Caracterização de microRNAs reguladores da diferenciação de linfócitos T gd na sua resposta à infeção por malária

gd T cells are known to play important roles in malaria, but their effector potential is yet to be translated into innovative therapeutics. On the path towards that ultimate goal, this project focuses on an original (yet unanswered) question: how is the distinct functional potential of effector gd T cell subsets in malaria regulated by miRNAs? This overarching objective will be tackled through 4 complementary tasks: Aim/ Task 1 – Characterize the impact of candidate microRNAs in vitro and in vivo, in the context of experimental malaria in mice. We will focus on miR-181a, miR-128, miR-139 and miR-322, none of them previously implicated in gd T cell biology. Aim/ Task 2 – Identify direct mRNA targets of selected miRNAs that impact gd T cell differentiation in order to establish their downstream regulatory mechanism. Aim/ Task 3 – Functionally validate the candidate miRNA:mRNA networks in gd T cell subsets: investigate the dynamics and manipulate the expression of mRNA targets in parallel with the candidate miRNAs. Aim/ Task 4 – Extend our key findings to in vitro systems of human gd T cells upon Plasmodium stimulation; candidate miRNAs (and mRNAs) will be modulated in P. falciparum-activated human gd T cells to evaluate their impact on effector functions. The key approaches to develop these objectives will be: (i) To select and validate candidate miRNAs based on differential expression and tissue-specific (liver/ spleen) profiles upon infection; (ii) To manipulate candidate gene expression in vitro using primary cultures of mouse gd T cell subsets under various stimulation (inflammatory) conditions; (iii) To modulate candidate gene expression in vivo using miRNA interference methodologies or transgenic (gene-deficient or overexpressing) mice; (iv) To identify candidate miRNA target genes and determine miRNA:mRNA networks in the context of malaria infection; (v) To validate the main findings in cultures of human gd T cells challenged with Plasmodium falciparum antigens. This global approach will go beyond the state-of-the-art in multiple regards. First, our unbiased analyses will characterize novel post-transcriptional determinants of effector gd T cell subsets that play distinct roles in responses to infection by malaria. Second, the identification of mRNA targets for each candidate microRNA will allow the establishment of specific miRNA:mRNA networks that govern gd T cell differentiation in the context of malaria, thus contributing to improving basic knowledge in this field. Third, the validation in human gd T cell cultures will open new avenues to translate the key findings and develop novel gd T cell-mediated therapies towards benefit for many individuals affected by malaria, in line with the UN sustainable goal of good health and well-being focused on fighting communicable diseases. All together, we expect to provide a novel dissection of the role of miRNAs in the differentiation and activation of gd17 versus gdIFN cells upon malaria infection, thus improving basic knowledge; and to identify molecular targets for therapeutic intervention, thus adding translational impact to our proposal.