Papel dos receptores inatos TLR na formação de memória humoral e linfócitos B de longa vida: ação das proteases natterinas, toxinas majoritárias do veneno de Thalassophryne nattereri.

KOMEGAE, E. N. Role of innate TLR receptors in formation of humoral memory and long-life lymphocytes B: Action of Natterins proteases, majority toxins of Thalassophryne nattereri venom. 2010. Master thesis (Immunology) – Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2010. The contribution of B cells for the immunological memory feels for two different populations of cells: memory B cells and long lived antibodies secreting cells (ASC). However, the interrelation among these cells as well as the mechanisms involved for the maintenance of these it has been little understood. The venom of Thalassophryne nattereri has if shown capable to induce an intense memory immune response. In this sense, we intended to evaluate the effect of Natterins, majority toxins of the venom and that they don't present homology with any present protein in database, in the induction and maintenance of the immune memory response of cells B, focusing the importance of the proteases activity. As well as, to evaluate the paper of Toll like receptors and MyD88 signalling in the regulation of this process. For this, mice were immunized with the Natterins activates or inactive adsorbed in alum, after 7, 21 and 28 days the mice were just injected with Natterins . After 48, 74 and 120 days, the animals were bled for obtaining of the plasma for dosages of antibodies for Elisa or PCA. Soon afterwards, the animals were killed for obtaining of the cells of the effector compartment (Peritoneum), of important compartments for maintenance of the immune responses (Spleen and bone marrow). Our results showed that the proteases activity of Natterins is important not only for the induction of the profile Th2 response (high titles of IgG1 and IgE), but for the maintenance of the chronicity of response of different subtypes of B cells (B1a, B1b, B2, effector/memory B cell, ASC positive and negative B220 expression) in the three analyzed compartments. The inactive Natterins tend to the profile Th1 responses (IgG2a), inducing a better precocious responses and mainly in effector compartment. Using deficient mice of TLR4 or MyD88, we observed that the memory response induced by Natterins is regulated by dependent mechanisms of TLR4/MyD88 in the negative regulation of effector/memory B cells during whole the course of the response and suppression of IgE in the late period. A positive regulation of B1a and B2 cells in the recent period are also mediated by this road. And the most important, TLR4 through MyD88 are responsible for the maintenance of conventional ASCs in the effector compartment for long periods. During the anti-Natterinas response, we also observed dependent mechanisms of TLR4, however independent of MyD88, suggesting the other road of signalling of TLR4, through TRIF, that seem to be the responsible for the: induction of antibodies IgG1 and IgE in the recent period, for ASCs B220 induction and suppression of B2cells during the chronic response, or even controlling the migration of B1a and B1b cells of the peritoneum. Still, dependent mechanisms of MyD88, however independent TLR4 were also observed in the regulation of the anti-Natterins response, what indicates that Natterins can mediate responses road other Toll like receptors, that control the negative regulation of profile Th2 antibodies and they are the responsible for the maintenance ASCs for long periods in the spleen and in the bone marrow. Therefore, the study with the proteases Natterins besides allowing a larger explanation of the humoral memory response induced by the venom of the fish T. nattereri, it allowed the study of the complex organization of the compartment of memory B cells , mainly of the subtype of long lived (ASC). Also, allowed to evidence us the importance of the proteases activity of Natterins for the maintenance of the chronicity of B cells response in the three analyzed compartments, as we verify that the activation of Toll like receptors is decisive for the generation and maintenance of cells ASCs B220 in response to Natterins, dependent of the signaling MyD88 or TRIF. These signallings control the quality and the long duration of the humoral memory response through the action in different subtypes of B cells, in different differentiation apprenticeships and in different environment. Support: FAPESP.