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Doctoral Thesis
DOI
https://doi.org/10.11606/T.60.2021.tde-26112021-165017
Document
Author
Full name
Felipe Teixeira Lima
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2021
Supervisor
Committee
Frantz, Fabiani Gai (President)
Cunha, Larissa Dias da
Guazzaroni, María Eugenia
Soares, Luana Silva
Title in Portuguese
Identificação de um novo conjunto de biomarcadores para predição do curso da infecção pelo Mycobacterium tuberculosis
Keywords in Portuguese
Biomarcadores
Epigenética
Mycobacterium tuberculosis
Resistência
RNAseq
Susceptibilidade
Transcriptoma
Abstract in Portuguese
O desenvolvimento de alternativas para conter a tuberculose (TB) aliado à necessidade de ampla compreensão de mecanismos de ação da resposta imune à infecção impulsionam a investigação de novas vias intracelulares, as quais poderiam estar associadas com novos biomarcadores de prognóstico da doença. Estudos mostram que dentre os possíveis órgãos afetados na infecção experimental em camundongos, com Mtb (Mtb), o fígado apresenta uma resposta eficiente contra a infecção quando comparado ao pulmão e baço. Nossa hipótese é que no fígado, mecanismos efetores da resposta imune são eficazes para o controle da infecção e poderiam não ser ativados nos órgãos onde o bacilo subverte à resposta efetora. Nosso objetivo foi analisar in silico dados já obtidos do transcriptoma em busca de genes e enzimas epigenéticas diferencialmente expressas em células totais do fígado e do pulmão de camundongos com TB experimental, que estivessem associados à regulação da resposta imune em ambos os órgãos, de forma a permitir a disseminação do bacilo no pulmão, enquanto promovem o seu controle no fígado. Na análise in silico, foram identificados 7 genes, Creb3l1, Trim17, Myo7b, Cyyr1, Cbs, Krt23 e Cyp3a43 como diferencialmente expressos e relacionados ao controle ou suscetibilidade. O objetivo seguinte foi validar in vitro os alvos identificados, através da infecção de monócitos humanos com Mtb, para que novos biomarcadores sejam propostos. Por meio da utilização de RNA de interferência, observamos que o perfil transcricional pôde ser revertido, o que induziu inibição nas funções efetoras de monócitos. Demonstramos que a expressão de Creb3l1, Trim17, Cyyr1, Myo7b e Hdac9, e a repressão de Cbs, Krt23 e Cyp3A43, induz produção de citocinas como IFN-?, IL-2 e CCL5/Rantes, e como consequência a indução da apoptose e resistência à infecção. Entretanto, quando há redução na expressão dos genes Creb3l1, Trim17, Cyyr1, Myo7b com concomitante aumento da expressão de Cbs, Krt23 e Cyp3A43, há maior produção de IL-4 e morte celular por necrose, o que resulta na persistência da micobactéria e consequente perfil de suscetibilidade. A expressão destes genes está sobre controle epigenético, podendo ser modulado in vitro por meio do tratamento com epidrogas. Assim, concluímos que o conjunto de genes aqui identificados pode integrar uma bioassinatura de predição do curso da infecção, e a modulação diferencial destes 7 genes pode favorecer um cenário de resistência ou suscetibilidade na tuberculose humana.
Title in English
Identification of a novel combination of biomarkers for predicting Mtb infection course
Keywords in English
Biomarkers
Epigenetics
Mycobacterium tuberculosis
Resistance
RNAseq
Susceptibility
Transcriptome
Abstract in English
The development of alternatives to curb tuberculosis (TB), combined with the need for a broad understanding of the mechanisms of action of the immune response to the infection promotes the investigation of new intracellular pathways, which could be associated with potential prognostic biomarkers of the disease. Studies show that infection in experimental mice with Mtb (Mtb), among the possible organs affected, the liver presents an efficient response against infection when compared to the lung and spleen. Our hypothesis is that in the liver, some effector mechanism of the immune response is effective in eliminating the bacillus and consequently controls the disease, while it could not be activated in organs where the bacillus subverts the effector response. Our aim was to analyze in silico data already obtained from the transcriptome in search of genes and epigenetic enzymes differentially expressed in total liver and lung cells of mice experimentally infected with Mtb, which were associated with the regulation of the immune response in both organs, in order to allow the spread of the bacillus in the lung, while promoting its control in the liver. In the in silico analysis, 7 genes, Creb3l1, Trim17, Myo7b, Cyyr1, Cbs, Krt23 and Cyp3a43 were identified as differentially expressed and related to control or susceptibility. The next aim was to validate the identified targets were validated in vitro by infecting human monocytes with Mtb, to identify potential biomarkers. Through the knockdown of selected mRNA by RNA interference, we observed that the transcriptional profile could be reversed, which induced inhibition of the effector functions of monocytes. We demonstrate that the the expression of Creb3l1, Trim17, Cyyr1, Myo7b and Hdac9 and repression of Cbs, Krt23 and Cyp3A43, induces the production of cytokines such as IFN-?, IL-2 and CCL5/Rantes, and as a consequence the induction of apoptosis and resistance to infection. However, when there is a reduction in the expression of genes Creb3l1, Trim17, Cyyr1, Myo7b with a concomitant increase in the expression of Cbs, Krt23 and Cyp3A43, there is greater production of IL-4 and cell death by necrosis, which results in the persistence of mycobacteria and consequent profile of susceptibility profile. The expression of these genes are under epigenetic control and can be modulated in vitro through treatment with epidrugs. Thus, we conclude that the set of genes identified here can integrate a biosignature to predict the course of infection in tuberculosis, and the differential modulation of these 7 genes can favor can favor a scenario of resistance or susceptibility in human tuberculosis.
 
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Release Date
2023-09-20
Publishing Date
2022-01-06
 
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