| Summary: | MicroRNAs are small RNA molecules, approximately 22 nucleotides in length, employed by cells to regulate gene expression. They act post-transcriptionally, destabilizing or inhibiting the translation of mRNA molecules that harbour a corresponding binding site. MicroRNAs are essential for cell function as their depletion in mice is embryonically lethal. MicroRNAs are required for the correct development and function of immune cells, where, in particular, they have been implicated in the control of cytokine secretion. The control of the production of cytokines, such as pro-inflammatory interferon (IFN)-γ and interleukin (IL)-17, is essential for the defence against pathogens and for homeostasis maintenance. IFN-γ production is critical for the immune response against viruses, intracellular bacteria and tumour cells whilst IL-17 is necessary in the fight against extracellular bacteria and fungi. However, an excessive or deregulated secretion of these cytokines can lead to the development of chronic inflammation and autoimmune diseases, such as multiple sclerosis, type I diabetes, rheumatoid arthritis, colitis or psoriasis. Different cells of the immune system have the ability to produce these cytokines. In a typical adaptive response to intracellular pathogens, the major sources of IFN-γ are CD8+ T cells and their CD4+ (T helper type 1, Th1) counterparts, taking on from innate-like NK and γδ T cells that provide most IFN-γ during the earlier stages. As for IL-17, the lymphocyte subsets that account for most of its production during immune responses to extracellular pathogens are γδ T cells (at early stages) and CD4+ T helper type 17 (Th17) cells. Given the importance of a regulated expression of these pro-inflammatory cytokines, the molecular mechanisms responsible for such regulation are of utmost relevance. It is within this conceptual frame that the work presented in this thesis was developed. In the first part of this thesis, we aimed to identify microRNAs involved in the regulation of IFN-γ production by CD8+ T cells. We started by demonstrating that microRNAs are globally important in this process by showing that CD8+ T cells lacking microRNAs (due to genetic deficiency in the microRNA-processing enzyme Dicer) display exacerbated IFN-γ production compared to controls. By analysing the expression levels of microRNAs in CD8+ T cells from a mouse strain with a fluorescent reporter for IFN-γ (bicistronicconstruction of IFN-γ with the fluorescent protein YFP), we identified 29 microRNAs differentially expressed between IFN-γ producing (YFP+ ) versus non-producing (YFP- ) CD8+ T cells. From these, we showed in gain-of-function experiments that microRNAs miR-181a-5p and miR-451a inhibit IFN-γ expression in CD8+ T cells, by targeting and destabilizing mRNA molecules of genes such as Id2, Akt2 and Map2k1. We further showed that a reduction in Id2 levels leads to a reduction in IFN-γ production in CD8+ T cells, thus phenocopying the effects of overexpressing miR-181-5p. These observations support the hypothesis that miR-181a-5p reduces IFN-γ production in CD8+ T cells by, at least partially, reducing Id2 mRNA levels. When present in low amounts, Id2 cannot inhibit the ligation of proteins E2/E47 to the Tbx21 locus, which leads to the transcriptional silencing of this gene, which is an important transcription factor for the expression of IFN-γ in CD8+ T cells. In the second part of this thesis, we characterized, through Next Generation Sequencing (NGS), the transcriptome and miRNome of the two main effector subpopulations of γδ T cells: IL-17 producers and IFN-γ producers. These were isolated from a reporter mouse strain for both cytokines (with bicistronic constructions for both GFP and YFP respectively). We thereby identified the genes differentially expressed between the two γδ T cell effector populations aiming at improving our knowledge on their cellular identity and microRNA-mediated gene regulation. Our analysis revealed 103 differentially expressed microRNAs from which we selected 8 candidates to study in more detail. In vitro polarization experiments showed that miR-326-3p, miR-7a-5p and miR-1949 limit IFN-γ expression; that miR-128-3p inhibits IFN-γ expression and promotes IL-17 expression; and that miR-322-5p promotes co-production IFN-γ together with IL-17 . Further work is required to determine the impact these microRNAs may have in vivo, namely in the context of infection or inflammatory disease. In turn, our transcriptome analysis revealed 7882 genes differentially expressed between effector γδ T cell subsets, with 4341 enriched in IL-17+ cells and 3541 enriched in IFN-γ + cells. The data provided a global view on the biology of γδ T lymphocytes, namely on the pathways and processes enriched in each of the effector subpopulations. We identified 62 pathways enriched in the IL-17 producing subpopulation and 27 in their IFN-γ producing counterparts. Differences in the ability to integrate external cues, including some not typically associated with the immune system, such as neuronal mediators or hormone responsiveness, and metabolic differences are amongst the most interesting and novel ones, which warrant further functional investigation. In sum, the work presented in this thesis allowed the identification of various microRNAs involved in the regulation of cytokine expression in CD8+ or γδ T cells; and highlighted several unanticipated differences between effector γδ T cell populations, thus constituting a useful resource for the scientific community to formulate new hypotheses on the cellular identity and physiology of γδ T cells.
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