Usando interferência CRISPR e transcriptômica de célula única para examinar sistematicamente estados reativos inflamatórios
Os astrócitos ou astroglia, a maior população de células gliais no sistema nervoso central (SNC), desempenham inúmeras funções vitais. Entre outras coisas, essas células gliais (ou seja, células que suportam as funções do sistema nervoso) estão....
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)
Imunocoloração de GFAP de um astrócitos derivados de iPSC exibindo uma morfologia ramificada estrelada em cocultura com neurônios derivados de iPSC (não visualizados). Crédito: Leng et al.
Os astrócitos ou astroglia, a maior população de células gliais no sistema nervoso central (SNC), desempenham inúmeras funções vitais. Entre outras coisas, essas células gliais (ou seja, células que suportam as funções do sistema nervoso) estão envolvidas na regulação do fluxo de sangue no cérebro e na reparação do cérebro ou da medula espinhal após infecções ou lesões traumáticas.
Quando o SNC está ferido ou inflamado, astrócitos tornam-se altamente reativos, seguindo assinaturas celulares únicas que são específicas para um contexto ou evento neural. Embora as assinaturas celulares reativas dos astrócitos estejam agora bem documentadas, os mecanismos moleculares subjacentes a elas ainda são pouco compreendidos.
Pesquisadores da Universidade da Califórnia, da Universidade Vanderbilt e da Escola de Medicina Icahn no Monte Sinai realizaram recentemente um estudo com o objetivo de entender melhor os fundamentos moleculares dos distintos padrões de reação dos astrócitos com diferentes estados inflamatórios, usando uma combinação de técnicas genéticas. Suas descobertas, publicadas em Nature Neuroscience , permitiram identificar diferentes assinaturas reativas inflamatórias que foram reguladas por uma proteína específica.
"Meu projeto foi construído em torno da ideia de usar genômica funcional baseada em CRISPR (ou seja, uma técnica de perturbação genética) para estudar sistematicamente os processos celulares que impulsionam a reatividade dos astrócitos, que pode ser amplamente entendido como como os astrócitos respondem a insultos ao sistema nervoso central . " Kun Leng, o primeiro autor do artigo do laboratório Kampmann na UCSF, disse ao Medical Xpress. "Como os astrócitos reativos são induzidos sob uma grande variedade de contextos patológicos que podem ser difíceis de modelar in vitro, optei por me concentrar na reatividade dos astrócitos induzida por citocinas inflamatórias (ou seja, moléculas sinalizadoras que promovem a inflamação)."
Os métodos experimentais usados ??por Leng e seus colegas foram inspirados em um artigo de 2017 de Shane Liddelow publicado na Nature . Neste trabalho, Liddelow mostrou que as citocinas IL-1?+TNF+C1q, que são liberadas pela micróglia ativada in vivo, induzem uma forma de reatividade inflamatória in vitro que é patológica e pode estar envolvida em certas doenças neurodegenerativas e neuroinflamatórias humanas.
"Uma vez que um estado celular é em grande parte determinado pelo perfil de expressão gênica de uma célula, uma ferramenta crítica que nos permitiu identificar distintos estados reativos inflamatórios induzidos por IL-1?+TNF+C1q foi o sequenciamento de RNA de célula única, que captura o perfil de expressão gênica de células individuais", explicou Leng. "Isso nos permitiu identificar marcadores desses estados (ou seja, genes expressos que eram relativamente distintos de qualquer um dos dois estados inflamatórios que observamos)".
Imunocoloração de marcadores de estado reativo inflamatório (verde-C3,
vermelho-IFIT3) em astrócitos derivados de iPSC tratados
com IL-1?+TNF+C1q. Crédito: Leng et al.
Leng e seus colegas observaram dois estados reativos inflamatórios diferentes induzidos pelas citocinas identificadas por Liddelow e, em seguida, identificaram marcadores genéticos desses estados. Posteriormente, eles realizaram experimentos de citometria de fluxo, que também permitem a observação de células individuais. Nesses experimentos, eles usaram anticorpos contra esses marcadores, para determinar como a perturbação de certos fatores de transcrição de genes, quinases ou receptores usando perturbação genética afetava o equilíbrio dos dois estados.
"Do ponto de vista metodológico, nosso estudo foi o primeiro a aplicar a triagem CRISPRi, que requer um grande número de células (na faixa de centenas de milhões), a astrócitos derivados de iPSC", disse Leng. "Conseguimos produzir astrócitos derivados de iPSC de forma escalável, com base em trabalhos anteriores identificando um meio de cultura de astrócitos comercial adequado, bem como trabalhos que mostraram aceleração da diferenciação de astrócitos através da superexpressão de fatores de transcrição gliogênicos. Embora existam limitações para estudar astrócitos in vitro (como para qualquer tipo de célula), acreditamos que a aplicação de triagem baseada em CRISPR a astrócitos derivados de iPSC será útil para estudar aspectos importantes da biologia dos astrócitos."
O trabalho recente desta equipe de pesquisadores fornece novas informações valiosas sobre os marcadores genéticos que regulam diferentes estados reativos inflamatórios no SNC. Essencialmente, Leng e seus colegas mostraram que uma mistura de citocinas pode induzir estados celulares distintos por meio de vias de sinalização mutuamente contra-reguladoras.
Notavelmente, os marcadores genéticos que influenciam as assinaturas que eles identificaram foram previamente regulados nos cérebros de pessoas afetadas por várias condições neurológicas, incluindo doença de Alzheimer e encefalopatia hipóxico-isquêmica (ou seja, uma lesão cerebral que ocorre como resultado da diminuição do fluxo de oxigênio para o cérebro). No futuro, seu trabalho poderá informar o desenvolvimento de novas ferramentas terapêuticas para modular seletivamente aspectos específicos da reatividade dos astrócitos inflamatórios, melhorando potencialmente a compreensão e o tratamento atuais de algumas condições neurológicas.
Em seus próximos estudos, os pesquisadores gostariam de investigar as funções dos dois estados de astrócitos reativos inflamatórios que observaram em seus experimentos. Além disso, eles planejam traduzir suas estratégias para regular esses estados de reatividade em ferramentas que permitiriam aos pesquisadores médicos amplificá-los ou inibi-los seletivamente.
“A próxima fronteira para nós agora é estabelecer telas CRISPR para astrócitos e outros tipos de células em camundongos vivos”, disse Martin Kampmann, autor sênior do artigo. “Isso nos permitirá entender o que controla os estados celulares no contexto do cérebro na saúde e na doença, e descobrir como podemos reprogramar os estados celulares para benefício terapêutico”.