Produção de citocinas inflamatórias e indução da transição epitelial-mesenquimal de células amnióticas no cenário de infecção polimicrobiana
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Data
2022-09-14
Autores
Silva, Mariana de Castro
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Universidade Estadual Paulista (Unesp)
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Introdução: O parto pré-termo espontâneo (PP) é considerado um dos principais problemas da clínica obstétrica e tem como uma das principais causas associada a infecção polibacteriana e consequente inflamação dos tecidos da interface materno-fetal. Esse desfecho gestacional desfavorável resulta da combinação de vários mecanismos, incluindo a elevada produção de mediadores inflamatórios e a senescência celular. O objetivo desse estudo foi avaliar a produção e cinética de mediadores inflamatórios em modelos in vitro de infecção polimicrobiana de células amnióticas na presença de diferentes espécies de lactobacilos, além de avaliar fatores indutores da transição epitelial-mesenquimal em células amnióticas. Metodologia: Células amnióticas epiteliais imortalizadas foram cultivadas em modelos in vitro e submetidas a diferentes estímulos compostos por espécies bacterianas comumente identificadas em gestações complicadas por PP. Os sobrenadantes da cultura foram coletados em cinco momentos diferentes: 0h, 6h, 12h, 18h e 24h. As Interleucinas (IL) -6, IL-8, IL-10, IL-13, fator estimulador de colônias de granulócitos e macrófagos (GM-CSF) e fator de necrose tumoral α (TNF-α) foram mensurados empregando-se ensaio imunoenzimático (ELISA) e a análise morfológica foi realizada por meio de análise de cell shape index. Resultados: A produção de citocinas pró-inflamatórias foi maior nos grupos estimulados com Lactobacillus iners em combinações com Mycoplasma hominis e diferentes bactérias. A resposta anti-inflamatória foi maior nos grupos de bactérias anaeróbias estimulados junto com L. crispatus. A transição epitelial-mesenquimal foi significativamente maior no grupo estimulado com L. iners e M. hominis, assim como em combinação com Fusobacterium nucleatum, depois de 24h de estímulo. Esse aumento na transição epitelial-mesenquimal não foi observado quando as células eram tratadas com LPS, mas houve um aumento significativo da quantificação de células mesenquimais quando o estímulo era inflamatório, sendo esse tempo e dose dependente. Conclusão: O cenário induzido por estimulação, das células epiteliais amnióticas, com bactérias Gram-variáveis, na presença de L. iners, apresentou maiores níveis de citocinas pró-inflamatórias, assim como a transição epitelial-mesenquimal esteve associada à presença de mediador inflamatório (TNFα) e às combinações específicas de bactérias.
Introduction: Preterm birth (PTB) is one of the main problems in the obstetric clinic. Polybacterial invasion and consequent inflammation on the maternal-fetal interface are one of the major causes associated to PTB, resulting in elevated production of inflammatory mediators, as well as cellular senescence. The aim of this study was to evaluate the production and kinetics of inflammatory mediators in in vitro models of polybacterial infection of amniotic cells in the presence of different species of lactobacilli. Methods: Immortalized amnion epithelial cells were cultured in in vitro models and subjected to different stimuli composed of bacterial species commonly identified in pregnancies complicated by PTB. Culture supernatants were collected at five different times-points: 0h, 6h, 12h, 18h and 24h. Interleukins (IL) -6, IL-8, IL-10, IL-13, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and tumor necrosis factor α (TNF-α) were measured by Enzyme-Linked Immunosorbent Assay (ELISA) and quantification of cell morphology was performed using cell shape index analysis. Results: The production of pro-inflammatory cytokines was higher in groups stimulated with L. iners in combinations with M. hominis and different bacteria. The anti-inflammatory response was greater in the groups of anaerobic bacteria stimulated together with L. crispatus. The epithelial-mesenchymal transition was significantly higher in the group stimulated with L. iners and M. hominis, as well as in combination with F. nucleatum after 24h of stimulation. This increase in the epithelial-mesenchymal transition was not observed when the cells were treated with LPS, but there was a significant increase in the quantification of mesenchymal cells when the stimulus was inflammatory (TNFα), being this time and dose dependent. Conclusion: The scenario induced by stimulation of amnion epithelial cells, with Gram-variable bacteria, in the presence of L. iners, showed higher levels of pro-inflammatory cytokines, as well as the epithelial-mesenchymal transition was associated with the presence of inflammatory mediator (TNFα) and specific combinations of bacteria.
Introduction: Preterm birth (PTB) is one of the main problems in the obstetric clinic. Polybacterial invasion and consequent inflammation on the maternal-fetal interface are one of the major causes associated to PTB, resulting in elevated production of inflammatory mediators, as well as cellular senescence. The aim of this study was to evaluate the production and kinetics of inflammatory mediators in in vitro models of polybacterial infection of amniotic cells in the presence of different species of lactobacilli. Methods: Immortalized amnion epithelial cells were cultured in in vitro models and subjected to different stimuli composed of bacterial species commonly identified in pregnancies complicated by PTB. Culture supernatants were collected at five different times-points: 0h, 6h, 12h, 18h and 24h. Interleukins (IL) -6, IL-8, IL-10, IL-13, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and tumor necrosis factor α (TNF-α) were measured by Enzyme-Linked Immunosorbent Assay (ELISA) and quantification of cell morphology was performed using cell shape index analysis. Results: The production of pro-inflammatory cytokines was higher in groups stimulated with L. iners in combinations with M. hominis and different bacteria. The anti-inflammatory response was greater in the groups of anaerobic bacteria stimulated together with L. crispatus. The epithelial-mesenchymal transition was significantly higher in the group stimulated with L. iners and M. hominis, as well as in combination with F. nucleatum after 24h of stimulation. This increase in the epithelial-mesenchymal transition was not observed when the cells were treated with LPS, but there was a significant increase in the quantification of mesenchymal cells when the stimulus was inflammatory (TNFα), being this time and dose dependent. Conclusion: The scenario induced by stimulation of amnion epithelial cells, with Gram-variable bacteria, in the presence of L. iners, showed higher levels of pro-inflammatory cytokines, as well as the epithelial-mesenchymal transition was associated with the presence of inflammatory mediator (TNFα) and specific combinations of bacteria.
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Células amnióticas epiteliais, Lactobacilos, Micoplasmas genitais, Desfecho gestacional adverso, Infecção polibacteriana, Immortalized amnion epithelial cells, Lactobacilli, Genital mycoplasmas, Pregnancy outcome, Polybacterial infection