RESUMO

Equine asthma is an airway disease that affects a large number of horses annually leading to considerable economic losses in the horse industry. Despite advances in research in this area, there is still a lack of information on its etiology and molecular characterization in pasture associated asthma. The objective of the current study was to characterize the inflammatory disease of lower airways in horses maintained on pasture through cytologic and immunologic profile during the summer in a tropical environment by analysis of the gene expression of Th1 cytokines (IFN- λ, IL-8), Th2 cytokines (IL-4 and IL-5), and pro-inflammatory cytokines (IL-1, TNF-α) in the bronchoalveolar lavage (BAL) fluid in healthy and asthma horses on pasture. A group 39 of clinically healthy horses maintained on native pasture and supplemented with concentrate was evaluated by BAL analyzed for differential cellular count and assigned into a control and an asthma group. The gene expression of pro-inflammatory cytokines was analyzed in the BAL by reverse time PCR (RT-PCR) (IL-1α (alpha), IL-4, IL-5, IL-8, TNF-α alpha and IFN-λ), using ß-actin as housekeeping gene. Higher gene expression of IL-1, IL-4, IL-5, IL-8, IFN-λ in the BAL of asthma horses was found. Current results indicate an increase in Th2, characterizing an allergic inflammatory reaction due to the significant increase in IL-5 in asthmatic horses (10.3 ± 1.13), when compared to the values ​​obtained in normal horses (3.27 ± 0.46). The only down regulated cytokine in the asthma group was TNF-α, suggesting a chronic antigenic reaction.

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Background: Summer Pasture Associated Obstructive Pulmonary Disease (SPAOPD), or Equine Pasture Asthma (EPA),has been described as an environmentally-induced respiratory disease that occurs during the warmer and more humidmonths, leading to reversible airway obstruction, persistent and non-specific airway hyper-responsiveness, and chronicneutrophilic airway inflammation. Exacerbation of clinical signs vary according to warm seasons and range from mildto severe episodes of wheezing, coughing, and laboured breathing, being highly debilitating to the equine. This reportdescribed two cases of Equine Pasture Asthma that showed clinical and environmental similarities with Summer PastureAssociated Obstructive Pulmonary Disease.Cases: The patients were crossbreed geldings that have never been stabled and were used for cattle management in a farmin southeastern Brazil. They presented poor performance and a persistent cough for over 3 years. Initially, the respiratorysigns were only observed after exercise but, over the years, it gradually progressed to being observed when the horses wereat rest. Both animals had a history of regular deworming and were previously treated by other veterinarians with antibiotics,clenbuterol, and mucokinetics. Little improvement was noticed by the owner and the signs returned over time as treatmentwas often discontinued. Clinical findings were compatible with the grade 3 mucus classification as well as with score 2for Severe Asthma. BALF cytology was done according to routine procedure. Animal 1 presented slides with free yeast,macrophages, and mucus with Curschmanns spiral and counting of 29.7% of neutrophils (NE), 43.7% of lymphocytes(LP), 25.3% of macrophages (MC) and 1.3% of eosinophils (EO). Animal 2 presented slides with phagocytized yeast,mucus and counting of 27% of NE, 38.5% of LP, 33% of MC and 1.5% of EO...(AU)

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RESUMO

Background: Summer Pasture Associated Obstructive Pulmonary Disease (SPAOPD), or Equine Pasture Asthma (EPA),has been described as an environmentally-induced respiratory disease that occurs during the warmer and more humidmonths, leading to reversible airway obstruction, persistent and non-specific airway hyper-responsiveness, and chronicneutrophilic airway inflammation. Exacerbation of clinical signs vary according to warm seasons and range from mildto severe episodes of wheezing, coughing, and laboured breathing, being highly debilitating to the equine. This reportdescribed two cases of Equine Pasture Asthma that showed clinical and environmental similarities with Summer PastureAssociated Obstructive Pulmonary Disease.Cases: The patients were crossbreed geldings that have never been stabled and were used for cattle management in a farmin southeastern Brazil. They presented poor performance and a persistent cough for over 3 years. Initially, the respiratorysigns were only observed after exercise but, over the years, it gradually progressed to being observed when the horses wereat rest. Both animals had a history of regular deworming and were previously treated by other veterinarians with antibiotics,clenbuterol, and mucokinetics. Little improvement was noticed by the owner and the signs returned over time as treatmentwas often discontinued. Clinical findings were compatible with the grade 3 mucus classification as well as with score 2for Severe Asthma. BALF cytology was done according to routine procedure. Animal 1 presented slides with free yeast,macrophages, and mucus with Curschmann’s spiral and counting of 29.7% of neutrophils (NE), 43.7% of lymphocytes(LP), 25.3% of macrophages (MC) and 1.3% of eosinophils (EO). Animal 2 presented slides with phagocytized yeast,mucus and counting of 27% of NE, 38.5% of LP, 33% of MC and 1.5% of EO...

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The present work characterized the metabolomic profile of bronchoalveolar lavage fluid (BALF) in healthy horses, experimentally-induced airway inflammation by lipopolysaccharide (LPS) nebulization, and naturally-occurring asthma (n = 3 in each group). All animals underwent clinical and upper airway endoscopic examinations, and bronchoalveolar lavage. BALF supernatant samples were subjected to metabolic analysis based on gas chromatography-mass spectrometry (GC-MS). Overall, 67 peaks were obtained from BALF GC-MS analysis, corresponding to 53 metabolites which were categorized according to chemical class, such as organic acids, fatty acids, nucleosides or their derivatives, amino acids, peptides or their derivatives, carbohydrates, and other compounds. Our results showed that the airway inflammation induction model with LPS produced the same pattern of metabolite changes as in horses with naturally occurring asthma. Metabolic pathway analysis was done by means of Fisher's exact test, for detection of metabolites over-represented in asthma affected-horses and LPS-induced airway inflammation as compared with healthy horses. The most significant altered metabolic pathways were fatty acid biosynthesis, galactose metabolism and citrate cycle. These results suggest that the airway inflammation induction model with LPS is a good study model for asthma-affected horses, due to the similarity of the profile of inflammatory cells (specifically neutrophils) and similar metabolic alterations found in BALF that occur during the inflammatory process of the airways. Further research may increase understanding of metabolomics disturbances and their significance in the pathogenesis of equine asthma.

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The term "equine asthma syndrome" (EAS) was recently proposed due to the resemblance of the equine disease to human asthma. Leukotrienes cause constriction of the bronchi, especially in the lower airways and increase mucus secretion in the respiratory system. Leukotriene B4 (LTB4) has been discovered as a strong chemotactic factor, which plays a role in neutrophil migration. The immunologic background of EAS remains not fully elucidated despite many studies on the pathogenesis. This study aimed to evaluate the LTB4 concentration in the bronchoalveolar lavage fluid (BALF) of horses with and without pulmonary inflammatory disease. Thirty-five mixed breed horses were studied and LTB4 was determined by using specific ELISA Kit. The horses were grouped by 2 different criteria for statistical analysis of data: 1) according to the values for BALF citology and 2) according to the detection of LTB4 in BALF. There was significant difference of effect of age on the LTB4 detection in equine BALF. Younger animals were the majority where it was possible to detect LTB4 values in LBA. In conclusion, there was an effect of age on the detection of LTB4 in equine BALF, where LTB4 levels were more easily detected in younger animals than older animals and the results of this study raise the possibility of considering future studies with the objective of establishing the real role and the best moment to detect LTB4 in BALF of the equine asthma syndrome.(AU)

Recentemente, o termo "síndrome da asma equina" (SAE) foi proposto devido à semelhança da doença equina à asma humana. Os leucotrienos causam constrição dos brônquios, especialmente nas vias aéreas posteriores e aumentam a secreção de muco no sistema respiratório. O leucotrieno B4 (LTB4) foi descoberto como um forte fator quimiotático, que desempenha um papel na migração de neutrófilos. O fundo imunológico do SAE permanece não completamente elucidado apesar de muitos estudos sobre a patogênese. Este estudo teve como objetivo avaliar a concentração de LTB4 no lavado broncoalveolar (LBA) de equinos com e sem doença inflamatória pulmonar. Trinta e cinco cavalos de raças mistas foram estudados e o LTB4 foi determinado usando o kit ELISA específico. Os animais foram agrupados por dois critérios diferentes para análise estatística dos dados: 1) de acordo com os valores para citologia do LBA e 2) de acordo com a detecção do LTB4 no LBA. Houve diferença significativa do efeito da idade na detecção do LTB4 no LBA equino. Os animais mais jovens foram a maioria onde foi possível detectar os valores de LTB4 no LBA. Em conclusão, houve um efeito da idade na detecção de LTB4 em LBA equino, onde os níveis de LTB4 foram mais facilmente detectados em animais jovens do que em animais mais velhos e foi possível detectar a concentração de LTB4 no LBA equino e os resultados deste estudo levantam a possibilidade de considerar futuros estudos com o objetivo de estabelecer o real papel e o melhor momento para detectar LTB4 no LBA da síndrome asmática equina.(AU)

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RESUMO

The term “equine asthma syndrome” (EAS) was recently proposed due to the resemblance of the equine disease to human asthma. Leukotrienes cause constriction of the bronchi, especially in the lower airways and increase mucus secretion in the respiratory system. Leukotriene B4 (LTB4) has been discovered as a strong chemotactic factor, which plays a role in neutrophil migration. The immunologic background of EAS remains not fully elucidated despite many studies on the pathogenesis. This study aimed to evaluate the LTB4 concentration in the bronchoalveolar lavage fluid (BALF) of horses with and without pulmonary inflammatory disease. Thirty-five mixed breed horses were studied and LTB4 was determined by using specific ELISA Kit. The horses were grouped by 2 different criteria for statistical analysis of data: 1) according to the values for BALF citology and 2) according to the detection of LTB4 in BALF. There was significant difference of effect of age on the LTB4 detection in equine BALF. Younger animals were the majority where it was possible to detect LTB4 values in LBA. In conclusion, there was an effect of age on the detection of LTB4 in equine BALF, where LTB4 levels were more easily detected in younger animals than older animals and the results of this study raise the possibility of considering future studies with the objective of establishing the real role and the best moment to detect LTB4 in BALF of the equine asthma syndrome.(AU)

Recentemente, o termo "síndrome da asma equina" (SAE) foi proposto devido à semelhança da doença equina à asma humana. Os leucotrienos causam constrição dos brônquios, especialmente nas vias aéreas posteriores e aumentam a secreção de muco no sistema respiratório. O leucotrieno B4 (LTB4) foi descoberto como um forte fator quimiotático, que desempenha um papel na migração de neutrófilos. O fundo imunológico do SAE permanece não completamente elucidado apesar de muitos estudos sobre a patogênese. Este estudo teve como objetivo avaliar a concentração de LTB4 no lavado broncoalveolar (LBA) de equinos com e sem doença inflamatória pulmonar. Trinta e cinco cavalos de raças mistas foram estudados e o LTB4 foi determinado usando o kit ELISA específico. Os animais foram agrupados por dois critérios diferentes para análise estatística dos dados: 1) de acordo com os valores para citologia do LBA e 2) de acordo com a detecção do LTB4 no LBA. Houve diferença significativa do efeito da idade na detecção do LTB4 no LBA equino. Os animais mais jovens foram a maioria onde foi possível detectar os valores de LTB4 no LBA. Em conclusão, houve um efeito da idade na detecção de LTB4 em LBA equino, onde os níveis de LTB4 foram mais facilmente detectados em animais jovens do que em animais mais velhos e foi possível detectar a concentração de LTB4 no LBA equino e os resultados deste estudo levantam a possibilidade de considerar futuros estudos com o objetivo de estabelecer o real papel e o melhor momento para detectar LTB4 no LBA da síndrome asmática equina.(AU)

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Alveolar macrophages (AMs) are an essential part of defense mechanisms within the lungs and their phagocytic activity is important for organ homeostasis. The phagocytic ability of AMs obtained from bronchoalveolar lavage from 17 mature mixed-breed pleasure horses (8 healthy and 9 diagnosed with mild equine asthma) was studied through assays with Leishmania (Viannia) braziliensis promastigotes, which enabled the calculation of a phagocytic index (PI) and a survival index (SI). Results indicate that phagocytic activity of AMs in asthma affected horses is similar to healthy horses, while leishmanicidal activity is significantly increased in horses with asthma.(AU)

Os macrófagos alveolares (MAs) são uma parte essencial dos mecanismos de defesa dentro dos pulmões e sua atividade fagocítica é importante para a homeostase desse órgão. A capacidade fagocitária dos MAs obtidos do lavado broncoalveolar de 17 equinos adultos, sem raça definida (oito saudáveis e nove com diagnóstico de asma equina leve), foi estudada por meio de ensaios com promastigotas de Leishmania (Viannia) braziliensis. Foi calculado o índice fagocítico e o índice de sobrevivência. Os resultados indicam que a atividade fagocítica de MAs em cavalos com asma é semelhante a cavalos saudáveis, enquanto a atividade leishmanicida está significativamente aumentada em cavalos com essa enfermidade.(AU)

Assuntos

RESUMO

Alveolar macrophages (AMs) are an essential part of defense mechanisms within the lungs and their phagocytic activity is important for organ homeostasis. The phagocytic ability of AMs obtained from bronchoalveolar lavage from 17 mature mixed-breed pleasure horses (8 healthy and 9 diagnosed with mild equine asthma) was studied through assays with Leishmania (Viannia) braziliensis promastigotes, which enabled the calculation of a phagocytic index (PI) and a survival index (SI). Results indicate that phagocytic activity of AMs in asthma affected horses is similar to healthy horses, while leishmanicidal activity is significantly increased in horses with asthma.(AU)

Os macrófagos alveolares (MAs) são uma parte essencial dos mecanismos de defesa dentro dos pulmões e sua atividade fagocítica é importante para a homeostase desse órgão. A capacidade fagocitária dos MAs obtidos do lavado broncoalveolar de 17 equinos adultos, sem raça definida (oito saudáveis e nove com diagnóstico de asma equina leve), foi estudada por meio de ensaios com promastigotas de Leishmania (Viannia) braziliensis. Foi calculado o índice fagocítico e o índice de sobrevivência. Os resultados indicam que a atividade fagocítica de MAs em cavalos com asma é semelhante a cavalos saudáveis, enquanto a atividade leishmanicida está significativamente aumentada em cavalos com essa enfermidade.(AU)

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Neutrophilic bronchiolitis is the primary lesion in asthma-affected horses. Neutrophils are key actors in host defense, migrating toward sites of inflammation and infection, where they act as early responder cells toward external insults. However, neutrophils can also mediate tissue damage in various non-infectious inflammatory processes. Within the airways, these cells likely contribute to bronchoconstriction, mucus hypersecretion, and pulmonary remodeling by releasing pro-inflammatory mediators, including the cytokines interleukin (IL)-8 and IL-17, neutrophil elastase, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs). The mechanisms that regulate neutrophil functions in the tissues are complex and incompletely understood. Therefore, the inflammatory activity of neutrophils must be regulated with exquisite precision and timing, a task achieved through a complex network of mechanisms that regulates neutrophil survival. The discovery and development of compounds that can help regulate ROS, NET formation, cytokine release, and clearance would be highly beneficial in the design of therapies for this disease in horses. In this review, neutrophil functions during inflammation will be discussed followed by a discussion of their contribution to airway tissue injury in equine asthma.

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BACKGROUND: It is accepted that T regulatory cells (Treg) control different types of immune responses. In connection with this role, we have recently described an important increase in CD4+, CD25high, Foxp3+ lymphocytes in the airway system of horses coursing with an exacerbation of severe equine asthma (EA). To explore the potential role of this population in the resolution of EA inflammation, we used a murine experimental model in which airway neutrophilic inflammation, which is similar to that observed in EA, is induced in mice by continual exposure to Aspergillus fumigatus contaminated hay. This model has the advantage that in mice we may induce a reduction of the Treg population using low doses of cyclophosphamide (Cy). RESULTS: The results indicated that the percentage of Treg cells increased with allergen exposure, as in horses; and animals partially depleted of Treg cells by treatment with Cy showed increased airway inflammation, demonstrated by an increased percentage of neutrophils and specific immunoglobulins in bronchoalveolar lavage fluid (BALF). Furthermore, a histopathologic study of animals that were pretreated with Cy before antigenic challenge showed higher cellular infiltration in the lung and deeper remodeling changes in the bronchi, including epithelial and goblet cell hyperplasia as well as airway smooth muscle hypertrophy. CONCLUSION: In this murine model of EA, the reduced number and function of Treg induced by low doses of Cy, which directly correlates with increased airway inflammation and lung infiltration, indicates that Treg may play a major role in the regulation and resolution of EA.

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