gene expression and protein concentrations of IL-4, IL-8 and IFN-gamma

In diagnosing inflammatory airway disease (IAD) in performance horses, a histamine bronchoprovocation (HBP) test is often performed. In previously published studies, HBP is usually undertaken prior to cytological examination of the bronchoalveolar lavage (BAL) cells. The purpose of this study was to determine if HBP alters (1) the total nucleated cell numbers and distribution in BAL fluid (BALF) and (2) the mRNA and protein concentrations of selected cytokines in BAL cells and BALF, respectively. BALF was initially collected endoscopically from the right middle or diaphragmatic lung lobe in eight healthy young Standardbred horses. Five to six days later, HBP was performed by aerosolization of histamine (8mg) over a 2min period. BALF was again collected within 2-4h of the HBP from the left middle or diaphragmatic lung lobe. In both samples, total and differential WBC counts were obtained. The gene expressions of interleukin-4 (IL-4), IL-8, interferon-gamma (IFN-gamma) and beta-actin in BAL cells were measured using real-time RT-PCR. The cytokine protein concentrations were measured in the BALF using ELISA. HBP was not associated with either a change in the total BAL cell number or in the distribution of the BAL cells. BAL cell expression of IL-4, IL-8 and IFN-gamma, detected in all samples with the exception of IL-4 in one horse (post-HBP), was not altered as a result of HBP. HBP was not associated with a significant change in IL-8 or IFN-gamma concentrations in the BALF. IL-4 protein was undetectable in BALF either prior to or following HBP. We conclude that HBP can precede BALF collection performed within 2-4h of the former without affecting selected parameters analysed in the BAL cells or BALF.     

Serum concentration of surfactant protein D in horses with lower airway inflammation

Reasons for performing study: Surfactant protein D (SP‐D), mainly synthesised by alveolar type II cells and nonciliated bronchiolar cells, is one important component of innate pulmonary immunity. In man, circulating concentrations of SP‐D are routinely used as biomarkers for pulmonary injury. To date, serum SP‐D levels have only been investigated in horses in an experimental model of bacterial airway infection. Objectives: To compare serum SP‐D concentrations at rest and after exercise in horses with and without inflammatory airway disease (IAD). Methods: Venous blood samples were collected from 42 Standardbred racehorses at rest and 60 min after performing a standardised treadmill exercise test. Tracheal wash and bronchoalveolar lavage fluid (BALF) samples were collected after exercise. Based on BALF cytology, 22 horses were defined as IAD‐affected and 20 classified as controls. Serum SP‐D concentrations were assessed using a commercially available ELISA kit and statistically compared between groups of horses and sampling times. Results: Serum concentrations of SP‐D in IAD‐affected horses were significantly higher than those of control horses, both at rest and after exercise. Within the IAD‐affected group, no significant correlation was found between serum SP‐D concentrations and BALF cytology. Within each group of horses (IAD and control), no significant influence of exercise was found on serum SP‐D levels. Conclusions: This is the first study determining serum SP‐D concentrations in a noninfectious, naturally occurring form of lower airway inflammation in horses. The results highlight that IAD is associated with a detectable, though moderate, increase of circulating SP‐D levels. Potential relevance: Serum concentration of surfactant protein D could represent a potentially valuable and readily accessible blood biomarker of equine lower airway inflammation.     

The role of alveolar macrophages in the pathogenesis of recurrent airway obstruction in horses

When challenged with allergens and pro-inflammatory agents, such as Aspergillus fumigatus (AF), hay dust solution (HDS) and lipopolysaccharide (LPS), the innate immune response will not only activate the immune system but also increase the amount of pro-inflammatory cytokines in the bronchoalveolar space. The aim of this study was to assess the response of equine alveolar macrophages to different aerosolized challenges and to investigate the differences in this response between horses susceptible or nonsusceptible to recurrent airway obstruction (RAO). Seven susceptible and 5 nonsusceptible horses were challenged with saline, LPS, HDS, or AF, and bronchoalveolar lavage (BAL) cytology, total cell counts, and lung function were assessed. In addition, alveolar macrophages were isolated 6 and 24 hours after challenge, and macrophage mRNA expression of tumor necrosis factor (TNF)-alpha and interleukins (IL) IL-1beta, IL-6, IL-8, and IL-10 were measured by means of real-time (RT) polymerase chain reaction (PCR). There was a significant difference in lung function, neutrophil ratios, and total cell counts in the bronchoalveolar lavage fluid between RAO-susceptible and nonsusceptible horses. In addition, the expression of TNF-alpha, IL-1beta, and IL-8 by alveolar macrophages after challenges were higher in susceptible horses, than in nonsusceptible horses. In contrast, I1-6, considered an anti-inflammatory cytokine, showed a higher expression in nonsusceptible horses 6 hours after inhalation challenge with allergens and pro-inflammatory antigens. These data suggest that the differences between susceptible and nonsusceptible horses to RAO are not only dependent on adaptive immunity but also start with an innate immune response.     

IgG antibody responses to an inhaled antigen in horses with "heaves" (recurrent airway obstruction).

A controlled experimental system for the evaluation of pulmonary immune responses in horses with "heaves" (recurrent airway obstruction) has been developed. We hypothesized that the humoral immune response to an inhaled antigen in diseased horses would be different from that of healthy horses and that chronic pulmonary inflammation would bias the production of IgG isotypes in diseased horses as compared to healthy horses. Healthy and affected horses were housed in a natural challenge environment (stabled, fed dusty hay) and exposed by inhalation, to a nebulized solution of keyhole limpet hemocyanin (KLH). Sera and bronchoalveolar lavage fluids (BALFs) were collected from horses prior to and following their inhalation exposure to the antigen. Differential cell counts were performed on the cells in the BALF. An enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of IgGa, IgGb, IgG(T) and combined IgG specific for KLH in the sera and BALF. The percentages of neutrophils in the BALF of diseased horses were increased 4-6-fold over healthy horses. Combined IgG specific for KLH was significantly greater in BALF and serum from healthy compared to diseased horses. Differences in isotypes were also evident; however, only IgGb specific for KLH in the BALF was significantly increased in healthy versus diseased horses. Possible explanations for this difference include: (1) increased destruction of antigen before it could interact with lymphocytes, (2) down-regulation of IgGb production by inhibitory cytokines in diseased horses, or (3) binding of IgGb to Fc receptors on the large numbers of neutrophils in the lungs of diseased horses. In contrast to the prevailing notion that horses with heaves have exaggerated immune responses, our data suggest that diseased horses exposed to an aerosolized protein mount weaker IgG responses compared to healthy horses.     

Antioxidant and inflammatory responses of healthy horses and horses affected by recurrent airway obstruction to inhaled ozone

REASONS FOR PERFORMING STUDY: Inhaled ozone can induce oxidative injury and airway inflammation. Horses affected by recurrent airway obstruction (RAO) have a decreased pulmonary antioxidant capacity, which may render them more susceptible to oxidative challenge. It is currently unknown whether RAO-affected horses are more susceptible to oxidative stress than those unaffected by RAO. OBJECTIVES: To determine whether ozone exposure induces greater oxidative stress and airway inflammation in RAO-affected horses in remission than in healthy horses. METHODS: Seven healthy control horses and 7 RAO-affected horses were exposed to 0.8 ppm ozone for 2 h at rest. RESULTS: At baseline, bronchoalveolar lavage fluid (BALF) ascorbic acid concentrations were lower in RAO-affected horses than healthy controls. Ozone appeared to preferentially oxidise glutathione rather than ascorbic acid 6 h after exposure. Individual healthy and RAO-affected horses demonstrated oxidation of BALF glutathione after ozone exposure. Overall, RAO-affected horses did not demonstrate increased oxidative stress following ozone exposure, compared with healthy horses. Ozone did not induce significant airway inflammation in either group. CONCLUSIONS: RAO-affected horses in remission are not more sensitive to ozone despite a decreased pulmonary antioxidant capacity. Sensitivity to ozone appears to be independent of initial pulmonary antioxidant status. POTENTIAL RELEVANCE: Horses with high susceptibility to oxidative stress may benefit from antioxidant supplementation.     

Lung function and airway cytologic profiles in horses with recurrent airway obstruction maintained in low-dust environments

BACKGROUND: The effects of long-term environmental management on airway obstruction and inflammation in horses with recurrent airway obstruction (RAO) are unknown. HYPOTHESIS: Horses with RAO maintained in low-dust environments have persistent airway obstruction and neutrophilic inflammation. ANIMALS: Study horses were treated for RAO and then maintained in low-dust environments with no medical management. Horses were classified into 3 groups by years after diagnosis: 1 year (time 1, n = 9), 2-3 years (time 2, n = 7), and 5-6 years (time 3, n = 8). The comparison groups were age-matched healthy horses. METHODS: In this cross-sectional study, a clinical examination was performed, and the clinical score was calculated. Standard lung function, forced expiratory maneuvers, and the cytology of bronchoalveolar lavage fluid (BALF) were evaluated. RESULTS: The clinical scores of the RAO horses were higher than those of the non-RAO horses at time 2 (P = .018). Standard lung function data were not different between the groups at any time point. The forced expiratory flow between 75-95% of exhaled vital capacity was lower in RAO horses than in non-RAO horses at all time points (P < .02), indicating persistent peripheral airway obstruction. Cytologic evaluation of BALF revealed no difference in total nucleated cell numbers or differential cell counts between RAO and non-RAO horses at any time point. CONCLUSIONS AND CLINICAL IMPORTANCE: The peripheral airway obstruction detected in horses with RAO maintained in low-dust environments likely is due to irreversible airway remodeling but is not associated with cytologic evidence of airway inflammation.     

Influence of subclinical inflammatory airway disease on equine respiratory function evaluated by impulse oscillometry

Reasons for performing study: Inflammatory airway disease (IAD) is a nonseptic condition of the lower respiratory tract. Its negative impact on respiratory function has previously been described using either forced expiration or forced oscillations techniques. However, sedation or drug‐induced bronchoconstriction were usually required. The impulse oscillometry system (IOS) is a noninvasive and sensitive respiratory function test validated in horses, which could be useful to evaluate IAD‐affected horses without further procedures. Objectives: To determine the sensitivity of IOS in detecting alterations of the respiratory function in subclinically IAD‐affected horses without inducing bronchoprovocation and to characterise their respiratory impedance according to frequency for each respiratory phase. Methods: Pulmonary function was evaluated at rest by IOS in 34 Standardbred trotters. According to the cytology of bronchoalveolar lavage fluid (BALF), 19 horses were defined as IAD‐affected and 15 horses were used as control (CTL). Total respiratory resistance (Rrs) and reactance (Xrs) from 1–20 Hz as well as their inspiratory and expiratory components were compared between groups. Results: A significant increase of Rrs at the lower frequencies (R_{1–10 Hz}) as well as a significant decrease of Xrs beyond 5 Hz (X_{5–20 Hz}) was observed in IAD compared to CTL horses. IOS‐data was also significantly different between inspiration and expiration in IAD‐affected horses. In the whole population, both BALF eosinophil and mast cell counts were significantly correlated with IOS measurements. Conclusions: Functional respiratory impairment may be measured, even in the absence of clinical signs of disease. In IAD‐affected horses, the different parameters of respiratory function (Rrs or Xrs) may vary depending on the inflammatory cell profiles represented in BALF. Potential relevance: Impulse oscillometry could be used in a routine clinical setting as a noninvasive method for early detection of subclinical respiratory disease and of the results of treatment in horses.     

Cytokine profiles of peripheral blood and airway CD4 and CD8 T lymphocytes in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is thought to result from an aberrant immune response to inhaled antigens, modulated by T lymphocytes via the secretion of pro-inflammatory cytokines. However data relating to the phenotypes of the T lymphocytes present in peripheral blood and bronchoalveolar lavage fluid of RAO horses and their cytokine profiles are contradictory. The aim of this study was to further investigate the cytokine (IL-4, IL-5, IL-13 and INF-gamma) mRNA expression profile in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes from RAO and control horses, before and at 48 h after horses were exposed to hay/straw. In contrast to previous studies, cytokine expression was quantified in populations of CD4 and CD8 T lymphocytes which were purified using magnetic bead antibody cell separation. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and airway lymphocytosis in RAO horses, and, induced a mild, but significant, airway neutrophilia in controls. However, hay/straw exposure had no significant effect on peripheral blood lymphocyte or bronchoalveolar lavage lymphocyte cytokine expression in either group. In conclusion, RAO was not associated with alterations in lymphocyte cytokine expression that are consistent with Th1 or Th2 responses, but rather with a general down-regulation in expression of the measured cytokines in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes.     

Effect of airway disease on blood gas exchange in racehorses

Inflammatory Airway Disease (IAD), exercise-induced pulmonary hemorrhage (EIPH), and upper airway obstruction (UAO) are common respiratory tract diseases that can decrease performance. The purpose of this retrospective study was to compare bronchoalveolar lavage fluid cytology and arterial blood gas analysis during a treadmill test by poorly performing racehorses presented to Purdue University. One hundred thirty-two horses with a history of poor performance were included in this study. Ten horses with no history or diagnosis of EIPH, IAD, or UAO served as controls. Horses were evaluated by rhinolaryngoscopy for upper airway abnormalities and underwent a standardized treadmill test, and samples were collected for blood gas analysis. Horses with IAD or EIPH had a more severe exercise-induced hypoxemia, (mean+/-SD; 84.8+/-1.5 and 86.0+/-1.7 mm Hg average Pao2, respectively), than horses in the control group (92.8+/-2.1 mm Hg). The average Pao2 of horses with only UAO (88.3+/-3.3 mm Hg) was not significantly different from control horses. Gas exchanges were the most severely impaired in horses affected with both EIPH and UAO because they exhibited the lowest Pao2 and highest Paco2 values (66.5+/-15.2 and 52.2+/-6.3 mm Hg, respectively).     

Expression of toll‐like receptor 2 mRNA in bronchial epithelial cells is not induced in RAO‐affected horses

Reasons for performing study: Airway inflammation in recurrent airway obstruction (RAO) is triggered by housing affected horses in stables. It has been suggested that RAO is an allergic condition, but innate immune mechanisms are also involved. Fungal products activate innate immune mechanisms through toll‐like receptor 2 (TLR2). In human airway epithelium, TLR2 activation leads to interleukin (IL)‐8 production. This pathway is negatively regulated by the zinc finger protein A20. This study was performed to enhance understanding of innate immune mechanisms in RAO. Hypothesis: TLR2 and IL‐8 mRNA are elevated in RAO during stabling compared with controls. A20 mRNA is negatively associated with the numbers of airway inflammatory cells. Objectives: To determine TLR 2 , IL‐8 and A20 mRNA expression in lungs of stabled and pastured RAO‐affected and control horses. Methods: Airway obstruction and inflammatory cell counts in bronchoalveolar lavage were measured, and TLR 2 , IL‐8 and A20 mRNA expression quantified by qRT‐PCR in 6 RAO‐affected and 6 control horses, during and after exposure to hay and straw. Results: Airway obstruction and neutrophils were increased in RAO‐affected horses during stabling. While stabling increased IL‐ 8 , TLR2 and A20 mRNA were unaffected. TLR2 and A20 were significantly correlated (r = 0.83) and A20 mRNA was negatively associated with inflammatory cells. Potential relevance: Stabling does not lead to an increase in TLR2 expression. Other molecules or processes in the TLR2 cascade might be important in fungal‐induced airway inflammation. Equine epithelial‐derived A20 may be involved in modulation of airway inflammation.     

Interleukin-4 and interferon-gamma gene expression in summer pasture-associated obstructive pulmonary disease affected horses

We hypothesised that horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) react to an allergen or allergens in their summer environment that is either absent or present at lower levels in their winter environment; and that such allergens stimulate SPAOPD-affected horses to produce a different T helper lymphocyte cytokine profile from that of control horses. The primary objective of this study was to determine the cytokine mRNA profile of T helper lymphocytes obtained from summer pasture-associated obstructive pulmonary disease (SPAOPD) affected horses when 1) the horses were showing signs of disease (summer) and 2) they were in clinical remission (winter). A further objective was to determine the differences between cytokine mRNA T helper lymphocyte profiles of control and affected horses in the summer and winter seasons. Interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression levels were increased in bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cell (PBMC) samples of affected horses during disease expression. No significant amounts of IL-5 mRNA were detected in any of the samples. These results suggest that there is an allergic component to SPAOPD of horses and that appropriate manipulation of the immune system could offer hope for treatment and prevention of the disease in the future. Further research studies will be needed to determine the most appropriate treatments to use to alter the antigen-stimulated cytokine profile being expressed by SPAOPD-affected horses or to alter the effects that these cytokines produce.     

All three classes of CpG ODNs up-regulate IP-10 gene in pigs

The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR^@ green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-γ, IL-12 (P40), IL-6, IL-4 and TNF-α mRNA, C-class CpG ODN induced significant level of IFN-γ, IFN-α and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12 h post stimulation. These in vitro and in vivo observations suggest that interferon-γ inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.