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Trypanosoma brucei effect on leukocyte differentiation and activation
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Tripanossomíase Africana ou doença do sono é uma doença zoonótica causada por Trypanosoma brucei, um protozoário parasita transmitido pela mosca tsetse ou Glossina. A introdução do parasita no hospedeiro vertebrado provoca uma sucessão de eventos que envolvem a imunidade inata e adaptativa. Os macrófagos (MΦ) apresentam um papel fundamental na defesa inata por serem células apresentadoras profissionais de antigénio (APC) e células fagocíticas, importantes na eliminação dos tripanossomas. A imunidade adaptativa é assegurada pelos linfócitos, nomeadamente pelos linfócitos T. Contudo, o papel desempenhado por estas células imunitárias ainda não se encontra completamente clarificado. Assim, este estudo teve como objetivo analisar a atividade dos MΦ na infeção por T. brucei, avaliando os níveis de ureia e óxido nítrico (NO) e a expressão membranar das moléculas de classe I (MHCI) e classe II (MHCII) do complexo principal de histocompatibilidade. A diferenciação das populações linfocitárias, T helper (Th), T citotóxicas (Tc) e T reguladoras foi também avaliada. MΦ de murganho foram expostos a tripomastigotas de T. brucei e estimulados com antigénio total e exossomas de T. brucei. A caracterização da ativação macrofágica através de ensaios colorimétricos demonstrou que ambos os fenótipos M1 e M2 foram expressos e foi evidenciada correlação positiva entre a produção de ureia e de NO. Adicionalmente, os resultados da citometria de fluxo indicaram que o parasita prejudica a diferenciação das subpopulações de MΦMHCI+ e MΦMHCII+ mas induz o aumento de moléculas MHCI. Contrariamente, os exossomas demonstraram estimular as funções APC através das moléculas MHCI e MHCII. Os rácios MHCI/MHCII indicaram que o contacto com o parasita favorece a apresentação antigénica às células TCD4+, enquanto que os exossomas direcionam a apresentação antigénica para as subpopulações de linfócitos TCD4+ e TCD8+. A caraterização das populações linfocitárias através da citometria de fluxo demonstrou que T. brucei causa a diminuição das populações de linfócitos Th e Tc. Contrariamente, a estimulação com o antigénio total ou com os exossomas induziram a expansão de ambas as subpopulações linfocitárias. T. brucei parece promover a expansão das subpopulações linfocitárias CD8-CD25+FoxP3- e CD8+CD25+FoxP3-. A expansão da subpopulação celular CD25-FoxP3+ foi observada na fração celular CD8- após estimulação do antigénio e na fração celular CD8+ estimulada por exossomas. Além disso, os exossomas também promoveram a expansão da subpopulação de linfócitos T CD8+CD25+FoxP3+. Curiosamente, apesar da contração celular T. brucei induziu o aumento das moléculas FoxP3. Em conjunto, os resultados obtidos indicam que o parasita e os exossomas parecem exercer efeitos opostos nas células analisadas. Os parasitas parecem minimizar a apresentação antigénica e evitam induzir a expansão das subpopulações de linfócitos T, facilitando a sua permanência no hospedeiro. Por outro lado, os exossomas segregados por T. brucei parecem estimular a apresentação antigénica e mediar a expansão dos linfócitos Th, possivelmente deslocando o foco da atividade do sistema imunitário dos parasitas para os exossomas. Estes resultados permitiram clarificar alguns dos princípios subjacentes à resposta imunitária inata e adaptativa na fase inicial da infeção. A compreensão destes mecanismos pode vir a contribuir para o desenvolvimento de novas estratégias de controlo e eliminação da Tripanossomíase Africana.
African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by tsetse fly or Glossina. Parasite introduction into mammal hosts, triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defense, since they are antigen-presenting cells (APC) and have a phagocytosis function essential for trypanosomes clearance. Adaptive immune defense is carried out by lymphocytes, in particular by T lymphocytes. However, the exact role of these immune cells remains not completely understood. Thus, this study aimed to assess the role of MΦ in T. brucei infection by measuring the urea and nitric oxide (NO) levels, and by evaluating membrane expression of class I (MHCI) and class II (MHCII) molecules of major histocompatibility complex. The differentiation of T helper (Th), T cytotoxic (Tc) and T regulatory cell subsets was assessed. Mouse MΦ were exposed to T. brucei trypomastigotes and stimulated by T. brucei extract and T. brucei exosomes. Characterization of MΦ activation with colorimetric assays have indicated that both M1 and M2 phenotypes were expressed, evidencing a positive correlation between urea and NO levels produced. Additionally, results of flow cytometry indicated that T. brucei impairs the expansion of both MHCI+ and MHCII+ MΦ subsets, but enhanced MHCI molecules. On the contrary, T. brucei exosomes stimulated APC functions through MHCI and MHCII molecules. MHCI/MHCII rates indicated that T. brucei shift the antigen presentation to CD4+ T cells, while exosomes directed the antigen presentation to both CD4+ and CD8+ T cells. Characterization of lymphocyte subsets by flow cytometry demonstrated that T. brucei impairs both Th and Tc lymphocytes. On the contrary, cell stimulation by extract and exosomes promote the expansion of both T cell subpopulations. T. brucei seem to promote the expansion of CD8-CD25+FoxP3- and CD8+CD25+FoxP3- T cell subsets. The expansion of CD25-FoxP3+ T cell subset was observed in CD8- cell fraction antigen stimulated and in the CD8+ cell fraction exosome stimulated. Moreover, exosomes also induced the expansion of CD8+ CD25+FoxP3+ T cell subset. Interestingly, despite cell decrease T. brucei seemed to increase FoxP3 molecules. Taken together, these findings indicate that parasite and parasite exosomes seem to have opposite effects on the evaluated cells. Parasites seem to minimize antigen presentation and avoid inducing the expansion of T cell subsets, facilitating its permanence in the host. On the other hand, T. brucei secreted exosomes seems to induce APC functions and mediate the expansion of Th lymphocytes, probably focusing the immune activity on the exosomes and not on the parasites. These findings allowed to understand some underlying principles of the innate and adaptive immune response in the early-stage of infection. Comprehension of these mechanisms can endorse the development of new strategies for control and elimination of African Trypanosomiasis.
African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by tsetse fly or Glossina. Parasite introduction into mammal hosts, triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defense, since they are antigen-presenting cells (APC) and have a phagocytosis function essential for trypanosomes clearance. Adaptive immune defense is carried out by lymphocytes, in particular by T lymphocytes. However, the exact role of these immune cells remains not completely understood. Thus, this study aimed to assess the role of MΦ in T. brucei infection by measuring the urea and nitric oxide (NO) levels, and by evaluating membrane expression of class I (MHCI) and class II (MHCII) molecules of major histocompatibility complex. The differentiation of T helper (Th), T cytotoxic (Tc) and T regulatory cell subsets was assessed. Mouse MΦ were exposed to T. brucei trypomastigotes and stimulated by T. brucei extract and T. brucei exosomes. Characterization of MΦ activation with colorimetric assays have indicated that both M1 and M2 phenotypes were expressed, evidencing a positive correlation between urea and NO levels produced. Additionally, results of flow cytometry indicated that T. brucei impairs the expansion of both MHCI+ and MHCII+ MΦ subsets, but enhanced MHCI molecules. On the contrary, T. brucei exosomes stimulated APC functions through MHCI and MHCII molecules. MHCI/MHCII rates indicated that T. brucei shift the antigen presentation to CD4+ T cells, while exosomes directed the antigen presentation to both CD4+ and CD8+ T cells. Characterization of lymphocyte subsets by flow cytometry demonstrated that T. brucei impairs both Th and Tc lymphocytes. On the contrary, cell stimulation by extract and exosomes promote the expansion of both T cell subpopulations. T. brucei seem to promote the expansion of CD8-CD25+FoxP3- and CD8+CD25+FoxP3- T cell subsets. The expansion of CD25-FoxP3+ T cell subset was observed in CD8- cell fraction antigen stimulated and in the CD8+ cell fraction exosome stimulated. Moreover, exosomes also induced the expansion of CD8+ CD25+FoxP3+ T cell subset. Interestingly, despite cell decrease T. brucei seemed to increase FoxP3 molecules. Taken together, these findings indicate that parasite and parasite exosomes seem to have opposite effects on the evaluated cells. Parasites seem to minimize antigen presentation and avoid inducing the expansion of T cell subsets, facilitating its permanence in the host. On the other hand, T. brucei secreted exosomes seems to induce APC functions and mediate the expansion of Th lymphocytes, probably focusing the immune activity on the exosomes and not on the parasites. These findings allowed to understand some underlying principles of the innate and adaptive immune response in the early-stage of infection. Comprehension of these mechanisms can endorse the development of new strategies for control and elimination of African Trypanosomiasis.
Descrição
Palavras-chave
Ciências biomédicas Biologia molecular Tripamossomíases Doença do sono Tripanossoma brucei brucei Exossomas Macrófagos Linfócitos T Células T Reguladoras
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical