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Characterization and functional analysis of cell-mediated immunity to Leishmania infantum
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Leishmania infantum é o agente responsável pela leishmaniose visceral zoonótica, uma parasitose frequentemente caracterizada por alterações específicas da imunidade celular e parasitismo progressivo. Sabe-se actualmente que as células T reguladoras (Treg) são na verdade linfócitos T que se encontram directamente envolvidos na indução de mecanismos de imunossupressão da resposta imunológica durante infecções crónicas, como por exemplo Leishmania. Deste modo, este estudo tem como objectivo analisar o papel das Treg durante a infecção com L. infantum em modelo animal susceptível. Os resultados obtidos indicam que os linfócitos T CD4+CD25+ estão presentes em murganhos BALB/c infectados com L. infantum e exibem características fenotípicas e funcionais de Treg. A detecção de níveis elevados de expressão do gene foxp3 e do marcador de superfície E7 integrina (CD103) sugere a predisposição para a retenção de Treg nos locais preferenciais de infecção por L. infantum, como é o caso do baço e dos gânglios linfáticos, influenciando a resposta local e induzindo susceptibilidade. No entanto, apesar de neste modelo de infecção se ter observado parasitismo crónico no baço e no fígado não parece ter havido uma resposta Th polarizada o que pode estar relacionado com a expansão de Treg. Linfócitos T CD4+CD25+ que expressam Foxp3 demonstraram ter capacidade de produzir TGF-, contribuindo para a imunossupressão e controlo da imunopatologia induzida pelo parasita. Supreendentemente, linfócitos T CD4+CD25-Foxp3- produtores de IL-10 também foram identificados como fonte adicional de IL-10, representando uma sub-população de linfócitos T reguladores do tipo 1 (Tr1) cuja diferenciação foi induzida pelo parasita. Estes resultados sugerem que diferentes tipos de células T reguladoras podem ser estimuladas durante a resposta imunológica à infecção por L. infantum, contribuindo para a persistência do parasita e o estabelecimento da infecção crónica neste modelo experimental. Tendo demonstrado que a imunossupressão mediada por Treg é evidente no modelo susceptível de L. infantum, o próximo passo seria verificar se num modelo experimental de resistência, a supressão evidenciada pelas Treg seria regulada ou não pelo parasita, representando deste modo o desenvolvimento pelo parasita de uma estratégia para promover a expansão de células imunossupressivas e a inibição da resposta efectora do hospedeiro, isto é regulando os reguladores. Assim para estudar a função imunossupressora das Treg induzida por L. infantum e os mecanismos envolvidos na interacção hospedeiro-parasita e na regulação imunológica, a segunda parte deste estudo avalia o papel do receptor TLR-2 na função das Treg durante a infecção experimental por L. infantum e analisa a influência deste receptor na cinética das Treg, na resposta imunológica e na patologia. Para tal, murganhos mutantes C57BL/6 para o gene TLR-2 (TLR-2-/-) e os murganhos ―wild-type‖ C57BL/6 foram infectados com L. infantum. A análise comparativa foi efectuada de modo a verificar se a presença ou ausência de TLR-2 produz um efeito diferencial nos parâmetros do hospedeiro associados à dinâmica das Treg e à imunidade protectora. A ausência de sinalização TLR-2 teve um efeito visível no desenrolar da infecção. Elevadas taxas de multiplicação do parasita foram observados no baço e fígado dos murganhos TLR-2-/- apesar de se ter evidenciado a presença de granulomas hepáticos aparentemente bem estruturados e definidos. Estas formas granulatomosas são, aparentemente, ineficazes na eliminação do parasita comparativamente aos murganhos ‖wild-type‖. A ausência de sinalização TLR-2 induziu a retenção tardia de Treg de memória, associada à elevada carga parasitária e níveis reduzidos de IFN- O TLR-2 poderá desempenhar um papel na regulação das Treg e consequentemente na patogénese por L. infantum. Nos murganhos ―wild-type‖, a sinalização via TLR-2 pode ser importante no controlo das populações de Treg FOXP3+ , na regulação negativa das Tregs e no desenvolvimento de imunidade protectora mais eficaz contra o parasita. A presença ou ausência de TLR-2 não influenciou a expressão de IL-10 ou de TGF-e não está relacionado com a detecção de Treg CD103+ FOXP3+ durante a fase tardia da infecção nos murganhos TLR-2-/-. Elevados níveis de Treg nestes murganhos não foram acompanhados pela indução de citocinas imunossupressoras. A presença de níveis elevados de Treg no baço de animais infectados, na ausência de TLR-2, sugere que este receptor poderá desempenhar um papel importante na regulação dos reguladores, mediando desta forma a imunidade inata e adquirida desenvolvida pelo hospedeiro durante a infecção por L. infantum.
Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis (ZVL), a disease frequently characterized by specific impairment of cell-mediated immune responses and uncontrolled parasitization. Regulatory T cells (Treg) have been shown to be involved in the direct induction of immunosuppresion of effector immune response during chronic Leishmania infections. The present study aims firstly to investigate the possible involvement of Treg cells during L. infantum infection in a susceptible animal model. Results indicated that CD4+CD25+ regulatory T cells are present in L. infantum-infected BALB/c mice and exhibit phenotypic and functional characteristics of Treg. The presence of high levels of foxp3 gene expression and surface expression ofE7 integrin (CD103) suggest a predisposition for Treg retention within sites of L. infantum infection, as is the case of the spleen and draining lymph nodes, consequently influencing local immune response and increased susceptibility. However, no evident Th polarization despite chronic parasitism in both spleen and liver was observed during L. infantum infection in this model. Th1 and Th2 effector immune responses seemed inadequate, perhaps due to Treg expansion. Foxp3-expressing-CD4+CD25+ T cells are indeed capable of producing TGF- and may contribute to immunosuppression and better control of parasite-mediated-immunopathology during infection. Surprisingly, IL-10 producing-CD4+CD25-Foxp3- T cells were also identified as an additional source of IL-10 and represent a type 1 regulatory T (Tr1) cell subset that is being induced in vivo by L. infantum parasites. These findings suggest that distinct regulatory T cells develop in response to L. infantum and may play a possible role in promoting parasite persistence and the establishment of chronic infection in this particular experimental model of infection. Having demonstrated that Treg-mediated immunosuppression is evident in L. infantum susceptible infection model, the next step would be to verify if in a resistant experimental model of infection, immunosuppresive Treg can or cannot be modulated by the parasite. This would represent the development of a parasite strategy able to upregulate immunosuppressive cells, dampen effector immune response in their favour, and promote expansion, ultimately regulating the regulator. So to elucidate on immunosuppressive Treg function induced by L. infantum parasites and the underlying mechanisms involved in the direct host-parasite interactions and immune regulation, the second part of this study focuses on the role of TLR-2 on Treg function during L. infantum experimental murine infection by investigating the influence of TLR-2 on Treg kinetics, immune response, parasite-associated pathology and the outcome of L. infantum infection. To achieve this, TLR-2 deficient mice (TLR-2-/-) and their wild-type C57BL/6 mice (WT) were infected with L. infantum parasites and comparative analysis was done to see whether or not the presence or absence of TLR-2 produces any differential effect on the host parameters related to Treg dynamics and protective immunity. Defective TLR-2 signalling had a visible effect on outcome of L. infantum infection. Higher rates of parasite multiplication were observed in both spleen and liver of TLR-2-/- knock-out mice, despite the ability in forming well-defined and structured liver granulomas. These granulomas were apparently ineffective in parasite clearance, when compared to wild-type mice. Defective TLR-2 signalling did induce during late infection high retention of memory Treg which seemed to be associated to high parasite load and low IFN- levels. TLR-2 signalling pathway may play a role in Treg modulation and consequently in L. infantum pathogenesis. Functional TLR-2 signalling in WT may be important in providing tight control over FOXP3+ committed Treg populations, negative Treg regulation and more protective immunity, giving rise to enhanced immunity and more effective response against infection. The presence or absence of TLR-2 did not seem to influence IL-10 or TGF- expression, and it did not seem to correlate with CD103+ FOXP3+ Treg detected late during infection in TLR-2-/- mice. Detection of high levels of suppressive Treg in L. infantum-infected TLR-2 deficient mice was not accompanied by associated inductions of immunosuppressive cytokines. The presence of high levels of immunosuppressive Treg in infected spleen, in the absence of TLR-2, suggests that this receptor in particular plays an important role in regulating the regulators, thus orchestrating effective innate and acquired immunity against L. infantum infection.
Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis (ZVL), a disease frequently characterized by specific impairment of cell-mediated immune responses and uncontrolled parasitization. Regulatory T cells (Treg) have been shown to be involved in the direct induction of immunosuppresion of effector immune response during chronic Leishmania infections. The present study aims firstly to investigate the possible involvement of Treg cells during L. infantum infection in a susceptible animal model. Results indicated that CD4+CD25+ regulatory T cells are present in L. infantum-infected BALB/c mice and exhibit phenotypic and functional characteristics of Treg. The presence of high levels of foxp3 gene expression and surface expression ofE7 integrin (CD103) suggest a predisposition for Treg retention within sites of L. infantum infection, as is the case of the spleen and draining lymph nodes, consequently influencing local immune response and increased susceptibility. However, no evident Th polarization despite chronic parasitism in both spleen and liver was observed during L. infantum infection in this model. Th1 and Th2 effector immune responses seemed inadequate, perhaps due to Treg expansion. Foxp3-expressing-CD4+CD25+ T cells are indeed capable of producing TGF- and may contribute to immunosuppression and better control of parasite-mediated-immunopathology during infection. Surprisingly, IL-10 producing-CD4+CD25-Foxp3- T cells were also identified as an additional source of IL-10 and represent a type 1 regulatory T (Tr1) cell subset that is being induced in vivo by L. infantum parasites. These findings suggest that distinct regulatory T cells develop in response to L. infantum and may play a possible role in promoting parasite persistence and the establishment of chronic infection in this particular experimental model of infection. Having demonstrated that Treg-mediated immunosuppression is evident in L. infantum susceptible infection model, the next step would be to verify if in a resistant experimental model of infection, immunosuppresive Treg can or cannot be modulated by the parasite. This would represent the development of a parasite strategy able to upregulate immunosuppressive cells, dampen effector immune response in their favour, and promote expansion, ultimately regulating the regulator. So to elucidate on immunosuppressive Treg function induced by L. infantum parasites and the underlying mechanisms involved in the direct host-parasite interactions and immune regulation, the second part of this study focuses on the role of TLR-2 on Treg function during L. infantum experimental murine infection by investigating the influence of TLR-2 on Treg kinetics, immune response, parasite-associated pathology and the outcome of L. infantum infection. To achieve this, TLR-2 deficient mice (TLR-2-/-) and their wild-type C57BL/6 mice (WT) were infected with L. infantum parasites and comparative analysis was done to see whether or not the presence or absence of TLR-2 produces any differential effect on the host parameters related to Treg dynamics and protective immunity. Defective TLR-2 signalling had a visible effect on outcome of L. infantum infection. Higher rates of parasite multiplication were observed in both spleen and liver of TLR-2-/- knock-out mice, despite the ability in forming well-defined and structured liver granulomas. These granulomas were apparently ineffective in parasite clearance, when compared to wild-type mice. Defective TLR-2 signalling did induce during late infection high retention of memory Treg which seemed to be associated to high parasite load and low IFN- levels. TLR-2 signalling pathway may play a role in Treg modulation and consequently in L. infantum pathogenesis. Functional TLR-2 signalling in WT may be important in providing tight control over FOXP3+ committed Treg populations, negative Treg regulation and more protective immunity, giving rise to enhanced immunity and more effective response against infection. The presence or absence of TLR-2 did not seem to influence IL-10 or TGF- expression, and it did not seem to correlate with CD103+ FOXP3+ Treg detected late during infection in TLR-2-/- mice. Detection of high levels of suppressive Treg in L. infantum-infected TLR-2 deficient mice was not accompanied by associated inductions of immunosuppressive cytokines. The presence of high levels of immunosuppressive Treg in infected spleen, in the absence of TLR-2, suggests that this receptor in particular plays an important role in regulating the regulators, thus orchestrating effective innate and acquired immunity against L. infantum infection.
Descrição
Palavras-chave
Parasitologia médica Leishmania infantum Análises Imunologia
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical