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Espiroquetídeos do complexo Borrelia burgdorferi s.l. e Leptospira spp: avaliação da atividade microbicida de macrófagos infetados
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Resumo(s)
Os macrófagos (MΦ) fagocitam e destroem agentes patogénicos através de diversos
mecanismos, desempenhando um papel importante na resposta imune inata. A virulência de
diferentes espécies de espiroquetas do complexo Borrelia burgdorferi s.l., e Leptospira spp, agentes
da borreliose de Lyme e Leptospirose, respetivamente, podem influenciar os mecanismos
bactericidas dos MΦ, levando a uma diversidade de manifestações clínicas. Este trabalho teve
como objetivo, analisar os mecanismos microbicidas de MΦ na presença de espécies patogénicas
de borrélias e leptospiras com diferentes níveis de patogenicidade, com enfoque na acidificação e
maturação de fagossomas, e produção de espécies reativas de oxigénio (ROS) e azoto, e ureia.
MΦ THP-1 e de sangue periférico foram infetados com as espécies B. garinii e B. lusitaniae,
e L. biflexa e L. interrogans. A taxa de infeção foi calculada pela quantificação de MΦ com borrélias
e leptospiras intracelulares por imunofluorescência. Por microscopia de fluorescência, ROS foi
detetado com CM-H2DCFDA, vesículas acídicas com LysoTracker Red e lisossomas com anticorpo
anti-LAMP-1. A concentração de nitritos e ureia foi medida por ensaios colorimétricos.
Em T0, a taxa de MΦ infetados por B. garinii foi superior à de B. lusitaniae. B. garinii
localizou-se maioritariamente no citoplasma mantendo a sua morfologia, enquanto B. lusitaniae
dentro de vacúolos. Não se observou co-localização de borrélias e lisossomas. Após 24h de
infeção, o número e tamanho de vesículas acídicas aumentou, especialmente com B. garinii. Ambas
as bactérias induziram produção de ROS. Não houve diferenças na produção de nitritos e ureia.
B. garinii apresentou uma maior infeciosidade, permanecendo livre no citoplasma da célula
sem aparente formação de fagolisossomas. B. lusitaniae localizou-se dentro de fagossomas
imaturos, demonstrando um possível mecanismo de sobrevivência. ROS, parece contribuir para a
destruição intracelular das borrélias. Os ensaios com Leptospira spp foram igualmente realizados,
tendo sido verificadas taxas de infeção elevadas
Macrophages (MΦ) are involved in phagocytosis and destruction of pathogens through several mechanisms, playing an important role in the innate immune response. The virulence of different species of spirochetes of the Borrelia burgdorferi s.l. complex and Leptospira spp, agents of Lyme Borreliosis and Leptospirosis, respectively, can influence the bactericidal mechanisms of MΦ, leading to a variety of clinical manifestations. This work aimed to analyze the microbicidal mechanisms of MΦ in the presence of pathogenic species of Borrelia and Leptospira with different levels of pathogenicity, focusing on acidification and maturation of phagosomes, and production of reactive oxygen (ROS) and nitrogen species, and urea. THP-1 MΦ and peripheral blood MΦ were infected with the species B. garinii and B. lusitaniae, and L. biflexa and L. interrogans. The infection rate was calculated by quantifying MΦ with intracellular borrelia and leptospires by immunofluorescence. By fluorescence microscopy, ROS was detected with CM-H2DCFDA, acidic vesicles with LysoTracker Red and lysosomes with anti-LAMP-1 antibody. The concentration of nitrites and urea was measured by colorimetric assays. At T0, the rate of MΦ infected by B. garinii was higher than the rate of MΦ infected by B. lusitaniae. B. garinii was mostly located in the cytoplasm, maintaining its morphology, while B. lusitaniae was within vacuoles. No co-location of Borrelia and lysosomes was observed. After 24h of infection, the number and size of acidic vesicles increased, especially with B. garinii. Both bacteria induced ROS production. There were no differences in nitrite and urea production. B. garinii showed higher infectivity, remaining free in the cell cytoplasm without apparent formation of phagolysosomes. B. lusitaniae was located within immature phagosomes, demonstrating a possible survival mechanism. ROS appears to contribute to the intracellular destruction of Borrelia. Tests with Leptospira spp were also performed and demonstrated high rates of infection.
Macrophages (MΦ) are involved in phagocytosis and destruction of pathogens through several mechanisms, playing an important role in the innate immune response. The virulence of different species of spirochetes of the Borrelia burgdorferi s.l. complex and Leptospira spp, agents of Lyme Borreliosis and Leptospirosis, respectively, can influence the bactericidal mechanisms of MΦ, leading to a variety of clinical manifestations. This work aimed to analyze the microbicidal mechanisms of MΦ in the presence of pathogenic species of Borrelia and Leptospira with different levels of pathogenicity, focusing on acidification and maturation of phagosomes, and production of reactive oxygen (ROS) and nitrogen species, and urea. THP-1 MΦ and peripheral blood MΦ were infected with the species B. garinii and B. lusitaniae, and L. biflexa and L. interrogans. The infection rate was calculated by quantifying MΦ with intracellular borrelia and leptospires by immunofluorescence. By fluorescence microscopy, ROS was detected with CM-H2DCFDA, acidic vesicles with LysoTracker Red and lysosomes with anti-LAMP-1 antibody. The concentration of nitrites and urea was measured by colorimetric assays. At T0, the rate of MΦ infected by B. garinii was higher than the rate of MΦ infected by B. lusitaniae. B. garinii was mostly located in the cytoplasm, maintaining its morphology, while B. lusitaniae was within vacuoles. No co-location of Borrelia and lysosomes was observed. After 24h of infection, the number and size of acidic vesicles increased, especially with B. garinii. Both bacteria induced ROS production. There were no differences in nitrite and urea production. B. garinii showed higher infectivity, remaining free in the cell cytoplasm without apparent formation of phagolysosomes. B. lusitaniae was located within immature phagosomes, demonstrating a possible survival mechanism. ROS appears to contribute to the intracellular destruction of Borrelia. Tests with Leptospira spp were also performed and demonstrated high rates of infection.
Descrição
Palavras-chave
Espiroquetas Borrelia burgdorferi s.l. Macrófagos Fagolisossoma ROS