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1.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1869(7): 159528, 2024 Jun 25.
Article in English | MEDLINE | ID: mdl-38936507

ABSTRACT

Inflammatory Bowel Disease (IBD) comprises a heterogeneous group of chronic inflammatory conditions of the gastrointestinal tract that include ulcerative colitis (UC) and Crohn's disease. Although the etiology is not well understood, IBD is characterized by a loss of the normal epithelium homeostasis that disrupts the intestinal barrier of these patients. Previous work by our group demonstrated that epithelial homeostasis along the colonic crypts involves a tight regulation of lipid profiles. To evaluate whether lipidomic profiles conveyed the functional alterations observed in the colonic epithelium of IBD, we performed matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) analyses of endoscopic biopsies from inflamed and non-inflamed segments obtained from UC patients. Our results indicated that lipid profiling of epithelial cells discriminated between healthy and UC patients. We also demonstrated that epithelial cells of the inflamed mucosa were characterized by a decrease in mono- and di-unsaturated fatty acid-containing phospholipids and higher levels of arachidonic acid-containing species, suggesting an alteration of the lipid gradients occurring concomitantly to the epithelial differentiation. This result was reinforced by the immunofluorescence analysis of EPHB2 and HPGD, markers of epithelial cell differentiation, sustaining that altered lipid profiles were at least partially due to a faulty differentiation process. Overall, our results showed that lipid profiling by MALDI-MSI faithfully conveys molecular and functional alterations associated with the inflamed epithelium, providing the foundation for a novel molecular characterization of UC patients.

2.
Am J Transplant ; 2024 Apr 29.
Article in English | MEDLINE | ID: mdl-38692412

ABSTRACT

In this proof-of-concept study, spatial transcriptomics combined with public single-cell ribonucleic acid-sequencing data were used to explore the potential of this technology to study kidney allograft rejection. We aimed to map gene expression patterns within diverse pathologic states by examining biopsies classified across nonrejection, T cell-mediated acute rejection, interstitial fibrosis, and tubular atrophy. Our results revealed distinct immune cell signatures, including those of T and B lymphocytes, monocytes, mast cells, and plasma cells, and their spatial organization within the renal interstitium. We also mapped chemokine receptors and ligands to study immune cell migration and recruitment. Finally, our analysis demonstrated differential spatial enrichment of transcription signatures associated with kidney allograft rejection across various biopsy regions. Interstitium regions displayed higher enrichment scores for rejection-associated gene expression patterns than tubular areas, which had negative scores. This implies that these signatures are primarily driven by processes unfolding in the renal interstitium. Overall, this study highlights the value of spatial transcriptomics for revealing cellular heterogeneity and immune signatures in renal transplant biopsies and demonstrates its potential for studying the molecular and cellular mechanisms associated with rejection. However, certain limitations must be borne in mind regarding the development and future applications of this technology.

3.
Biomed Pharmacother ; 174: 116492, 2024 May.
Article in English | MEDLINE | ID: mdl-38537579

ABSTRACT

Targeting epigenetic mechanisms has emerged as a potential therapeutic approach for the treatment of kidney diseases. Specifically, inhibiting the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1 has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, its clinical translation faces challenges due to issues with poor pharmacokinetics and side effects. Here, we developed engineered liposomes loaded with JQ1 with the aim of enhancing kidney drug delivery and reducing the required minimum effective dose by leveraging cargo protection. These liposomes efficiently encapsulated JQ1 in both the membrane and core, demonstrating superior therapeutic efficacy compared to freely delivered JQ1 in a mouse model of kidney ischemia-reperfusion injury. JQ1-loaded liposomes (JQ1-NPs) effectively targeted the kidneys and only one administration, one-hour after injury, was enough to decrease the immune cell (neutrophils and monocytes) infiltration to the kidney-an early and pivotal step to prevent damage progression. By inhibiting BRD4, JQ1-NPs suppress the transcription of pro-inflammatory genes, such as cytokines (il-6) and chemokines (ccl2, ccl5). This success not only improved early the kidney function, as evidenced by decreased serum levels of BUN and creatinine in JQ1-NPs-treated mice, along with reduced tissue expression of the damage marker, NGAL, but also halted the production of extracellular matrix proteins (Fsp-1, Fn-1, α-SMA and Col1a1) and the fibrosis development. In summary, this work presents a promising nanotherapeutic strategy for AKI treatment and its progression and provides new insights into renal drug delivery.


Subject(s)
Azepines , Bromodomain Containing Proteins , Disease Progression , Kidney , Liposomes , Mice, Inbred C57BL , Nuclear Proteins , Renal Insufficiency, Chronic , Reperfusion Injury , Triazoles , Animals , Azepines/pharmacology , Azepines/administration & dosage , Reperfusion Injury/drug therapy , Reperfusion Injury/pathology , Triazoles/pharmacology , Triazoles/administration & dosage , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/pathology , Mice , Kidney/drug effects , Kidney/pathology , Kidney/metabolism , Male , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Acute Kidney Injury/drug therapy , Acute Kidney Injury/prevention & control , Disease Models, Animal , Nanoparticles , Cell Cycle Proteins/antagonists & inhibitors
4.
Int J Biol Sci ; 20(5): 1547-1562, 2024.
Article in English | MEDLINE | ID: mdl-38481808

ABSTRACT

Renal ischemia-reperfusion injury (IRI) leads to endoplasmic reticulum (ER) stress, thereby initiating the unfolded protein response (UPR). When sustained, this response may trigger the inflammation and tubular cell death that acts to aggravate the damage. Here, we show that knockdown of the BET epigenetic reader BRD4 reduces the expression of ATF4 and XBP1 transcription factors under ER stress activation. BRD4 is recruited to the promoter of these highly acetylated genes, initiating gene transcription. Administration of the BET protein inhibitor, JQ1, one hour after renal damage induced by bilateral IRI, reveals reduced expression of ATF4 and XBP1 genes, low KIM-1 and NGAL levels and recovery of the serum creatinine and blood urea nitrogen levels. To determine the molecular pathways regulated by ATF4 and XBP1, we performed stable knockout of both transcription factors using CRISPR-Cas9 and RNA sequencing. The pathways triggered under ER stress were mainly XBP1-dependent, associated with an adaptive UPR, and partially regulated by JQ1. Meanwhile, treatment with JQ1 downmodulated most of the pathways regulated by ATF4 and related to the pathological processes during exacerbated UPR activation. Thus, BRD4 inhibition could be useful for curbing the maladaptive UPR activation mechanisms, thereby ameliorating the progression of renal disease.


Subject(s)
Antineoplastic Agents , Reperfusion Injury , Humans , Transcription Factors/genetics , Transcription Factors/metabolism , Nuclear Proteins/genetics , Endoplasmic Reticulum Stress/genetics , Unfolded Protein Response , Antineoplastic Agents/pharmacology , Reperfusion Injury/drug therapy , Reperfusion Injury/genetics , Reperfusion Injury/metabolism , Bromodomain Containing Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism
5.
Front Immunol ; 12: 709164, 2021.
Article in English | MEDLINE | ID: mdl-34489960

ABSTRACT

Operational tolerance after kidney transplantation is defined as stable graft acceptance without the need for immunosuppression therapy. However, it is not clear which cellular and molecular pathways are driving tolerance in these patients. We performed genome-wide analysis of DNA methylation in peripheral blood mononuclear cells from kidney transplant recipients with chronic rejection and operational tolerance from the Genetic Analysis of Molecular Biomarkers of Immunological Tolerance (GAMBIT) study. Our results showed that both clinical stages diverge in 2737 genes, indicating that each one has a specific methylation signature associated with transplant outcome. We also observed that tolerance is associated with demethylation in genes involved in immune function, including B and T cell activation and Th17 differentiation, while in chronic rejection it is associated with intracellular signaling and ubiquitination pathways. Using co-expression network analysis, we selected 12 genomic regions that are specifically hypomethylated or hypermethylated in tolerant patients. Analysis of these genes in transplanted patients with low dose of steroids showed that these have a similar methylation signature to that of tolerant recipients. Overall, these results demonstrate that methylation analysis can mirror the immune status associated with transplant outcome and provides a starting point for understanding the epigenetic mechanisms associated with tolerance.


Subject(s)
DNA Methylation , Kidney Transplantation , Transplantation Tolerance , Adult , Aged , Aged, 80 and over , Graft Rejection , Humans , Immunosuppression Therapy , Kidney Transplantation/adverse effects , Middle Aged , Th17 Cells/immunology , Young Adult
6.
Oncoimmunology ; 10(1): 1897294, 2021 03 25.
Article in English | MEDLINE | ID: mdl-33796404

ABSTRACT

B7-H6, a ligand for the NK activating receptor NKp30, has been identified as a biomarker of poor prognosis in several solid cancers. However, little is known about the role of B7-H6 and the mechanisms that control its expression in acute myeloid leukemia (AML). Epigenome modulation, including epigenomic reader dysregulation, is one of the hallmarks of AML. Bromodomain-containing protein 4 (BRD4), the best-known member of the BET family of epigenetic readers, is overexpressed in AML cells and regulates the transcription of genes involved in the pathogenesis of AML, as MYC oncogene. Here, we analyze the role of BRD4 in regulating B7-H6 in AML cells. Results demonstrated that the specific inhibition of BRD4 drastically reduces the expression of B7-H6 in AML cells. Histone acetylation mediated by CBP30/P300 facilitates the binding of BRD4 to the B7-H6 promoter, which recruits the P-TEFb elongation factor that phosphorylates RNA polymerase II, thereby activating B7-H6 transcription. BRD4 also co-bounded with JMJD6 at the distal enhancer of the B7-H6 gene. Metabolic modulation with metformin modifies the acetylation pattern in the B7-H6 promoter, impairing BRD4 binding, thereby inhibiting B7-H6 expression. B7-H6 knockdown induces the apoptosis in HEL-R cell line. Moreover, a high level of B7-H6 expression in AML patients is related to increased BRD4 levels, myelodysplastic-derived AML, and del5q, the two latter being associated with poor prognosis. Our data show that BRD4 is a positive regulator of the pro-tumorigenic molecule B7-H6 and that the blockage of the B7-H6 is a potential therapeutic target for the treatment of AML.


Subject(s)
B7 Antigens , Cell Cycle Proteins , Leukemia, Myeloid, Acute , Transcription Factors , Cell Cycle Proteins/genetics , Cell Line, Tumor , Epigenesis, Genetic/genetics , Humans , Jumonji Domain-Containing Histone Demethylases , Leukemia, Myeloid, Acute/genetics , Natural Cytotoxicity Triggering Receptor 3/genetics , Transcription Factors/genetics
7.
Int J Mol Sci ; 21(15)2020 Aug 01.
Article in English | MEDLINE | ID: mdl-32752264

ABSTRACT

Integration of the tumor microenvironment as a fundamental part of the tumorigenic process has undoubtedly revolutionized our understanding of cancer biology. Increasing evidence indicates that neoplastic cells establish a dependency relationship with normal resident cells in the affected tissue and, furthermore, develop the ability to recruit new accessory cells that aid tumor development. In addition to normal stromal and tumor cells, this tumor ecosystem includes an infiltrated immune component that establishes complex interactions that have a critical effect during the natural history of the tumor. The process by which immune cells modulate tumor progression is known as immunoediting, a dynamic process that creates a selective pressure that finally leads to the generation of immune-resistant cells and the inability of the immune system to eradicate the tumor. In this context, the cellular and functional characterization of the immune compartment within the tumor microenvironment will help to understand tumor progression and, ultimately, will serve to create novel prognostic tools and improve patient stratification for cancer treatment. Here we review the impact of the immune system on tumor development, focusing particularly on its clinical implications and the current technologies used to analyze immune cell diversity within the tumor.


Subject(s)
Biomarkers, Tumor/immunology , Immunotherapy/methods , Neoplasms/therapy , Tumor Microenvironment/immunology , Animals , Cell Communication/immunology , Humans , Immune System/cytology , Immune System/immunology , Neoplasms/diagnosis , Neoplasms/immunology , Neoplastic Stem Cells/immunology , Prognosis
8.
Cell Rep ; 29(4): 860-872.e5, 2019 10 22.
Article in English | MEDLINE | ID: mdl-31644909

ABSTRACT

In recent years, the macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF) cytokines have been identified as opposing regulators of the inflammatory program. However, the two cytokines are simultaneously present in the inflammatory milieu, and it is not clear how cells integrate these signals. In order to understand the regulatory networks associated with the GM/M-CSF signaling axis, we analyzed DNA methylation in human monocytes. Our results indicate that GM-CSF induces activation of the inflammatory program and extensive DNA methylation changes, while M-CSF-polarized cells are in a less differentiated state. This inflammatory program is mediated via JAK2 associated with the GM-CSF receptor and the downstream extracellular signal-regulated (ERK) signaling. However, PI3K signaling is associated with a negative regulatory loop of the inflammatory program and M-CSF autocrine signaling in GM-CSF-polarized monocytes. Our findings describe the regulatory networks associated with the GM/M-CSF signaling axis and how they contribute to the establishment of the inflammatory program associated with monocyte activation.


Subject(s)
DNA Methylation , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Macrophage Colony-Stimulating Factor/metabolism , Monocytes/metabolism , Signal Transduction , Adult , Cells, Cultured , Humans , Inflammation/genetics , Inflammation/metabolism , Janus Kinase 2/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphatidylinositol 3-Kinases/metabolism
9.
Nucleic Acids Res ; 47(10): 5016-5037, 2019 06 04.
Article in English | MEDLINE | ID: mdl-30923829

ABSTRACT

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.


Subject(s)
Apoptosis , Cell Differentiation , Chromatin/metabolism , Histones/metabolism , Lysine/metabolism , Myeloid Cells/metabolism , Acetylation , Animals , Cells, Cultured , Chromatin/genetics , Epigenesis, Genetic , Humans , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/cytology , Protein Processing, Post-Translational , Transcription, Genetic
10.
Front Immunol ; 10: 2951, 2019.
Article in English | MEDLINE | ID: mdl-31998288

ABSTRACT

Macrophage activation and polarization are closely linked with metabolic rewiring, which is required to sustain their biological functions. These metabolic alterations allow the macrophages to adapt to the microenvironment changes associated with inflammation or tissue damage (hypoxia, nutrient imbalance, oxidative stress, etc.) and to fulfill their highly energy-demanding proinflammatory and anti-microbial functions. This response is integrated via metabolic sensors that coordinate these metabolic fluxes with their functional requirements. Here we review how the metabolic and phenotypic plasticity of macrophages are intrinsically connected with the hypoxia stress sensors and the unfolded protein response in the endoplasmic reticulum, and how these molecular pathways participate in the maladaptive polarization of macrophages in human pathology and chronic inflammation.


Subject(s)
Endoplasmic Reticulum Stress/physiology , Hypoxia/physiopathology , Macrophages/physiology , Animals , Humans , Inflammation/physiopathology , Macrophage Activation/physiology , Signal Transduction/physiology
11.
Front Immunol ; 9: 2181, 2018.
Article in English | MEDLINE | ID: mdl-30319636

ABSTRACT

The positive long-term effects of conversion to everolimus (EVL) after heart transplantation (HT) have been evaluated in several studies. However, the timing of EVL initiation, the best way to combine it with other immunosuppressive treatments, and the impact of these combinations on the immune response are poorly understood aspects. Here, we analyzed the immune phenotype and function of HT patients (n = 56) at short and long terms (prospective and retrospective cohorts), taking into account the time of EVL initiation: early (3 months post-transplant, EVL-E group) or late (>1 year post-transplant, EVL-L group) compared with mycophenolate mofetil treatment (MMF group). We show that early EVL conversion from MMF allows the increase of cytotoxic (CD56dim CD16+) NK and effector-memory (EM, CD45RA- CCR7-) CD8+ T cell subsets, which show a significantly higher level of expression of cytotoxic molecules, IFN-γ production and degranulation ability under activation. NK cell expansion is accompanied by an altered balance of receptor expression, increasing the activation state, and lytic activity of those cells. Those changes are detected after as little as 1 month after EVL conversion in association with the expansion of regulatory T cells and the decrease in B cell frequency. However, no changes in the immune cells subsets were observed after late EVL initiation (EVL-L) compared with the MMF group. Our results imply that only early EVL conversion induces key changes in the post-transplant immune response, preserving an efficient anti-viral response, but simultaneously showing a limited ability to counteract the cytotoxic response to the allograft.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Everolimus/administration & dosage , Graft Rejection/prevention & control , Heart Transplantation/adverse effects , Immunosuppressive Agents/administration & dosage , Killer Cells, Natural/immunology , Adolescent , Adult , Aged , Allografts/drug effects , Allografts/immunology , Cardiomyopathy, Dilated/surgery , Female , Graft Rejection/immunology , Heart/drug effects , Humans , Male , Middle Aged , Mycophenolic Acid/administration & dosage , Myocardium/immunology , Prospective Studies , Retrospective Studies , Time Factors , Treatment Outcome , Young Adult
13.
Oncotarget ; 8(19): 31959-31976, 2017 05 09.
Article in English | MEDLINE | ID: mdl-28404876

ABSTRACT

Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza®, AZA) and decitabine (Dacogen®, DAC) have been approved for the treatment of AML patients, but their mechanisms of action are poorly understood. Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.


Subject(s)
DNA Methylation/drug effects , Gene Expression Regulation, Leukemic/drug effects , Histocompatibility Antigens Class I/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Tissue Inhibitor of Metalloproteinase-3/genetics , ADAM17 Protein/metabolism , Adult , Aged , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Azacitidine/therapeutic use , Cell Line, Tumor , Chromosome Aberrations , Decitabine , Female , GPI-Linked Proteins/metabolism , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/immunology , Male , Middle Aged , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Prognosis
14.
J Immunol ; 198(2): 937-949, 2017 01 15.
Article in English | MEDLINE | ID: mdl-27974453

ABSTRACT

Epigenetic mechanisms play a critical role during differentiation of T cells by contributing to the formation of stable and heritable transcriptional patterns. To better understand the mechanisms of memory maintenance in CD8+ T cells, we performed genome-wide analysis of DNA methylation, histone marking (acetylated lysine 9 in histone H3 and trimethylated lysine 9 in histone), and gene-expression profiles in naive, effector memory (EM), and terminally differentiated EM (TEMRA) cells. Our results indicate that DNA demethylation and histone acetylation are coordinated to generate the transcriptional program associated with memory cells. Conversely, EM and TEMRA cells share a very similar epigenetic landscape. Nonetheless, the TEMRA transcriptional program predicts an innate immunity phenotype associated with genes never reported in these cells, including several mediators of NK cell activation (VAV3 and LYN) and a large array of NK receptors (e.g., KIR2DL3, KIR2DL4, KIR2DL1, KIR3DL1, KIR2DS5). In addition, we identified up to 161 genes that encode transcriptional regulators, some of unknown function in CD8+ T cells, and that were differentially expressed in the course of differentiation. Overall, these results provide new insights into the regulatory networks involved in memory CD8+ T cell maintenance and T cell terminal differentiation.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Epigenesis, Genetic , Gene Expression Regulation/immunology , Immunologic Memory/genetics , Blotting, Western , Cell Separation , Chromatin Immunoprecipitation , DNA Methylation , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation/genetics , Humans , Immunologic Memory/immunology , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Transcription, Genetic , Transcriptome
15.
Aging Cell ; 16(2): 293-303, 2017 04.
Article in English | MEDLINE | ID: mdl-28026094

ABSTRACT

Aging is associated with a progressive loss of the CD28 costimulatory molecule in CD4+ lymphocytes (CD28null T cells), which is accompanied by the acquisition of new biological and functional properties that give rise to an impaired immune response. The regulatory mechanisms that govern the appearance and function of this cell subset during aging and in several associated inflammatory disorders remain controversial. Here, we present the whole-genome DNA methylation and gene expression profiles of CD28null T cells and its CD28+ counterpart. A comparative analysis revealed that 296 genes are differentially methylated between the two cell subsets. A total of 160 genes associated with cytotoxicity (e.g. GRZB, TYROBP, and RUNX3) and cytokine/chemokine signaling (e.g. CX3CR1, CD27, and IL-1R) are demethylated in CD28null T cells, while 136 de novo-methylated genes matched defects in the TCR signaling pathway (e.g. ITK, TXK, CD3G, and LCK). TCR-landscape analysis confirmed that CD28null T cells have an oligo/monoclonal expansion over the polyclonal background of CD28+ T cells, but feature a Vß family repertoire specific to each individual. We reported that CD28null T cells show a preactivation state characterized by a higher level of expression of inflammasome-related genes that leads to the release of IL-1ß when activated. Overall, our results demonstrate that CD28null T cells have a unique DNA methylation landscape, which is associated with differences in gene expression, contributing to the functionality of these cells. Understanding these epigenetic regulatory mechanisms could suggest novel therapeutic strategies to prevent the accumulation and activation of these cells during aging.


Subject(s)
CD28 Antigens/metabolism , CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Cellular Senescence/genetics , Cellular Senescence/immunology , DNA Methylation/genetics , Genome, Human , Apoptosis/genetics , CpG Islands/genetics , Cytotoxicity, Immunologic , Gene Expression Regulation , Humans , Immunity , Inflammasomes/metabolism , Phenotype , Receptors, Antigen, T-Cell/metabolism , Reproducibility of Results , Signal Transduction
16.
J Am Soc Nephrol ; 28(2): 504-519, 2017 02.
Article in English | MEDLINE | ID: mdl-27436852

ABSTRACT

Renal inflammation has a key role in the onset and progression of immune- and nonimmune-mediated renal diseases. Therefore, the search for novel anti-inflammatory pharmacologic targets is of great interest in renal pathology. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) proteins, was previously found to preserve renal function in experimental polycystic kidney disease. We report here that JQ1-induced BET inhibition modulated the in vitro expression of genes involved in several biologic processes, including inflammation and immune responses. Gene silencing of BRD4, an important BET protein, and chromatin immunoprecipitation assays showed that JQ1 alters the direct association of BRD4 with acetylated histone-packaged promoters and reduces the transcription of proinflammatory genes (IL-6, CCL-2, and CCL-5). In vivo, JQ1 abrogated experimental renal inflammation in murine models of unilateral ureteral obstruction, antimembrane basal GN, and infusion of Angiotensin II. Notably, JQ1 downregulated the expression of several genes controlled by the NF-κB pathway, a key inflammatory signaling pathway. The RelA NF-κB subunit is activated by acetylation of lysine 310. In damaged kidneys and cytokine-stimulated renal cells, JQ1 reduced the nuclear levels of RelA NF-κB. Additionally, JQ1 dampened the activation of the Th17 immune response in experimental renal damage. Our results show that inhibition of BET proteins reduces renal inflammation by several mechanisms: chromatin remodeling in promoter regions of specific genes, blockade of NF-κB pathway activation, and modulation of the Th17 immune response. These results suggest that inhibitors of BET proteins could have important therapeutic applications in inflammatory renal diseases.


Subject(s)
Azepines/pharmacology , Azepines/therapeutic use , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Kidney Diseases/drug therapy , Nuclear Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Triazoles/pharmacology , Triazoles/therapeutic use , Animals , Chromosomal Proteins, Non-Histone/physiology , Disease Models, Animal , Kidney Diseases/etiology , Male , Mice , Mice, Inbred C57BL , Nuclear Proteins/physiology , Transcription Factors/physiology
17.
Int J Biochem Cell Biol ; 67: 75-85, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25975824

ABSTRACT

T cell lymphopoiesis is a complex, stepwise process in which the transcriptional program of the progenitor cells is progressively adapted in order to generate mature phenotypes. This transcriptional program in differentiated cells is also very flexible, allowing the silencing or activation of critical genes in response to extrinsic or intrinsic stimuli, or, in the case of progenitors, to developmental signals. Thus, progenitor and mature cells must maintain a balance between stability, to preserve their phenotypic identity, and plasticity, to respond and adapt to stimuli. A long-standing question is, therefore, how the transcriptional program is regulated to allow both controlled differentiation and a flexible response. Here we review the contribution of epigenetic mechanisms to transcriptional control during CD4(+) T cell differentiation and the ways in which these mechanisms interact with key transcription factors to ensure proper maturation and maintenance of cell identity. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.


Subject(s)
Epigenesis, Genetic/immunology , Forkhead Transcription Factors/genetics , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Regulatory/immunology , Cell Differentiation , Cell Lineage/immunology , DNA Methylation , Forkhead Transcription Factors/immunology , Gene Expression , Humans , Lymphopoiesis/genetics , Lymphopoiesis/immunology , Promoter Regions, Genetic , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Regulatory/cytology , Transcription, Genetic
18.
Nucleic Acids Res ; 43(2): 760-74, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25539926

ABSTRACT

Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.


Subject(s)
DNA Methylation , Gene Expression Regulation , Receptors, Antigen, T-Cell, alpha-beta/analysis , T-Lymphocytes/immunology , Transcription, Genetic , Cell Differentiation/genetics , Gene Expression , Humans , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Thymocytes/cytology , Thymus Gland/cytology , Thymus Gland/immunology , Transcription Factors/genetics , Transcription Factors/metabolism
19.
Epigenetics ; 9(4): 566-78, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24445267

ABSTRACT

The bromodomain and extra terminal (BET) protein family member BRD4 is a transcriptional regulator, critical for cell cycle progression and cellular viability. Here, we show that BRD4 plays an important role in embryonic stem cell (ESC) regulation. During differentiation of ESCs, BRD4 expression is upregulated and its gene promoter becomes demethylated. Disruption of BRD4 expression in ESCs did not induce spontaneous differentiation but severely diminished hematoendothelial potential. Although BRD4 regulates c-Myc expression, our data show that the role of BRD4 in hematopoietic commitment is not exclusively mediated by c-Myc. Our results indicate that BRD4 is epigenetically regulated during hematopoietic differentiation ESCs in the context of a still unknown signaling pathway.


Subject(s)
Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Cell Cycle Proteins , Cell Differentiation , Cell Line , DNA Methylation , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Fetal Blood/cytology , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Humans , Infant, Newborn , Nuclear Proteins/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Transcription Factors/genetics
20.
J Mol Med (Berl) ; 91(8): 939-50, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23475283

ABSTRACT

The basic mechanisms underlying promoter DNA hypermethylation in cancer are still largely unknown. It has been proposed that the levels of the methyl donor group in DNA methylation reactions, S-adenosylmethionine (SAMe), might be involved. SAMe levels depend on the glycine-N-methyltransferase (GNMT), a one-carbon group methyltransferase, which catalyzes the conversion of SAMe to S-adenosylhomocysteine in hepatic cells. GNMT has been proposed to display tumor suppressor activity and to be frequently repressed in hepatocellular carcinoma (HCC). In this study, we show that GNMT shows aberrant DNA hypermethylation in some HCC cell lines and primary tumors (20 %). GNMT hypermethylation could contribute to gene repression and its restoration in cell lines displaying hypermethylation-reduced tumor growth in vitro. In agreement, human primary tumors expressing GNMT were of smaller size than tumors showing GNMT hypermethylation. Genome-wide analyses of gene promoter methylation identified 277 genes whose aberrant methylation in HCC was associated with GNMT methylation/expression. The findings in this manuscript indicate that DNA hypermethylation plays an important role in the repression of GNMT in HCC and that loss of GNMT in human HCC could promote the establishment of aberrant DNA methylation patterns at specific gene promoters.


Subject(s)
Carcinoma, Hepatocellular/genetics , DNA Methylation , Glycine N-Methyltransferase/genetics , Liver Neoplasms/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival , Epigenetic Repression , Glycine N-Methyltransferase/metabolism , Histones/metabolism , Humans , Liver Neoplasms/metabolism , RNA, Messenger/metabolism , S-Adenosylmethionine/metabolism
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