Ativação das STAT-1, STAT-3 e STAT-6 na modulação da coinfecção com duas cepas de Trypanosoma cruzi em macrófagos humanos polarizados M1 e M2
Arquivos
Data
2022-02-08
Autores
Oliveira, Melissa Martins de 
Orientadores
Mortara, Renato Arruda
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A interação do parasito Trypanosoma cruzi (T. cruzi), agente etiológico da doença de Chagas, com o sistema imunológico do indivíduo infectado é um fator determinante no curso da doença. A resposta imune é, em geral, apta para a defesa frente ao parasito, que, por sua vez, dispõe de mecanismos de evasão que visam sua sobrevivência e permanência no ambiente intracelular. O objetivo deste trabalho foi avaliar a coinfecção com duas cepas de Trypanosoma cruzi (G e CL) em comparação com as monoinfecções de cada uma, em macrófagos derivados da linhagem tumoral THP-1 e ativados pelas vias clássica e alternativa. Para isso, obtivemos macrófagos a partir da linhagem THP-1 e realizamos as polarizações para os perfis M1 e M2. Esses macrófagos foram infectados com formas tripomastigotas de cultura (TCTs) ou amastigotas extracelulares (AEs) de T. cruzi em mono- ou coinfecção. Nós avaliamos a porcentagem de células infectadas através de citometria de fluxo, produção de óxido nítrico por quimiluminescência, produção de espécies reativas de oxigênio (ROS) por microscopia de fluorescência, secreção de citocinas por beads magnéticas e ativação dos fatores de transcrição STAT-1, STAT-3 e STAT-6 por citometria de fluxo. Nossos dados demonstraram que ambas as cepas foram capazes de infectar macrófagos não estimulados (M0), os estimulados com IFN-γ + LPS demonstraram resistência à infecção independente da cepa e com IL-4 ou IL-6 houve maior suscetibilidade à infecção com a cepa CL, independente da forma do parasito. A quantidade de citocinas secretadas variou conforme os modelos de infecção, sendo que as infecções com AEs diminuíram os níveis de IL-1β, IFN-γ, TGF-β e TNF-α, e com TCTs algumas diminuíram (TNF-α, IL-6 e IL-10) e outras aumentaram (TGF-β e IL-4). Houve maior produção de ROS em macrófagos estimulados com IFN-γ + LPS, principalmente associadas à presença da cepa G na monoinfecção tanto com AEs como TCTs e na coinfecção apenas com AEs. O padrão de ativação das STATs avaliadas diferiu entre as condições, mas em geral aumentou na coinfecção, apresentando também desfosforilação, especialmente na população de células não infectadas de cada grupo. Essas observações demonstram que embora haja responsividade dos macrófagos frente à infecção, ambas as cepas, isoladas ou não, são capazes de modular o microambiente dos macrófagos, visando sua permanência intracelular.
The interaction of the parasite Trypanosoma cruzi (T. cruzi), Chagas’ disease aetiological agent, with the immune system of the infected individual is a determining factor in disease course. The immune response is generally able to defend itself against the parasite, which, in turn, has evasion mechanisms that aims its survival and persistence in the intracellular environment. This work’s objective was to evaluate the co-infection with two Trypanosoma cruzi strains (G and CL) in comparison to monoinfections (G or CL), in THP-1 tumor cell line derived macrophages activated either by classical or alternative pathways. Thus, we obtained macrophages from the THP-1 lineage and performed the polarizations for M1 and M2 profiles. These macrophages were infected with T. cruzi cultured trypomastigote forms (TCTs) or extracellular amastigotes (AEs) in mono- or coinfections. We evaluated the infected cells percentage by flow cytometry, nitric oxide production by chemiluminescence, reactive oxygen species (ROS) production by fluorescence microscopy, cytokines secretion by magnetic beads and transcription factors STAT-1, STAT-3 and STAT-6 activation by flow cytometry. Our data showed that both strains were able to infect unstimulated macrophages (M0), and IFN-γ + LPS stimulated macrophages demonstrated resistance to infection independent of the strain and stimuli with IL-4 or IL-6 resulted in higher susceptibility to infection with the CL strain, independent of the parasite’s evolutive form. Cytokines levels varied according to the infection models, whereas AEs infection decreased IL-1β, IFN-γ, TGF-β and TNF-α levels, and TCTs infection led some to decrease (TNF-α, IL-6 and IL-10) and others to increase (TGF-β and IL-4). There was greater production of ROS in IFN-γ + LPS stimulated macrophages, mainly associated with the presence of the G strain in monoinfection with either AEs or TCTs and in coinfection only with AEs. The activation pattern of the evaluated STATs differed between the conditions, but generally it was higher in coinfection, also showing dephosphorylation especially in non-infected population of each group. Thus, although there is a responsiveness of macrophages to infection, both parasite strains, isolated or not, are able to modulate the macrophage microenvironment, in order to achieve their intracellular permanence.
The interaction of the parasite Trypanosoma cruzi (T. cruzi), Chagas’ disease aetiological agent, with the immune system of the infected individual is a determining factor in disease course. The immune response is generally able to defend itself against the parasite, which, in turn, has evasion mechanisms that aims its survival and persistence in the intracellular environment. This work’s objective was to evaluate the co-infection with two Trypanosoma cruzi strains (G and CL) in comparison to monoinfections (G or CL), in THP-1 tumor cell line derived macrophages activated either by classical or alternative pathways. Thus, we obtained macrophages from the THP-1 lineage and performed the polarizations for M1 and M2 profiles. These macrophages were infected with T. cruzi cultured trypomastigote forms (TCTs) or extracellular amastigotes (AEs) in mono- or coinfections. We evaluated the infected cells percentage by flow cytometry, nitric oxide production by chemiluminescence, reactive oxygen species (ROS) production by fluorescence microscopy, cytokines secretion by magnetic beads and transcription factors STAT-1, STAT-3 and STAT-6 activation by flow cytometry. Our data showed that both strains were able to infect unstimulated macrophages (M0), and IFN-γ + LPS stimulated macrophages demonstrated resistance to infection independent of the strain and stimuli with IL-4 or IL-6 resulted in higher susceptibility to infection with the CL strain, independent of the parasite’s evolutive form. Cytokines levels varied according to the infection models, whereas AEs infection decreased IL-1β, IFN-γ, TGF-β and TNF-α levels, and TCTs infection led some to decrease (TNF-α, IL-6 and IL-10) and others to increase (TGF-β and IL-4). There was greater production of ROS in IFN-γ + LPS stimulated macrophages, mainly associated with the presence of the G strain in monoinfection with either AEs or TCTs and in coinfection only with AEs. The activation pattern of the evaluated STATs differed between the conditions, but generally it was higher in coinfection, also showing dephosphorylation especially in non-infected population of each group. Thus, although there is a responsiveness of macrophages to infection, both parasite strains, isolated or not, are able to modulate the macrophage microenvironment, in order to achieve their intracellular permanence.
Descrição
Citação
OLIVEIRA, M. M. de. Ativação das STAT-1, STAT-3 e STAT-6 na modulação da coinfecção com duas cepas de Trypanosoma cruzi em macrófagos humanos polarizados M1 e M2. São Paulo, 2022. 68 f. Trabalho de Conclusão de Curso (Graduação em Biomedicina) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2022.