Anouk Lavoie-Lamoureux

Heaves, an asthma-like disease of horses

Animal models have been developed to investigate specific components of asthmatic airway inflammation, hyper‐responsiveness or remodelling. However, all of these aspects are rarely observed in the same animal. Heaves is a naturally occurring disease of horses that combines these features. It is characterized by stable dust‐induced inflammation, bronchospasm and remodelling. The evaluation of horses during well‐controlled natural antigen exposure and avoidance in experimental settings allows the study of disease mechanisms in the asymptomatic and symptomatic stages, an approach rarely feasible in humans. Also, the disease can be followed over several years to observe the cumulative effect of repeated episodes of clinical exacerbation or to evaluate long‐term treatment, contrasting most murine asthma models. This model has shown complex gene and environment interactions, the involvement of both innate and adaptive responses to inflammation, and the contribution of bronchospasm and tissue remodelling to airway obstruction, all occurring in a natural setting. Similarities with the human asthmatic airways are well described and the model is currently being used to evaluate airway remodelling and its reversibility in ways that are not possible in people for ethical reasons. Tools including antibodies, recombinant proteins or gene arrays, as well as methods for sampling tissues and assessing lung function in the horse are constantly evolving to facilitate the study of this animal model. Research perspectives that can be relevant to asthma include the role of neutrophils in airway inflammation and their response to corticosteroids, systemic response to pulmonary inflammation, and maintaining athletic capacities with early intervention.

Corticosteroids and Antigen Avoidance Decrease Airway Smooth Muscle Mass in an Equine Asthma Model

Recent studies suggest that airway smooth muscle remodeling is an early event in the course of asthma. Little is known of the effects of long-term antigen avoidance and inhaled corticosteroids on chronically established airway remodeling. We sought to measure the effects of inhaled corticosteroids and antigen avoidance on airway remodeling in the peripheral airways of horses with heaves, a naturally occurring asthma-like disease. Heaves-affected adult horses with ongoing airway inflammation and bronchoconstriction were treated with fluticasone propionate (with and without concurrent antigen avoidance) (n = 6) or with antigen avoidance alone (n = 5). Lung function and bronchoalveolar lavage were performed at multiple time points, and peripheral lung biopsies were collected before and after 6 and 12 months of treatment. Lung function improved more quickly with inhaled corticosteroids, but eventually normalized in both groups. Inflammation was better controlled with antigen avoidance. During the study period, corrected smooth muscle mass decreased from 12.1 ± 2.8 × 10(-3) and 11.3 ± 1.2 × 10(-3) to 8.3 ± 1.4 × 10(-3) and 7.9 ± 1.0 × 10(-3) in the antigen avoidance and fluticasone groups, respectively (P = 0.03). At 6 months, smooth muscle mass was significantly smaller compared with baseline only in the fluticasone-treated animals. The subepithelial collagen area was lower at 12 months than at baseline in both groups. During the study period, airway smooth muscle remodeling decreased by approximately 30% in both groups, although the decrease was faster in horses receiving inhaled corticosteroids. Inhaled corticosteroids may accelerate the reversal of smooth muscle remodeling, even if airway inflammation is better controlled with antigen avoidance.

IL-4 activates equine neutrophils and induces a mixed inflammatory cytokine expression profile with enhanced neutrophil chemotactic mediator release ex vivo

Neutrophils are potent contributors to the lung pathophysiological changes occurring in allergic airway inflammation, which typically involve T helper type 2 (Th2) cytokine overexpression. We have previously reported that equine pulmonary endothelial cells are activated by the Th2 cytokine IL-4 and express chemotactic factors for neutrophils after stimulation. We have further explored the possible mechanisms linking Th2-driven inflammation and neutrophilia by studying the effects of recombinant equine IL-4, a prototypical Th2 cytokine, on peripheral blood neutrophils (PBN) isolated from normal animals and from horses with asthmatic airway inflammation (equine heaves). We found that IL-4 induced morphological changes in PBN, dose- and time-dependent expression of IL-8 mRNA, as well as the release of chemotactic factors for neutrophils in culture supernatants. Also, IL-4 induced a mixed inflammatory response in PBN from control and asthmatic-animals with increased expression of proinflammatory IL-8 and TNF-α but a marked inhibition of IL-1β. IL-4 type I receptor (IL-4Rα) and CD23 (FcεRII) expression were also upregulated by IL-4. Importantly, disease as well as chronic antigenic exposure modified gene expression by PBN. Finally, we found that activation of equine neutrophils with IL-4 involved STAT6 phosphorylation and p38 MAPK and phosphatidylinositol 3-kinase (PI3K); the pharmacological inhibitors, SB-203580 and LY-294002, respectively, significantly reversed IL-4-induced gene modulation in PBN. Overall, results from this study add to the link between Th2-driven inflammation and neutrophilia in the equine model and further extend the characterization of IL-4 effects on neutrophils.

Markers of Systemic Inflammation in Horses with Heaves

BACKGROUND Systemic inflammation in horses with heaves is poorly characterized. OBJECTIVES To assess acute phase proteins (APP) and inflammatory cytokine profiles in serum of healthy horses and horses with heaves. ANIMALS Six healthy horses and 6 heaves-affected horses belonging to the University of Montreal. METHODS Prospective, observational study. Healthy and heaves-affected control horses were exposed to a 30-day natural challenge with hay and straw to induce clinical exacerbation of heaves. Serum samples were obtained by venipuncture before (T0) as well as after 7 (T7) and 30 days (T30) of stabling. Serum APP (haptoglobin, serum amyloid A protein [SAA] and C-reactive protein [CRP]) and cytokines (IL-2, IL-4, IFN-α, IL-10, IFN-γ, and CCL-2) were measured using singleplex or multiplex ELISA. RESULTS Serum haptoglobin concentrations were significantly higher in heaves-affected horses at all time points with no overlap with those of healthy controls. They were also significantly increased by antigen challenge in both controls (T7) and horses with heaves (T7 and T30). Serum SAA was detected more frequently in heaves-affected horses compared with healthy controls at T7. There was no difference in serum concentrations of CRP, IL-10, IFN-γ, and CCL-2 between groups, whereas IL-2, IL-4, and IFN-α remained undetectable in all samples. CONCLUSIONS AND CLINICAL IMPORTANCE In heaves, haptoglobin is a marker of both acute and chronic systemic inflammation, whereas high concentrations of SAA indicate acute inflammation.

Effect of Antigenic Exposure on Airway Smooth Muscle Remodeling in an Equine Model of Chronic Asthma

Recent studies suggest that airway smooth muscle remodeling is an early event in asthma, but whether it remains a dynamic process late in the course of the disease is unknown. Moreover, little is known about the effects of an antigenic exposure on chronically established smooth muscle remodeling. We measured the effects of antigenic exposure on airway smooth muscle in the central and peripheral airways of horses with heaves, a naturally occurring airway disease that shares similarities with chronic asthma. Heaves-affected horses (n = 6) and age-matched control horses (n = 5) were kept on pasture before being exposed to indoor antigens for 30 days to induce airway inflammation and bronchoconstriction. Peripheral lung and endobronchial biopsies were collected before and after antigenic exposure by thoracoscopy and bronchoscopy, respectively. Immunohistochemistry and enzymatic labeling were used for morphometric analyses of airway smooth muscle mass and proliferative and apoptotic myocytes. In the peripheral airways, heaves-affected horses had twice as much smooth muscle as control horses. Remodeling was associated with smooth muscle hyperplasia and in situ proliferation, without reduced apoptosis. Further antigenic exposure had no effect on the morphometric data. In central airways, proliferating myocytes were increased compared with control horses only after antigenic exposure. Peripheral airway smooth muscle mass is stable in chronically affected animals subjected to antigenic exposure. This increased mass is maintained in a dynamic equilibrium by an elevated cellular turnover, suggesting that targeting smooth muscle proliferation could be effective at decreasing chronic remodeling.

Neutrophils Are Not Less Sensitive Than Other Blood Leukocytes to the Genomic Effects of Glucocorticoids

Background Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out. Objective We assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes. Methods Blood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10−8 M and 10−6 M). IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations. Results We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils. Conclusions Glucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some chronic neutrophilic diseases such as severe asthma or COPD is not explained by a relative lack of inhibition of these drugs on pro-inflammatory cytokines expression in neutrophils.

Airway collagen and elastic fiber content correlates with lung function in equine heaves

The consequences on lung function and inflammation of alterations in the extracellular matrix affecting the peripheral airway wall in asthma are largely unknown. We hypothesized that remodeling of collagen and elastic fibers in the peripheral airway wall leads to airway obstruction and contributes to neutrophilic airway inflammation. Animals used were six heaves-affected horses and five controls. Large peripheral lung biopsies were obtained from horses with heaves in clinical remission (Baseline) and during disease exacerbation and from age-matched controls. The area of collagen and elastic fiber content in the lamina propria was measured by histological staining techniques and corrected for airway size. Collagen type 1 and type 3 content was further assessed from additional horses after postmortem lung samples by immunohistochemistry. The collagen breakdown products proline-glycine-proline (PGP) and N-acetylated-PGP (N-α-PGP) were also measured in bronchoalveolar lavage fluids (BALF) by mass spectrometry. Compared with controls, heaves-affected horses had an increase in collagen (P = 0.05) and elastic fiber contents (P = 0.04) at baseline. Collagen types 1 and 3 content was also significantly increased in diseased horses (P = 0.015) when both collagen types were combined. No further change in collagen content was observed after a 30-day antigenic challenge. Airway collagen at baseline was positively correlated with pulmonary resistance in asthmatic horses (r(2) = 0.78, P = 0.03) and elastic fiber content was positively associated with pulmonary elastance in controls (r(2) = 0.95, P = 0.02). No difference between groups was appreciated in PGP and N-α-PGP peptides in BALF. Increased airway wall collagen and elastic fiber content may contribute to residual obstruction in the asthmatic airways.

Effect of long-term fluticasone treatment on immune function in horses with heaves.

BACKGROUND Corticosteroids currently are the most effective pharmacological treatment available to control heaves in horses. Systemically administered corticosteroids have been shown to alter immune response in horses, humans, and other species. Aerosolized administration theoretically minimizes systemic adverse effects, but the effect of inhaled corticosteroids on immune function has not been evaluated in horses. OBJECTIVES To evaluate the effects of prolonged administration of inhaled fluticasone on the immune system of heaves-affected horses. ANIMALS Heaves-affected horses were treated with inhaled fluticasone (n = 5) for 11 months or received environmental modifications only (n = 5). METHODS Prospective analysis. Clinical parameters and CBC, lymphocyte subpopulations and function, and circulating neutrophil gene expression were sequentially measured. Primary and anamnestic immune responses also were evaluated by measuring antigen-specific antibodies in response to vaccination with bovine viral antigen and tetanus toxoid, respectively. RESULTS No clinical adverse effects were observed and no differences in immune function were detected between treated and untreated horses. CONCLUSIONS AND CLINICAL IMPORTANCE The treatment of heaves-affected horses with inhaled fluticasone at therapeutic dosages for 11 months has no significant detectable effect on innate and adaptive (both humoral and cell-mediated) immune parameters studied. These results suggest that prolonged administration of fluticasone would not compromise the systemic immune response to pathogens nor vaccination in adult horses.

Bronchoalveolar Lavage Fluid Cytology and Cytokine Messenger Ribonucleic Acid Expression of Racehorses with Exercise Intolerance and Lower Airway Inflammation

BACKGROUND There is limited information relating bronchoalveolar lavage (BAL) cytology and cytokine messenger ribonucleic acid (mRNA) expression in racehorses with inflammatory airway disease (IAD). HYPOTHESIS AND OBJECTIVE: We hypothesize that cytokine expression in BAL cells would correlate with cytology. Thus, we evaluated the mRNA expression of selected cytokines in BAL cells in racehorses with exercise intolerance and lower airway inflammation. ANIMALS Thirty-one client-owned Standardbred racehorses with exercise intolerance. METHODS Prospective, observational study. Cells were obtained by BAL, and mRNA expression of interleukin (IL)-1β, IL-4, IL-8, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS Nine horses had normal BAL cell differential cytology (Controls), while 22 horses had evidence of IAD based on BAL fluid cytology. Relative expressions of TNF-α/glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 0.0092 ± 0.010 versus 0.0045 ± 0.005, P= .034), IL-4/GAPDH (0.001 ± 0.002 versus 0.0003 ± 0.0003, P= .029), and IFN-γ/GAPDH (0.0027 ± 0.003 versus 0.0009 ± 0.001, P= .028) were greater in horses with IAD compared with controls. Furthermore, IL-4/GAPDH (0.001 ± 0.002 versus 0.0002 ± 0.0003, P < .0001) and IFN-γ/GAPDH (0.003 ± 0.003 versus 0.001 ± 0.001, P= .002) mRNA expression was increased in horses with increased metachromatic cell counts compared with horses with normal metachromatic cell counts. Only the mRNA expression of IL-1β/GAPDH (1.1 ± 0.7 versus 0.3 ± 0.3, P= .045) was increased with airway neutrophilia. CONCLUSIONS AND CLINICAL IMPORTANCE Differences in gene expression were associated with the presence of IAD and with specific cell types present in airway secretions of Standardbred racehorses with poor performance. These findings suggest that different pathophysiological pathways are implicated in IAD.

Profiling of differentially expressed genes using suppression subtractive hybridization in an equine model of chronic asthma.

Background Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. Objective To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. Methods Eleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay. Results Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways. Conclusions Pathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies.