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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Exploring the pathogenic potential of myelin-reactive Th1 and Th17 cells in central nervous system autoimmune disease

Prendergast, Catriona Taguma January 2011 (has links)
The activation of naïve T cells results in their proliferation and differentiation into a particular T-helper (Th) phenotype, namely Th1, Th2 or Th17 cells. This thesis focuses on the role of pro-inflammatory Th1 and Th17 cells in the induction of autoimmune disease of the central nervous system (CNS), using murine experimental autoimmune encephalomyelitis (EAE) as the model. Classically, EAE has been considered to be a Th1-mediated disease. However, since the emergence of the Th17 cells, there has been a paradigm shift towards Th17 cells being the key pathogenic subset in autoimmune disease. This thesis established robust protocols for the differentiation of naïve T cells into myelin-reactive Th1 or Th17 cells, producing ‘clean’ populations devoid of any contaminating cells. Passive T cell-transfer experiments revealed that myelin-reactive Th1 cells could induce EAE, whereas Th17 cells could not. This lack of disease correlated with the inability of the Th17 cells to accumulate in the non-inflamed CNS. Myelin-reactive Th1 cells did have this capability and only once inflammation was established, could Th17 cells be identified in the CNS, potentially exacerbating the disease. After these differences were observed, the project investigated two main aims: 1) to identify differences in homing molecule expression on Th1 and Th17 cells which could explain the difference in their ability to home to the CNS, and to investigate the functional significance of such differences, by molecular blockade; 2) to investigate the requirements for three key cytokines in EAE pathogenesis in passive T cell transfer models, investigating IFN-γ,IL-17 and TNF-α. P-selectin glycoprotein ligand-1 appeared to be important for the initial entry of inflammatory T cells into the CNS. Th1 cells deficient in IFN-γ were capable of IFNinducing EAE. A proportion of the mice developed “atypical” clinical signs, which correlated with T cell infiltration predominantly of the brain, rather than the spinal cord. This atypical EAE may be IL-17 dependent. In conclusion, this thesis indicates the importance of not focusing all resources and therapeutic approaches on Th17- induced inflammation as Th17 cells may not play such a major role as previously thought.
2

New Insights into the Regulation of Intestinal Immunity by Nod1 and Nod2

Rubino, Stephen 02 April 2014 (has links)
Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate immune responses. Importantly, mutations in the human NOD2 gene have been associated with increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease. However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear. In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-) exhibited delayed induction of intestinal inflammation at early timepoints after infection with Citrobacter rodentium compared to wild-type mice, which correlated with compromised control of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17 and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response the innate Th17 (iTh17) response. The iTh17 cells exhibited an effector memory phenotype and required priming from the enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major histocompatibility complex (MHC) class II expression in hematopoietic cells was required for the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa. In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP. Together, the data presented in this thesis expand our current understanding of the roles of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and the therapeutic potential of novel Nod2 agonists.
3

New Insights into the Regulation of Intestinal Immunity by Nod1 and Nod2

Rubino, Stephen 02 April 2014 (has links)
Nod1 and Nod2 are intracellular pattern recognition receptors that detect specific moieties of peptidoglycan, a critical component of the bacterial cell wall, to initiate host innate immune responses. Importantly, mutations in the human NOD2 gene have been associated with increased risk to develop mucosal auto-inflammatory disorders such as Crohn’s Disease. However, how Nod1 and Nod2 mediate mucosal homeostasis still remains unclear. In Chapter 2, I determined that mice deficient for both Nod1 and Nod2 (Nod1-/-Nod2-/-) exhibited delayed induction of intestinal inflammation at early timepoints after infection with Citrobacter rodentium compared to wild-type mice, which correlated with compromised control of the pathogen at later timepoints. Notably, I determined that induction of the cytokines IL-17 and IL-22 in the cecal lamina propria (LP) was blunted in Nod1-/-Nod2-/- mice after infection with either C. rodentium or Salmonella enterica serovar Typhimurium. Importantly, I found that Th17 cells were the principal producers of IL-17 and IL-22 after infection. Due to the rapid kinetics of activation and the regulation by Nod1 and Nod2, I termed this early mucosal response the innate Th17 (iTh17) response. The iTh17 cells exhibited an effector memory phenotype and required priming from the enteric microbiota for full induction. Therefore, in Chapter 3, I next determined that major histocompatibility complex (MHC) class II expression in hematopoietic cells was required for the induction of LP Th17 responses after infection. Interestingly, I found that the percentage IL-17+CD8+ T cells was strongly upregulated when MHCII signaling was ablated, suggesting a dynamic compensatory mechanism of IL-17-producing T cell responses in the mucosa. In Chapter 4, I identified MDP(D-Glu2)-OCH3 as a synthetic Nod2 agonist that exhibited increased stimulatory ability of Nod2-dependent NF-B activation compared to MDP in an unbiased screen. Moreover, I determined that MDP(D-Glu2)-OCH3 induced more potent inflammatory responses both in vitro and in vivo and was a better adjuvant than MDP. Together, the data presented in this thesis expand our current understanding of the roles of Nod1 and Nod2 in the intestinal LP, the regulation of IL-17 producing T cells in the gut and the therapeutic potential of novel Nod2 agonists.
4

Prostaglandina E2 inibe a diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas / Prostaglandina E2 inhibts the differentiation of Th17 cells on the context of phagocytosis of infected apoptotic cells

Silva, Felipe Fortino Verdan da 16 November 2015 (has links)
A fagocitose de células apoptóticas, também denominada eferocitose, é um processo dinâmico e de fundamental importância para homeostase dos tecidos após uma injúria. Estudos demonstraram previamente que a fagocitose de células apoptóticas promove a síntese de mediadores anti-inflamatórios como PGE2, TGF-? e IL-10, podendo resultar num microambiente supressor e aumento da susceptibilidade do hospedeiro contra agentes infecciosos. Entretanto, a fagocitose de células apoptóticas infectadas por células dendríticas promove a geração não apenas de citocinas anti-inflamatórias como TGF-?, mas também de IL-6 e IL-23, levando a um efeito imunoestimulador, a diferenciação de células Th17. A atuação da PGE2 na imunidade adaptativa vem sendo investigada quanto à diferenciação e ativação de linfócitos Th1, Treg e Th17. Nossos resultados demonstram que a fagocitose de células apoptóticas infectadas com E. coli promove a ativação e migração de células dendríticas, assim como a produção de citocinas pró- e anti-inflamatórias e altos níveis de PGE2. No entanto, diferente da hipótese inicial, a presença de altas concentrações de PGE2 inibe drasticamente a diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas com E. coli por células dendríticas, in vitro. O tratamento de linfócitos T CD4+naive com antagonistas e agonistas de EP2/EP4 demonstram que o efeito supressor de PGE2 é mediado primordialmente pelo receptor EP4. Por fim, nossos resultados in vivo comprovam os resultados obtidos in vitro, demonstrando o papel supressor de PGE2 na diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas em modelo de infecção pulmonar. / The phagocytosis of apoptotic cells, also called efferocytosis, is a dynamic process critical for tissue homeostasis after injury. We and other groups previously have shown that phagocytosis of apoptotic cells promotes the synthesis of anti-inflammatory mediators such as PGE2, TGF-? and IL-10, that may result in the suppression of host defense against microorganisms. However, an elegant study using infected apoptotic cells showed that phagocytosis of these cells promote not only the generation of anti-inflammatory cytokines such as TGF-? but also IL-6 and IL-23, resulting in an immunostimulatory effect, the differentiation of Th17 cells. The role of PGE2 in adaptive immunity has been investigated regarding differentiation and activation of Th1, Th17 and Treg. Our results demonstrate that engulfment of E.coli infected apoptotic cells promotes the activation and migration of dendritic cells as well as production of pro and anti-inflammatory cytokines together with high levels of PGE2. However, differing from our hypothesis, high levels of PGE2 inhibits drastically the differentiation of Th17 cells on the context of engulfment of E.coli infected apoptotic cells by dendritic cells in vitro. The treatment of T CD4+naive cells with antagonist or agonists of EP2/EP4 receptors demonstrates the suppressor effect is mainly mediated by EP4 receptor. Finally, the instillation of E.coli infected apoptotic cells in E.coli infected animals resulted on modest Th17 increase but treatment with cox inhibitor increased Th17 cell differentiation. Therefore, our in vivo results prove the in vitro results.
5

Prostaglandina E2 inibe a diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas / Prostaglandina E2 inhibts the differentiation of Th17 cells on the context of phagocytosis of infected apoptotic cells

Felipe Fortino Verdan da Silva 16 November 2015 (has links)
A fagocitose de células apoptóticas, também denominada eferocitose, é um processo dinâmico e de fundamental importância para homeostase dos tecidos após uma injúria. Estudos demonstraram previamente que a fagocitose de células apoptóticas promove a síntese de mediadores anti-inflamatórios como PGE2, TGF-? e IL-10, podendo resultar num microambiente supressor e aumento da susceptibilidade do hospedeiro contra agentes infecciosos. Entretanto, a fagocitose de células apoptóticas infectadas por células dendríticas promove a geração não apenas de citocinas anti-inflamatórias como TGF-?, mas também de IL-6 e IL-23, levando a um efeito imunoestimulador, a diferenciação de células Th17. A atuação da PGE2 na imunidade adaptativa vem sendo investigada quanto à diferenciação e ativação de linfócitos Th1, Treg e Th17. Nossos resultados demonstram que a fagocitose de células apoptóticas infectadas com E. coli promove a ativação e migração de células dendríticas, assim como a produção de citocinas pró- e anti-inflamatórias e altos níveis de PGE2. No entanto, diferente da hipótese inicial, a presença de altas concentrações de PGE2 inibe drasticamente a diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas com E. coli por células dendríticas, in vitro. O tratamento de linfócitos T CD4+naive com antagonistas e agonistas de EP2/EP4 demonstram que o efeito supressor de PGE2 é mediado primordialmente pelo receptor EP4. Por fim, nossos resultados in vivo comprovam os resultados obtidos in vitro, demonstrando o papel supressor de PGE2 na diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas em modelo de infecção pulmonar. / The phagocytosis of apoptotic cells, also called efferocytosis, is a dynamic process critical for tissue homeostasis after injury. We and other groups previously have shown that phagocytosis of apoptotic cells promotes the synthesis of anti-inflammatory mediators such as PGE2, TGF-? and IL-10, that may result in the suppression of host defense against microorganisms. However, an elegant study using infected apoptotic cells showed that phagocytosis of these cells promote not only the generation of anti-inflammatory cytokines such as TGF-? but also IL-6 and IL-23, resulting in an immunostimulatory effect, the differentiation of Th17 cells. The role of PGE2 in adaptive immunity has been investigated regarding differentiation and activation of Th1, Th17 and Treg. Our results demonstrate that engulfment of E.coli infected apoptotic cells promotes the activation and migration of dendritic cells as well as production of pro and anti-inflammatory cytokines together with high levels of PGE2. However, differing from our hypothesis, high levels of PGE2 inhibits drastically the differentiation of Th17 cells on the context of engulfment of E.coli infected apoptotic cells by dendritic cells in vitro. The treatment of T CD4+naive cells with antagonist or agonists of EP2/EP4 receptors demonstrates the suppressor effect is mainly mediated by EP4 receptor. Finally, the instillation of E.coli infected apoptotic cells in E.coli infected animals resulted on modest Th17 increase but treatment with cox inhibitor increased Th17 cell differentiation. Therefore, our in vivo results prove the in vitro results.
6

Participação do Nlrp12 na diferenciação de linfócitos Th17 e no desenvolvimento da artrite experimental / Role of Nlrp12 on Th17 differentiation and experimental arthritis development

Douglas da Silva Prado 28 January 2016 (has links)
A Artrite reumatoide é uma doença autoimune que acomete cerca de 1% da população mundial adulta, sendo caracterizada pela participação de linfócitos Th17 no seu desenvolvimento. Na busca por novos alvos terapêuticos e pela compreensão da fisiopatologia, se destacam os inflamassomas que são plataformas proteicas, caracterizados pela produção de citocinas pró-inflamatórias por células do sistema imune inato. De forma interessante, foi demonstrado que linfócitos T CD4 também expressam alguns sensores dessas plataformas, como o Nlrp12. Adicionalmente, este sensor é responsável pela modulação negativa do NF-?B, demonstrando outra característica atípica em relação aos outros inflamassomas. Nesse sentido, foi avaliada a participação do Nlrp12 no desenvolvimento da artrite experimental e na diferenciação linfócitos Th17. Foi verificado nesse estudo que o Nlrp12 é regulado positivamente durante o desenvolvimento da artrite experimental, sendo um modulador negativo desse processo. Isso se deve a uma redução na resposta inflamatória inata do modelo e pela modulação negativa na resposta Th17. Nesse sentido, o controle da resposta Th17 e o desenvolvimento da artrite experimental ocorre por um mecanismo dependente da fosforilação do fator de transcrição Stat3, que é crítico na diferenciação de linfócitos Th17. Desta forma, este estudo demonstra uma nova função para o sensor Nlrp12 no desenvolvimento da artrite experimental, por modular a resposta imune adaptativa de forma direta nos linfócitos T CD4 / Rheumatoid Arthritis is an autoimmune disease that occurs in approximately 1% of the adult population worldwide, with critical role of Th17 cells in your development. In the search for new therapeutic targets and the understanding of the pathophysiology, there are inflammasomes which are protein platforms, characterized through pro-inflammatory cytokines production by innate immune system cells. Interestingly, it was demonstrated that CD4 T cells express some inflammasome sensors, as Nlrp12. Additionally, this sensor is responsible for downregulation of NF-?B, showing another atypical feature in relation to other inflammasomes. Thereby, it was evaluated the role of Nlrp12 on experimental arthritis development and Th17 differentiation. It was found in this study that Nlrp12 is upregulated during experimental arthritis development, working as negative regulator of this process. Thus, Nlrp12 downregulates innate inflammatory response from experimental model and Th17 response. Therefore, experimental arthritis development and Th17 differentiation control occurs in a Stat3 phosphorylation dependente-manne, which is a critical transcription factor on Th17 differentiation. Thus, this study demonstrates a new function for Nlrp12 on experimental arthritis development, by directly to modulate adaptive immune response in CD4 cells
7

Participação do Nlrp12 na diferenciação de linfócitos Th17 e no desenvolvimento da artrite experimental / Role of Nlrp12 on Th17 differentiation and experimental arthritis development

Prado, Douglas da Silva 28 January 2016 (has links)
A Artrite reumatoide é uma doença autoimune que acomete cerca de 1% da população mundial adulta, sendo caracterizada pela participação de linfócitos Th17 no seu desenvolvimento. Na busca por novos alvos terapêuticos e pela compreensão da fisiopatologia, se destacam os inflamassomas que são plataformas proteicas, caracterizados pela produção de citocinas pró-inflamatórias por células do sistema imune inato. De forma interessante, foi demonstrado que linfócitos T CD4 também expressam alguns sensores dessas plataformas, como o Nlrp12. Adicionalmente, este sensor é responsável pela modulação negativa do NF-?B, demonstrando outra característica atípica em relação aos outros inflamassomas. Nesse sentido, foi avaliada a participação do Nlrp12 no desenvolvimento da artrite experimental e na diferenciação linfócitos Th17. Foi verificado nesse estudo que o Nlrp12 é regulado positivamente durante o desenvolvimento da artrite experimental, sendo um modulador negativo desse processo. Isso se deve a uma redução na resposta inflamatória inata do modelo e pela modulação negativa na resposta Th17. Nesse sentido, o controle da resposta Th17 e o desenvolvimento da artrite experimental ocorre por um mecanismo dependente da fosforilação do fator de transcrição Stat3, que é crítico na diferenciação de linfócitos Th17. Desta forma, este estudo demonstra uma nova função para o sensor Nlrp12 no desenvolvimento da artrite experimental, por modular a resposta imune adaptativa de forma direta nos linfócitos T CD4 / Rheumatoid Arthritis is an autoimmune disease that occurs in approximately 1% of the adult population worldwide, with critical role of Th17 cells in your development. In the search for new therapeutic targets and the understanding of the pathophysiology, there are inflammasomes which are protein platforms, characterized through pro-inflammatory cytokines production by innate immune system cells. Interestingly, it was demonstrated that CD4 T cells express some inflammasome sensors, as Nlrp12. Additionally, this sensor is responsible for downregulation of NF-?B, showing another atypical feature in relation to other inflammasomes. Thereby, it was evaluated the role of Nlrp12 on experimental arthritis development and Th17 differentiation. It was found in this study that Nlrp12 is upregulated during experimental arthritis development, working as negative regulator of this process. Thus, Nlrp12 downregulates innate inflammatory response from experimental model and Th17 response. Therefore, experimental arthritis development and Th17 differentiation control occurs in a Stat3 phosphorylation dependente-manne, which is a critical transcription factor on Th17 differentiation. Thus, this study demonstrates a new function for Nlrp12 on experimental arthritis development, by directly to modulate adaptive immune response in CD4 cells
8

Envolvimento da enzima Piruvato Quinase M2 (PKM2) na diferenciação de linfócitos Th17 e patogênese da encefalomielite autoimune experimental / Involvement of the enzyme Pyruvate Kinase M2 (PKM2) in the differentiation of Th17 lymphocytes and pathogenesis of experimental autoimmune encephalomyelitis

Damasceno, Luis Eduardo Alves 30 January 2018 (has links)
Nos últimos anos, um importante destaque tem sido dado aos linfócitos Th17 para o desenvolvimento e manutenção da inflamação associada à autoimunidade. A esclerose múltipla é uma doença autoimune desmielinizante do SNC, cuja patogênese está associada à resposta do padrão Th17. Evidências têm demonstrado que estas células são submetidas a uma reprogramação metabólica após serem ativadas, sendo essa adequação essencial para sua completa diferenciação e aquisição de funções efetoras. A enzima Piruvato Quinase M2 (PKM2) participa da etapa final da glicólise convertendo fosfoenolpiruvato em piruvato. Estudos recentes demonstraram que a fosforilação de PKM2 a torna capaz de translocar para o núcleo, onde adquire um papel no controle da expressão gênica. Nesse sentido, o objetivo deste estudo foi avaliar o envolvimento da PKM2 na diferenciação de linfócitos Th17, bem como sua participação no desenvolvimento da encefalomielite autoimune experimental (EAE), um modelo animal de esclerose múltipla. Observou-se que durante o processo de diferenciação, os linfócitos Th17 aumentam a expressão gênica de PKM2 bem como a sua forma fosforilada (Y105). De forma interessante, tanto a inibição farmacológica como a deleção gênica da PKM2 especificamente em linfócitos T promoveram uma redução da diferenciação e expansão da subpopulação Th17, que foi associada com diminuição na expressão de moléculas efetoras e fatores de transcrição chave para o estabelecimento do fenótipo Th17. Em um contexto de resposta autoimune, notou-se que PKM2 é superexpressa nos órgãos linfóides periféricos e sistema nervoso central de animais com EAE, sendo correlacionada com o infiltrado de células inflamatórias. Corroborando com os dados in vitro, a deficiência de PKM2 em linfócitos T promoveu redução dos sinais clínicos da EAE, acompanhada de baixa frequência de linfócitos Th17 e menor expressão de moléculas inflamatórias do perfil Th17. Adicionalmente, o tratamento farmacológico com o inibidor da PKM2 atenuou a progressão e gravidade da EAE. Portanto, esses achados implicam um importante papel para PKM2 em doenças autoimunes por regular o desenvolvimento e função de linfócitos Th17. / Over the past few years, an important highlight has been given to Th17 lymphocytes for the development and maintenance of autoimmunity-associated inflammation. Multiple sclerosis is a CNS demyelinating autoimmune disease that is associated to Th17-mediated response. Some evidences have demonstrated that those cells undergo metabolic reprogramming after being activated, which is essential for their complete differentiation and acquisition of effector functions. The enzyme Pyruvate kinase M2 (PKM2) participates at the final step of glycolysis by converting phosphoenolpyruvate into pyruvate. Recent studies have demonstrated that PKM2 phosphorylation allows its translocation into the nucleus, where it plays a role in controlling gene expression. Thus, the aim of this study was to evaluate the involvement of PKM2 in Th17 lymphocytes differentiation, as well as its role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. It was perceived that during differentiation process, Th17 lymphocytes increase PKM2 gene expression, and also its phosphorylated form (Y105). Interestingly, both pharmacological inhibition and T-lymphocyte-specific PKM2 gene deletion promoted a reduction in differentiation and expansion of Th17 subpopulation, being associated to diminished expression of effector molecules and key transcription factors for the establishment of Th17 phenotype. In the context of an autoimmune response, it was noticed that PKM2 is overexpressed in peripheral lymphoid organs and central nervous system of EAE-bearing mice, which was correlated with the inflammatory cell infiltration. Corroborating with in vitro data, the deficiency of PKM2 in T lymphocytes led to a reduction of EAE clinical score along with low Th17 frequency and diminished expression of Th17-related inflammatory molecules. Additionally, pharmacological treatment with the PKM2 inhibitor attenuated EAE progression and severity. Therefore, these findings imply an important role for PKM2 in autoimmune diseases by regulating the development and function of Th17 lymphocytes.
9

Envolvimento da enzima Piruvato Quinase M2 (PKM2) na diferenciação de linfócitos Th17 e patogênese da encefalomielite autoimune experimental / Involvement of the enzyme Pyruvate Kinase M2 (PKM2) in the differentiation of Th17 lymphocytes and pathogenesis of experimental autoimmune encephalomyelitis

Luis Eduardo Alves Damasceno 30 January 2018 (has links)
Nos últimos anos, um importante destaque tem sido dado aos linfócitos Th17 para o desenvolvimento e manutenção da inflamação associada à autoimunidade. A esclerose múltipla é uma doença autoimune desmielinizante do SNC, cuja patogênese está associada à resposta do padrão Th17. Evidências têm demonstrado que estas células são submetidas a uma reprogramação metabólica após serem ativadas, sendo essa adequação essencial para sua completa diferenciação e aquisição de funções efetoras. A enzima Piruvato Quinase M2 (PKM2) participa da etapa final da glicólise convertendo fosfoenolpiruvato em piruvato. Estudos recentes demonstraram que a fosforilação de PKM2 a torna capaz de translocar para o núcleo, onde adquire um papel no controle da expressão gênica. Nesse sentido, o objetivo deste estudo foi avaliar o envolvimento da PKM2 na diferenciação de linfócitos Th17, bem como sua participação no desenvolvimento da encefalomielite autoimune experimental (EAE), um modelo animal de esclerose múltipla. Observou-se que durante o processo de diferenciação, os linfócitos Th17 aumentam a expressão gênica de PKM2 bem como a sua forma fosforilada (Y105). De forma interessante, tanto a inibição farmacológica como a deleção gênica da PKM2 especificamente em linfócitos T promoveram uma redução da diferenciação e expansão da subpopulação Th17, que foi associada com diminuição na expressão de moléculas efetoras e fatores de transcrição chave para o estabelecimento do fenótipo Th17. Em um contexto de resposta autoimune, notou-se que PKM2 é superexpressa nos órgãos linfóides periféricos e sistema nervoso central de animais com EAE, sendo correlacionada com o infiltrado de células inflamatórias. Corroborando com os dados in vitro, a deficiência de PKM2 em linfócitos T promoveu redução dos sinais clínicos da EAE, acompanhada de baixa frequência de linfócitos Th17 e menor expressão de moléculas inflamatórias do perfil Th17. Adicionalmente, o tratamento farmacológico com o inibidor da PKM2 atenuou a progressão e gravidade da EAE. Portanto, esses achados implicam um importante papel para PKM2 em doenças autoimunes por regular o desenvolvimento e função de linfócitos Th17. / Over the past few years, an important highlight has been given to Th17 lymphocytes for the development and maintenance of autoimmunity-associated inflammation. Multiple sclerosis is a CNS demyelinating autoimmune disease that is associated to Th17-mediated response. Some evidences have demonstrated that those cells undergo metabolic reprogramming after being activated, which is essential for their complete differentiation and acquisition of effector functions. The enzyme Pyruvate kinase M2 (PKM2) participates at the final step of glycolysis by converting phosphoenolpyruvate into pyruvate. Recent studies have demonstrated that PKM2 phosphorylation allows its translocation into the nucleus, where it plays a role in controlling gene expression. Thus, the aim of this study was to evaluate the involvement of PKM2 in Th17 lymphocytes differentiation, as well as its role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. It was perceived that during differentiation process, Th17 lymphocytes increase PKM2 gene expression, and also its phosphorylated form (Y105). Interestingly, both pharmacological inhibition and T-lymphocyte-specific PKM2 gene deletion promoted a reduction in differentiation and expansion of Th17 subpopulation, being associated to diminished expression of effector molecules and key transcription factors for the establishment of Th17 phenotype. In the context of an autoimmune response, it was noticed that PKM2 is overexpressed in peripheral lymphoid organs and central nervous system of EAE-bearing mice, which was correlated with the inflammatory cell infiltration. Corroborating with in vitro data, the deficiency of PKM2 in T lymphocytes led to a reduction of EAE clinical score along with low Th17 frequency and diminished expression of Th17-related inflammatory molecules. Additionally, pharmacological treatment with the PKM2 inhibitor attenuated EAE progression and severity. Therefore, these findings imply an important role for PKM2 in autoimmune diseases by regulating the development and function of Th17 lymphocytes.
10

Avaliação da capacidade de indução de diferenciação de células TH17 por células dendríticas estimuladas com células leveduriformes de Paracoccidioides brasiliensis e mecanismos de sinalização intracelular envolvidos / Evaluation of the capability of differentiation of Th17 cells by dendritic cells stimulated with Paracoccidioides brasiliensis yeast cells and intracellular signaling mechanisms involved

Ferreira, Maria Carolina, 1983- 23 August 2018 (has links)
Orientadores: Ronei Luciano Mamoni, Maria Heloisa de Souza Lima Blotta / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T00:10:57Z (GMT). No. of bitstreams: 1 Ferreira_MariaCarolina_D.pdf: 3693187 bytes, checksum: 65dcd416d067ae66b391e67841a7b240 (MD5) Previous issue date: 2013 / Resumo: A paracoccidioidomicose (PCM), causada pelo fungo dimórfico Paracoccidioides brasiliensis (Pb), é a micose sistêmica de maior incidência no Brasil. Estudos anteriores demonstraram que a resistência ou suscetibilidade a essa infecção podem ser associadas, respectivamente, a padrões de resposta Th1 ou Th2. Recentemente, foram descritas novas subpopulações de linfócitos T, dentre elas as células Th17, que tem se mostrado importantes na proteção contra infecções fúngicas, mas cujo papel ainda não foi estudado na PCM humana. A diferenciação de células T CD4+ é modulada após o reconhecimento do patógeno por células dendríticas (DCs) por meio de vários receptores de reconhecimento padrão (PRRs), os quais modulam a diferenciação de células Th1, Th2 e Th17. Neste trabalho investigamos o papel dos receptores TLR2, TLR4 e Dectina-1 no reconhecimento de células leveduriformes de P. brasiliensis (Pb) por DCs derivadas de monócitos, assim como, a capacidade dessas DCs em modular a diferenciação de linfócitos T CD4+. Observamos que DCs expostas a células leveduriformes de Pb apresentam um fenótipo maduro (expressão de CCR7, CD83, CD86 e MHC II) e produzem citocinas (IL- ?, IL-6, IL-23, TNF-? e TFG- ?), levando à diferenciação de linfócitos T CD4+ produtores de IL-17, IFN-?, IL-22, IL-17/IL-22 e IL-17/IFN- ?. Também observamos que DCs estimuladas com células leveduriformes de Pb apresentam ativação de moléculas envolvidas na sinalização via TLRs e Dectina-1 (JNK, p38, Akt e ERK para os TLRs e Syk para Dectina-1), e que o bloqueio de Dectina-1 e/ou TLR2 resultou em um número menor de células Th17. Ao analisar a produção de citocinas por células mononucleares do sangue periférico, observamos que pacientes com a FJ da PCM apresentam produção aumentada de IL-4 e que pacientes com a FA apresentam produção elevada de IL-17 e IFN- ?. Em conjunto nossos resultados demonstram que a Dectina-1 e TLR2 são os receptores mais importantes para o reconhecimento de leveduras de Pb por células dendríticas e que esse reconhecimento pode levar à diferenciação de linfócitos T efetores produtores de IL-17 e IFN- ?. Além disso, podemos concluir que a FA da PCM apresenta uma resposta imunológica mista, com participação de linfócitos Th17 / Abstract: Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycosis in Brazil. Previous studies showed that resistance or susceptibility to PCM can be associated to a Th1 or Th2 responses respectively. More recently, Th17 response was showed to be protective in fungal infections, but there is no data about Th17 cells in human PCM. T CD4+ differentiation is elicited and shaped after pathogen recognition by dendritic cells (DCs) through several PRRs, which modulate Th1, Th2 and Th17 generation. In this study we evaluated the role of TLR-2, TLR-4 and Dectin-1 in the recognition of Pb yeast cells by monocytes derived DCs, as well as, the capability of these DCs to modulate T CD4+ lymphocytes differentiation. We observed that DCs exposed to Pb presented a mature phenotype (expression of CCR7, CD83, CD80, CD86 and MHC II) and produce cytokines (IL-1, IL-6, IL-23, TNF-? and TGF- ) that promote the differentiation of TCD4+ lymphocytes producing IL- 7, IFN-?, IL-22, IL-17/IL-22 and IL-17/IFN-?. We also observed that DCs stimulated by Pb presented activation of molecules involved in the TLRs and dectin-1 signaling (JNK, p38, AKT, ERK - for TLRs and Syk for dectin- The blockage of dectin-1 and/or TLR2 diminished the differentiation of Th17 cells. We also analyzed PBMCs from patients with the acute/juvenile form (JF) or the chronic/adult form (AF) of PCM. Our results showed that patients with JF present elevated production of IL-4 and AF patients present higher production of IL-17. Altogether our results showed that Dectin-1 and TLR-2 are the most important receptors for human DCs to recognize Pb yeast cells and to produce cytokines such as IL- , IL- ? and TNF-?. After DC activation and cytokine production these cells are able to induce Th1 and Th17 differentiation through these receptors. Also we showed that Th17 cells are a major population in AF patients / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

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