The research topic of our group concerns the study of transcriptional and post-transcriptional mechanisms of regulation of gene expression in cells of the immune system. The general goal is to extend our knowledge of the mechanisms that regulate immune cell differentiation, proliferation and functions and to disclose networks of interaction between epigenetic modifications, transcription factors, microRNAs (miRNAs) and their targets. Epigenetic modifications include all those sustained changes in transcription programs and cell physiology that do not involve irreversible genetic changes in the DNA. The methylation of the genomic DNA represents an important epigenetic mechanism of regulation of gene expression. The process of DNA methylation must be carefully regulated for example during embryonic development, but it is also important in the immune response to pathogenic or noxious agents. For instance, we recently found that the absence of an enzyme responsible for DNA methylation leads to the hyper-activation of cells of the immune system, which in turn can lead to tissue damage and disease (Leoni C. et al. PNAS 2017). We also determined the dynamics of these modifications in the genomic DNA, and addressed whether DNA demethylation processes linked to T cell activation occur primarily through active or passive processes. We found that activation of human T lymphocytes led to a global reduction of genomic modifications, which was crucial for the proper acquisition of T cell effector functions and that was primarily linked to a proliferation-dependent process of dilution of DNA modifications (Vincenzetti L. et al. European Journal of Immunology 2019; Monticelli S. Trends in Biochemical Sciences 2019).
Other regulatory molecular mechanisms are linked to the cellular expression of miRNAs. MiRNAs are small non-coding RNAs that have emerged as key post-transcriptional regulators in a wide variety of organisms and biological processes, by acting through the modulation of levels of protein expression. Because each miRNA can regulate expression of a distinct set of genes, miRNA expression can shape the repertoire of proteins that are expressed during development, differentiation or disease. Accordingly, genetic ablation of the miRNA machinery, as well as loss or dysregulation of certain individual miRNAs, severely compromises immune development and might lead to disease. In our lab we are studying the role of DNA methylation, transcription factors and miRNAs in the differentiation and function of cells of the immune system, with a special focus on T lymphocytes and mast cells.
Efficient immune responses orchestrated by CD4+ T lymphocytes require both lineage commitment and phenotypic flexibility, allowing the development of responses tailored to invading pathogens. With this project we aim at comprehensively investigating the role of DNA modifications and DNA-modifying enzymes in human T cell responses. Specifically, we want to address fundamental questions about the gene regulatory networks controlling the balance between commitment, phenotypic stability and plasticity of T cells. This will be performed by combining genome-wide analyses of DNA modifications and genetic manipulation of primary human T cells. Indeed, the stability of DNA methylation and its heritability across mitosis make it particularly apt to mediate the maintenance of transcriptional networks and cellular phenotypes. In the case of T cells, however, stability in the expression of subset-selective genes (notably cytokine genes) must be reconciled with mechanisms enabling plastic phenotypic changes in response to environmental clues. The recent discovery that methylated DNA can be dynamically modified, impacting gene expression directly or via erasure of DNA methylation, suggests its possible role in T cell plasticity. Our study will thoroughly describe dynamics in the methylation landscape in primary human T cells in response to specific pathogens and antigens, and assess the effects of methylation dynamics in T cell functions, leading to novel insights in immunity against pathogens and in disease.
Mast cell activation is involved in the response to a variety of pathogens and allergens, making these cells an important effector type not only in innate immunity but also in allergic reactions and asthma. In addition, alterations in the number, localization, and reactivity of mast cells are typical features of systemic mastocytosis, a myeloproliferative disorder characterized by an increase in mast cell burden.
|Mast cell responses in health and disease. Mast cells act as important sentinels against danger signals in innate immunity, but alterations in the number, localization, and reactivity of mast cells are features of mast cell-related diseases such as systemic mastocytosis or allergy and asthma.|
Multiple genetic and epigenetic mechanisms can contribute to the onset and severity of all types of mast cell-related diseases. Methylation of the genomic DNA is an epigenetic process in which a methyl group is covalently linked to a cytosine base in the DNA, and such modification in our genome has a critical impact in the control of gene expression. Indeed, enzymes involved in catalyzing this process are implicated in the pathogenesis of a variety of diseases and in regulating the function of immune cells. The enzyme TET2 is responsible for the oxidation of 5-methylcytosine (5mC) in genomic DNA to 5-hydroxymethylcytosine (5hmC), and such modification contributes to gene transcriptional regulation and in some cases to tumorigenic transformation (Leoni, 2015).
We found that overall levels of genomic 5hmC and the activity of the TET enzymes were crucial in regulating mast cell differentiation from hematopoietic progenitors (Montagner, 2016), while appropriate patterns of DNA methylation and sufficient levels of DNA methyltransferase enzyme activity were critical to restrain mast cell inflammatory responses in vivo and in vitro, in response to both acute and chronic stimulation (Leoni, 2017). In other words, mast cells with a normal hydroxymethylation/ methylation pattern can differentiate and respond adequately to stimuli from the environment, while mast cells with an altered methylation pattern show abnormal proliferation and respond with exaggerated responses to normal stimuli, leading to unrestrained inflammation.
Mechanisms that regulate the threshold of T cell activation, as well as the magnitude and inflammatory potential of T cell responses are likely to be crucial in autoimmunity, and may become relevant therapeutic targets. Among the factors that can modulate T cell activation and responses, it is becoming increasingly clear that dysregulation of microRNA (miRNA) expression is involved in autoimmunity, implying that a detailed knowledge of miRNA-regulated gene expression networks is critical to gain understanding of normal and disease states. Utilizing a combination of cutting-edge cellular immunology techniques, in conjunction with the extensive investigation of the network of miRNA:mRNA interactions, we will ultimately determine the importance of miRNAs in regulating T cell pathogenicity in autoimmunity.
Group leaders: Silvia Monticelli
Status: In progress
Multiple Sclerosis (MS) is a chronic inflammatory disease with an autoimmune etiology mediated at least in part by CD4+T lymphocytes producing the pro-inflammatory cytokine Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). Indeed, GM-CSF was shown to be necessary and sufficient to induce disease in several models of experimental autoimmunity. Levels of GM-CSF were also shown to be increased in patients with MS and to be associated with disease severity. We are investigating mechanisms that regulate the pathogenic potential of T lymphocytes, including the acquired ability to express high levels of GM-CSF. We identified optimal conditions to functionally separate primary human CD4+ T lymphocytes based on their ability to produce high levels of inflammatory cytokines, such as GM-CSF, and we profiled both the transcriptome and miRnome of the cytokine-producing and non-producing populations. We successfully identified candidate genes and miRNAs specifically associated with either the cytokine-producing or non-producing phenotype, and we now initiated studies to understand the biological relevance of such candidates in primary human T lymphocytes from both healthy donors and people with MS.