Publicação
MicroRNA determinants of the balance between effector and regulatory CD4+ T cells
| Resumo: | CD4+ T cells play a central role in orchestrating immune responses. They have the capacity to help B cells in producing antibodies, they induce macrophage activation, enhance and maintain CD8+ T cell-mediated responses and are capable of immune cell suppression. After encountering an antigen, CD4+ T cells undergo differentiation into specific subtypes according to the cytokine milieu of the microenvironment. These include effector T helper (Th) 1 and Th17 cells, and anti-inflammatory regulatory T (Treg) cells. In order to avoid autoimmune and inflammatory diseases, it is extremely important to maintain the balance between the effector and regulatory CD4+ T cell populations. Therefore, the differentiation of these CD4+ T cell subsets must be tightly regulated. MicroRNAs (miRNAs) are negative post-transcriptional regulators of gene expression that have been involved in regulating and maintaining the fate decisions of CD4+ T cell populations and, consequently, affect the balance between them. While several studies showed the role of specific miRNAs in the differentiation of individual CD4+ T cell subsets, we aimed at further dissecting which miRNAs regulate the balance between effector and regulatory CD4+ T cell populations in an immune response in vivo. We identified 6 miRNAs that were differentially expressed between Th1, Th17 and Treg cells isolated from a triple reporter mouse with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. While two candidates were initially subjected to functional studies in vitro, the remaining four were studied in mice bearing EAE as their expression in in vitro-differentiated CD4+ T cell subsets did not reproduce the in vivo data. Our data showed that overexpression of miR-126a-5p limits IL-17a production in Th17 cells polarized in vitro. Additionally, inhibition of miR-122-5p promoted an earlier EAE onset while mice treated with miR-1247 antagomiR develop a less severe disease. In this thesis, to confirm the overexpression results obtained by the host lab, we aimed to study the impact of inhibiting miR-126a-5p and miR-467a-5p in in vitro-differentiated CD4+ T cell subsets. Also, we aimed to dissect the external cues responsible for the expression of all functionally relevant miRNAs for CD4+ T cell differentiation, including miR-122 and miR-1247. Our results reveal that inhibiting miR-126a increases the frequency of IL-17+ cells, further confirming that miR-126a limits the production of IL-17 by Th17 cells. We show that the IL-6 signalling pathway is involved in inducing not only miR-126a expression, but also the expression of miR-122 when associated with TGF−β signalling. On the contrary, IL-23 and IL-1β were shown to inhibit miR-122 expression, which allowed us to hypothesize that this miRNA is expressed in a context of non-pathogenic Th17 cells. Moreover, we observed that miR-1247 is induced downstream of the TGF−β signalling pathway. Relying on a bioinformatic approach, we identified transcription factors from each signalling pathway that can be directly regulating the expression of each candidate miRNAs. More specifically, Maf, Ets1 and Ets2 were identified as candidate transcription factors that might induce miR-126a expression; STAT3, RORα, IRF1 and SMAD2:SMAD3:SMAD4 complex as inducers of miR-122 expression; and Bach1:MafK as potential regulators of miR-1247 expression. Overall, our results show that both miR-126a and miR-122, act as molecular brakes of cytokine production on Th17 cells, possibly as an attempt to control excessive inflammation. Contrary to miR-126a, miR-122 expression appears to be context-specific, acting in non-pathogenic Th17 cells to maintain their phenotype. miR-1247, was shown to be important to maintain the inflammatory phenotype of effector Th1 cells in the presence of TGF−β, an anti-inflammatory cytokine. The data described here reveals new insights on three critical miRNAs for the differentiation of CD4+ T cell subsets, highlighting their potential to regulate the balance between effector and regulatory populations in inflammatory conditions in vivo and revealing their potential use as therapeutic targets in EAE and other autoimmune diseases. |
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| Autores principais: | Pais, Ana Teresa da Silva |
| Assunto: | Subtipos de células T CD4+ microRNAs regulação da expressão de microRNAs vias de sinalização fatores de transcrição Teses de mestrado - 2021 |
| Ano: | 2021 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | CD4+ T cells play a central role in orchestrating immune responses. They have the capacity to help B cells in producing antibodies, they induce macrophage activation, enhance and maintain CD8+ T cell-mediated responses and are capable of immune cell suppression. After encountering an antigen, CD4+ T cells undergo differentiation into specific subtypes according to the cytokine milieu of the microenvironment. These include effector T helper (Th) 1 and Th17 cells, and anti-inflammatory regulatory T (Treg) cells. In order to avoid autoimmune and inflammatory diseases, it is extremely important to maintain the balance between the effector and regulatory CD4+ T cell populations. Therefore, the differentiation of these CD4+ T cell subsets must be tightly regulated. MicroRNAs (miRNAs) are negative post-transcriptional regulators of gene expression that have been involved in regulating and maintaining the fate decisions of CD4+ T cell populations and, consequently, affect the balance between them. While several studies showed the role of specific miRNAs in the differentiation of individual CD4+ T cell subsets, we aimed at further dissecting which miRNAs regulate the balance between effector and regulatory CD4+ T cell populations in an immune response in vivo. We identified 6 miRNAs that were differentially expressed between Th1, Th17 and Treg cells isolated from a triple reporter mouse with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. While two candidates were initially subjected to functional studies in vitro, the remaining four were studied in mice bearing EAE as their expression in in vitro-differentiated CD4+ T cell subsets did not reproduce the in vivo data. Our data showed that overexpression of miR-126a-5p limits IL-17a production in Th17 cells polarized in vitro. Additionally, inhibition of miR-122-5p promoted an earlier EAE onset while mice treated with miR-1247 antagomiR develop a less severe disease. In this thesis, to confirm the overexpression results obtained by the host lab, we aimed to study the impact of inhibiting miR-126a-5p and miR-467a-5p in in vitro-differentiated CD4+ T cell subsets. Also, we aimed to dissect the external cues responsible for the expression of all functionally relevant miRNAs for CD4+ T cell differentiation, including miR-122 and miR-1247. Our results reveal that inhibiting miR-126a increases the frequency of IL-17+ cells, further confirming that miR-126a limits the production of IL-17 by Th17 cells. We show that the IL-6 signalling pathway is involved in inducing not only miR-126a expression, but also the expression of miR-122 when associated with TGF−β signalling. On the contrary, IL-23 and IL-1β were shown to inhibit miR-122 expression, which allowed us to hypothesize that this miRNA is expressed in a context of non-pathogenic Th17 cells. Moreover, we observed that miR-1247 is induced downstream of the TGF−β signalling pathway. Relying on a bioinformatic approach, we identified transcription factors from each signalling pathway that can be directly regulating the expression of each candidate miRNAs. More specifically, Maf, Ets1 and Ets2 were identified as candidate transcription factors that might induce miR-126a expression; STAT3, RORα, IRF1 and SMAD2:SMAD3:SMAD4 complex as inducers of miR-122 expression; and Bach1:MafK as potential regulators of miR-1247 expression. Overall, our results show that both miR-126a and miR-122, act as molecular brakes of cytokine production on Th17 cells, possibly as an attempt to control excessive inflammation. Contrary to miR-126a, miR-122 expression appears to be context-specific, acting in non-pathogenic Th17 cells to maintain their phenotype. miR-1247, was shown to be important to maintain the inflammatory phenotype of effector Th1 cells in the presence of TGF−β, an anti-inflammatory cytokine. The data described here reveals new insights on three critical miRNAs for the differentiation of CD4+ T cell subsets, highlighting their potential to regulate the balance between effector and regulatory populations in inflammatory conditions in vivo and revealing their potential use as therapeutic targets in EAE and other autoimmune diseases. |
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