Efeitos do eugenol e deidrodieugenol na modulação da imunidade adaptativa (Th17/Treg) em modelo experimental de asma
Arquivos
Data
2021-11-26
Autores
Lourenço, Luiz Otávio 
Orientadores
Prado, Carla Máximo
Tipo
Dissertação de mestrado
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ISSN da Revista
Título de Volume
Resumo
Introdução: A asma é um problema de saúde pública, particularmente em sua forma mais grave e é definida como uma doença inflamatória crônica de vias aéreas. Apesar do avanço dos tratamentos e de vários novos fármacos, ainda existe uma porcentagem dos pacientes que não melhoram com o uso de corticosteroides e que evoluem a óbito ou apresentam alta morbidade. Nosso grupo demonstrou previamente que o eugenol (EUG) e deidrodieugenol (DHDE), este último um composto fenólico dímero do eugenol, reduziu a inflamação pulmonar em modelo de asma experimental eosinofílico e também em modelo que simula a forma mais grave da asma, o neutrofílico ou misto. Assim, o presente estudo busca entender quais mecanismos estão envolvidos nos efeitos do deidrodieugenol em modelo de asma experimental, e nossa hipótese é que este composto possa melhorar a resposta imune. Objetivos: Avaliar se o tratamento com o eugenol ou deidrodieugenol modulam a imunidade inata e adaptativa (Th17/Treg) em modelo de asma em camundongos. Metodologia: Animais do grupo controle receberam apenas solução salina 0,9%, enquanto o grupo ovoalbumina (OVA) e aqueles tratados com EUG e DHDE foram sensibilizados com injeção intraperitoneal de ovoalbumina + hidróxido de alumínio nos dias 0 e 14 do protocolo, e posteriormente foram submetidos ao desafio inalatório nos dias 22, 24, 26 e 28 com ovoalbumina 1%, o grupo controle apenas solução salina. O eugenol e o deidrodieugenol foram administrados na concentração de 10 mg/kg do dia 22 ao dia 29. Os grupos controle e OVA receberam veículo como tratamento. No 29o dia do protocolo, os animais foram eutanasiados e o pulmão coletado para análise de células inflamatórias e remodelamento pulmonar por coloração de hematoxilina e eosina (H&E) e picrosírius, respectivamente, e células positivas para CD20, CD8, IL-17, IL-10, IL-4, TNF- α, FOXP3-Treg, STAT3 e NF-κB ao redor de vias aéreas, espaço peribroncovascular e parênquima pulmonar por meio de imunohistoquímica. Além disso foi conduzido a obtenção do lavado broncoalveolar e citometria de fluxo em painel de células e fatores de transcrição, sendo feita ainda a concatenação destes dados em agrupamentos de células de acordo com seus marcadores. Resultados: Animais do grupo OVA apresentaram aumento de células mononucleares e polimorfonucleares e fibras colágenas e células positivas para CD20, CD8, para as citocinas de perfil Th1 (TNF-α), Th2 (IL-4), Th17 (IL-17) e Treg (IL-10) e positivas para fatores de transcrição STAT3, NF-κB e FOXP3 tanto em vias aéreas quanto no espaço peribroncovascular e no parênquima tecidual. Na citometria de fluxo detectamos que a sensibilização com OVA induziu aumento células inatas (macrófagos, eosinófilos, linfócitos e células dendríticas) e adaptativas (linfócitos Th2, Th17, CD8 e Treg). Os
tratamentos com EUG e DHDE foram capazes de reduzir diversos marcadores inflamatórios como células positivas para CD20, CD8, IL-17, IL-4, TNF-α, STAT3 e NF-κB nos diferentes compartimentos pulmonares. Além disso, o tratamento com DHDE ainda aumentou o número de células positivas para FOXP3 e reduziu as células positivas para IL-17 no parênquima pulmonar, sugerindo que estes compostos podem interferir com o equilíbrio Th17/Treg neste modelo. Estes dados sugerem que estes compostos tem um potencial anti-inflamatório em modelo de asma por redução das respostas Th1, Th2 e Th17 e pelo menos em parte, aumento de células T regulatórias, melhorando assim o equilíbrio entre estes perfis.
Introduction: Asthma is a public health problem, particularly in its most severe form, and is defined as a chronic inflammatory disease of the airways. Despite advances in treatments and several new drugs, there is still a percentage of patients who do not improve with the use of corticosteroids and who evolve to death or have high morbidity. Our group has previously demonstrated that eugenol (EUG) and dehydrodieugenol (DHDE), the latter a dimer phenolic compound of eugenol, reduced lung inflammation in an eosinophilic experimental asthma model and also in a model that simulates the most severe form of asthma, the neutrophilic or mixed. Thus, the question of the present study is to understand which mechanisms are involved in the effects of dehydrodieugenol in an experimental asthma model, and our hypothesis is that this compound can improve the immune response. Objectives: To evaluate whether treatment with eugenol or dehydrodieugenol modulates innate and adaptive immunity (Th17/Treg) in a mouse model of asthma. Methodology: Animals in the ovalbumin group (OVA) and those treated with EUG and DHDE were sensitized with an intraperitoneal injection of ovalbumin + aluminum hydroxide on days 0 and 14 of the protocol, and were subsequently subjected to inhalation challenge on days 22, 24, 26 and 28 with 1% ovalbumin. The control group received only 0.9% saline solution following the same protocol used in the other groups. Eugenol and dehydrodieugenol were administered at a concentration of 10 mg/kg from day 22 to day 29. Control and OVA groups received vehicle as treatment. On the 29th day of the protocol, the animals were euthanized and the lungs were collected for analysis of inflammatory cells and lung remodeling by staining with hematoxylin and eosin (H&E) and picrosirius, respectively, and positive cells for CD20, CD8, IL-17, IL- 10, IL-4, TNF-α, FOXP3-Treg, STAT3 and NF-κB around the airways, peribronchovascular space and lung parenchyma using immunohistochemistry. In addition, bronchoalveolar lavage and flow cytometry were performed in a panel of cells and transcription factors, and the concatenation of these data into cell clusters according to their markers was carried out. Results: Animals in the group showed an increase in mononuclear and polymorphonuclear cells and collagen fibers and cells positive for CD20, CD8, Th1 (TNF-α), Th2 (IL-4), Th17 (IL-17) and Treg (IL) cytokines -10) and positive for STAT3, NFκB and FOXP3 transcription factors both in the airways and in the peribronchovascular space and tissue parenchyma. In flow cytometry we detected that sensitization with OVA induced an increase in innate cells (macrophages, eosinophils, lymphocytes and dendritic cells) and adaptive (Th2, Th17, CD8 and Treg lymphocytes). Treatments with EUG and DHDE were able to reduce several inflammatory markers such as positive cells for CD20, CD8, IL-17, IL-4, TNF-α, STAT3 and NF-κB in different lung compartments. Furthermore, DHDE treatment further increased the number of FOXP3 positive cells and reduced IL-17 positive cells in the lung parenchyma, suggesting that these compounds may interfere with the Th17/Treg balance in this model. These data suggest that these compounds have antiinflammatory potential in an asthma model by reducing Th1, Th2 and Th17 responses and, at least in part, increasing regulatory T cells, thus improving the balance between these profiles
Introduction: Asthma is a public health problem, particularly in its most severe form, and is defined as a chronic inflammatory disease of the airways. Despite advances in treatments and several new drugs, there is still a percentage of patients who do not improve with the use of corticosteroids and who evolve to death or have high morbidity. Our group has previously demonstrated that eugenol (EUG) and dehydrodieugenol (DHDE), the latter a dimer phenolic compound of eugenol, reduced lung inflammation in an eosinophilic experimental asthma model and also in a model that simulates the most severe form of asthma, the neutrophilic or mixed. Thus, the question of the present study is to understand which mechanisms are involved in the effects of dehydrodieugenol in an experimental asthma model, and our hypothesis is that this compound can improve the immune response. Objectives: To evaluate whether treatment with eugenol or dehydrodieugenol modulates innate and adaptive immunity (Th17/Treg) in a mouse model of asthma. Methodology: Animals in the ovalbumin group (OVA) and those treated with EUG and DHDE were sensitized with an intraperitoneal injection of ovalbumin + aluminum hydroxide on days 0 and 14 of the protocol, and were subsequently subjected to inhalation challenge on days 22, 24, 26 and 28 with 1% ovalbumin. The control group received only 0.9% saline solution following the same protocol used in the other groups. Eugenol and dehydrodieugenol were administered at a concentration of 10 mg/kg from day 22 to day 29. Control and OVA groups received vehicle as treatment. On the 29th day of the protocol, the animals were euthanized and the lungs were collected for analysis of inflammatory cells and lung remodeling by staining with hematoxylin and eosin (H&E) and picrosirius, respectively, and positive cells for CD20, CD8, IL-17, IL- 10, IL-4, TNF-α, FOXP3-Treg, STAT3 and NF-κB around the airways, peribronchovascular space and lung parenchyma using immunohistochemistry. In addition, bronchoalveolar lavage and flow cytometry were performed in a panel of cells and transcription factors, and the concatenation of these data into cell clusters according to their markers was carried out. Results: Animals in the group showed an increase in mononuclear and polymorphonuclear cells and collagen fibers and cells positive for CD20, CD8, Th1 (TNF-α), Th2 (IL-4), Th17 (IL-17) and Treg (IL) cytokines -10) and positive for STAT3, NFκB and FOXP3 transcription factors both in the airways and in the peribronchovascular space and tissue parenchyma. In flow cytometry we detected that sensitization with OVA induced an increase in innate cells (macrophages, eosinophils, lymphocytes and dendritic cells) and adaptive (Th2, Th17, CD8 and Treg lymphocytes). Treatments with EUG and DHDE were able to reduce several inflammatory markers such as positive cells for CD20, CD8, IL-17, IL-4, TNF-α, STAT3 and NF-κB in different lung compartments. Furthermore, DHDE treatment further increased the number of FOXP3 positive cells and reduced IL-17 positive cells in the lung parenchyma, suggesting that these compounds may interfere with the Th17/Treg balance in this model. These data suggest that these compounds have antiinflammatory potential in an asthma model by reducing Th1, Th2 and Th17 responses and, at least in part, increasing regulatory T cells, thus improving the balance between these profiles
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Citação
LOURENÇO, Luiz Otávio. Efeitos do eugenol e deidrodieugenol na modulação da imunidade adaptativa (Th17/Treg) em modelo experimental de asma. 2021. 86 f. Dissertação (Mestrado Interdisciplinar em Ciências da Saúde) - Instituto de Saúde e Sociedade, Universidade Federal de São Paulo, Santos, 2021.