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Made available in DSpace on 2014-06-11T19:25:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-03Bitstream added on 2014-06-13T20:14:13Z : No. of bitstreams: 1 000739847.pdf: 1497375 bytes, checksum: 323121923378634f36af730ba11e938a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Proposição: As células epiteliais das vias respiratórias desempenham uma importante função na patogênese da asma. Elas são responsáveis pela liberação de mediadores químicos ativadores do sistema imunológico na resposta inflamatória das vias aéreas. Citocinas e quimiocinas produzidas por leucócitos ativam as células estruturais dos brônquios e incitam a síntese de outros mediadores químicos que potencializam a inflamação no local. A interleucina-9 (IL-9) é uma importante citocina ativadora das células epiteliais, regula a produção de muco e induz a expressão de quimiocinas e citocinas. Os objetivos deste estudo foram investigar se células epiteliais de pulmão murino (LA-4) estimuladas com a citocina IL-9 produzem a quimiocina do timo regulada por ativação (CCL17/TARC) e o mediador lipídico leucotrieno-C4 (LTC4), e quais vias de sinalização intracelular estariam envolvidas nesse processo. Julga-se que as proteínas quinases desempenhem uma função primordial na expressão e ativação de citocinas e quimiocinas nas vias aéreas, portanto, foi investigada a função da p38 proteína quinase ativada por mitógeno (MAPK), MAPK p42/44, e fosfatidilinositol 3-quinase (PI3K) sobre o efeito da IL-9 na expressão da quimiocina CCL17/TARC em células LA-4. Abordagem experimental: a expressão de CCL17/TARC por LA-4 foi avaliada utilizando Reação em Cadeia da Polimerase Transcriptase Reversa (RT-PCR). A produção de leucotrieno C4 (LTC4) e CCL17/TARC foi avaliada por ELISA. As células LA-4 foram pré-tratados com o antagonista do receptor de cisteinil leucotrieno (CysLT) (MK 571) (10 μM), com o inibidor da p42/44 MAPK (PD98059) (30 μM), inibidor... / Background and propose: The airway epithelial cells play an important role in the pathogenesis of asthma, are responsible for the release of chemical mediators of the immune system triggers inflammation in the airways. Cytokines and chemokines produced by leukocytes activate structural bronchial cells and induce the synthesis of other chemical mediators which enhance the site inflammation. Interleukin-9 (IL-9) is an important cytokine activating epithelial cells, regulating mucus production and induces the expression of chemokines and cytokines. The objectives of this study were to investigate whether murine lung epithelial cells (LA-4) IL-9-stimulated produce the thymus and activation-regulated chemokines (CCL17/TARC) and lipid mediator leukotriene-C4 (LTC4) and what intracellular signaling pathways would be involved in this process. Kinases are believed to play a crucial role in the expression and activation of cytokines and chemokines in the airways. Therefore the role of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and phosphatidylinositol 3-kinase (PI3K) upon the effect of IL-9 on the CCL17/TARC expression in murine lung alveolar epithelial cells (LA-4) was investigated. Experimental approach: CCL17/TARC expression in LA-4 was evaluated using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Leukotriene C4 (LTC4) and CCL17/TARC production were assessed by ELISA. LA-4 had been pre-treated with cysteinyl leukotriene receptor antagonist (CysLT) (MK 571) (10 μM), p42/44 MAPK inhibitor (PD98059) (30 M), p38 MAPK inhibitor (SB203580) (30 M), and PI3K inhibitor (Wortmannin) (0.1 M) 30 min prior to IL-9 (40 ng.mL-1) stimulation. Key results: IL-9-stimulated LA-4 induced CCL17/TARC mRNA expression after 24 hours. PD98059 and...

Orientador: Sandra Helena Penha de Oliveira / Banca: Lucia Helena Faccioli / Banca: Ana Paula Campanelli / Resumo: Proposição: As células epiteliais das vias respiratórias desempenham uma importante função na patogênese da asma. Elas são responsáveis pela liberação de mediadores químicos ativadores do sistema imunológico na resposta inflamatória das vias aéreas. Citocinas e quimiocinas produzidas por leucócitos ativam as células estruturais dos brônquios e incitam a síntese de outros mediadores químicos que potencializam a inflamação no local. A interleucina-9 (IL-9) é uma importante citocina ativadora das células epiteliais, regula a produção de muco e induz a expressão de quimiocinas e citocinas. Os objetivos deste estudo foram investigar se células epiteliais de pulmão murino (LA-4) estimuladas com a citocina IL-9 produzem a quimiocina do timo regulada por ativação (CCL17/TARC) e o mediador lipídico leucotrieno-C4 (LTC4), e quais vias de sinalização intracelular estariam envolvidas nesse processo. Julga-se que as proteínas quinases desempenhem uma função primordial na expressão e ativação de citocinas e quimiocinas nas vias aéreas, portanto, foi investigada a função da p38 proteína quinase ativada por mitógeno (MAPK), MAPK p42/44, e fosfatidilinositol 3-quinase (PI3K) sobre o efeito da IL-9 na expressão da quimiocina CCL17/TARC em células LA-4. Abordagem experimental: a expressão de CCL17/TARC por LA-4 foi avaliada utilizando Reação em Cadeia da Polimerase Transcriptase Reversa (RT-PCR). A produção de leucotrieno C4 (LTC4) e CCL17/TARC foi avaliada por ELISA. As células LA-4 foram pré-tratados com o antagonista do receptor de cisteinil leucotrieno (CysLT) (MK 571) (10 μM), com o inibidor da p42/44 MAPK (PD98059) (30 μM), inibidor... / Abstract: Background and propose: The airway epithelial cells play an important role in the pathogenesis of asthma, are responsible for the release of chemical mediators of the immune system triggers inflammation in the airways. Cytokines and chemokines produced by leukocytes activate structural bronchial cells and induce the synthesis of other chemical mediators which enhance the site inflammation. Interleukin-9 (IL-9) is an important cytokine activating epithelial cells, regulating mucus production and induces the expression of chemokines and cytokines. The objectives of this study were to investigate whether murine lung epithelial cells (LA-4) IL-9-stimulated produce the thymus and activation-regulated chemokines (CCL17/TARC) and lipid mediator leukotriene-C4 (LTC4) and what intracellular signaling pathways would be involved in this process. Kinases are believed to play a crucial role in the expression and activation of cytokines and chemokines in the airways. Therefore the role of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and phosphatidylinositol 3-kinase (PI3K) upon the effect of IL-9 on the CCL17/TARC expression in murine lung alveolar epithelial cells (LA-4) was investigated. Experimental approach: CCL17/TARC expression in LA-4 was evaluated using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Leukotriene C4 (LTC4) and CCL17/TARC production were assessed by ELISA. LA-4 had been pre-treated with cysteinyl leukotriene receptor antagonist (CysLT) (MK 571) (10 μM), p42/44 MAPK inhibitor (PD98059) (30 M), p38 MAPK inhibitor (SB203580) (30 M), and PI3K inhibitor (Wortmannin) (0.1 M) 30 min prior to IL-9 (40 ng.mL-1) stimulation. Key results: IL-9-stimulated LA-4 induced CCL17/TARC mRNA expression after 24 hours. PD98059 and... / Mestre

Indiana University-Purdue University Indianapolis (IUPUI) / Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. The majority of the T cells in tissues such as the lung are tissue-resident memory (Trm) cells, defined as cells that maintain long-lasting presence in the tissue and have rapid functional recall following challenge. Allergen-specific CD4 T helper cells that secrete the cytokine IL-9 have been shown to be a necessary component of asthma pathogenesis. However, the precise characterization and function of IL-9-secreting CD4+ cells (Th9 cells) are unknown. Here we demonstrate that IL-9 production is progressively lost in Th9 cells over several rounds of culture and that environmental cues dictate the instability or effector function of the Th9 phenotype. We show Th9 cells are long-lived tissue-resident cells with the capacity to rapidly respond to secondary allergen challenge causing allergic airway disease (AAD). We found in a memory model of Aspergillus fumigatus challenge, Th9 cells maintain tissue residency throughout a 12-week period of antigen-free rest. Additionally, we demonstrated increased frequency of IL-9-producing cells and quantity of IL-9 upon rechallenge, characteristic of a secondary response. Antibody blockade of IL-9 immediately prior to the recall challenge significantly reduced overall allergic lung inflammation, suggesting that IL-9 plays an obligate role in the allergic memory response following pulmonary allergen challenge. The protection afforded by IL-9 antibody blockade was not seen in a chronic model asthma-like disease demonstrating IL-9 has a specific role in allergic memory responses. Interestingly, IL-9-secreting cells have a polyfunctional multi-cytokine phenotype demonstrating a highly pathogenic state that we reproduced in culture. These observations suggest that IL-9 from Trm cell populations and Th9 cells play a novel role in allergen recall responses and are potential therapeutic targets for patients suffering from chronic intermittent asthma. / 2022-05-05

Indiana University-Purdue University Indianapolis (IUPUI) / CD4 T cells are critical regulators of inflammatory diseases and play an important role in allergic airway diseases (AAD) by producing type 2 cytokines including IL-4, IL- 13, IL-5 and IL-9. In chronic AAD models, IL-9 producing CD4 T-helper (TH9) cells lead to accumulation of eosinophils and mast cells in the airway, increase levels of type-2 cytokines, stimulate ILC2 cell proliferation, and induce mucus production from airway epithelium. However, the transcriptional network that governs the development of TH9 cells and their function during allergic responses is not clearly understood. Naïve CD4 T cells differentiate into TH9 cells in the presence of IL-2, IL-4 and TGFβ, activating a complex network of transcription factors that restricts their development to TH9 lineage. In this study a variety of approaches were utilized, including characterizing Il9 reporter mice, to identify an additional Ets-transcription factor termed ERG (Ets-related gene) that is expressed preferentially in the TH9 subset. Knock-down of Erg during TH9 polarization led to a decrease in IL-9 production in TH9 cells in vitro. Deletion of Erg at the later stage of TH9 induced pathogenesis resulted in reduced IL-9 production in the airways in chronic AAD. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during TH9 polarization. In the absence of PU.1 and ETV5, ERG regulated IL-9 production independent of other Ets-transcription factors and the deletion of Erg further lead to a decrease in IL-9 production by lung-derived CD4-T cells in chronic AAD model. Lastly, I also identified IL-9 secreting CD4 tissue resident memory cell population that play an instrumental role in allergic recall responses. In summary, in this study novel transcription factors were identified that can regulate TH9 function and the role of IL-9 in allergic airway recall responses. / 2022-12-28

Indiana University-Purdue University Indianapolis (IUPUI) / Among the cytokines regulating immunity, interleukin 9 (IL-9) has gained considerable attention for its role in inflammation, immune tolerance, and tumor immunity. IL-9 has a broad array of functions and acts on multiple cell types to regulate immune responses. IL-9 receptor is expressed on both non-hematopoietic cells and hematopoietic cells in the innate and adaptive immune system. IL-9 demonstrates a remarkable degree of tissue-specific functionality that varies by tissue site and the context of the inflammatory milieu. In this dissertation, we investigate the biological activities of IL-9 and identify distinct IL-9-responsive cell type in the immune pathogenesis of disease models including allergic airway disease, lung cancer, and urinary tract infection. When examining airway hyperreactivity, we found IL-9-dependent mast cell function was critical. Using adoptive transfer models and newly generated mice with an inactivation of the Il9 gene restricted to T cells generated by CD4-cre/LoxP-mediated targeting, we demonstrate that T cell secreted IL-9 promotes mast cell progenitor proliferation and CCR2-dependent mast cell migration during allergic airway inflammation. In IL-9-mediated pro-tumor responses, interstitial macrophages, but not mast cells, respond to T cell IL-9 to enhance B16 metastatic tumor growth. In the context of urinary tract infection, IL-9 contributes to protection against E. coli bladder infection potentially by enhancing CCL20 production in epithelial cells to recruit macrophages and neutrophils. Altogether, IL-9 can exert cell type-specific effects that identify its roles in immunity and disease. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial, and where it has the potential to complicate clinical outcomes.

The thesis focuses on the investigation of the role of tick salivary components in the course of Lyme disease in mice. It includes studies on the saliva-facilitated transmission of Borrelia burgdorferi in vivo and the effect of tick cysteine protease inhibitors (cystatins) both on murine immune cells and the transmission of B. burgdorferi spirochetes in mice. The thesis also reveals practical applications of salivary cystatins for the development of anti-tick vaccine and the application of the pharmacological action of a tick salivary cystatin for the therapy of the disease symptoms in a mouse model of experimental asthma.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Chagas disease, caused by the protozoan Trypanosoma cruzi, is ranked as the most serious parasitic disease in Latin America and has become a worldwide problem. A bulk of studies substantiates that Th1-associated cytokines are essential elements in early resistance against the parasite and are associated with the development of the chronic cardiac form. Although cytokines have a key role in the immune response against T. cruzi, little is known about IL-3, IL-7 and IL-9 in this context. Then the aim of this study was to analyze the role of IL-3, IL-7 and IL-9 in the acute phase of T. cruzi experimental infection. For this purpose, parameters indicative of improvement in clinical status of the animals both infected and treated as just treated were studied, such as: morbidity, mortality and histopathology. The amount of cardiac mast cells and the serum cytokines profile were also evaluated. Our data revealed that the treatment with IL-3, IL-7 or IL-9 did not alter the clinical parameters analyzed or the amount of cardiac mast cells in mice infected with T. cruzi. However the treatment with IL-3 decreased the cardiac T. cruzi-induced inflammation and the treatment with IL-7 increased serum levels of IL-5 in infected animals. In addition, the treatment with IL-9 increased the serum levels of Th1 cytokine profile (IL-2, IFN-y and TNF-α) and decreased cardiac fibrosis in infected animals, suggesting a possible protective role of this cytokine in this context. Taken together, our results underline the importance of these cytokines in modulation of T. cruzi infection. Since studies involving IL-3, IL-7 and IL-9 activity during Chagas disease are critical in understanding the parasite control process and the protective and/or harmful action of these cytokines in the host. In addition, understanding of the immunological mechanisms mediated by these cytokines that are involved in disease development may contribute to the establishment of new therapeutic interventions to prevent Chagas disease and treat their symptomatic forms. / A Doença de Chagas, causada pelo protozoário Trypanosoma cruzi, é classificada como a doença parasitária mais grave da América Latina e tornou-se um problema mundial. A maior parte dos estudos confirma que as citocinas de perfil Th1 são elementos essenciais na resistência precoce contra o parasita e estão associadas ao desenvolvimento da forma cardíaca crônica. Embora as citocinas tenham um papel chave na resposta imune contra T. cruzi, pouco se sabe a respeito da função de IL-3, IL-7 e IL-9 neste contexto. Assim, o objetivo deste estudo foi analisar o papel destas citocinas na fase aguda da infecção experimental com T. cruzi. Para isto, parâmetros indicativos de melhora clínica dos animais tanto infectados e tratados como apenas tratados foram estudados, tais como: morbidade, mortalidade e histopatologia. A quantidade de mastócitos cardíacos e o perfil de citocinas séricas também foram avaliados. Os dados revelaram que o tratamento com IL-3, IL-7 ou IL-9 não alterou os parâmetros clínicos analisados nem a quantidade de mastócitos cardíacos nos camundongos infectados com T. cruzi. No entanto, o tratamento com IL-3 diminuiu a inflamação cardíaca T. cruzi-induzida e o tratamento com IL-7 aumentou os níveis séricos de IL-5 nos animais infectados. Além disso, o tratamento com IL-9 aumentou os níveis séricos das citocinas de perfil Th1 (IL-2, IFN-y e TNF-α) e diminuiu a fibrose cardíaca nos animais infectados, sugerindo um possível papel protetor desta citocina neste contexto. Em suma, os nossos resultados demonstram a importância destas citocinas na modulação da infecção por T. cruzi. Já que estudos envolvendo a atividade de IL-3, IL-7 e IL-9 durante a doença de Chagas são fundamentais na compreensão sobre o processo de controle parasitário e da ação protetora e/ou prejudicial dessas citocinas no hospedeiro. Além disso, o entendimento dos mecanismos imunológicos mediados por estas citocinas que estão envolvidos no desenvolvimento da doença pode contribuir para o estabelecimento de novas intervenções terapêuticas a fim de prevenir a doença de Chagas e tratar suas formas sintomáticas / Doutor em Imunologia e Parasitologia Aplicadas