Low levels of microRNA-21 in neutrophil-derived exosomes may contribute to airway smooth muscle hyperproliferation in horses with severe asthma.

OBJECTIVE: Neutrophilic inflammation is associated with the degree of airway obstruction in severe equine asthma (SEA), but the contribution of these leukocytes to bronchial remodeling remains ill defined. Neutrophils could cause structural alterations of the airways by the release of exosomes, a type of cell-derived nanoparticles that can modify the biology of local and distant cells. Neutrophil-derived exosomes have been shown to increase airway smooth muscle (ASM) cell proliferation in humans and horses. Therefore, this study aimed to identify neutrophil exosomal microRNAs (miRs) implicated in the regulation of ASM biology in SEA. ANIMALS: 6 horses with SEA and 6 healthy controls. METHODS: The expression of selected miRs in exosomes from peripheral neutrophils was studied by quantitative PCR. The effects of miR-21 transfection in ASM cells were evaluated by gene expression analysis and proliferation studies. RESULTS: The miR-21 was downregulated in neutrophil exosomes from SEA horses, and it attenuated the proliferation of ASM cells stimulated with lipopolysaccharide. CLINICAL RELEVANCE: The lower level of miR-21 in neutrophil-derived exosomes could contribute to ASM hyperproliferation, which could, in turn, promote the thickening of the bronchial wall in SEA.

Characterization of fungal exposure and dectin-1 expression in healthy horses and horses with severe asthma.

OBJECTIVE: To quantify dectin-1 expression in bronchoalveolar lavage fluid (BALF), create polyclonal antibodies against equine dectin-1 and localize it in tissues, and quantify fungal exposure in pastured and stabled asthmatic and nonasthmatic horses. SAMPLES: BALF samples from 6 controls and 6 horses with severe asthma. Stored lung and nasal wash samples. PROCEDURES: Dectin-1 expression was quantified by quantitative PCR (qPCR). Purified peptide from equine dectin-1 was used to generate polyclonal antibodies and was confirmed with immunological testing. Fungal exposure was quantified in BALF samples by counting fungal-like intracellular particles in phagocytic cells, by qPCR quantification of the "universal" 18S rRNA fungal gene, and by quantifying 36 specific fungi in equine and dust samples using qPCR assays. RESULTS: Equine dectin-1 was localized in tissues and cells, and functional isoforms were upregulated significantly in BALF after stabling. Pastured horses from both groups had low levels of fungi in BALF, and there was a significant increase in some specific fungi, most notably for Eurotium amstelodami, Wallemia sebi, and Aspergillus niger after stabling. However, stabled asthmatic horses had fewer phagocytized particles, less 18S rRNA signal, and fewer specific fungi compared to nonasthmatic horses. CLINICAL RELEVANCE: Stabling increases exposure to fungi, but asthmatic horses had fewer fungi reaching their lower airways, presumably resulting from congestion and narrowing of the airways. Exposure to fungi could contribute to airway inflammation by increasing dectin-1 functional isoforms, and exposure to indoor molds should be avoided.

Effects of azithromycin on bronchial remodeling in the natural model of severe neutrophilic asthma in horses.

Steroid resistance in asthma has been associated with neutrophilic inflammation and severe manifestations of the disease. Macrolide add-on therapy can improve the quality of life and the exacerbation rate in refractory cases, possibly with greater effectiveness in neutrophilic phenotypes. The mechanisms leading to these beneficial effects are incompletely understood and whether macrolides potentiate the modulation of bronchial remodeling induced by inhaled corticosteroids (ICS) is unknown. The objective of this study was to determine if adding azithromycin to ICS leads to further improvement of lung function, airway inflammation and bronchial remodeling in severe asthma. The combination of azithromycin (10 mg/kg q48h PO) and inhaled fluticasone (2500 µg q12h) was compared to the sole administration of fluticasone for five months in a randomized blind trial where the lung function, airway inflammation and bronchial remodeling (histomorphometry of central and peripheral airways and endobronchial ultrasound) of horses with severe neutrophilic asthma were assessed. Although the proportional reduction of airway neutrophilia was significantly larger in the group receiving azithromycin, the lung function and the peripheral and central airway smooth muscle mass decreased similarly in both groups. Despite a better control of airway neutrophilia, azithromycin did not potentiate the other clinical effects of fluticasone.

Studies of molecular pathways associated with blood neutrophil corticosteroid insensitivity in equine asthma.

Severe equine asthma is characterized by airway hyperresponsiveness, neutrophilic inflammation and structural alterations of the lower airways. In asthmatic horses with neutrophilic inflammation, there is insensitivity to corticosteroids characterized by the persistence of neutrophils within the airways with therapy. We hypothesized that hypoxia or oxidative stress in the microenvironment of the lung contributes to this insensitivity of neutrophils to corticosteroids in asthmatic horses. Blood neutrophils isolated from horses with severe asthma (N = 8) and from healthy controls (N = 8) were incubated under different cell culture conditions simulating hypoxia and oxidative stress and, in the presence, or absence of dexamethasone. The pro-inflammatory gene and protein expression of neutrophils were studied. In both groups, pyocyanin-induced oxidative stress increased the mRNA expression of IL-8, IL-1ß, and TNF-α. While IL-1ß and TNF-α were downregulated by dexamethasone under these conditions, IL-8 was not. Simulated hypoxic conditions did not enhance pro-inflammatory gene expression in neutrophils from either group of horses. In conclusion, oxidative stress but not hypoxia may contribute to corticosteroid insensitivity via a selective gene regulation pathway. Equine neutrophil responses were similar in both heathy and asthmatic horses, indicating that it is not specific to asthmatic inflammation.

Recellularization of Bronchial Extracellular Matrix With Primary Bronchial Smooth Muscle Cells.

Severe asthma is associated with an increased airway smooth muscle (ASM) mass and altered composition of the extracellular matrix (ECM). Studies have indicated that ECM-ASM cell interactions contribute to this remodeling and its limited reversibility with current therapy. Three-dimensional matrices allow the study of complex cellular responses to different stimuli in an almost natural environment. Our goal was to obtain acellular bronchial matrices and then develop a recellularization protocol with ASM cells. We studied equine bronchi as horses spontaneously develop a human asthma-like disease. The bronchi were decellularized using Triton/Sodium Deoxycholate. The obtained scaffolds retained their anatomical and histological properties. Using immunohistochemistry and a semi-quantitative score to compare native bronchi to scaffolds revealed no significant variation for matrixial proteins. DNA quantification and electrophoresis revealed that most DNA was 29.6 ng/mg of tissue ± 5.6, with remaining fragments of less than 100 bp. Primary ASM cells were seeded on the scaffolds. Histological analysis of the recellularizations showed that ASM cells migrated and proliferated primarily in the decellularized smooth muscle matrix, suggesting a chemotactic effect of the scaffolds. This is the first report of primary ASM cells preferentially repopulating the smooth muscle matrix layer in bronchial matrices. This protocol is now being used to study the molecular interactions occurring between the asthmatic ECMs and ASM to identify effectors of asthmatic bronchial remodeling.

Tracheal microbial populations in horses with moderate asthma.

BACKGROUND: There are limited data on potential dysbiosis of the airway microbiota in horses with asthma. HYPOTHESIS/OBJECTIVES: We hypothesized that the respiratory microbiota of horses with moderate asthma is altered. Our objectives were (a) to quantify tracheal bacterial populations using culture and qPCR, (2) to compare aerobic culture and qPCR, and (c) to correlate bacterial populations with bronchoalveolar lavage fluid (BALF) cytology. ANIMALS: Eighteen horses with moderate asthma from a hospital population and 10 controls. METHODS: Prospective case-control study. Aerobic culture was performed on tracheal aspirates, and streptococci, Pasteurella multocida, Chlamydophila spp., Mycoplasma spp., as well as 16S (bacterial) and 18S (fungal) rRNA subunits were quantified by qPCR. RESULTS: Potential pathogens such as Streptococcus spp., Actinobacillus spp., and Pasteurellaceae were isolated from 8, 5, and 6 horses with asthma and 3, 0, and 2 controls, respectively. There was a positive correlation between Streptococcus spp. DNA and 16S rRNA gene (r ≥ 0.7, P ≤ 0.02 in both groups), but the overall bacterial load (16S) was lower in asthma (1.5 ± 1.3 versus 2.5 ± 0.8 × 104 copy/µL, P < 0.05). There was no association between microbial populations and clinical signs, tracheal mucus or BALF inflammation. CONCLUSIONS AND CLINICAL IMPORTANCE: This study does not support that bacterial overgrowth is a common feature of chronic moderate asthma in horses. Lower bacterial load could suggest dysbiosis of the lower airways, either as a consequence of chronic inflammation or previous treatments, or as a perpetuating factor of inflammation.

Cryptococcus gattii pneumonia in an adult horse which had travelled in an endemic area.

A gelding from eastern Canada was presented for cough and exercise intolerance 14 months after it had travelled on Vancouver Island. Cryptococcus gattii pneumonia was diagnosed based on cytology, antigen titers, and polymerase chain reaction (PCR). The horse was treated with fluconazole for 10 months. Delayed C. gattii infection can occur after travel in an endemic area.

Pneumonie à Cryptococcus gattii chez un cheval adulte ayant voyagé dans une région endémique. Un cheval hongre de l'est canadien fut présenté pour de la toux et de l'intolérance à l'exercice 14 mois après avoir voyagé sur l'Île de Vancouver. Une pneumonie à Cryptococcus gattii fut diagnostiquée sur la base de la cytologie, des titres antigéniques, et de la réaction d'amplification en chaîne par la polymérase (PCR). Le cheval fut traité avec du fluconazole pendant 10 mois. Une infection à retardement par C. gattii peut survenir à la suite d'un voyage dans une région endémique.(Traduit par Dr Serge Messier).

MicroRNA-221 is overexpressed in the equine asthmatic airway smooth muscle and modulates smooth muscle cell proliferation.

Airway wall remodeling, including hyperplasia and hypertrophy of smooth muscle (ASM) cells leading to an increased smooth muscle mass, is considered central to asthma. However, molecular pathways responsible for ASM remodeling remain poorly understood. MicroRNAs (miRNAs) have emerged as key regulators of inflammatory and repair processes affecting the lungs and can downregulate protein expression by inhibiting target mRNA translation. We therefore hypothesized that miRNAs are involved in ASM remodeling in asthma by modulating ASM proliferation. We have analyzed the expression of miRNAs in bronchial smooth muscle from asthmatic horses during disease exacerbation and remission and from controls. Their involvement in ASM cell proliferation was then studied. Our results shown that miR-26a, miR-133, and miR-221 were upregulated in ASM from horses with asthma exacerbation compared with asthma remission and controls. MiR-221 induced cell hyperproliferation and reduced the expression of contractile gene markers in ASM cells. These changes were associated with the decreased mRNA expression of cell cycle regulatory genes (p53, p21, and p27). In conclusion, we demonstrated for the first time an upregulation of miR-221 in asthmatic airway smooth muscle and confirm the involvement of miR-221 in ASM cell proliferation by regulation of the cell cycle arrest genes. Targeting miR-221 network genes may represent a novel approach for the treatment of ASM remodeling in asthma.

Lung Microbiome Is Influenced by the Environment and Asthmatic Status in an Equine Model of Asthma.

There is evidence that the lung microbiome differs between patients with asthma and healthy humans, but the effect of environmental conditions and medication is unknown and difficult to study. Equine asthma is a naturally occurring chronic airway disease characterized by reversible airway inflammation and bronchoconstriction upon exposure to inhaled antigens. In the present study, we evaluated the effect that environmental conditions and disease status have on pulmonary, nasal, and oral microbiomes. Six asthmatic and six healthy horses were studied while at pasture ("low antigen exposure"), as well as when being housed indoors and fed good-quality hay ("moderate exposure") and poor-quality hay ("high exposure"). At each time point, lung function was recorded; BAL, oral, and nasal rinses were collected; and 16S rRNA gene sequencing was performed. Asthmatic horses developed airway obstruction and inflammation under moderate and high antigen exposure conditions, whereas nonasthmatic horses showed mild inflammation under high antigen exposure, without bronchoconstriction. Lung, oral, and nasal communities clustered by environmental condition, but only lung communities were different between healthy and asthmatic horses. The association between asthma and lung microbiome was strongest in horses under moderate antigen exposure. Pulmonary, oral, and nasal microbiomes are influenced by environmental conditions, but only the pulmonary microbiome differs between horses with and without asthma. This difference, seen mainly when airway inflammation was present in horses with asthma but not in control animals, suggests that the altered lung microbiome in asthma might not be inherent but coincident with inflammation.

Adherence to treatment recommendations and short-term outcome of pleasure and sport horses with equine asthma.

Treatments for mild forms of equine asthma are extrapolated from those recommended for severe equine asthma (heaves), but little is known about owner's adherence to recommendations and treatment efficacy. The objective was to determine which recommendations are implemented by owners and their perception of the clinical response to treatment. Medical records of 43 horses diagnosed with moderate asthma between 2010 and 2012 were retrieved from the Université de Montréal database. Treatments and perceived responses were recorded by telephone survey, 2 to 35 months after diagnosis. All 33 owners who completed the survey attempted to decrease exposure to dust and half had also administered medication. Twenty-four owners (73%) described a > 50% improvement in the clinical signs. There was no association between a specific treatment and outcome. A majority of owners of pleasure and sport horses with equine asthma perceived improvement when limiting exposure to hay and barn dust (alone or with medications).

Respect des recommandations de traitement et résultats à court terme pour les chevaux d'agrément et de sport atteints d'asthme des équidés. Les traitements pour des formes bénignes d'asthme des équidés sont extrapolés de ceux recommandés pour le traitement de l'asthme des équidés grave (emphysème chronique), mais on en sait encore peu à propos de l'observance des recommandations par les propriétaires et de l'efficacité du traitement. L'objectif consistait à déterminer quelles recommandations sont mises en oeuvre par les propriétaires et leur perception de la réponse clinique au traitement. Les dossiers médicaux de 43 chevaux diagnostiqués avec un asthme modéré entre 2010 et 2012 ont été récupérés de la base de données de l'Université de Montréal. Les traitements et les réactions perçues ont été consignés lors d'un sondage par téléphone, de 2 à 35 mois après le diagnostic. Les 33 propriétaires qui ont répondu au sondage avaient tenté de réduire l'exposition à la poussière et la moitié avaient aussi administré des médicaments. Vingt-quatre propriétaires (73 %) ont décrit une amélioration de > 50 % des signes cliniques. Il n'y avait aucune association entre un traitement spécifique et un résultat. La plupart des propriétaires possédant des chevaux d'agrément et de sport atteints d'asthme modéré ont perçu une amélioration lorsqu'ils limitaient l'exposition à la poussière de foin et de grange (comme seule mesure ou avec des médicaments). lorsqu'ils limitaient l'exposition à la poussière de foin et de grange (comme seule mesure ou avec des médicaments).(Traduit par Isabelle Vallières).

SRSF6 is upregulated in asthmatic horses and involved in the MYH11 SMB expression.

Smooth muscle has a central role in bronchospasm-induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis-regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT-PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis-acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.

Glucocorticosteroids administration is associated with increased regulatory T cells in equine asthmatic lungs.

Recurrent inflammation in severe equine asthma causes a remodeling of the airways leading to incompletely reversible airway obstruction. Despite the improvement of clinical signs and lung function with glucocorticoids (GC), inflammation, translated by an increased percentage of neutrophils, persists in the airways. Regulatory T cells (Treg) have been shown to have anti-inflammatory properties and play an important role in balancing the immune response by suppressing effector lymphocyte activity. However, interactions between Treg, neutrophils and glucocorticosteroids in vivo are unclear, particularly in asthma. Furthermore, the effects of GC on Treg in the airway of asthmatic horses have not been investigated. We hypothesized that horses with severe asthma display a decreased population of pulmonary Treg when compared to heathy controls, and that treatment with GC lead to an increased pulmonary Treg cell population only in affected horses. Using lung function measurements and flow cytometry with surface antigens CD4 and FoxP3, we investigated Treg in airway luminal cells obtained by bronchoalveolar lavage fluid (BALF) from 6 asthmatic horses in exacerbation of the disease and 6 aged-match controls, kept in the same environment, before and following a 2-week treatment with dexamethasone. Results showed that the number of Treg increases only in the lungs of asthmatic horses following GC therapy, despite continued presence of increased numbers of neutrophils. Our results support the complexity of the interaction between Treg, neutrophils and GC.

Neutrophil extracellular traps are downregulated by glucocorticosteroids in lungs in an equine model of asthma.

BACKGROUND: Severe neutrophilic asthma is poorly responsive to glucocorticosteroids (GC). Neutrophil extracellular traps (NETs) within the lungs have been associated with the severity of airway obstruction and inflammation in asthma, and were found to be unaffected by GC in vitro. As IL-17 is overexpressed in neutrophilic asthma and contributes to steroid insensitivity in different cell types, we hypothesized that NETs formation in asthmatic airways would be resistant to GC through an IL-17 mediated pathway. METHODS: Six neutrophilic severe asthmatic horses and six healthy controls were studied while being treated with dexamethasone. Lung function, bronchoalveolar lavage fluid (BALF) cytology and NETs formation, as well as the expression of CD11b and CD13 by blood and airway neutrophils were evaluated. The expression of IL-17 and its role in NETs formation were also studied. RESULTS: Airway neutrophils from asthmatic horses, as opposed to blood neutrophils, enhanced NETs formation, which was then decreased by GC. GC also tended to decrease the expression of CD11b in blood neutrophils, but not in airway neutrophils. IL-17 mRNA was increased in BALF cells of asthmatic horses and was unaffected by GC. However, both GC and IL-17 inhibited NETs formation in vitro. CONCLUSION: GC decreased NETs formation in vitro and also in vivo in the lungs of asthmatic horses. However, airway neutrophil activation during asthmatic inflammation was otherwise relatively insensitive to GC. The contribution of IL-17 to these responses requires further study.

Inhaled corticosteroids modulate the (+)insert smooth muscle myosin heavy chain in the equine asthmatic airways.

RATIONALE: Overexpression of the (+)insert smooth muscle myosin heavy chain (SMMHC) isoform could contribute to airway bronchospasm by increasing the velocity of contraction. Whether the (+)insert isoform is present in the small airways and its expression is reversible in asthma are unknown. OBJECTIVES: To determine the anatomical location and the expression kinetics of the (+)insert SMMHC isoform in airways of horses with heaves and to evaluate its modulation in response to disease status. METHODS: We evaluated the (+)insert SMMHC isoform in the airways of horses with heaves during disease exacerbation and remission, and in controls. The expression kinetics of the SMMHC (+)insert was then assessed at multiple time points in two studies: first, in horses with heaves treated for a 1-year period with antigen avoidance alone, inhaled corticosteroids alone or both; second, in horses with heaves before and after a 30-day natural antigen exposure. Gene expression analysis was assessed by quantitative PCR and protein expression was confirmed by targeted mass spectrometry. MEASUREMENTS AND MAIN RESULTS: The (+)insert SMMHC isoform was significantly increased in central and peripheral airways, but not in the trachea of heaves-affected horses in clinical exacerbation when compared horses with heaves in remission and controls. Both corticosteroid administration and antigen avoidance led to a significant reduction of the (+)insert expression in the airways. The (+)insert SMMHC isoform was not significantly increased in airways after 1 month of antigenic re-exposure. CONCLUSIONS: The (+)insert SMMHC expression is increased throughout the bronchial tree in horses with heaves and reversible by corticosteroids administration and antigen avoidance.