ABSTRACT
High cholesterol levels are a risk factor for the development of Alzheimer's disease. Experiments investigating the influence of cholesterol on the proteolytic processing of the amyloid precursor protein (APP) by the ß-secretase Bace1 and on their proximity in cells have led to conflicting results. By using a fluorescence bioassay coupled with flow cytometry we found a direct correlation between the increase in membrane cholesterol amount and the degree of APP shedding in living human neuroblastoma cells. Analogue results were obtained for cells overexpressing an APP mutant that cannot be processed by α-secretase, highlighting the major influence of cholesterol enrichment on the cleavage of APP carried out by Bace1. By contrast, the cholesterol content was not correlated with changes in membrane dynamics of APP and Bace1 analyzed with single molecule tracking, indicating that the effect of cholesterol enrichment on APP processing by Bace1 is uncoupled from changes in their lateral diffusion.
ABSTRACT
Within the immune system, microRNAs (miRNAs) exert key regulatory functions. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9-mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Cell Proliferation/genetics , Gene Expression Regulation , Intercellular Signaling Peptides and Proteins/genetics , MicroRNAs/genetics , Regulatory Factor X Transcription Factors/genetics , 3' Untranslated Regions/genetics , Blotting, Western , CD4-Positive T-Lymphocytes/cytology , Cells, Cultured , Chromatin Immunoprecipitation Sequencing/methods , HEK293 Cells , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Jurkat Cells , Lymphocyte Activation/genetics , Regulatory Factor X Transcription Factors/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/geneticsABSTRACT
The appropriate regulation of T lymphocyte functions is key to achieve protective immune responses, while at the same time limiting the risks of tissue damage and chronic inflammation. Deciphering the mechanisms underpinning T cell responses in humans may therefore be beneficial for a range of infectious and chronic diseases. Recently, the development of methods based on CRISPR-Cas9 gene-editing has greatly expanded the available tool-box for the mechanistic studies of primary human T cell responses. While the deletion of a surface protein has become a relatively straightforward task, as long as an antibody for detection is available, the identification and selection of cells lacking an intracellular protein, a non-coding RNA or a protein for which no antibody is available, remain more problematic. Here, we discuss the options currently available to scientists interested in performing gene-editing in primary human T lymphocytes and we describe the optimization of a workflow for the screening and analysis of lymphocytes following gene-editing with CRISPR-Cas9 based on T cell cloning and T7 endonuclease I cleavage assay.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Membrane Proteins/genetics , Transcription Factors/genetics , Cell Cycle Proteins/deficiency , Cell Cycle Proteins/genetics , Cells, Cultured , Endoribonucleases/deficiency , Endoribonucleases/genetics , Humans , Membrane Proteins/deficiency , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Receptors, Antigen, T-Cell, alpha-beta/deficiency , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Box Domain Proteins/deficiency , T-Box Domain Proteins/genetics , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Transcription Factors/deficiencyABSTRACT
A mechanistic understanding of the regulatory circuits that control the effector responses of memory T helper lymphocytes, and in particular their ability to produce pro-inflammatory cytokines, may lead to effective therapeutic interventions in all immune-related diseases. Activation of T lymphocytes induces robust immune responses that in most cases lead to the complete eradication of invading pathogens or tumor cells. At the same time, however, such responses must be both highly controlled in magnitude and limited in time to avoid unnecessary damage. To achieve such sophisticated level of control, T lymphocytes have at their disposal an array of transcriptional and post-transcriptional regulatory mechanisms that ensure the acquisition of a phenotype that is tailored to the incoming stimulus while restraining unwarranted activation, eventually leading to the resolution of the inflammatory response. Here, we will discuss some of these cell-intrinsic mechanisms that control T cell responses and involve transcription factors, microRNAs, and RNA-binding proteins. We will also explore how the same mechanisms can be involved both in anti-tumor responses and in autoimmunity.
Subject(s)
Gene Expression Regulation , T-Lymphocytes , Autoimmunity , Cytokines/metabolism , T-Lymphocytes/metabolism , Transcription FactorsABSTRACT
IFN-ß treatment is a commonly used therapy for relapsing-remitting multiple sclerosis (MS), while vitamin D deficiency correlates with an increased risk of MS and/or its activity. MS is a demyelinating chronic inflammatory disease of the central nervous system, in which activated T lymphocytes play a major role, and may represent direct targets of IFN-ß and vitamin D activities. However, the underlying mechanism of action of vitamin D and IFN-ß, alone or in combination, remains incompletely understood, especially when considering their direct effects on the ability of T lymphocytes to produce inflammatory cytokines. We profiled the expression of immune-related genes and microRNAs in primary human T lymphocytes in response to vitamin D and IFN-ß, and we dissected the impact of these treatments on cytokine production and T cell proliferation. We found that the treatments influenced primarily memory T cell plasticity, rather than polarization toward a stable phenotype. Moreover, our data revealed extensive reprogramming of the transcriptional output of primary T cells in response to vitamin D and IFN-ß and provide the bases for further mechanistic insights into these commonly used treatments.
Subject(s)
Interferon-beta/pharmacology , T-Lymphocytes/drug effects , Vitamin D/pharmacology , Vitamins/pharmacology , Cell Proliferation/drug effects , Cells, Cultured , Gene Expression Regulation/drug effects , Humans , Inflammation/genetics , Multiple Sclerosis, Relapsing-Remitting/immunology , T-Lymphocytes/immunologyABSTRACT
Understanding the mechanisms that modulate helper T lymphocyte functions is crucial to decipher normal and pathogenic immune responses in humans. To identify molecular determinants influencing the pathogenicity of T cells, we separated ex vivo-isolated primary human memory T lymphocytes on the basis of their ability to produce high levels of inflammatory cytokines. We found that the inflammatory, cytokine-producing phenotype of memory T lymphocytes was defined by a specific core gene signature and was mechanistically regulated by the constitutive activation of the NF-κB pathway and by the expression of the transcriptional repressor BHLHE40. BHLHE40 attenuated the expression of anti-inflammatory factors, including miR-146a, a negative regulator of NF-κB activation and ZC3H12D, an RNase of the Regnase-1 family able to degrade inflammatory transcripts. Our data reveal a molecular network regulating the proinflammatory phenotype of human memory T lymphocytes, with the potential to contribute to disease.
Subject(s)
Gene Expression Regulation/immunology , Immunologic Memory/immunology , Inflammation/immunology , Cell Line , Cell Line, Tumor , Cytokines/immunology , HEK293 Cells , Humans , Jurkat Cells , Lymphocyte Activation/immunology , NF-kappa B/immunology , Phenotype , T-Lymphocytes/immunologyABSTRACT
Very small embryonic-like cells (VSELs) are a population of very rare pluripotent stem cells isolated in adult murine bone marrow and many other tissues and organs, including umbilical cord blood (UCB). VSEL existence is still not universally accepted by the scientific community, so for this purpose, we sought to investigate whether presumptive VSELs (pVSELs) could be isolated from human UCB with an improved protocol based on the isolation of enriched progenitor cells by depletion of nonprogenitor cells with magnetic separation. Progenitor cells, likely including VSELs, cultured with retinoic acid were able to form dense colonies and cystic embryoid bodies and to differentiate toward the ecto-meso-endoderm lineages as shown by the positivity to specific markers. VSEL differentiative potential toward mesodermal lineage was further demonstrated in vitro upon exposure to an established inductive protocol, which induced the acquisition of renal progenitor cell phenotype. VSEL-derived renal progenitors showed regenerative potential in a cisplatin model of acute kidney injury by restoring renal function and tubular structure through induction of proliferation of endogenous renal cells. The data presented here foster the great debate that surrounds VSELs and, more in general, the existence of cells endowed with pluripotent features in adult tissues. In fact, the possibility to find and isolate subpopulations of cells that fully fit all the criteria utilized to define pluripotency remains, nowadays, almost unproven. Thus, efforts to better characterize the phenotype of these intriguing cells are crucial to understand their possible applications for regenerative and precision medicine purposes.