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Neutrophil-Leishmania interaction during the initial phase of Leishmania infantum infection in dogs
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A leishmaniose canina (LCan) é uma doença zoonótica causada pelo protozoário Leishmania infantum e transmitida por insetos do género Phlebotomus e Lutzomyia, respetivamente no Velho e Novo Mundos. Trata-se de uma doença grave, sistémica e potencialmente fatal. Embora estejam disponíveis algumas medidas profiláticas e terapêuticas, a sua eficácia é questionável. Apesar dos neutrófilos (PMN) serem a primeira linha de defesa do organismo, fagocitando rapidamente o parasita após inoculação, os macrófagos (MØ) são as células hospedeiras definitivas, na medida em que suportam a sua multiplicação e disseminação. Este trabalho teve como objetivos
identificar os mecanismos efetores ativados pelos PMN em resposta à exposição ao parasita e o impacto da interação PMN-MØ na fase inicial da infeção canina, determinando a sua contribuição no controlo da infeção ou, pelo contrário, no estabelecimento da doença clínica. Foram selecionados animais clinica e analiticamente saudáveis que testaram negativamente para as principais patologias infeciosas e
parasitárias e estabeleceram-se culturas PMN-Leishmania. Verificou-se por citometria e observação microscópica que promastigotas de L. infantum foram eficazmente fagocitados por PMN na maioria dos canídeos avaliados, activando os mecanismos oxidativos (produção de superóxido) e não oxidativos (exocitose de elastase neutrofílica), mas prevenindo a libertação de armadilhas extracelulares neutrofílicas (neutrophil extracelular traps, NET). Para além disso, promastigotas e sobrenadantes de cultura induziram a migração de PMN, mas o contacto prévio com Leishmania inibiu a quimiotaxia, o que contribui para a retenção dos PMN no local da inoculação. A interação com o parasita teve um impacto negativo na viabilidade dos PMN, tendo induzido a necrose secundária. A interação PMN-parasita resultou numa diminuição da viabilidade parasitária, embora alguns promastigotas intracelulares tenham sobrevivido e mantido a
capacidade de proliferar, assegurando o estabelecimento da infeção. MØ diferenciados a partir de monócitos sanguíneos sofreram alterações morfológicas importantes com o intuito de conter o parasita e também libertaram Histona H1, cujo efeito leishmanicida não foi ainda provado em promastigotas de L. infantum. A transferência do parasita para os MØ foi confirmada por citometria em co-culturas de MØ+PMN infetados aos quais foram removidos os parasitas extracelulares. A observação microscópica destas coculturas mostrou que a eferocitose de PMN infetados e provavelmente a fagocitose de parasitas libertados por PMN apoptóticos são importantes na transferência do parasita
para a célula hospedeira definitiva. Inesperadamente, a interação PMN-MØ ativou a produção de óxido nítrico e induziu a libertação de NET, o que pode contribuir para a contenção do parasita e para o controlo da infeção numa fase precoce. Os resultados obtidos ampliam o conhecimento da infeção por L. infantum no cão e podem contribuir
para o desenvolvimento de novas ferramentas terapêuticas e profiláticas que conduzam à redução acentuada da LCan.
Canine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan Leishmania infantum transmitted by insects of the genus Phlebotomus and Lutzomyia in the Old and New Worlds, respectively. It is a severe, systemic and potentially fatal disease. Despite the availability of some prophylactic and therapeutic tools, their effectiveness is questionable. Although neutrophils (PMN) are the first line of defense of the organism, rapidly phagocytizing the parasite after inoculation, macrophages (MØ) are the definitive host cells, supporting their multiplication and spread. This study aimed to identify the effector mechanisms activated by PMN in response to L. infantum promastigotes and the impact of PMN-MØ interaction in the initial phase of canine infection by determining its contribution to infection control or, on the contrary, the establishment of clinical disease. Clinically and analytically healthy animals who tested negative for the main infectious and parasitic diseases were selected and PMN-Leishmania cultures were established. It was found by flow cytometry and microscopic observation that L. infantum promastigotes are efficiently phagocytized by PMN in the majority of the dogs, activating oxidative (superoxide production) and non-oxidative (neutrophil elastase exocytosis) mechanisms, but preventing the release of neutrophil extracellular traps (NET). Furthermore, promastigotes and culture supernatants induced PMN migration, but the prior contact with Leishmania inhibited chemotaxis, which contributes to PMN retention at the inoculation site. The interaction with the parasite had a negative impact on PMN viability, promoting secondary necrosis. PMN-parasite interaction resulted in a decrease in parasite viability, although some intracellular promastigotes survived and maintained their proliferative capacity, contributing to the establishment of the infection. MØ differentiated from blood monocytes underwent major morphological changes in order to contain the parasite and also released Histone H1, but its leishmanicidal effect was not yet proven in L. infantum promastigotes. Parasite transfer to MØ was confirmed by flow cytometry in co-cultures of MØ-infected PMN whose extracellular parasites were removed. The microscopic observation of these co-cultures showed that infected PMN efferocytosis and probably phagocytosis of parasites released from apoptotic PMN are crucial for parasite transfer to the definitive host cell. Unexpectedly, PMN-MØ interaction activated nitric oxide production and induced NET release, which can contribute to parasite containment and to the early control of the infection. These findings broaden the knowledge of L. infantum infection in the dog and can shed light in the design of new therapeutic and prophylactic tools, leading to a marked reduction in CanL.
Canine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan Leishmania infantum transmitted by insects of the genus Phlebotomus and Lutzomyia in the Old and New Worlds, respectively. It is a severe, systemic and potentially fatal disease. Despite the availability of some prophylactic and therapeutic tools, their effectiveness is questionable. Although neutrophils (PMN) are the first line of defense of the organism, rapidly phagocytizing the parasite after inoculation, macrophages (MØ) are the definitive host cells, supporting their multiplication and spread. This study aimed to identify the effector mechanisms activated by PMN in response to L. infantum promastigotes and the impact of PMN-MØ interaction in the initial phase of canine infection by determining its contribution to infection control or, on the contrary, the establishment of clinical disease. Clinically and analytically healthy animals who tested negative for the main infectious and parasitic diseases were selected and PMN-Leishmania cultures were established. It was found by flow cytometry and microscopic observation that L. infantum promastigotes are efficiently phagocytized by PMN in the majority of the dogs, activating oxidative (superoxide production) and non-oxidative (neutrophil elastase exocytosis) mechanisms, but preventing the release of neutrophil extracellular traps (NET). Furthermore, promastigotes and culture supernatants induced PMN migration, but the prior contact with Leishmania inhibited chemotaxis, which contributes to PMN retention at the inoculation site. The interaction with the parasite had a negative impact on PMN viability, promoting secondary necrosis. PMN-parasite interaction resulted in a decrease in parasite viability, although some intracellular promastigotes survived and maintained their proliferative capacity, contributing to the establishment of the infection. MØ differentiated from blood monocytes underwent major morphological changes in order to contain the parasite and also released Histone H1, but its leishmanicidal effect was not yet proven in L. infantum promastigotes. Parasite transfer to MØ was confirmed by flow cytometry in co-cultures of MØ-infected PMN whose extracellular parasites were removed. The microscopic observation of these co-cultures showed that infected PMN efferocytosis and probably phagocytosis of parasites released from apoptotic PMN are crucial for parasite transfer to the definitive host cell. Unexpectedly, PMN-MØ interaction activated nitric oxide production and induced NET release, which can contribute to parasite containment and to the early control of the infection. These findings broaden the knowledge of L. infantum infection in the dog and can shed light in the design of new therapeutic and prophylactic tools, leading to a marked reduction in CanL.
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Ciência biomédica Biologia molecular Leishmaniose canina Neutrófilos Interação neutrófilo-macrófago