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Canine Leishmaniasis: Explore modulation of T lymphocytes activity by autologous dendritic cells
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A leishmaniose canina é uma doença zoonótica endémica em várias regiões, causada por
Leishmania infantum. O cão, como reservatório principal, tem um papel central na
transmissão do parasita. As células dendríticas (CDs) são fundamentais na apresentação
de antigénios, estabelecendo a ponte entre a imunidade inata e adaptativa. Durante a
resposta imune adaptativa, as células T CD4+
e CD8+
imaturas são estimuladas por CDs,
diferenciando-se e proliferando em células T efetoras. O estabelecimento da infeção e o
desenvolvimento da doença dependem de vários fatores intrínsecos ao parasita e
hospedeiro. Assim, a interação entre hospedeiro e parasita, a imunidade inata e adaptativa
e os mecanismos de defesa do parasita desempenham um papel crítico no desfecho da
doença. O presente estudo teve como objetivo examinar a capacidade de CDs caninas,
diferenciadas in vitro e estimuladas por L. infantum, de direcionar a atividade de linfócitos
T autólogos. CDs derivadas de monócitos (moCDs) geradas a partir de células
mononucleares de sangue periférico foram expostas a promastigotas de L. infantum,
estimuladas com antigénios solúveis do parasita (Ag) e por vesículas extracelulares (VEs)
libertadas por L. infantum. Co-culturas de moCDs estimuladas e linfócitos autólogos
foram estabelecidas. As vias utilizadas por L. infantum para a sinalização das CDs foram
analisadas através da expressão génica de recetores toll-like e citocinas. A apresentação
antigénica foi indiretamente avaliada através da expressão de moléculas de classe I
(MHCI) e classe II (MHCII) do complexo maior de histocompatibilidade. A
diferenciação de subpopulações de células T [células T CD4+
, T CD8+
, T reguladoras
(Treg) e T de memória] foi investigada por imunofenotipagem e a actividade imunitária
e citotóxica analisada, respetivamente, pela expressão génica de citocinas e produção de
granzima B e perforina. Durante a infeção por L. infantum, os resultados obtidos indicam
que as moCDs foram sinalizadas através de TLR2, TLR4 e TLR9, gerando citocinas pró inflamatórias (TNF-α e IL-12p40) e apresentando antigénios aos linfócitos T através do
MHCI e MHCII. Promoveu a expansão de células T CD4+
e CD8+
e a resposta imunitária
incluiu a expressão de citocinas pró-inflamatórias (TNF-α e IL-12p40) e anti inflamatórias (IL-4). Nos sobrenadantes das co-culturas foi também detetada granzima B.
Foi igualmente observada resposta imunossupressora mediada por células Treg CD4+
e
CD8+
e geração de IL-10. Simultaneamente, foi verificado a expansão de células T de
memória efetora e central, sugerindo que a ativação de linfócitos T por moCDs
estimuladas com L. infantum promove o aumento das células T de memória. Também foi
possível perceber que as VEs libertadas pelo parasita têm um enorme potencial
imunomodulador, promovendo o desenvolvimento de inflamação, mas simultaneamente
uma resposta anti-inflamatória. Em conjunto, os resultados do estudo sugerem que as
moCDs caninas são sinalizadas por L. infantum através de sensores intramembranares de
superfície e intracelulares, ficando ativadas e podendo modular atividade linfocitária
através de apresentação antigénica, induzindo a expansão e ativação das células T helper
e T citotóxicas. A ativação dos linfócitos T e a resposta inflamatória gerada parece induzir
a regulação da resposta celular e o estabelecimento de memória celular. Abordagens
imunoterapêuticas baseadas em CDs direcionadas por estímulos antigénicos precisos
podem conduzir ao desenvolvimento de estratégias inovadoras aplicadas ao controlo da
leishmaniose.
Canine leishmaniasis is a zoonotic disease endemic in many regions caused by Leishmania infantum. The dog, as the main reservoir, has a central role in parasite transmission. Dendritic cells (DCs) are potent antigen-presenting cells that form a bridge between innate and adaptive immunity. During the adaptive immune response, naïve CD4+ and CD8+ T cells are stimulated by DCs and differentiate and proliferate into effector T cells. The establishment of infection and the development of the disease rely on several factors intrinsic to the parasite and host. Thus, the interaction between the host and parasite, the innate and adaptive immunity and the parasite defense mechanisms play a critical role in the disease outcome. Therefore, the main objective of the present study was to examine if canine DCs that were differentiated in vitro and primed by L. infantum could direct the activation of autologous T lymphocytes. Monocyte-derived DCs (moDCs) generated from peripheral blood mononuclear cells were exposed to L. infantum promastigotes and stimulated with parasite-soluble antigens (Ag) and extracellular vesicles (EVs) shed by L. infantum. Co-cultures of primed moDCs and autologous lymphocytes were established. DC signaling and immunological activity were evaluated by gene expression of toll-like receptors and cytokines. The antigenic presentation was indirectly assessed through the expression of class I (MHCI) and class II (MHCII) molecules of major histocompatibility complex. The differentiation of T cell subpopulations [CD4+ T cells, CD8+ T cells, regulatory T cells (Treg) and memory T cells] was investigated by immunophenotyping and the immune and cytotoxic activity by cytokine gene expression and production of granzyme B and perforin, respectively. During L. infantum infection, the results indicate that moDCs were signalized through TLR2, TLR4 and TLR9, generating pro-inflammatory cytokines (TNF-α and IL-12p40) and presenting antigens to T lymphocytes through MHCI and MHCII. Contact with moDCs promoted the expansion of CD4+ and CD8+ T cells and the immune response included the expression of pro-inflammatory (TNF-α and IL-12p40) and anti inflammatory (IL-4) cytokines. Granzyme B was also detected in the supernatants of the co-cultures. The immunosuppressive response mediated by CD4+ and CD8+ Treg cells and the generation of IL-10 was observed as well as the expansion of effector memory and central memory T cells, suggesting that the activation of T lymphocytes by L. infantum primed moDCs promotes the establishment of memory T cells. It was also possible to perceive that the EVs shed by the parasite have an enormous immunomodulatory potential, promoting the development of inflammation, but simultaneously an anti-inflammatory response. The findings of the current study suggest that canine moDCs are signaled by L. infantum through surface intramembrane and intracellular sensors, become activated and are able to modulate lymphocyte activity through the antigenic presentation, inducing the expansion and activation of helper and cytotoxic T cells. T cell activation and the generation of inflammatory response seem to induce the differentiation of regulatory immune response and the establishment of memory. Thus, cell-based immunotherapy approaches based on primed-directed DCs can lead to the development of innovative strategies for leishmaniasis control.
Canine leishmaniasis is a zoonotic disease endemic in many regions caused by Leishmania infantum. The dog, as the main reservoir, has a central role in parasite transmission. Dendritic cells (DCs) are potent antigen-presenting cells that form a bridge between innate and adaptive immunity. During the adaptive immune response, naïve CD4+ and CD8+ T cells are stimulated by DCs and differentiate and proliferate into effector T cells. The establishment of infection and the development of the disease rely on several factors intrinsic to the parasite and host. Thus, the interaction between the host and parasite, the innate and adaptive immunity and the parasite defense mechanisms play a critical role in the disease outcome. Therefore, the main objective of the present study was to examine if canine DCs that were differentiated in vitro and primed by L. infantum could direct the activation of autologous T lymphocytes. Monocyte-derived DCs (moDCs) generated from peripheral blood mononuclear cells were exposed to L. infantum promastigotes and stimulated with parasite-soluble antigens (Ag) and extracellular vesicles (EVs) shed by L. infantum. Co-cultures of primed moDCs and autologous lymphocytes were established. DC signaling and immunological activity were evaluated by gene expression of toll-like receptors and cytokines. The antigenic presentation was indirectly assessed through the expression of class I (MHCI) and class II (MHCII) molecules of major histocompatibility complex. The differentiation of T cell subpopulations [CD4+ T cells, CD8+ T cells, regulatory T cells (Treg) and memory T cells] was investigated by immunophenotyping and the immune and cytotoxic activity by cytokine gene expression and production of granzyme B and perforin, respectively. During L. infantum infection, the results indicate that moDCs were signalized through TLR2, TLR4 and TLR9, generating pro-inflammatory cytokines (TNF-α and IL-12p40) and presenting antigens to T lymphocytes through MHCI and MHCII. Contact with moDCs promoted the expansion of CD4+ and CD8+ T cells and the immune response included the expression of pro-inflammatory (TNF-α and IL-12p40) and anti inflammatory (IL-4) cytokines. Granzyme B was also detected in the supernatants of the co-cultures. The immunosuppressive response mediated by CD4+ and CD8+ Treg cells and the generation of IL-10 was observed as well as the expansion of effector memory and central memory T cells, suggesting that the activation of T lymphocytes by L. infantum primed moDCs promotes the establishment of memory T cells. It was also possible to perceive that the EVs shed by the parasite have an enormous immunomodulatory potential, promoting the development of inflammation, but simultaneously an anti-inflammatory response. The findings of the current study suggest that canine moDCs are signaled by L. infantum through surface intramembrane and intracellular sensors, become activated and are able to modulate lymphocyte activity through the antigenic presentation, inducing the expansion and activation of helper and cytotoxic T cells. T cell activation and the generation of inflammatory response seem to induce the differentiation of regulatory immune response and the establishment of memory. Thus, cell-based immunotherapy approaches based on primed-directed DCs can lead to the development of innovative strategies for leishmaniasis control.
Descrição
Palavras-chave
Microbiologia médica Biologia molecular Saúde pública Leishmaniose canina Imunidade inata Células dendríticas Imunidade adaptativa Linfócitos T