Avaliação do inflamassoma NLRP3 e autofagia em placentas de gestantes portadoras de pré-eclâmpsia
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Data
2016-09-01
Autores
Weel, Ingrid Cristina [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A pré-eclâmpsia (PE) é uma síndrome clinicamente identificada por hipertensão arterial e proteinúria e está associada à produção excessiva de citocinas proinflamatórias, deficiência na produção de citocinas reguladoras e aumento nos níveis séricos de anticorpos antifosfolípides (aPLs) em pacientes com PE grave. Os aPLs são uma família de autoanticorpos que reagem com proteínas ligantes de fosfolipídios, sendo seu principal alvo a beta-2 glicoproteina I (β2GPI). Estes anticorpos são responsáveis por inibir a diferenciação do sinciciotrofoblasto e restringir a migração trofoblástica, resultando em remodelação anormal das arteríolas espiraladas, alteração característica da PE. O inflamassoma é um complexo proteico que promove a secreção das citocinas proinflamatórias interleucina-1 beta (IL-1β) e interleucina 18 (IL-18) e, também a secreção da proteína “high-mobility group box 1” (HMGB1) após ativação por patógenos e/ou produtos endógenos associados ao dano celular. A autofagia é uma via de degradação celular ou de eliminação de organelas e proteínas através de processos lisossomais que são importantes para a manutenção da homeostase celular, promovendo a sobrevivência das células em resposta ao estresse. O presente trabalho teve como objetivos: 1. Investigar as proteinas relacionadas ao inflamassoma e à autofagia em placenta de gestantes portadoras de pré-eclâmpsia e de normotensas; 2. Avaliar a relação existente entre inflamassoma e autofagia em células de trofoblasto extraviloso humano (SW.71), induzida por aPL. Fragmentos placentários foram coletados de 20 gestantes normotensas e de 20 gestantes com pré-eclâmpsia para avaliar a expressão gênica e proteica de NLRP3, caspase-1, IL-1β, fator de necrose tumoral-alpha (TNF-α), IL-18, HMGB1, proteína de cadeia leve (LC3B-II), beclin-1 e de “mammalian target of rapamycin” (mTOR). As respostas autofágica e inflamatória e, a expressão de fatores antiangiogênicos também foram analizadas em células SW.71 induzidas por aPL. Os resultados mostraram níveis elevados de RNAm de NLRP3, caspase-1, IL-1β, TNF-α e HMGB1 em tecido placentário, bem como aumento significativo dos níveis de caspase-1, IL-1β, TNF-α e HMGB1 em homogenato de placenta de gestantes portadoras de PE quando comparadas às normotensas. A expressão gênica e proteica de IL-18 mostrou-se significativamente diminuida no grupo de gestantes pré-eclâmpticas. A expressão das proteínas relacionadas à autofagia, LC3B-II e beclin-1 e os níveis de RNAm destas proteinas mostraram-se significativamente diminuídos em placenta de gestantes com PE comparados aos de gestantes normotensas. Por outro lado, o RNAm e a proteína de mTOR foram mais elevados nas gestantes com PE em comparação com as normotensas. Nas células SW.71 observou-se que o tratamento com aPL por 8h induziu diminuição significativa da razão LC3B-II/LC3BI seguida de aumento significativo após 72 hr do estímulo. Esses resultados não foram observados na expressão de beclin-1 e p62. Em cultura das células submetidas à inibição do processo autofágico detectou-se aumento na secreção de IL-1β, IL-8 e endoglina solúvel (sEng), enquanto nas culturas em que a autofagia não foi inibida observou-se diminuição da secreção de sEng e de fms-like tyrosine-kinase-1 solúvel (sFlt-1). Além disso, a permanência do processo autofágico reduziu significativamente a secreção de IL-1β e IL-8 em células induzidas por aPL. Em conclusão, os resultados obtidos mostram que placenta de gestantes com PE apresentam ativação do inflamassoma NLRP3 e diminuição da resposta autofágica, sugerindo que estes processos podem contribuir para a patogênese da PE. O estímulo de células de trofoblasto extraviloso com aPL é capaz de diminuir a resposta autofágica nessas células por meio de ativação do inflamassoma. O restabelecimento tardio da autofagia nessas células poderia ocorrer, provavelmente, devido à tentativa desse processo em controlar a ativação do inflamassoma.
Preeclampsia (PE) is a syndrome clinically identified by hypertension and proteinuria and is associated with excessive production of proinflammatory cytokines, deficiency in the production of regulatory cytokines, and increased serum levels of antiphospholipid antibodies (aPLs) in patients with severe forms of PE. aPLs are a family of autoantibodies that react with phospholipid binding proteins, which the main target is beta 2 glycoprotein-I (β2GPI). These antibodies are responsible for inhibiting the differentiation of syncytiotrophoblast and restrict trophoblast migration, resulting in abnormal remodeling of the spiral arterioles, characteristic alteration in PE. The inflammasome is a protein complex that promotes the secretion of the proinflammatory cytokines interleukin-1 beta (IL-1β) and interleukin 18 (IL-18), and also the secretion of high-mobility group box 1 (HMGB1) protein after activation by pathogens and/or endogenous products associated with cellular damage. Autophagy is a pathway of cell degradation or elimination of organelles and proteins by lysosomal processes that are important for the maintenance of cellular homeostasis by promoting the survival of cells in response to stress. The objectives of the present study are: 1. To investigate the proteins related to inflammasome and autophagy in placenta from pregnant women with PE and from normotensive control; 2. To evaluate the relationship between inflammasome and autophagy in human extravillous trophoblast cells (SW.71) induced by aPL. Placental fragments were collected from 20 normotensive pregnant women and from 20 pregnant women with PE to evaluate gene and protein expression of NLRP3, caspase-1, IL-1β, tumor necrosis factor-alpha (TNF-α), IL-18, HMGB1, light chain protein (LC3B- II), beclin-1 and mammalian target of rapamycin (mTOR). The autophagic and inflammatory responses as well as the expression of antiangiogenic factors were also analyzed in SW.71 cells induced by aPL. The results showed higher levels of mRNA for NLRP3, caspase-1, IL-1β, TNF-α and HMGB1 in placental tissue as well as significant increase in caspase-1, IL-1β, TNF-α and HMGB1 levels in placental homogenate from women with PE compared with the normotensive group. Gene and protein expression of IL-18 was significantly decreased in the group of preeclamptic women. The expression of proteins related to autophagy, LC3B-II and beclin-1 and the mRNA levels of these proteins were significantly decreased in placenta of women with PE compared with the normotensive ones. On the other hand, mRNA and protein for mTOR were higher in women with PE. In SW.71 cells it was observed that the treatment with aPL for 8h induced significant decrease in LC3B-II/LC3BI ratio, followed by its significant increase after 72hr of aPL stimulation. These results were not observed in the expression of beclin-1 and p62. When SW.71 cells were submitted to autophagy inhibition a significant increase in IL-1β, IL-8 and soluble endoglin (sEng) was detected, whereas the maintenance of autophagy lead to reduced secretion of sEng and fms-like tyrosine kinase-1-soluble (sFlt-1). Furthermore, the maintenance of the autophagic process significantly reduced the secretion of IL-1β and IL-8 in cells induced by aPL. In conclusion, the results demonstrated that placenta from preeclamptic women shows NLRP3 inflammasome activation and decreased autophagic response suggesting that these processes may contribute to the pathogenesis of PE. The stimulus of extravillous trophoblast cells with aPL caused decrease in the autophagic response in these cells by inflammasome activation, leading to delayed recovery of autophagy, which may probably occur due to an attempted of inflammasome control by the autophagy process.
Preeclampsia (PE) is a syndrome clinically identified by hypertension and proteinuria and is associated with excessive production of proinflammatory cytokines, deficiency in the production of regulatory cytokines, and increased serum levels of antiphospholipid antibodies (aPLs) in patients with severe forms of PE. aPLs are a family of autoantibodies that react with phospholipid binding proteins, which the main target is beta 2 glycoprotein-I (β2GPI). These antibodies are responsible for inhibiting the differentiation of syncytiotrophoblast and restrict trophoblast migration, resulting in abnormal remodeling of the spiral arterioles, characteristic alteration in PE. The inflammasome is a protein complex that promotes the secretion of the proinflammatory cytokines interleukin-1 beta (IL-1β) and interleukin 18 (IL-18), and also the secretion of high-mobility group box 1 (HMGB1) protein after activation by pathogens and/or endogenous products associated with cellular damage. Autophagy is a pathway of cell degradation or elimination of organelles and proteins by lysosomal processes that are important for the maintenance of cellular homeostasis by promoting the survival of cells in response to stress. The objectives of the present study are: 1. To investigate the proteins related to inflammasome and autophagy in placenta from pregnant women with PE and from normotensive control; 2. To evaluate the relationship between inflammasome and autophagy in human extravillous trophoblast cells (SW.71) induced by aPL. Placental fragments were collected from 20 normotensive pregnant women and from 20 pregnant women with PE to evaluate gene and protein expression of NLRP3, caspase-1, IL-1β, tumor necrosis factor-alpha (TNF-α), IL-18, HMGB1, light chain protein (LC3B- II), beclin-1 and mammalian target of rapamycin (mTOR). The autophagic and inflammatory responses as well as the expression of antiangiogenic factors were also analyzed in SW.71 cells induced by aPL. The results showed higher levels of mRNA for NLRP3, caspase-1, IL-1β, TNF-α and HMGB1 in placental tissue as well as significant increase in caspase-1, IL-1β, TNF-α and HMGB1 levels in placental homogenate from women with PE compared with the normotensive group. Gene and protein expression of IL-18 was significantly decreased in the group of preeclamptic women. The expression of proteins related to autophagy, LC3B-II and beclin-1 and the mRNA levels of these proteins were significantly decreased in placenta of women with PE compared with the normotensive ones. On the other hand, mRNA and protein for mTOR were higher in women with PE. In SW.71 cells it was observed that the treatment with aPL for 8h induced significant decrease in LC3B-II/LC3BI ratio, followed by its significant increase after 72hr of aPL stimulation. These results were not observed in the expression of beclin-1 and p62. When SW.71 cells were submitted to autophagy inhibition a significant increase in IL-1β, IL-8 and soluble endoglin (sEng) was detected, whereas the maintenance of autophagy lead to reduced secretion of sEng and fms-like tyrosine kinase-1-soluble (sFlt-1). Furthermore, the maintenance of the autophagic process significantly reduced the secretion of IL-1β and IL-8 in cells induced by aPL. In conclusion, the results demonstrated that placenta from preeclamptic women shows NLRP3 inflammasome activation and decreased autophagic response suggesting that these processes may contribute to the pathogenesis of PE. The stimulus of extravillous trophoblast cells with aPL caused decrease in the autophagic response in these cells by inflammasome activation, leading to delayed recovery of autophagy, which may probably occur due to an attempted of inflammasome control by the autophagy process.
Descrição
Palavras-chave
Placenta, Autofagia, Pré-eclâmpsia, Sindrome anti-fosfolípide, Inflamassoma, Autophagy, Inflammasome, Preeclampsia, Antiphospholipid syndrome