Publicação
Epigenetic mechanisms of effector y&T cell differentiation : focus on histone modifications and microRNAs
| Resumo: | T cell differentiation involves the establishment of distinct transcriptomes that are preserved throughout cell divisions. Many inheritable changes in gene expression are not due to alterations in the DNA sequence (genes and regulatory elements) but rather attributed to epigenetic mechanisms. There are several layers of epigenetic regulation, from histone modifications that control gene loci accessibility, to microRNAs (miRNAs) that act posttranscriptionally to induce mRNA decay. Effector T cell subsets can be functionally distinguished by the signature cytokines they produce, and interferon-gamma (IFN-γ) and interleukin-17 (IL-17) are critical pro-inflammatory mediators in contexts of infection, cancer and autoimmunity. Among the various cellular sources of these two cytokines, it is now clear that innate-like gamma-delta (γδ) T cells are major providers especially at early stages of the immune response. Our laboratory has recently shown that murine γδ T cells contain two thymic-derived subsets, segregated on the basis of CD27 expression, that produce either IFN-γδ (CD27+) or IL-17 (CD27-). While the molecular mechanisms that control the production of these cytokines have been extensively studied in CD4+ T cells, they remain poorly understood for γδ T cells. As this is particularly notable concerning epigenetic mechanisms, these have been the major focus of my thesis. In the first part of my thesis, we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing (γδIFNγ) or IL-17- producing (γδ17) γδ T cells. Given that these populations are present in lymphoid organs of naïve mice, we were able to analyse in vivo derived γδIFNγ (CD27+ ) or γδ17 (CD27-) cells. We found that CD27+ γδ cells are epigenetically committed to express Ifng but not Il17, whereas CD27- γδ cells spontaneously make IL-17 but can be induced to produce IFN-γ under specific inflammatory conditions in vitro and in vivo. This “plastic” behaviour of CD27- γδ cells associates with permissive histone H3 marks at loci encoding Ifng and upstream IFN-γ-driving transcription factors. By contrast, Il17 and related IL17-driving transcription factors are epigenetically and transcriptionally active in CD27- but silenced in CD27+ γδ cells. Hence, stable versus plastic behaviours of γδ cell subsets are controlled by integrated epigenetic and transcriptional mechanisms that regulate the expression of “master” transcription factors and effector cytokine genes. In the second part of my thesis, I explored another epigenetic level of gene regulation that is mediated by miRNAs. miRNAs are an evolutionarily conserved family of small non-coding RNAs that posttranscriptionally repress gene expression by targeting mRNA stability and/or blocking translation. Our analysis of T cell-specific miRNAdeficient mice (LckCre Dicer mice) revealed a global role of miRNAs in the differentiation of γδ T cells. Thus, we observed decreased numbers of γδ17 cells in the periphery (lymph node and spleen) and in the thymus of miRNA-deficient mice when compared to wild-type controls. To identify individual miRNAs implicated in γδ T cell differentiation, we undertook a transcriptome-wide analysis of miRNA expression in peripheral CD27+ and CD27- γδ T cell subsets and detected 35 differently expressed miRNAs. Based on the individual patterns of miRNA expression, we concentrated our analysis on miR-146a which was enriched in CD27- γδ T cells. We employed miR146a-deficient mice to investigate the impact of this miRNA on IL-17 and IFN-γ production by γδ T cells in naïve and parasite-infected mice. We detected a reduced frequency of γδ17 cells and an increased frequency of γδIFNγ cells in miR-146a-deficient mice during parasitic infections. Moreover, the retroviral-mediated over expression of miR-146a in wild-type γδ T cells led to a reduction in γδIFNγ cells. Therefore, miR-146a is the first miRNA shown to impact on γδ T cell differentiation. Future experiments will address the molecular mechanisms by which miR-146a controls IFN-γ and IL-17 expression in γδ T cells. Collectively, the data presented in this thesis provide novel insights into the epigenetic control of effector γδ T cell subsets that make key contributions to immune responses against infections and tumours, as well as to the pathogenesis of autoimmune diseases. |
|---|---|
| Autores principais: | Schmolka, Nina |
| Assunto: | Linfócitos T Diferenciação celular Histonas MicroRNAs Interleucina-7 Teses de doutoramento - 2014 |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | T cell differentiation involves the establishment of distinct transcriptomes that are preserved throughout cell divisions. Many inheritable changes in gene expression are not due to alterations in the DNA sequence (genes and regulatory elements) but rather attributed to epigenetic mechanisms. There are several layers of epigenetic regulation, from histone modifications that control gene loci accessibility, to microRNAs (miRNAs) that act posttranscriptionally to induce mRNA decay. Effector T cell subsets can be functionally distinguished by the signature cytokines they produce, and interferon-gamma (IFN-γ) and interleukin-17 (IL-17) are critical pro-inflammatory mediators in contexts of infection, cancer and autoimmunity. Among the various cellular sources of these two cytokines, it is now clear that innate-like gamma-delta (γδ) T cells are major providers especially at early stages of the immune response. Our laboratory has recently shown that murine γδ T cells contain two thymic-derived subsets, segregated on the basis of CD27 expression, that produce either IFN-γδ (CD27+) or IL-17 (CD27-). While the molecular mechanisms that control the production of these cytokines have been extensively studied in CD4+ T cells, they remain poorly understood for γδ T cells. As this is particularly notable concerning epigenetic mechanisms, these have been the major focus of my thesis. In the first part of my thesis, we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing (γδIFNγ) or IL-17- producing (γδ17) γδ T cells. Given that these populations are present in lymphoid organs of naïve mice, we were able to analyse in vivo derived γδIFNγ (CD27+ ) or γδ17 (CD27-) cells. We found that CD27+ γδ cells are epigenetically committed to express Ifng but not Il17, whereas CD27- γδ cells spontaneously make IL-17 but can be induced to produce IFN-γ under specific inflammatory conditions in vitro and in vivo. This “plastic” behaviour of CD27- γδ cells associates with permissive histone H3 marks at loci encoding Ifng and upstream IFN-γ-driving transcription factors. By contrast, Il17 and related IL17-driving transcription factors are epigenetically and transcriptionally active in CD27- but silenced in CD27+ γδ cells. Hence, stable versus plastic behaviours of γδ cell subsets are controlled by integrated epigenetic and transcriptional mechanisms that regulate the expression of “master” transcription factors and effector cytokine genes. In the second part of my thesis, I explored another epigenetic level of gene regulation that is mediated by miRNAs. miRNAs are an evolutionarily conserved family of small non-coding RNAs that posttranscriptionally repress gene expression by targeting mRNA stability and/or blocking translation. Our analysis of T cell-specific miRNAdeficient mice (LckCre Dicer mice) revealed a global role of miRNAs in the differentiation of γδ T cells. Thus, we observed decreased numbers of γδ17 cells in the periphery (lymph node and spleen) and in the thymus of miRNA-deficient mice when compared to wild-type controls. To identify individual miRNAs implicated in γδ T cell differentiation, we undertook a transcriptome-wide analysis of miRNA expression in peripheral CD27+ and CD27- γδ T cell subsets and detected 35 differently expressed miRNAs. Based on the individual patterns of miRNA expression, we concentrated our analysis on miR-146a which was enriched in CD27- γδ T cells. We employed miR146a-deficient mice to investigate the impact of this miRNA on IL-17 and IFN-γ production by γδ T cells in naïve and parasite-infected mice. We detected a reduced frequency of γδ17 cells and an increased frequency of γδIFNγ cells in miR-146a-deficient mice during parasitic infections. Moreover, the retroviral-mediated over expression of miR-146a in wild-type γδ T cells led to a reduction in γδIFNγ cells. Therefore, miR-146a is the first miRNA shown to impact on γδ T cell differentiation. Future experiments will address the molecular mechanisms by which miR-146a controls IFN-γ and IL-17 expression in γδ T cells. Collectively, the data presented in this thesis provide novel insights into the epigenetic control of effector γδ T cell subsets that make key contributions to immune responses against infections and tumours, as well as to the pathogenesis of autoimmune diseases. |
|---|