The brain's resistance to ischemic injury can be transiently augmented by prior exposure to a sub-lethal stress stimulus, i.e. preconditioning. It has been reported that Toll-like receptors (TLRs) are involved in the preconditioning-induced protective effect against ischemic brain injury. In this study, we investigated the effect of preconditioning with a TLR2 specific ligand, Pam3CSK4, on focal cerebral ischemia/reperfusion (I/R) injury in mice. Pam3CSK4 was administered systemically 24 h before the mice were subjected to focal cerebral ischemia (1 h) followed by reperfusion. Cerebral infarct size was determined, blood brain barrier (BBB) permeability was evaluated, and expression of tight-junction proteins were examined after focal cerebral I/R. Results showed that pre-treatment with Pam3CSK significantly reduced brain infarct size (1.9 ± 0.5% vs 9.4 ± 2.2%) compared with the untreated I/R group. Pam3CSK4 pre-treatment also significantly reduced acute mortality (4.3% vs 24.2%), preserved neurological function (8.22 ± 0.64 vs 3.91 ± 0.57), and attenuated brain edema (84.61 ± 0.08% vs 85.29 ± 0.09%) after cerebral I/R. In addition, Pam3CSK4 pre-treatment preserved BBB function as evidenced by decreased leakage of serum albumin (0.528 ± 0.026 vs 0.771 ± 0.059) and Evans Blue (9.23 ± 0.72 μg/mg vs 12.56 ± 0.65 μg/mg) into brain tissue. Pam3CSK4 pre-treatment also attenuated the loss of the tight junction protein occludin in response to brain I/R injury. These results suggest that TLR2 is a new target of ischemic preconditioning in the brain and preconditioning with a TLR2 specific ligand will protect the brain from I/R injury.

No description available.

O sistema imune neonatal tem sido considerado funcionalmente imaturo e recentes estudos sugerem que a suscetibilidade do neonato às infecções pode ser devido a alterações funcionais de células apresentadoras de antígenos que podem levar a deficiências secundárias nas respostas adaptativas. A ativação das células apresentadoras de antígenos é desencadeada pela estimulação de receptores, como os Toll-like Receptors (TLRs) e alterações na ativação desses receptores podem levar a uma subsequente redução da ativação de proteínas da via de sinalização intracelular e consequente alterações dos níveis das citocinas pró- e anti-inflamatórias, contribuindo assim, para uma resposta imune ineficiente do neonato. O Toll-like receptor 2 (TLR-2) é um receptor essencial para o reconhecimento seletivo de vários antígenos bacterianos e virais, em especial, o peptideoglicano, que compreende cerca de 50% da parede celular de bactérias Grampositivas, como os estafilococos, que são agentes infecciosos que prevalecem nas Unidades de Terapia Intensiva Neonatal. O objetivo deste estudo foi avaliar a ativação e resposta de monócitos de sangue do cordão umbilical de recém-nascidos pré-termo saudáveis < 34 semanas de gestação (Grupo 1), recém-nascidos pré-termo : 34 e < 37 semanas de gestação (Grupo 2) e recém-nascidos a termo (Grupo 3) e de adultos saudáveis, como controles, após a estimulação de TLR-2 ex-vivo com Pam3CSK4. Após a estimulação dos monócitos, foram determinados os níveis de expressão dos marcadores de ativação celular, os níveis das citocinas pró- e anti-inflamatórias e a expressão de moléculas envolvidas na sinalização intracelular. A caracterização das populações leucocitárias, bem como a capacidade fagocítica de Staphylococcus aureus e geração de burst oxidativo por monócitos e neutrófilos foram analisados por Citometria de Fluxo. Os resultados demonstraram que as células dendríticas e monócitos de neonatos expressam TLR-2 em níveis semelhantes aos de adultos. A expressão adequada de TLR-2 sugere um reconhecimento antigênico eficiente que é refletido em uma ativação apropriada das moléculas da cascata de sinalização e uma potente produção de citocinas pró-inflamatórias, apesar da reduzida produção de IL-10. Fagócitos neonatais apresentaram capacidade fagocítica de S. aureus reduzida em relação aos adultos e geração do burst oxidativo semelhante entre os grupos, no entanto neonatos prétermo apresentaram produção de peróxido de hidrogênio deficiente, o que poderia contribuir com uma reduzida morte intracelular deste microrganismo. Em conclusão, o recém-nascido não apresenta uma imaturidade funcional, mas sim, um desequilíbrio em sua resposta imune inata, com uma aparente menor produção de fatores antiinflamatórios, o que pode levar a predisposição à sepse / The neonatal immune system has been considered functionally immature and recent studies suggest that susceptibility of the neonate to infections may be due to functional alterations in antigen-presenting cells that can prompt to secondary deficiencies in adaptive responses. The activation of antigen-presenting cells is triggered by stimulation of receptors such as Toll-like receptors (TLRs) and changes in the activation of these receptors may lead to a subsequent reduction in the activation of intracellular signaling pathway proteins and consequent changes in pro- and anti-inflammatory cytokine levels, thus contributing to an inefficient immune response of the neonate. Toll-like Receptor 2 (TLR-2) is an essential receptor for the selective recognition of several bacterial and viral antigens, in particular, peptidoglycan, which comprises about 50% of the Gram-positive bacteria cell wall, such as staphylococci, which are infectious agents that prevail in Neonatal Intensive Care Units. The aim of this study was to evaluate the activation and response of monocytes derived from umbilical cord blood of healthy preterm newborns <34 weeks of gestation (Group 1), preterm newborns :34 and <37 weeks of gestation (Group 2) and term newborns (Group 3) and from healthy adults, as controls, after ex-vivo TLR-2 stimulation with Pam3CSK4. After monocyte stimulation, it was determined the expression levels of cellular activation markers, pro- and anti-inflammatory cytokine levels and the expression of molecules involved in downstream intracellular signaling. The characterization of leukocyte populations, as well as the phagocytic ability of Staphylococcus aureus and generation of oxidative burst by monocytes and neutrophils were analyzed by flow cytometry. The results demonstrated that neonatal dendritic cells and monocytes express TLR- 2 at similar levels to those of adults. The proper expression of TLR-2 suggests an efficient antigen recognition which is reflected in an appropriate activation of downstream signaling molecules and potent production of pro-inflammatory cytokines, in spite of the reduced production of IL-10. Neonatal phagocytes showed reduced phagocytic capacity of S. aureus compared to adults and similar generation of oxidative burst between groups, however preterm neonates showed deficient production of hydrogen peroxide, which could contribute to a reduced intracellular killing of this microorganism. In conclusion, the newborn does not present a functional immaturity, but an imbalance in its innate immune response, with an apparent lower production of antiinflammatory factors, which can lead to a predisposition to sepsis

O sistema imune neonatal tem sido considerado funcionalmente imaturo e recentes estudos sugerem que a suscetibilidade do neonato às infecções pode ser devido a alterações funcionais de células apresentadoras de antígenos que podem levar a deficiências secundárias nas respostas adaptativas. A ativação das células apresentadoras de antígenos é desencadeada pela estimulação de receptores, como os Toll-like Receptors (TLRs) e alterações na ativação desses receptores podem levar a uma subsequente redução da ativação de proteínas da via de sinalização intracelular e consequente alterações dos níveis das citocinas pró- e anti-inflamatórias, contribuindo assim, para uma resposta imune ineficiente do neonato. O Toll-like receptor 2 (TLR-2) é um receptor essencial para o reconhecimento seletivo de vários antígenos bacterianos e virais, em especial, o peptideoglicano, que compreende cerca de 50% da parede celular de bactérias Grampositivas, como os estafilococos, que são agentes infecciosos que prevalecem nas Unidades de Terapia Intensiva Neonatal. O objetivo deste estudo foi avaliar a ativação e resposta de monócitos de sangue do cordão umbilical de recém-nascidos pré-termo saudáveis < 34 semanas de gestação (Grupo 1), recém-nascidos pré-termo : 34 e < 37 semanas de gestação (Grupo 2) e recém-nascidos a termo (Grupo 3) e de adultos saudáveis, como controles, após a estimulação de TLR-2 ex-vivo com Pam3CSK4. Após a estimulação dos monócitos, foram determinados os níveis de expressão dos marcadores de ativação celular, os níveis das citocinas pró- e anti-inflamatórias e a expressão de moléculas envolvidas na sinalização intracelular. A caracterização das populações leucocitárias, bem como a capacidade fagocítica de Staphylococcus aureus e geração de burst oxidativo por monócitos e neutrófilos foram analisados por Citometria de Fluxo. Os resultados demonstraram que as células dendríticas e monócitos de neonatos expressam TLR-2 em níveis semelhantes aos de adultos. A expressão adequada de TLR-2 sugere um reconhecimento antigênico eficiente que é refletido em uma ativação apropriada das moléculas da cascata de sinalização e uma potente produção de citocinas pró-inflamatórias, apesar da reduzida produção de IL-10. Fagócitos neonatais apresentaram capacidade fagocítica de S. aureus reduzida em relação aos adultos e geração do burst oxidativo semelhante entre os grupos, no entanto neonatos prétermo apresentaram produção de peróxido de hidrogênio deficiente, o que poderia contribuir com uma reduzida morte intracelular deste microrganismo. Em conclusão, o recém-nascido não apresenta uma imaturidade funcional, mas sim, um desequilíbrio em sua resposta imune inata, com uma aparente menor produção de fatores antiinflamatórios, o que pode levar a predisposição à sepse / The neonatal immune system has been considered functionally immature and recent studies suggest that susceptibility of the neonate to infections may be due to functional alterations in antigen-presenting cells that can prompt to secondary deficiencies in adaptive responses. The activation of antigen-presenting cells is triggered by stimulation of receptors such as Toll-like receptors (TLRs) and changes in the activation of these receptors may lead to a subsequent reduction in the activation of intracellular signaling pathway proteins and consequent changes in pro- and anti-inflammatory cytokine levels, thus contributing to an inefficient immune response of the neonate. Toll-like Receptor 2 (TLR-2) is an essential receptor for the selective recognition of several bacterial and viral antigens, in particular, peptidoglycan, which comprises about 50% of the Gram-positive bacteria cell wall, such as staphylococci, which are infectious agents that prevail in Neonatal Intensive Care Units. The aim of this study was to evaluate the activation and response of monocytes derived from umbilical cord blood of healthy preterm newborns <34 weeks of gestation (Group 1), preterm newborns :34 and <37 weeks of gestation (Group 2) and term newborns (Group 3) and from healthy adults, as controls, after ex-vivo TLR-2 stimulation with Pam3CSK4. After monocyte stimulation, it was determined the expression levels of cellular activation markers, pro- and anti-inflammatory cytokine levels and the expression of molecules involved in downstream intracellular signaling. The characterization of leukocyte populations, as well as the phagocytic ability of Staphylococcus aureus and generation of oxidative burst by monocytes and neutrophils were analyzed by flow cytometry. The results demonstrated that neonatal dendritic cells and monocytes express TLR- 2 at similar levels to those of adults. The proper expression of TLR-2 suggests an efficient antigen recognition which is reflected in an appropriate activation of downstream signaling molecules and potent production of pro-inflammatory cytokines, in spite of the reduced production of IL-10. Neonatal phagocytes showed reduced phagocytic capacity of S. aureus compared to adults and similar generation of oxidative burst between groups, however preterm neonates showed deficient production of hydrogen peroxide, which could contribute to a reduced intracellular killing of this microorganism. In conclusion, the newborn does not present a functional immaturity, but an imbalance in its innate immune response, with an apparent lower production of antiinflammatory factors, which can lead to a predisposition to sepsis

Stress, either physical or psychological, can have a dramatic impact on our immune system. There has been little progress, however, in understanding chronic stress-induced immunosuppression. Naive CD4+ T cells could modulate immune responses via differentiation to T helper (Th) cells. In this study, we showed that stress promotes the release of the Th1 cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α, the Th2 cytokines interleukin (IL)-4 and IL-10 and the Th17 cytokine IL-17 of splenic naive CD4+ T cells. This suggests that stress promotes the differentiation of naive CD4+ T cells to Th1, Th2 and Th17 cells. Knockout strategies verified that TLR2 might modulate the differentiation of Th1/Th2 cells by inhibiting p38 mitogen-activated protein kinase (MAPK). Taken together, our data suggest that chronic stress induces immune suppression by targeting TLR2 and p38 MAPK in naive CD4+ T cells.

Grâce aux nombreuses études sur le sujet, nous savons qu’une stimulation inflammatoire vasculaire excessive entraîne un débalancement des fonctions homéostatiques de l’endothélium. Ce débalancement est à l’origine d’une dysfonction endothéliale définie comme étant l’étape clé contribuant au développement de l’athérosclérose. Le Toll-like receptor-2 (TLR2) est impliqué dans l’activation cellulaire via la transcription des gènes liés à l’inflammation. Il reconnaît des molécules microbiennes mais également des facteurs endogènes non-infectieux tels que sécrétés par les tissus endommagés provenant de la dysfonction endothéliale. Ainsi, l’activation et la signalisation du TLR2 sont en étroite relation avec le développement de l’athérosclérose. Les études épidémiologiques ont confirmé le rôle athéroprotecteur de l’œstrogène via de nombreux mécanismes d’action. Ainsi, nous avons cherché à identifier de nouvelles cibles moléculaires permettant de mieux interpréter les bénéfices potentiels de l’œstrogène sur le système cardiaque. Pour la première fois chez les cellules endothéliales (CE) vasculaires de souris, nos travaux ont confirmé l’effet anti-inflammatoire de l’œstrogène via la diminution de l’expression et de l’activité du TLR2. Nous avons également déterminé l’influence de l’œstrogène sur le profil de la réponse inflammatoire de ce récepteur en mesurant les potentiels endothéliaux de migration et d’adhésion. De plus, nous avons caractérisé les voies de signalisation impliquées en démontrant l’influence négative de l’œstrogène sur la phosphorylation des kinases activées par le TLR2; illustrant l’interaction entre l’œstrogène et la signalisation de ce récepteur. Nos travaux amènent ainsi de nouvelles connaissances sur la régulation endothéliale du TLR2 et mettent en lumière les effets anti-inflammatoires et vasculaires rapides de l’œstrogène. / Evidence supports the contribution of immune responses in atherosclerosis development in part by alterations in the endothelium activation status and by the recruitment of inflammatory cells triggered by cardiovascular risk factors. These alterations are the principal cause of endothelial dysfunction defined as the key step contributing to the development of atherosclerosis. Via the transcription of genes related to inflammation, the Toll-like receptor-2 (TLR2) is involved in endothelial cell activation. It generally recognizes microbial molecules but also non-infectious endogenous factors such as those secreted by damaged tissues from the endothelial dysfunction. Thus, activation and signalization of the TLR2 are closely linked with the development of atherosclerosis. Epidemiological studies have confirmed the atheroprotective role of estrogen through multiple mechanisms of action. Thus, to better interpret the potential benefits of estrogen on the cardiovascular system, we sought to identify new molecular targets such as TLR2 regulation. For the first time in mouse vascular endothelial cells (EC), our results have confirmed the anti-inflammatory effect of estrogen via the decreased expression and activity of TLR2. We also determined the influence of estrogen on the profile of the inflammatory response triggered through this receptor by measuring endothelial migration and adhesion potentials. Furthermore, we demonstrated the interaction between estrogen and TLR2 signalling pathways with a negative influence of estrogen on the phosphorylation level of kinases activated by this receptor. Thus, our study brings new insights into the endothelial regulation of TLR2 and highlights rapid anti-inflammatory and cardioprotective effects from estrogen.

Orientador: Juliana Regina Peiró / Coorientador:Sérgio Moraes Aoki / Banca:José Paes de Oliveira Filho / Banca:Flávia de Almeida Lucas / Resumo: Endotoxemia é distúrbio sistêmico que se origina da resposta do hospedeiro a um componente da membrana celular das bactérias Gram- negativas. Esta resposta se dá através da exposição dos receptores celulares TLR-4 e TLR-2 ao LPS. Os objetivos deste estudo foram investigar as alterações na expressão gênica da exposição ao LPS em leucócitos de equinos utilizando a técnica de microarray, avaliar a eficiência do modelo ex vivo para os estudos envolvendo a endotoxemia pela mesma técnica, avaliar a expressão global de genes em vias envolvidas, identificar componentes da cascata metabólica com potenciais para novas terapias, e fornecer subsídio para futuros estudos. Amostras de sangue total (15mL) de cavalos saudáveis (n=6) foram incubadas durante 4 horas a 37°C com 5% de CO2 na presença (1 ou 10 ng/mL) ou ausência de LPS (0 ng/mL). Alíquotas de 500µL de sangue foram coletadas nos momentos 0, 2 e 4 horas após o estímulo do LPS. O RNA, extraído das mostras, foi utilizado para a transcrição do cDNA. A hibridização do cDNA marcado com Cy-3 foi realizada em lâminas 4x44K v2 de humanos contendo sequências homólogas com a espécie equina para os genes de interesse. Após a leitura das lâminas, com filtro para genes 30x mais ou menos expressos, verificou-se um aumento da expressão do TLR-2 na concentração de 10 ng LPS/mL no momento 4 em relação ao momento 2. Este resultado sugere que, embora o receptor TLR-4 seja o principal receptor no reconhecimento do LPS, o receptor TLR-2 também tem um papel no reconhecimento destas moléculas / Abstract: Endotoxemia is systemic disturbance that origins from the host response to a component of the cellular membrane of Gram-negative bacterias. This response occurs through exposure of cellular receptors TLR-4 and TLR-2 to LPS. The objectives of this study were to investigate changes in gene expression of exposure to LPS in horses leukocytes using the microarray technique, to evaluate the efficiency of the ex vivo model for studies of endotoxemia by the same technique, to evaluate the global expression of genes in the metabolic pathways involved, identify components of the metabolic cascade with potential for new therapies, and provide allowance for future studies. Whole blood samples (15mL) of healthy horses (n=6) were incubated for 4 hours at 37°C with 5% of CO2 in the presence (1 or 10 ng/ml) or absence of LPS (0 ng/ml). Aliquots of 500μL of blood were collected at 0,2 and 4 hours after LPS stimulation. The RNAs, extracted from the samples, were used for the transcription of the cDNAs. Hybridization of labeled cDNAs with Cy-3 were performed in 4x44K v2 slides containing human sequences homologous to the equine species for the genes of interest. After the reading of the slides with filter for genes 30x up regulation or down regulation expression, it was observed an increased expression of the TLR-2 concentration of 10 ng LPS/mL at time 4 compared to time 2. This result suggests that, although the TLR-4 receptor is the main LPS recognition receptor, the receptor TLR-2 also plays a role in recognition of these molecules / Mestre

Grâce aux nombreuses études sur le sujet, nous savons qu’une stimulation inflammatoire vasculaire excessive entraîne un débalancement des fonctions homéostatiques de l’endothélium. Ce débalancement est à l’origine d’une dysfonction endothéliale définie comme étant l’étape clé contribuant au développement de l’athérosclérose. Le Toll-like receptor-2 (TLR2) est impliqué dans l’activation cellulaire via la transcription des gènes liés à l’inflammation. Il reconnaît des molécules microbiennes mais également des facteurs endogènes non-infectieux tels que sécrétés par les tissus endommagés provenant de la dysfonction endothéliale. Ainsi, l’activation et la signalisation du TLR2 sont en étroite relation avec le développement de l’athérosclérose. Les études épidémiologiques ont confirmé le rôle athéroprotecteur de l’œstrogène via de nombreux mécanismes d’action. Ainsi, nous avons cherché à identifier de nouvelles cibles moléculaires permettant de mieux interpréter les bénéfices potentiels de l’œstrogène sur le système cardiaque. Pour la première fois chez les cellules endothéliales (CE) vasculaires de souris, nos travaux ont confirmé l’effet anti-inflammatoire de l’œstrogène via la diminution de l’expression et de l’activité du TLR2. Nous avons également déterminé l’influence de l’œstrogène sur le profil de la réponse inflammatoire de ce récepteur en mesurant les potentiels endothéliaux de migration et d’adhésion. De plus, nous avons caractérisé les voies de signalisation impliquées en démontrant l’influence négative de l’œstrogène sur la phosphorylation des kinases activées par le TLR2; illustrant l’interaction entre l’œstrogène et la signalisation de ce récepteur. Nos travaux amènent ainsi de nouvelles connaissances sur la régulation endothéliale du TLR2 et mettent en lumière les effets anti-inflammatoires et vasculaires rapides de l’œstrogène. / Evidence supports the contribution of immune responses in atherosclerosis development in part by alterations in the endothelium activation status and by the recruitment of inflammatory cells triggered by cardiovascular risk factors. These alterations are the principal cause of endothelial dysfunction defined as the key step contributing to the development of atherosclerosis. Via the transcription of genes related to inflammation, the Toll-like receptor-2 (TLR2) is involved in endothelial cell activation. It generally recognizes microbial molecules but also non-infectious endogenous factors such as those secreted by damaged tissues from the endothelial dysfunction. Thus, activation and signalization of the TLR2 are closely linked with the development of atherosclerosis. Epidemiological studies have confirmed the atheroprotective role of estrogen through multiple mechanisms of action. Thus, to better interpret the potential benefits of estrogen on the cardiovascular system, we sought to identify new molecular targets such as TLR2 regulation. For the first time in mouse vascular endothelial cells (EC), our results have confirmed the anti-inflammatory effect of estrogen via the decreased expression and activity of TLR2. We also determined the influence of estrogen on the profile of the inflammatory response triggered through this receptor by measuring endothelial migration and adhesion potentials. Furthermore, we demonstrated the interaction between estrogen and TLR2 signalling pathways with a negative influence of estrogen on the phosphorylation level of kinases activated by this receptor. Thus, our study brings new insights into the endothelial regulation of TLR2 and highlights rapid anti-inflammatory and cardioprotective effects from estrogen.

Orientador: Juliana Regina Peiró / Coorientador: Sérgio Ribeiro Aoki / Banca:Lina Maria Wehrle Gomide / Banca: Flávia Lombardi Lopes / Banca:José Paes de Oliveira Filho / Banca:Glenda Nicioli da Silva / Resumo: A resposta imune inata é a principal responsável pela defesa do hospedeiro contra endotoxinas de bactérias Gram-negativas. Tal resposta se dá através dos receptores Toll-like-4 e -2, seja pela via MyD88-dependente, TRIF-dependente ou ambas. Entretanto, uma resposta exagerada pode resultar em muitos danos para o organismo e levar até mesmo ao choque séptico. Uma das formas de controle desta resposta se dá através da tolerância à endotoxina (TE). Por isso o presentes estudo tem por objetivo investigar a indução diferencial dos receptores TLR-4 e 2, suas vias de sinalização MyD88 e TRIF-dependentes e avaliar outros possíveis genes relacionados a estas vias após indução da TE. Foi coletado sangue total de cavalos saudáveis (n=6), o qual foram estimulados com diferentes doses (0, 1 ou 10ng de LSP/mL) LPS de E.coli no momento 0 e 4 horas após o primeiro estímulo e 4 horas após o segundo estímulo. O "pool" (n=6) de RNA das amostras, coletado 0, 2, 4 e 8 horas, foi utilizado para a transcrição do cDNA. A hibridização do cDNA marcado com Cy-3 foi realizada em uma lâmina 4x44 contendo sequências específicas da espécie equina. Foi identificado durante a TE, nas diferentes doses de LPS, um aumento de transcritos dos receptores TLR-4 e -2, TNFAIP3 e IL-10 e uma diminuição de outros transcritos de genes importantes, tais como: MyD88, IL1-B, TNF-a, TRAM2, o que caracteriza e esclarece, de certa forma, as alterações na sinalização do TLR nas condições de tolerância à endotoxina. / Abstract: The innate immune response is the main responsible for the host's defense against endotoxins from Gram-negative bacteria. Such response occurs via Toll-like-4 and -2 receptors, MyD88-dependent pathway, TRIF-dependent pathway or both. However, an exaggerated reponse can induce many damages to the organism, including septic shock. One way to control this exacerbated response is through endotoxin tolerance (ET). The goals of the present study were to investigate TLR-4 and 2 receptors, their signaling through MyD88 e TRIF-dependent pathways and evaluate other possible genes related to this ways after ET induction. Blood samples were collected from healthy horses (n=6) which were stimulated with different doses (0, 1 or 10ng of LSP/mL) of LPS from E.coli, for 0 and 4 hours after the first stimulus and 4 hours after second stimulus. The pool (n=6) of RNA, extracted from the samples collected at 0, 2, 4 e 8 hours, was used for the transcription of the cDNA. The hybridization of the cDNA marked with Cy-3 was done in one slide 4x44 containing specific sequences from the equine species. We identified during the ET, on the different doses of LPS, an increase of transcripts for the receptors TLR-4 and -2, TNFAIP3 and Il-10 and a reduction of transcripts of other important genes, such as: MyD88, IL1-B, TNF-a, TRAM2, what characterizes and enlightens the changes in TLR signaling during endotoxin tolerance. / Doutor

Introdução: As reações hansênicas caracterizam-se por eventos imunológicos agudos que ocorrem antes, durante ou após a Poliquimioterapia da hanseníase, sendo desencadeadas principalmente pela interação entre os antígenos do Mycobaterium leprae, PGL-1 e LAM, com os componentes da imunidade do hospedeiro, TLR1 e TLR2, que consequentemente induzem outras células a sintetizar citocinas que favorecem o controle ou disseminação bacilar. Objetivos: Estabelecer e quantificar as relações causais entre os antígenos de M. leprae (PGL-1 e LAM) e os receptores TLR1, TLR2 e seus efeitos na expressão de citocinas IFN-γ e IL-10 nas reações hansênicas. Material e métodos: Por meio da sorologia ELISA foi possível dosar os níveis de IgM anti-PGL-1 e IgG anti-LAM e com emprego da RT-qPCR procedeu-se a análise da expressão gênica de TLR1, TLR2, IFN-γ e IL-10 utilizando amostras do sangue de 17 pacientes com reação hansênica e 17 sem reação (caso-controle) que posteriormente foram subdivididos em 4 grupos. Na comparação entre as médias das variáveis nos diferentes grupos, utilizou-se a análise de variância (ANOVA) e na avaliação do grau de associação entre as principais variáveis, a matriz de correlação de Pearson. Ao final, elaborou-se a análise de trilha para verificar relações causais envolvendo os antígenos e receptores do tipo Toll tendo como efeitos direto e indireto a expressão de citocinas. Resultados: Do total de 34 pacientes, 76,4% (26/34) eram MB e 23,6% PB, sendo que 100% (17/17) dos casos reacionais eram MB e dos controles 53% (9/17). Analisando o grupo reacional, 41,2% (7/17) apresentaram reação tipo 1, enquanto que 58,8% (10/17) tiveram reação tipo 2, destacando que na reação tipo 1, 57,1% (4/7) dos pacientes tiveram reação antes do tratamento (downgrading), 14,2% (1/7) durante e 28,8% (2/7) após PQT. Na reação tipo 2, todos desenvolveram reação após alta. Os níveis de anti-PGl-1 estiveram elevados na reação tipo 1 e na reação tipo 2, com diferença significativa entre as médias desses grupos quando comparados aos controles PB (F=6,42; p=0,002). Em relação ao anti-LAM, esse esteve associado a reação do tipo 2, grupo cujas médias foram mais elevadas, e apresentaram diferenças significativas quando comparado aos demais grupos (F=15,27; p<0,001). Os grupos reacionais expressaram maiores níveis de mRNA de IL-10 (F=3,99; p=0,023), enquanto que o grupo controle expressou mais IFN-γ, especialmente aqueles PB (F=5,30; p=0,008). Não houve diferenças nos níveis de expressão de TLR1 e TLR2 nos diferentes grupos. No grupo reacional, a análise de trilha quantificou os efeitos diretos de anti-LAM (0,623) e anti-PGL-1 (0,605) sobre TLR2, maiores que o efeito residual desse modelo (0,255), indicando que esses marcadores antigênicos influenciaram somente a expressão de TLR2, que consequentemente associou-se a IL-10 nesse grupo. Um mecanismo oposto ocorreu no grupo controle, visto que, constatou-se uma via imunológica que regulou a expressão de IFN-γ dependente da associação entre TLR1-TLR2. Conclusão: Em conclusão, a análise de trilha permitiu demonstrar a relação causal entre antígenos, TLR2 e IL-10 nas reações hansênicas, que podem sofrer influência de falha no mecanismo de associação entre TLR1 e TLR2, objeto de futuras investigações. / Background: The leprosy reactions are acute immunological events that occur before, during or after MDT of leprosy, being triggered mainly by interaction between the antigens of Mycobacterium leprae, PGL-1 and LAM, with the components of immunity of the host, such as, TLR1 and TLR2, that induce cells to produce cytokines related to control or bacillary dissemination. Objectives: The main goal of this research is establish causal relations between antigens of M. leprae (PGL-1 and LAM) and receptors, TLR1, TLR2 and their effects on cytokines expression, IFN-γ and IL-10, in leprosy reactions. Methods: Through ELISA serology it was possible measure the levels of anti-PGL-1 IgM and anti-LAM IgG, and with the use of the RT-qPCR proceeded the analysis of gene expression of TLR1 TLR2, IFN-γ and IL-10 using blood samples from 17 reactional patients and 17 nonreactional patients (Case-control study) that posteriorly were subdivided into 4 groups. For comparison between means of the variables, it resorted to use of analysis of variance (ANOVA) and about the degree of association between the variables, it has applied the correlation matrix. At the end, it was used the path analysis to verify the causal relation evolving antigens and toll-like receptors, whose the direct and indirect effects were the cytokines expression. Results: Among 34 patients, 76.4% (26/34) were MB and 23.6% PB, being that 100% (17/17) of reactional cases were MB and among the controls 53% (9/17). Analyzing the reactional group, 41.2% (7/17) suffered type 1 reaction, while 58.8% (10/17) presented type 2 reaction, emphasizing that, among patients with type 1 reaction, 57.1% (4/7) had reaction before treatment (downgrading), 14.2% (1/7) during and 28.8% (2/7) after MDT, whereas all patients of the type 2 reaction group, developed this occurrence after treatment. The levels of anti-PGl-1 have been elevated in type 1 and type 2 reactions with significant differences between means of these groups when compared to PB controls (F=6.42; p=0.002). Regarding to anti-LAM, this has been associated to type 2 reaction, group whose the means were higher than others, and this data showed significant differences as well (F=15.27; p<0.001). The reactional groups expressed higher levels of IL-10 (F=3.99; p=0,023), whereas the control group produced high levels of IFN-γ, especially the PB control (F=5.30; p=0.008). There were no differences in the levels of TLR1 and TLR2 expression in the comparison among groups. In the reactional group, the path analysis quantified the direct effects of anti-LAM (0.623) e anti-PGL-1 (0.605) on TLR2, which were higher than the residual effect of this model (0.255), indicating that these antigenic markers influenced only the TLR2 expression, which consequently correlated with the IL-10 expression in this group. An opposing mechanism occurred in the control group, insofar as it was found an immunological pathway that regulated the IFN-γ expression dependent on TLR1 and TLR2 association. Conclusion: In conclusion, path analysis allowed to demonstrate the causal relation between TLR2 and IL-10 in leprosy reactions that may suffer influence of failure in the mechanism of association between TLR1 and TLR2, as such occurs in TLR2 polymorphisms, object of future investigations. / Tese (Doutorado)