Comparação das linhagens monocíticas U937 e THP-1 como modelos de macrófago para estudos in vitro
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Data
2022-03-03
Autores
Nascimento, Camyla Rodrigues
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Universidade Estadual Paulista (Unesp)
Resumo
Macrófagos são células mielóides do sistema imune, caracterizados pela atividade fagocítica e capacidade de apresentar antígenos via MHC-II. Em sua maior parte, podem ter monócitos como precursores. São células altamente responsivas a sinais do microambiente, podendo assumir diferentes fenótipos em um espectro que varia de um perfil pró-inflamatório (ou ‘clássico’) denominado M1, até um perfil anti-inflamatório (ou ‘alternativo’), denominado M2. Macrófagos M1 podem ser polarizados por estímulos inflamatórios e microbianos como LPS e IFN-gama, resultando no aumento da produção de citocinas inflamatórias, maior atividade microbicida e produção de ROS. Macrófagos M2 também conhecidos como pró-reparo, apresentam maior atividade fagocítica e propriedades angiogênicas podendo ser diferenciados por IL-4, IL-10 e IL-13. Macrófagos tem um papel central na homeostase, patogênese e reparo, assim há grande interesse nestas células para o entendimento de processos biológicos relacionados à diversas doenças e processos de reparo, além de serem um importante alvo terapêutico potencial. THP-1 e U937 são as linhagens monocíticas humanas mais comumente utilizadas como precursores de macrófagos, porém podem apresentar diferenças importantes na resposta biológica, influenciando as informações obtidas. Este trabalho tem o objetivo de caracterizar a resposta biológica de macrófagos derivados das linhagens monocíticas THP-1 e U937, contribuindo para a seleção da melhor linhagem celular segundo a área de interesse em pesquisa. Diferentemente de monócitos primários, M-CSF não induziu a diferenciação em macrófagos, indicada pela adesão ao substrato de cultura in vitro e por alterações morfológicas, tanto da linhagem de THP-1 quanto U937, sendo necessário o estímulo com forbol-miristil-acetato (PMA). A partir dos resultados observou-se que a condição M1 leva ao aumento da expressão de TNF e IL-6 e redução da expressão de Arg1 e IL-10 determinados por RT-qPCR assim como maior produção de ROS avaliada por citometria de fluxo e aumento da secreção de IL-6 e IL-1β determinada por ELISA. Condições M2 induziram maior atividade fagocítica apenas para macrófagos derivados da linhagem THP-1. Os resultados indicam que há diferenças entre as linhagens THP-1 e U937 na resposta a estímulos externos, mas existem similaridades com a resposta biológica de macrófagos primários. Por fim, macrófagos derivados da linhagem THP-1 tendem a ter maior responsividade à estímulos M1 e a assumirem um fenótipo relacionado ao perfil pró-inflamatório/clássico, enquanto macrófagos derivados da linhagem U937 apresentam maior responsividade à estímulos M2 e desvio fenotípico para o perfil alternativo/de reparo.
Macrophages are immune cells of myeloid origin, characterized by phagocytic activity and by capability of antigen presentation via MHC-II. Peripheral blood monocytes can differentiate into macrophages in the tissues. In response to multiple signals in microenvironment, macrophages can transiently and reversibly assume different phenotypes, ranging in a spectrum from varying from pro-inflammatory (or “classically activated”) also known as M1, to an anti-inflammatory (or “alternatively activated”) also known as M2. M1 macrophages can be induced by microbial (e.g., LPS) or endogenous (e.g., IFN-gamma) inflammatory stimuli and produce higher levels of inflammatory cytokines, have higher microbicidal activity and greater production of ROS. M2 macrophages are also known as pro-repair macrophages, have increased phagocytic activity and angiogenic properties. This phenotype can be induced by anti-inflammatory cytokines such as IL-4, IL-10 and IL-13. Macrophages play a central role in homeostasis, pathogenesis, and repair, justifying the interest in these cells for the comprehension of biological mechanisms associated with disease and repair processes, as well as an important potential therapeutic target. THP-1 and U937 are the two most used human monocytic cell lines. Albeit rarely the experimental choice for one cell line or another is rarely justified, these two monocytic cell lines have important differences in their biological response, which influences the information derived from these studies. The goal of the present study is to characterize the biological response of macrophages derived from THP-1 and U937 monocytic cell lines, contributing to an informed selection of the best cell line according to specific research goals. Unlike primary monocytes, M-CSF did not induce macrophage differentiation of neither THP-1 nor U937 cells, as observed by substrate adhesion and morphological changes, which required stimulation with phorbol-myristyl-acetate (PMA). Based on the results it was observed that the M1 condition leads to increased expression of TNF and IL-6 and reduced expression of Arg1, and IL-10 determined by RT-qPCR; as well as increased production of ROS detected by flow cytometry and higher secretion of IL-6 and IL-1β by ELISA. Under M2 conditions, only THP-1-derived macrophages had increased phagocytic activity. The results indicate that THP-1 and U937 cell lines do respond differently to the same external stimuli, but both mimic the response of primary macrophages. Finally, macrophages derived from THP-1 cells tend to have greater responsiveness to M1 stimuli and skew their phenotype to the pro-inflammatory/classical profile, whereas macrophages derived from U937 cells exhibit greater responsiveness to M2 stimuli and a phenotypic shift towards the alternative/repair profile.
Macrophages are immune cells of myeloid origin, characterized by phagocytic activity and by capability of antigen presentation via MHC-II. Peripheral blood monocytes can differentiate into macrophages in the tissues. In response to multiple signals in microenvironment, macrophages can transiently and reversibly assume different phenotypes, ranging in a spectrum from varying from pro-inflammatory (or “classically activated”) also known as M1, to an anti-inflammatory (or “alternatively activated”) also known as M2. M1 macrophages can be induced by microbial (e.g., LPS) or endogenous (e.g., IFN-gamma) inflammatory stimuli and produce higher levels of inflammatory cytokines, have higher microbicidal activity and greater production of ROS. M2 macrophages are also known as pro-repair macrophages, have increased phagocytic activity and angiogenic properties. This phenotype can be induced by anti-inflammatory cytokines such as IL-4, IL-10 and IL-13. Macrophages play a central role in homeostasis, pathogenesis, and repair, justifying the interest in these cells for the comprehension of biological mechanisms associated with disease and repair processes, as well as an important potential therapeutic target. THP-1 and U937 are the two most used human monocytic cell lines. Albeit rarely the experimental choice for one cell line or another is rarely justified, these two monocytic cell lines have important differences in their biological response, which influences the information derived from these studies. The goal of the present study is to characterize the biological response of macrophages derived from THP-1 and U937 monocytic cell lines, contributing to an informed selection of the best cell line according to specific research goals. Unlike primary monocytes, M-CSF did not induce macrophage differentiation of neither THP-1 nor U937 cells, as observed by substrate adhesion and morphological changes, which required stimulation with phorbol-myristyl-acetate (PMA). Based on the results it was observed that the M1 condition leads to increased expression of TNF and IL-6 and reduced expression of Arg1, and IL-10 determined by RT-qPCR; as well as increased production of ROS detected by flow cytometry and higher secretion of IL-6 and IL-1β by ELISA. Under M2 conditions, only THP-1-derived macrophages had increased phagocytic activity. The results indicate that THP-1 and U937 cell lines do respond differently to the same external stimuli, but both mimic the response of primary macrophages. Finally, macrophages derived from THP-1 cells tend to have greater responsiveness to M1 stimuli and skew their phenotype to the pro-inflammatory/classical profile, whereas macrophages derived from U937 cells exhibit greater responsiveness to M2 stimuli and a phenotypic shift towards the alternative/repair profile.
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Palavras-chave
Ativação de macrófagos, Células THP-1, Células U937, Fenótipo, Macrophage activation, THP-1 Cells, U937 Cells, Phenotype.