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Mechanisms of inhibition of Plasmodium liver infection by amino acid supplementation
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A malária é uma das doenças infeciosas mais prevalentes no mundo1. É causada por parasitas protozoários do género Plasmodium, os quais são transmitidos aos seus hospedeiros mamíferos através da picada de um mosquito Anopheles fêmea infetado1,2. A primeira fase da infeção, obrigatória e assintomática, ocorre no fígado e é iniciada quando os esporozoítos injetados invadem os hepatócitos do hospedeiro2,3. Aí, os parasitas diferenciam-se e desenvolvem-se até se formarem merozoitos, que são libertados na corrente sanguínea, infetando ciclicamente os eritrócitos e causando os sintomas da malária2,3.
A multiplicação dos parasitas durante a fase hepática da infeção é sustentada pela obtenção de nutrientes a partir do seu hospedeiro. Um desses nutrientes é a arginina (Arg), cujo metabolismo é crucial para o desenvolvimento intra-hepático do parasita4.
A Arg tem-se tornado cada vez mais popular na suplementação nutricional dada a sua capacidade de estimular o sistema imunitário5, sendo a única suplementação baseada em aminoácidos avaliada no contexto da malária. No entanto, embora a suplementação com Arg possa aumentar a produção de óxido nítrico (NO), diminuir a parasitemia, e melhorar a sobrevivência em modelos animais de infeção por Plasmodium, os resultados obtidos na clínica têm sido inconclusivos 6,7,8.
Resultados preliminares do laboratório de acolhimento demonstraram que a suplementação de murganhos C57Bl/6 com RKV, em que a administração de Arg (R) é combinada com a de Lisina (K) e Valina (V), dois aminoácidos descritos como inibidores da arginase, leva a uma diminuição acentuada da infeção hepática, sobretudo através da redução do número de hepatócitos infetados, sugerindo uma eliminação dos parasitas. No entanto, permanece por esclarecer se este fenótipo é recapitulado noutras estirpes de murganhos. Assim, o primeiro objetivo desta tese foi caracterizar a infeção hepática por Plasmodium em murganhos BALB/c após suplementação com Arg e RKV. Demonstrámos que a suplementação com Arg é suficiente para inibir a infeção hepática por Plasmodium em murganhos BALB/c, enquanto a suplementação com RKV pode levar tanto ao aumento como à diminuição da infeção hepática, em ambos os casos afetando principalmente o número de hepatócitos infetados. A razão desses resultados contraditórios permanece desconhecida.
O segundo objetivo desta tese foi elucidar o mecanismo de eliminação hepática do parasita em murganhos C57Bl/6 suplementados com RKV. Demonstrámos que a eliminação do parasita pela suplementação com RKV não depende nem da produção de NO, nem da estimulação da resposta de interferão tipo-I (IFN), anteriormente relatadas como cruciais para o controlo da infeção hepática9,10. Utilizando murganhos knockout e eliminando populações de células imunes específicas, identificámos as Células Linfóides Inatas (ILCs) como potenciais células imunes efetoras envolvidas na eliminação do parasita dependente de RKV. Adicionalmente, a sinalização através de MyD88 parece ser essencial para a eliminação hepática do parasita após suplementação com RKV, embora as células envolvidas nessa sinalização permaneçam desconhecidas.
Este projeto irá melhorar o nosso conhecimento quanto aos aspetos fundamentais da biologia de Plasmodium e quanto à resposta do hospedeiro face à infeção, abrindo caminho para o desenvolvimento de potenciais novas estratégias que possam vir a ser usadas para controlar a infeção por Plasmodium.
Malaria is an acute febrile illness and one of the most prevalent infectious diseases in the world1. It is caused by protozoan parasites of the genus Plasmodium that enter their mammalian host in the form of sporozoites, via the bite of an infected female Anopheles mosquito1,2. The first, obligatory and asymptomatic phase of infection occurs in the liver and is initiated when injected sporozoites invade their host’s hepatocytes2,3. There, parasites differentiate and develop until merozoites are formed and released into the bloodstream, cyclically infecting red blood cells and causing the malaria symptoms2,3. During the liver-stage of infection, Plasmodium parasites scavenge host nutrients to support their multiplication. One of these nutrients is arginine (Arg), whose metabolism is crucial for the parasite’s intrahepatic development4. Arg is becoming increasingly popular in nutritional supplementation for its ability to boost the immune system5. Arg is so far the only amino acid-based supplementation evaluated in the context of malaria. However, although Arg supplementation can enhance nitric oxide (NO) production, decrease parasitaemia and improve survival in animal models of Plasmodium infection, unclear results have been obtained in the clinic6,7,8. Preliminary results from the host laboratory have shown that supplementation of C57Bl/6 mice with RKV, which combines Arg (R) with Lysine (K) and Valine (V), two amino acids described as arginase inhibitors, but not Arg alone, leads to a striking decrease of hepatic infection, mostly by reducing the number of infected hepatocytes, suggesting that parasites are being eliminated. However, whether this phenotype was also recapitulated in other mouse strains remained unclarified. Thus, the first aim of this thesis was to characterize Plasmodium liver infection in BALB/c mice upon Arg and RKV supplementation. We found that Arg supplementation is sufficient to impair Plasmodium liver infection in BALB/c mice and, on the contrary, RKV supplementation can lead to either an increase or a decrease in Plasmodium hepatic infection, in both cases mostly by affecting the number of infected hepatocytes. The reason for these contradictory results remains unknown. The second aim of this thesis was to elucidate the mechanism of hepatic parasite elimination in C57Bl/6 mice upon RKV supplementation. We found that parasite elimination by RKV supplementation does not rely on NO production nor on a boost of the type-I interferon (IFN) response, previously reported as crucial to control liver-stage infection9,10. Employing knockout mice and depleting specific immune cell populations, we identified Innate Lymphoid Cells (ILCs) as potential effector immune cells involved in RKV-dependent parasite elimination. Additionally, signalling via MyD88, seems to be essential for hepatic parasite’s elimination upon RKV supplementation, although the cells in which this signalling occurs remain unidentified. This project will enhance our knowledge of fundamental aspects of Plasmodium biology and of the host’s response to infection, paving the way to the development of potential new strategies that may ultimately be employed to control Plasmodium infection.
Malaria is an acute febrile illness and one of the most prevalent infectious diseases in the world1. It is caused by protozoan parasites of the genus Plasmodium that enter their mammalian host in the form of sporozoites, via the bite of an infected female Anopheles mosquito1,2. The first, obligatory and asymptomatic phase of infection occurs in the liver and is initiated when injected sporozoites invade their host’s hepatocytes2,3. There, parasites differentiate and develop until merozoites are formed and released into the bloodstream, cyclically infecting red blood cells and causing the malaria symptoms2,3. During the liver-stage of infection, Plasmodium parasites scavenge host nutrients to support their multiplication. One of these nutrients is arginine (Arg), whose metabolism is crucial for the parasite’s intrahepatic development4. Arg is becoming increasingly popular in nutritional supplementation for its ability to boost the immune system5. Arg is so far the only amino acid-based supplementation evaluated in the context of malaria. However, although Arg supplementation can enhance nitric oxide (NO) production, decrease parasitaemia and improve survival in animal models of Plasmodium infection, unclear results have been obtained in the clinic6,7,8. Preliminary results from the host laboratory have shown that supplementation of C57Bl/6 mice with RKV, which combines Arg (R) with Lysine (K) and Valine (V), two amino acids described as arginase inhibitors, but not Arg alone, leads to a striking decrease of hepatic infection, mostly by reducing the number of infected hepatocytes, suggesting that parasites are being eliminated. However, whether this phenotype was also recapitulated in other mouse strains remained unclarified. Thus, the first aim of this thesis was to characterize Plasmodium liver infection in BALB/c mice upon Arg and RKV supplementation. We found that Arg supplementation is sufficient to impair Plasmodium liver infection in BALB/c mice and, on the contrary, RKV supplementation can lead to either an increase or a decrease in Plasmodium hepatic infection, in both cases mostly by affecting the number of infected hepatocytes. The reason for these contradictory results remains unknown. The second aim of this thesis was to elucidate the mechanism of hepatic parasite elimination in C57Bl/6 mice upon RKV supplementation. We found that parasite elimination by RKV supplementation does not rely on NO production nor on a boost of the type-I interferon (IFN) response, previously reported as crucial to control liver-stage infection9,10. Employing knockout mice and depleting specific immune cell populations, we identified Innate Lymphoid Cells (ILCs) as potential effector immune cells involved in RKV-dependent parasite elimination. Additionally, signalling via MyD88, seems to be essential for hepatic parasite’s elimination upon RKV supplementation, although the cells in which this signalling occurs remain unidentified. This project will enhance our knowledge of fundamental aspects of Plasmodium biology and of the host’s response to infection, paving the way to the development of potential new strategies that may ultimately be employed to control Plasmodium infection.
Descrição
Palavras-chave
Ciências biomédicas Malária Plasmodium Anopheles Parasitologia Infeção hepática Arginina Suplementação Resposta imune
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical