Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 28
Filter
Add more filters










Publication year range
1.
Biomedicines ; 12(7)2024 Jul 05.
Article in English | MEDLINE | ID: mdl-39062069

ABSTRACT

Recombinase-activating gene (RAG)-deficient SCID patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. The two RAG genes act as a required dimer to initiate gene recombination. Gene therapy is a valid treatment alternative for RAG-SCID patients who lack a suitable bone marrow donor, but developing such therapy for RAG1/2 has proven challenging. Using a clinically approved lentiviral vector with a codon-optimized RAG1 gene, we report here preclinical studies using CD34+ cells from four RAG1-SCID patients. We used in vitro T cell developmental assays and in vivo assays in xenografted NSG mice. The RAG1-SCID patient CD34+ cells transduced with the RAG1 vector and transplanted into NSG mice led to restored human B and T cell development. Together with favorable safety data on integration sites, these results substantiate an ongoing phase I/II clinical trial for RAG1-SCID.

2.
Int J Mol Sci ; 23(21)2022 Oct 22.
Article in English | MEDLINE | ID: mdl-36361533

ABSTRACT

The ex vivo expansion and maintenance of long-term hematopoietic stem cells (LT-HSC) is crucial for stem cell-based gene therapy. A combination of stem cell factor (SCF), thrombopoietin (TPO), FLT3 ligand (FLT3) and interleukin 3 (IL3) cytokines has been commonly used in clinical settings for the expansion of CD34+ from different sources, prior to transplantation. To assess the effect of IL3 on repopulating capacity of cultured CD34+ cells, we employed the commonly used combination of STF, TPO and FILT3 with or without IL3. Expanded cells were transplanted into NSG mice, followed by secondary transplantation. Overall, this study shows that IL3 leads to lower human cell engraftment and repopulating capacity in NSG mice, suggesting a negative effect of IL3 on HSC self-renewal. We, therefore, recommend omitting IL3 from HSC-based gene therapy protocols.


Subject(s)
Hematopoietic Stem Cell Transplantation , Interleukin-3 , Animals , Humans , Mice , Antigens, CD34 , Cells, Cultured , Cytokines/pharmacology , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells , Interleukin-3/pharmacology , Stem Cell Factor/pharmacology , Thrombopoietin/pharmacology
3.
Sci Adv ; 6(31): eaaw7313, 2020 07.
Article in English | MEDLINE | ID: mdl-32789164

ABSTRACT

T cell factor 1 (Tcf1) is the first T cell-specific protein induced by Notch signaling in the thymus, leading to the activation of two major target genes, Gata3 and Bcl11b. Tcf1 deficiency results in partial arrests in T cell development, high apoptosis, and increased development of B and myeloid cells. Phenotypically, seemingly fully T cell-committed thymocytes with Tcf1 deficiency have promiscuous gene expression and an altered epigenetic profile and can dedifferentiate into more immature thymocytes and non-T cells. Restoring Bcl11b expression in Tcf1-deficient cells rescues T cell development but does not strongly suppress the development of non-T cells; in contrast, expressing Gata3 suppresses their development but does not rescue T cell development. Thus, T cell development is controlled by a minimal transcription factor network involving Notch signaling, Tcf1, and the subsequent division of labor between Bcl11b and Gata3, thereby ensuring a properly regulated T cell gene expression program.

4.
Mol Ther Methods Clin Dev ; 17: 666-682, 2020 Jun 12.
Article in English | MEDLINE | ID: mdl-32322605

ABSTRACT

Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized RAG1 to ensure optimal expression. We used Rag1 -/- mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with RAG1 expression. Mice with low RAG1 expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34+ cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.

5.
Front Immunol ; 11: 607991, 2020.
Article in English | MEDLINE | ID: mdl-33584681

ABSTRACT

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen's flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.


Subject(s)
Branched DNA Signal Amplification Assay , DNA/biosynthesis , Flow Cytometry , Gene Expression Regulation , Genetic Therapy , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Single-Cell Analysis , Animals , Cells, Cultured , Genetic Vectors , Humans , Mice , Transduction, Genetic , Transgenes
6.
Bioinformatics ; 2019 Oct 30.
Article in English | MEDLINE | ID: mdl-31665245

ABSTRACT

SUMMARY: An effective immune system is characterized by a diverse immune repertoire. There is a strong demand for accurate and quantitative methods to assess the diversity of the immune repertoire for various (pre-)clinical applications, including the diagnosis and prognosis of primary immune deficiencies, or to assess the response to therapy. Current strategies for immune diversity assessment generally comprise the visual inspection of the length distribution of rearranged T- and B-cell receptors. Visual inspections, however, are prone to subjective assessments and thus lead to biases. Here, we introduce ImSpectR, a unified approach to quantify immunodiversity using either spectratype, repertoire sequencing or single cell RNA sequencing data. ImSpectR scores various types of deviations from the expected length distribution and integrates these into one measure, allowing for robust quantitative comparisons of immune diversity across individuals or conditions. AVAILABILITY: R-package is available for download on GitHub at https://github.com/martijn-cordes/ImSpectR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

7.
J Neuroinflammation ; 14(1): 257, 2017 Dec 21.
Article in English | MEDLINE | ID: mdl-29268771

ABSTRACT

BACKGROUND: Leukocyte infiltration into the central nervous system is an important feature of multiple sclerosis (MS) pathology. Among the infiltrating cells, monocytes comprise the largest population and are considered to play a dual role in the course of the disease. The enzyme tissue transglutaminase (TG2), produced by monocytes, plays a central role in monocyte adhesion/migration in animal models of MS. In the present study, we questioned whether TG2 expression is altered in monocytes from MS patients compared to healthy control (HC) subjects. Moreover, we determined the inflammatory status of these TG2-expressing monocytes, what inflammatory factor regulates TG2 expression, and whether TG2 can functionally contribute to their adhesion/migration processes. METHODS: Primary human monocytes from MS patients and HC subjects were collected, RNA isolated and subjected to qPCR analysis. Human THP-1 monocytes were lentivirally transduced with TG2 siRNA or control and treated with various cytokines. Subsequently, mRNA levels of inflammatory factors, adhesion properties, and activity of RhoA were analyzed in interleukin (IL)-4-treated monocytes. RESULTS: TG2 mRNA levels are significantly increased in monocytes derived from MS patients compared to HC subjects. In addition, correlation analyses indicated that TG2-expressing cells display a more anti-inflammatory, migratory profile in MS patients. Using THP-1 monocytes, we observed that IL-4 is a major trigger of TG2 expression in these cells. Furthermore, knockdown of TG2 expression leads to a pro-inflammatory profile and reduced adhesion/migration properties of IL-4-treated monocytes. CONCLUSIONS: TG2-expressing monocytes in MS patients have a more anti-inflammatory profile. Furthermore, TG2 mediates IL-4-induced anti-inflammatory status in THP-1 monocytes, adhesion, and cytoskeletal rearrangement in vitro. We thus propose that IL-4 upregulates TG2 expression in monocytes of MS patients, driving them into an anti-inflammatory status.


Subject(s)
GTP-Binding Proteins/metabolism , Inflammation/metabolism , Monocytes/metabolism , Multiple Sclerosis/metabolism , Transglutaminases/metabolism , Adult , Aged , Cell Adhesion/physiology , Cell Differentiation/physiology , Female , Humans , Male , Middle Aged , Protein Glutamine gamma Glutamyltransferase 2 , Young Adult
8.
Exp Hematol ; 44(9): 838-849.e9, 2016 09.
Article in English | MEDLINE | ID: mdl-27302866

ABSTRACT

Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse models suggest an accumulation of immature T-cell progenitors in the thymus as the main preleukemic event. The effects of LMO2 overexpression on human T-cell development in vivo are unknown. Here, we report studies of a humanized mouse model transplanted with LMO2-transduced human hematopoietic stem/progenitor cells. The effects of LMO2 overexpression were confined to the T-cell lineage; however, initially, multipotent cells were transduced. Three effects of LMO2 on human T-cell development were observed: (1) a block at the double-negative/immature single-positive stage, (2) an accumulation of CD4(+)CD8(+) double-positive CD3(-) cells, and (3) an altered CD8/CD4 ratio with enhanced peripheral T lymphocytes. Microarray analysis of sorted double-positive cells overexpressing LMO2 led to the identification of an LMO2 gene set that clustered with human T-ALL patient samples of the described "proliferative" cluster. In this article, we demonstrate previously unrecognized mechanisms by which LMO2 alters human T-cell development in vivo; these mechanisms correlate with human T-ALL leukemogenesis.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Gene Expression , LIM Domain Proteins/genetics , Proto-Oncogene Proteins/genetics , T-Lymphocytes/metabolism , Animals , Antigens, CD34/metabolism , Cell Differentiation/genetics , Cell Proliferation , Gene Expression Profiling , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Lymphoid Progenitor Cells/cytology , Lymphoid Progenitor Cells/metabolism , Mice , T-Lymphocytes/pathology , Transduction, Genetic
9.
Proc Natl Acad Sci U S A ; 112(44): E6020-7, 2015 Nov 03.
Article in English | MEDLINE | ID: mdl-26483497

ABSTRACT

The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ(-/-) xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.


Subject(s)
Bone Marrow Cells/cytology , T-Lymphocytes/cytology , Thymus Gland/cytology , Animals , Humans , Mice , Mice, Inbred NOD , Mice, SCID
10.
Hum Immunol ; 76(6): 431-7, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25843229

ABSTRACT

Monocytes play a key role in immune system function. Chromatin remodeling is crucial for various differentiation and gene regulation processes and is rather well studied in T cells. However, for monocytes not much is known regarding how the epigenetic machinery influences the differentiation into various effector cell types. In the work presented here, we explore the epigenetic underpinnings of monocyte differentiation. By transcriptional profiling we show that transcription of lysine methyltransferases (KMTs) and in particular KMT1c is markedly up regulated after differentiation of monocytes into immature dendritic cells (iDCs). Specifically inhibiting KMT1c function, using the small-molecule inhibitor BIX-01294, changes the transcription levels of the DC marker DC-SIGN, but does not affect surface protein expression. Blocking global KMT activity, using DZNep, does influence monocyte differentiation into iDCs, indicated by a loss of DC-SIGN surface expression. When BIX-01294 and DZNep treatment was combined DC-SIGN expression was almost lost completely. This work shows that the activities of KMTs are required for successful differentiation of monocyte-derived dendritic cells. Furthermore it shows the importance of KMT inhibitors in the field of epigenetic immune therapy, which is still much focused around HDAC inhibitors.


Subject(s)
Dendritic Cells/metabolism , Epigenesis, Genetic , Histocompatibility Antigens/genetics , Histone-Lysine N-Methyltransferase/genetics , Monocytes/metabolism , Acetylation , Adenosine/analogs & derivatives , Adenosine/pharmacology , Azepines/pharmacology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Differentiation/drug effects , Chromatin/chemistry , Chromatin/drug effects , Chromatin/metabolism , Chromatin Assembly and Disassembly/drug effects , Dendritic Cells/cytology , Dendritic Cells/drug effects , Gene Expression Profiling , Histocompatibility Antigens/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Humans , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Methylation , Monocytes/cytology , Monocytes/drug effects , Primary Cell Culture , Quinazolines/pharmacology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Signal Transduction , Transcription, Genetic
11.
Invest Ophthalmol Vis Sci ; 56(3): 1447-58, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-25593028

ABSTRACT

PURPOSE: Monosomy 3 (M3) or the presence of a specific RNA expression profile, known as class 2, is strongly associated with death from uveal melanoma (UM). Given the important role of epigenetic processes in cancer development and progression, we compared the transcriptional profiles of a selection of epigenetic regulators between primary UM with a good and a bad prognosis. METHODS: Transcriptional levels of 59 epigenetic regulator genes were measured by quantitative PCR (qPCR) in 20 UM, 12 with monosomy of chromosome 3 (M3) and 8 with disomy of chromosome 3 (D3). Validation was performed in an independent cohort. Expression levels were compared to clinicopathological characteristics, including class type. Bisulfite sequencing was used to evaluate the role of DNA methylation in gene silencing. RESULTS: In the first set of tumors, general downregulation of transcription of the genes encoding epigenetic regulatory enzymes was seen in association with M3. The 10 genes with the highest differential expression between M3 and D3 were selected and were analyzed in a second set of tumors. In the validation set, significantly lower levels of KAT2B (P = 0.008), HDAC11 (P = 0.009), KMT1C (P = 0.05), KDM4B (P = 0.003), KDM6B (P = 0.04), and BMI-1 (P = 0.001) transcripts were found in tumors with M3/class 2. Methylation of C-phosphate-G (CpG) residues was not observed on the putative regulatory regions of KAT2B, KDM4B, or KDM6B. CONCLUSIONS: Expression levels of a number of histone-modifying genes and polycomb family members are significantly lower in uveal melanoma with monosomy 3/class 2, supporting a general dysregulation of epigenetic modifiers in UM with a bad prognosis.


Subject(s)
Epigenesis, Genetic/genetics , Gene Expression Regulation, Neoplastic/genetics , Melanoma/genetics , Uveal Neoplasms/genetics , Adult , Aged , DNA Methylation/genetics , Disease Progression , Down-Regulation/genetics , Female , Gene Silencing , Genes, Regulator/genetics , Humans , Male , Melanoma/mortality , Melanoma/pathology , Middle Aged , Prognosis , Transcription, Genetic/genetics , Uvea/pathology , Uveal Neoplasms/mortality , Uveal Neoplasms/pathology
12.
Life Sci ; 129: 3-9, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25445221

ABSTRACT

AIMS: Alterations in epigenetic processes are frequently noted in human disease. These epigenetic processes involve methylation of DNA and post-translational modifications of histones. It is well established that in particular histone methylation plays a key role in gene transcription. In this study, we have investigated the relationship between triple methylation of lysine 27 in histone H3 (H3K27Me3) modifications and atherosclerotic plaque stage. MATERIALS AND METHODS: 28 peri-renal aortic tissue patches covering the entire spectrum of atherosclerotic plaque development were evaluated by immunohistochemistry for the levels of H3K27Me3, EZH2, JMJD3 and BMI1. KEY FINDINGS: The results of our studies are in support of a reduction in global levels of the H3K27Me3 modification in vessels with advanced atherosclerotic plaques. This reduction in H3K27Me3 levels is not accompanied by alterations in global levels of the corresponding histone methyltransferase EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2). Likewise no alterations in global levels of BMI1, a component of the PRC1 complex, which binds to H3K27Me3-modified histones or the global expression levels of the histone demethylase JMJD3, which removes the methyl marks on H3K27, were observed. SIGNIFICANCE: Together, our data show that in atherosclerosis development alterations in global levels of H3K27Me3 occur. The reduction in the number of nuclei in the tunica media that display the repressive H3K27Me3 mark in vessels with advanced atherosclerosis plaques therefore could be a reflection of the dynamic pattern of smooth muscle cell differentiation and proliferation associated with atherosclerotic disease.


Subject(s)
Aorta/pathology , DNA Methylation/physiology , Histones/metabolism , Plaque, Atherosclerotic/pathology , Analysis of Variance , Aorta/metabolism , Enhancer of Zeste Homolog 2 Protein , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Mitogen-Activated Protein Kinase 7/metabolism , Plaque, Atherosclerotic/metabolism , Polycomb Repressive Complex 2/metabolism
13.
J Immunol ; 193(11): 5480-7, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-25355919

ABSTRACT

The Wnt-responsive transcription factor T cell factor 1 (Tcf1) is well known for its role in thymic T cell development and the formation of memory CD8(+) T cells. However, its role in the initial phases of CD8(+) T effector cell formation has remained unexplored. We report that high levels of Wnt signaling and Tcf1 are operational in naive and memory CD8(+) T cells, whereas Wnt signaling and Tcf1 were low in effector CD8(+) T cells. CD8(+) T cells deficient in Tcf1 produce IFN-γ more rapidly, coinciding with increased demethylation of the IFN-γ enhancer and higher expression of the transcription factors Tbet and Blimp1. Moreover, virus-specific Tcf1(-/-) CD8(+) T cells show accelerated expansion in acute infection, which is associated with increased IFN-γ and TNF production and lower viral load. Genetic complementation experiments with various Tcf1 isoforms indicate that Tcf1 dosage and protein stability are critical in suppressing IFN-γ production. Isoforms lacking the ß-catenin binding domain are equally effective in inhibiting CD8(+) effector T cell formation. Thus, Tcf1 functions as a repressor of CD8(+) effector T cell formation in a ß-catenin/Wnt-independent manner.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Hepatocyte Nuclear Factor 1-alpha/metabolism , Interferon-gamma/metabolism , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , Animals , Cells, Cultured , Cytotoxicity, Immunologic , DNA Methylation , Gene Dosage , Hepatocyte Nuclear Factor 1-alpha/genetics , Immunologic Memory , Interferon-gamma/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Positive Regulatory Domain I-Binding Factor 1 , Protein Stability , Repressor Proteins/genetics , Repressor Proteins/metabolism , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolism , Up-Regulation , Viral Load , Virus Diseases
14.
Apoptosis ; 19(12): 1769-78, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25292014

ABSTRACT

The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.


Subject(s)
Antibodies/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/genetics , Chromatin/genetics , Colorectal Neoplasms/therapy , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor/drug effects , Cell Line, Tumor/radiation effects , Cisplatin/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/immunology , Colorectal Neoplasms/radiotherapy , Drug Resistance, Neoplasm , Histones/metabolism , Humans , Promoter Regions, Genetic , Protein Processing, Post-Translational , RNA, Messenger/metabolism , fas Receptor/immunology
15.
Hum Immunol ; 75(1): 10-4, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24135266

ABSTRACT

Statins exert anti-inflammatory characteristics, besides their lipid lowering properties, and may display beneficial effects for the treatment of inflammatory diseases. One possible explanation is that statins interfere in the deregulated gene transcription patterns associated with immune-mediated diseases, although the precise mechanism is not fully understood. Besides gene regulatory proteins, epigenetic mechanisms play an important role in the orchestration of gene expression. Disturbances in the tightly controlled epigenetic mechanisms influence the cellular portrait of expressed genes resulting in the protein dysfunctions found in many inflammatory diseases. In this study, we found that simvastatin reduces secretion and gene expression of CCL2 in monocyte-derived immature dendritic cells and in type 1 macrophages, which is accompanied by increased levels of the 3meK27H3 and 3meK9H3 repressive histone marks and decreased levels of the permissive histone marks AcH3 and 3meK4H3 in CCL2 promoter chromatin. The repressive chromatin status of the CCL2 promoter region affected recruitment of the NF-κB p65 subunit, which controls CCL2 transcription. The down-regulation of CCL2 in these immune cells may therefore impact their chemotactic activity and reduce their recruitment to sites of tissue injury.


Subject(s)
Chemokine CCL2/genetics , Chromatin/drug effects , Chromatin/genetics , Gene Expression Regulation/drug effects , Monocytes/drug effects , Monocytes/metabolism , Simvastatin/pharmacology , Cells, Cultured , Chemokine CCL2/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Macrophages/drug effects , Macrophages/metabolism , Monocytes/cytology , Promoter Regions, Genetic
16.
Mult Scler Relat Disord ; 3(2): 163-75, 2014 Mar.
Article in English | MEDLINE | ID: mdl-25878004

ABSTRACT

Multiple Sclerosis (MS) is a demyelinating disease characterized by chronic inflammation of the central nervous system (CNS) gray and white matter. Although the cause of MS is unknown, it is widely appreciated that innate and adaptive immune processes contribute to its pathogenesis. These include microglia/macrophage activation, pro-inflammatory T-cell (Th1) responses and humoral responses. Additionally, there is evidence indicating that MS has a neurodegenerative component since neuronal and axonal loss occurs even in the absence of overt inflammation. These aspects also form the rationale for clinical management of the disease. However, the currently available therapies to control the disease are only partially effective at best indicating that more effective therapeutic solutions are urgently needed. It is appreciated that in the immune-driven and neurodegenerative processes MS-specific deregulation of gene expressions and resulting protein dysfunction are thought to play a central role. These deviations in gene expression patterns contribute to the inflammatory response in the CNS, and to neuronal or axonal loss. Epigenetic mechanisms control transcription of most, if not all genes, in nucleated cells including cells of the CNS and in haematopoietic cells. MS-specific alterations in epigenetic regulation of gene expression may therefore lie at the heart of the deregulation of gene expression in MS. As such, epigenetic mechanisms most likely play an important role in disease pathogenesis. In this review we discuss a role for MS-specific deregulation of epigenetic features that control gene expression in the CNS and in the periphery. Furthermore, we discuss the application of small molecule inhibitors that target the epigenetic machinery to ameliorate disease in experimental animal models, indicating that such approaches may be applicable to MS patients.

17.
Eur J Hum Genet ; 21(11): 1219-25, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23486536

ABSTRACT

Immunodeficiency with centromeric instability and facial anomalies (ICF) syndrome is a primary immunodeficiency, predominantly characterized by agammaglobulinemia or hypoimmunoglobulinemia, centromere instability and facial anomalies. Mutations in two genes have been discovered to cause ICF syndrome: DNMT3B and ZBTB24. To characterize the clinical features of this syndrome, as well as genotype-phenotype correlations, we compared clinical and genetic data of 44 ICF patients. Of them, 23 had mutations in DNMT3B (ICF1), 13 patients had mutations in ZBTB24 (ICF2), whereas for 8 patients, the gene defect has not yet been identified (ICFX). While at first sight these patients share the same immunological, morphological and epigenetic hallmarks of the disease, systematic evaluation of all reported informative cases shows that: (1) the humoral immunodeficiency is generally more pronounced in ICF1 patients, (2) B- and T-cell compartments are both involved in ICF1 and ICF2, (3) ICF2 patients have a significantly higher incidence of intellectual disability and (4) congenital malformations can be observed in some ICF1 and ICF2 cases. It is expected that these observations on prevalence and clinical presentation will facilitate mutation-screening strategies and help in diagnostic counseling.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , Face/abnormalities , Genetic Heterogeneity , Genetic Predisposition to Disease , Immunologic Deficiency Syndromes/genetics , Mutation/genetics , Repressor Proteins/genetics , Adolescent , Adult , Child , Demography , Female , Hematopoietic Stem Cell Transplantation , Humans , Immunoglobulin G/blood , Immunologic Deficiency Syndromes/blood , Immunologic Deficiency Syndromes/therapy , Lymphocyte Count , Male , Middle Aged , Primary Immunodeficiency Diseases , Young Adult , DNA Methyltransferase 3B
18.
J Immunol ; 188(10): 4951-8, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22490869

ABSTRACT

Tight regulation of MHC class I gene expression is critical for CD8 T cell activation and host adaptive-immune responses. The promoters of MHC class I genes contain a well-conserved core module, the W/S-X-Y motif, which assembles a nucleoprotein complex termed MHC enhanceosome. A member of the nucleotide-binding domain, leucine-rich repeat (NLR) protein family, NLRC5, is a newly identified transcriptional regulator of MHC class I genes. NLRC5 associates with and transactivates the proximal promoters of MHC class I genes, although the molecular mechanism of transactivation has not been understood. In this article, we show that NLRC5-mediated MHC class I gene induction requires the W/S and X1, X2 cis-regulatory elements. The transcription factors RFX5, RFXAP, and RFXANK/B, which compose the RFX protein complex and associate with the X1 box, cooperate with NLRC5 for MHC class I expression. Coimmunoprecipitation experiments revealed that NLRC5 specifically interacts with the RFX subunit RFXANK/B via its ankyrin repeats. In addition, we show that NLRC5 can cooperate with ATF1 and the transcriptional coactivators CBP/p300 and general control nonderepressible 5, which display histone acetyltransferase activity. Taken together, our data suggest that NLRC5 participates in an MHC class I-specific enhanceosome, which assembles on the conserved W/S-X-Y core module of the MHC class I proximal promoters, including the RFX factor components and CREB/ATF1 family transcription factors, to promote MHC class I gene expression.


Subject(s)
DNA-Binding Proteins/physiology , HLA-B Antigens/genetics , Intracellular Signaling Peptides and Proteins/physiology , Transcription Factors/physiology , Activating Transcription Factor 1/genetics , Activating Transcription Factor 1/physiology , Amino Acid Motifs/genetics , Amino Acid Motifs/immunology , Cell Line , Cell Line, Tumor , DNA-Binding Proteins/genetics , HEK293 Cells , HLA-B Antigens/biosynthesis , Humans , Multigene Family , Promoter Regions, Genetic , Regulatory Factor X Transcription Factors , Regulatory Sequences, Nucleic Acid/immunology , Transcription Factors/genetics , Transcriptional Activation/immunology
19.
J Cell Mol Med ; 16(8): 1866-77, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22050776

ABSTRACT

Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study, we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4(+) T lymphocytes as well as in CD14(+) monocytes. In normal naïve T cells and CD14(+) monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5-expressing CD14(+) monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non-CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14(+) monocytes, this indicates a dominant role for AcH3 in CCR5 transcription. We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukaemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore, RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.


Subject(s)
CCR5 Receptor Antagonists , Epigenesis, Genetic/drug effects , Immunomodulation/drug effects , Lymphocytes/metabolism , Receptors, CCR5/genetics , Small Molecule Libraries/pharmacology , Cell Membrane/drug effects , Cell Membrane/metabolism , Chromatin Immunoprecipitation , Cyclic AMP Response Element Modulator/genetics , Cyclic AMP Response Element Modulator/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , DNA Methylation/drug effects , DNA Methylation/genetics , Histones/metabolism , Humans , Immunomodulation/genetics , Jurkat Cells , Lymphocytes/drug effects , Models, Immunological , Promoter Regions, Genetic/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Processing, Post-Translational/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, CCR5/metabolism , Reverse Transcriptase Polymerase Chain Reaction
20.
Adv Exp Med Biol ; 711: 36-49, 2011.
Article in English | MEDLINE | ID: mdl-21627041

ABSTRACT

This chapter describes recent advances in our understanding how epigenetic events control immune functions with emphasis on transcriptional regulation of major histocompatibility complex ClassI (MIC-I) and Class II (MHC-II) genes. MHC-I and MHC-II molecules play an essential role in the adaptive immune response by virtue of their ability to present peptides, respectively to CD8+ and CD4+ T cells. Central to the onset of an adequate immune response to pathogens is the presentation of pathogen-derived peptides in the context of MHC-II molecules by antigen presenting cells (APCs) to CD4+ T cells of the immune system. In particular dendritic cells are highly specialized APCs that are capable to activate naïve T cells. Given their central role in adaptive immunity, MHC-I and MHC-II genes are regulated in a tight fashion at the transcriptional level to meet with local requirements of an effective antigen-specific immune response. In these regulatory processes the MHC2TA encoded Class II transactivator (CIITA) plays a crucial role. CIITA is essential for transcriptional activation of all MHC-I genes, whereas it plays an ancillary function in the transcriptional control of MHC-I genes. The focus of this chapter therefore will be on the transcription factors that interact with conserved cis-acting promoter elements and epigenetic mechanisms that modulate cell type-specific regulation of MHC-I, MHC-I, and MHC2TA genes. Furthermore, we will also briefly discuss how genetic and epigenetic mechanisms contribute to T helper cell differentiation.


Subject(s)
Epigenesis, Genetic , Immunity , Nuclear Proteins/physiology , Trans-Activators/physiology , Animals , Antigen Presentation , Cell Differentiation , Gene Expression Regulation , Humans , Major Histocompatibility Complex , Nuclear Proteins/genetics , T-Lymphocytes, Helper-Inducer/cytology , Trans-Activators/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...