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1.
Nat Immunol ; 25(7): 1193-1206, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38834865

ABSTRACT

Immune cells experience large cell shape changes during environmental patrolling because of the physical constraints that they encounter while migrating through tissues. These cells can adapt to such deformation events using dedicated shape-sensing pathways. However, how shape sensing affects immune cell function is mostly unknown. Here, we identify a shape-sensing mechanism that increases the expression of the chemokine receptor CCR7 and guides dendritic cell migration from peripheral tissues to lymph nodes at steady state. This mechanism relies on the lipid metabolism enzyme cPLA2, requires nuclear envelope tensioning and is finely tuned by the ARP2/3 actin nucleation complex. We also show that this shape-sensing axis reprograms dendritic cell transcription by activating an IKKß-NF-κB-dependent pathway known to control their tolerogenic potential. These results indicate that cell shape changes experienced by immune cells can define their migratory behavior and immunoregulatory properties and reveal a contribution of the physical properties of tissues to adaptive immunity.


Subject(s)
Cell Movement , Dendritic Cells , Homeostasis , Lymph Nodes , Mice, Inbred C57BL , Receptors, CCR7 , Animals , Dendritic Cells/immunology , Dendritic Cells/metabolism , Lymph Nodes/immunology , Lymph Nodes/cytology , Receptors, CCR7/metabolism , Mice , Cell Movement/immunology , Cell Shape , NF-kappa B/metabolism , Mice, Knockout , Signal Transduction/immunology , I-kappa B Kinase/metabolism , Actin-Related Protein 2-3 Complex/metabolism
2.
Sci Adv ; 10(1): eadi1788, 2024 Jan 05.
Article in English | MEDLINE | ID: mdl-38170778

ABSTRACT

The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here, we report that septins participate in amoeboid motility of T cells, enabling the formation of F-actin and α-actinin-rich cortical rings at the sites of cell cortex-indenting collisions with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient "hourglass"-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results suggest that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots required for amoeboid motility of T cells in tissue-like microenvironments.


Subject(s)
Actomyosin , Amoeba , Actomyosin/metabolism , Septins/metabolism , Cell Movement , Amoeba/metabolism , T-Lymphocytes/metabolism
3.
Sci Adv ; 9(38): eadh1653, 2023 09 22.
Article in English | MEDLINE | ID: mdl-37729408

ABSTRACT

Migratory dendritic cells (migDCs) continuously patrol tissues and are activated by injury and inflammation. Extracellular adenosine triphosphate (ATP) is released by damaged cells or actively secreted during inflammation and increases migDC motility. However, the underlying molecular mechanisms by which ATP accelerates migDC migration is not understood. Here, we show that migDCs can be distinguished from other DC subsets and immune cells by their expression of the voltage-gated calcium channel subunit ß3 (Cavß3; CACNB3), which exclusively facilitates ATP-dependent migration in vitro and during tissue damage in vivo. By contrast, CACNB3 does not regulate lipopolysaccharide-dependent migration. Mechanistically, CACNB3 regulates ATP-dependent inositol 1,4,5-trisphophate receptor-controlled calcium release from the endoplasmic reticulum. This, in turn, is required for ATP-mediated suppression of adhesion molecules, their detachment, and initiation of migDC migration. Thus, Cacnb3-deficient migDCs have an impaired migration after ATP exposure. In summary, we identified CACNB3 as a master regulator of ATP-dependent migDC migration that controls tissue-specific immunological responses during injury and inflammation.


Subject(s)
Adenosine Triphosphate , Calcium Channels , Humans , Biological Transport , Inflammation , Dendritic Cells
4.
Nat Mater ; 22(11): 1409-1420, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37709930

ABSTRACT

The mechanical properties of the extracellular matrix dictate tissue behaviour. In epithelial tissues, laminin is a very abundant extracellular matrix component and a key supporting element. Here we show that laminin hinders the mechanoresponses of breast epithelial cells by shielding the nucleus from mechanical deformation. Coating substrates with laminin-111-unlike fibronectin or collagen I-impairs cell response to substrate rigidity and YAP nuclear localization. Blocking the laminin-specific integrin ß4 increases nuclear YAP ratios in a rigidity-dependent manner without affecting the cell forces or focal adhesions. By combining mechanical perturbations and mathematical modelling, we show that ß4 integrins establish a mechanical linkage between the substrate and keratin cytoskeleton, which stiffens the network and shields the nucleus from actomyosin-mediated mechanical deformation. In turn, this affects the nuclear YAP mechanoresponses, chromatin methylation and cell invasion in three dimensions. Our results demonstrate a mechanism by which tissues can regulate their sensitivity to mechanical signals.


Subject(s)
Keratins , Laminin , Laminin/metabolism , Cell Adhesion , Extracellular Matrix/metabolism , Fibronectins/metabolism , Cytoskeleton/metabolism , Integrins/metabolism
5.
PLoS Comput Biol ; 19(7): e1011250, 2023 07.
Article in English | MEDLINE | ID: mdl-37450544

ABSTRACT

Cell-matrix adhesion is a central mechanical function to a large number of phenomena in physiology and disease, including morphogenesis, wound healing, and tumor cell invasion. Today, how single cells respond to different extracellular cues has been comprehensively studied. However, how the mechanical behavior of the main individual molecules that form an adhesion complex cooperatively responds to force within the adhesion complex is still poorly understood. This is a key aspect of cell adhesion because how these cell adhesion molecules respond to force determines not only cell adhesion behavior but, ultimately, cell function. To answer this question, we develop a multi-scale computational model for adhesion complexes mechanics. We extend the classical clutch hypothesis to model individual adhesion chains made of a contractile actin network, a talin rod, and an integrin molecule that binds at individual adhesion sites on the extracellular matrix. We explore several scenarios of integrins dynamics and analyze the effects of diverse extracellular matrices on the behavior of the adhesion molecules and on the whole adhesion complex. Our results describe how every single component of the adhesion chain mechanically responds to the contractile actomyosin force and show how they control the traction forces exerted by the cell on the extracellular space. Importantly, our computational results agree with previous experimental data at the molecular and cellular levels. Our multi-scale clutch model presents a step forward not only to further understand adhesion complexes mechanics but also to impact, e.g., the engineering of biomimetic materials, tissue repairment, or strategies to arrest tumor progression.


Subject(s)
Actins , Integrins , Cell Adhesion/physiology , Integrins/metabolism , Actins/metabolism , Actin Cytoskeleton/metabolism , Extracellular Matrix/metabolism , Talin , Focal Adhesions/metabolism
6.
Nat Nanotechnol ; 18(4): 336-342, 2023 04.
Article in English | MEDLINE | ID: mdl-37037895

ABSTRACT

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.


Subject(s)
Image Enhancement , Kidney , Animals , Humans , Mice , Microscopy, Fluorescence/methods , Microscopy, Confocal/methods
7.
Eur J Immunol ; 2022 Dec 23.
Article in English | MEDLINE | ID: mdl-36563126

ABSTRACT

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

8.
Signal Transduct Target Ther ; 7(1): 226, 2022 07 09.
Article in English | MEDLINE | ID: mdl-35810162

Subject(s)
Macrophages
9.
Front Cell Dev Biol ; 10: 844297, 2022.
Article in English | MEDLINE | ID: mdl-35399514

ABSTRACT

Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.

10.
J Neurosci Methods ; 373: 109561, 2022 05 01.
Article in English | MEDLINE | ID: mdl-35301006

ABSTRACT

BACKGROUND: Intracranial hypertension (HI) is associated with worse neurological outcomes and higher mortality. Although there are several experimental models of HI, in this article we present a reproducible, reversible, and reliable model of intracranial hypertension, with continuous multimodal monitoring. NEW METHOD: A reversible intracranial hypertension model in swine with multimodal monitoring including intracranial pressure, arterial blood pressure, heart rate variation, brain tissue oxygenation, and electroencephalogram is developed to understand the relationship of ICP and EEG. By inflating and deflating a balloon, located 20 mm anterior to the coronal suture and a 15 mm sagittal suture, we generate intracranial hypertension events and simultaneously measure intracranial pressure and oxygenation in the contralateral hemisphere and the EEG, simulating the usual configuration in humans. RESULTS: We completed 5 experiments and in all of them, we were able to complete at least 6 events of intracranial hypertension in a stable and safe way. For events of 20-40 mmHg of ICP we need an median (IQR) of 4.2 (3.64) ml of saline solution into the Foley balloon, a median (IQR) infusion time of 226 (185) second in each event and for events of 40-50 mmHg of ICP we need a median (IQR) of 5.1 (4.66) ml of saline solution, a median (IQR) infusion time of 280 (48) seconds and a median (IQR). The median (IQR) maintenance time was 352 (77) seconds and 392 (166) seconds for 20-40 mmHg and 40-50 mmHg of ICP, respectively. COMPARISON WITH EXISTING METHOD(S): Existing methods do not include EEG measures and do not present the reversibility of intracranial hypertension. CONCLUSIONS: Our model is fully reproducible, it is capable of generating reversible focal intracranial hypertension through strict control of the injected volume, it is possible to generate different infusion rates of the volume in the balloon, in order to generate different scenarios, the data obtained are sufficient to determine the brain complacency in real time. and useful for understanding the pathophysiology of ICP and the relationship between ICP (CPP) and EEG.


Subject(s)
Intracranial Hypertension , Animals , Brain , Electroencephalography , Heart Rate , Intracranial Hypertension/etiology , Intracranial Pressure/physiology , Swine
11.
Sci Adv ; 7(27)2021 Jul.
Article in English | MEDLINE | ID: mdl-34215576

ABSTRACT

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration, and division. Because its thickness is below optical resolution, there is a tendency to consider the cortex as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time-resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension because of actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape-changing capacity of highly contractile cells that use amoeboid-like migration.

13.
Biochim Biophys Acta Mol Cell Res ; 1868(6): 118999, 2021 05.
Article in English | MEDLINE | ID: mdl-33711364

ABSTRACT

The first junior European Calcium Society online meeting, held October 20-21, 2020, aimed to promote junior researchers in the Ca2+ community. The meeting included four scientific sessions, covering Ca2+ research from molecular detail to whole organisms. Each session featured one invited speaker and three speakers selected based on submitted abstracts, with the overall aim of actively involving early-career researchers. Consequently, the meeting underlined the diversity of Ca2+ physiology, by showcasing research across scales and Kingdoms, as presented by a correspondingly diverse speaker panel across career stages and countries. In this meeting report, we introduce the visions of the junior European Calcium Society board and summarize the meeting content.


Subject(s)
Calcium Signaling , Calcium/metabolism , Humans , Professional Competence , Research Design
14.
Blood ; 137(24): 3416-3427, 2021 06 17.
Article in English | MEDLINE | ID: mdl-33619535

ABSTRACT

Orchestrated recruitment of neutrophils to inflamed tissue is essential during the initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it remains unclear how these factors influence the migration of neutrophils to and at the site of inflammation during their transmigration through the blood-endothelial cell barrier, as well as their motility in the interstitial space. Here, we reveal that activation of hypoxia-inducible factor 2 (HIF2α) as a result of a deficiency in HIF prolyl hydroxylase domain protein 2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNA sequencing analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation because it promotes neutrophil movement through highly confined tissue landscapes.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Movement , Cellular Microenvironment , Neutrophils/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Inflammation/genetics , Inflammation/metabolism , Mice , Mice, Knockout , RNA-Seq
15.
Front Immunol ; 12: 750480, 2021.
Article in English | MEDLINE | ID: mdl-34975840

ABSTRACT

The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes (i.e., T cells, dendritic cells) exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells (including fibroblasts, keratinocytes, astrocytes, and cancer cells). Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration.


Subject(s)
Cell Movement/physiology , Connexins/physiology , Dendritic Cells/physiology , Leukocytes/physiology , Phagocytes/physiology , Adenine Nucleotides/physiology , Aging/immunology , Aging/physiology , Animals , Astrocytes/physiology , Cell Polarity , Chemotaxis, Leukocyte/physiology , Cytoskeleton/physiology , Fibroblasts/physiology , Humans , Inflammation/immunology , Inflammation/physiopathology , Keratinocytes/physiology , Mechanotransduction, Cellular/physiology , Neoplasms/immunology , Nerve Degeneration/immunology , Nerve Degeneration/physiopathology , Nerve Tissue Proteins/physiology , Receptors, Purinergic/physiology
16.
Front Immunol ; 11: 696, 2020.
Article in English | MEDLINE | ID: mdl-32411134

ABSTRACT

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive T and B cells, autoantibody production, and immune complex deposition in various organs. Previous evidence showed abnormal accumulation of B cells in the thymus of lupus-prone mice, but the role of this population in the progression of the disease remains mostly undefined. Here we analyzed the spatial distribution, function, and properties of this thymic B cell population in the BWF1 murine model of SLE. We found that in diseased animals, thymic B cells proliferate, and cluster in structures that resemble ectopic germinal centers. Moreover, we detected antibody-secreting cells in the thymus of diseased-BWF1 mice that produce anti-dsDNA IgG autoantibodies. We also found that thymic B cells from diseased-BWF1 mice induced the differentiation of thymocytes to follicular helper T cells (TFH). These data suggest that the accumulation of B cells in the thymus of BWF1 mice results in the formation of germinal center-like structures and the expansion of a TFH population, which may, in turn, activate and differentiate B cells into autoreactive plasma cells. Therefore, the thymus emerges as an important niche that supports the maintenance of the pathogenic humoral response in the development of murine SLE.


Subject(s)
B-Lymphocytes/immunology , Germinal Center/immunology , Immunity, Humoral , Lupus Erythematosus, Systemic/immunology , T Follicular Helper Cells/immunology , Thymus Gland/immunology , Animals , Autoantibodies/immunology , Cell Differentiation , Cells, Cultured , Coculture Techniques , DNA/immunology , Disease Models, Animal , Female , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred NZB , Plasma Cells/immunology
17.
Sci Adv ; 6(11): eaay2103, 2020 03.
Article in English | MEDLINE | ID: mdl-32195344

ABSTRACT

Migration of cells can be characterized by two prototypical types of motion: individual and collective migration. We propose a statistical inference approach designed to detect the presence of cell-cell interactions that give rise to collective behaviors in cell motility experiments. This inference method has been first successfully tested on synthetic motional data and then applied to two experiments. In the first experiment, cells migrate in a wound-healing model: When applied to this experiment, the inference method predicts the existence of cell-cell interactions, correctly mirroring the strong intercellular contacts that are present in the experiment. In the second experiment, dendritic cells migrate in a chemokine gradient. Our inference analysis does not provide evidence for interactions, indicating that cells migrate by sensing independently the chemokine source. According to this prediction, we speculate that mature dendritic cells disregard intercellular signals that could otherwise delay their arrival to lymph vessels.


Subject(s)
Cell Communication , Cell Movement , Dendritic Cells/metabolism , Models, Biological , Wound Healing , Animals , HeLa Cells , Humans , Mice
18.
Front Immunol ; 10: 2703, 2019.
Article in English | MEDLINE | ID: mdl-31849935

ABSTRACT

Mast cells (MCs) release pro-inflammatory mediators through a process called degranulation response. The latter may be induced by several conditions, including antigen recognition through immunoglobulin E (IgE) or "cross-linking," classically associated with Type I hypersensitivity reactions. Early in this reaction, Ca2+ influx and subsequent increase of intracellular free Ca2+ concentration are essential for MC degranulation. Several membrane channels that mediate Ca2+ influx have been proposed, but their role remains elusive. Here, we evaluated the possible contribution of pannexin-1 channels (Panx1 Chs), well-known as ATP-releasing channels, in the increase of intracellular Ca2+ triggered during cross-linking reaction of MCs. The contribution of Panx1 Chs in the degranulation response was evaluated in MCs from wild type (WT) and Panx1 knock out (Panx1-/-) mice after anti-ovalbumin (OVA) IgE sensitization. Notably, the degranulation response (toluidine blue and histamine release) was absent in Panx1-/- MCs. Moreover, WT MCs showed a rapid and transient increase in Ca2+ signal followed by a sustained increase after antigen stimulation. However, the sustained increase in Ca2+ signal triggered by OVA was absent in Panx1-/- MCs. Furthermore, OVA stimulation increased the membrane permeability assessed by dye uptake, a prevented response by Panx1 Ch but not by connexin hemichannel blockers and without effect on Panx1-/- MCs. Interestingly, the increase in membrane permeability of WT MCs was also prevented by suramin, a P2 purinergic inhibitor, suggesting that Panx1 Chs act as ATP-releasing channels impermeable to Ca2+. Accordingly, stimulation with exogenous ATP restored the degranulation response and sustained increase in Ca2+ signal of OVA stimulated Panx1-/- MCs. Moreover, opening of Panx1 Chs in Panx1 transfected HeLa cells increased dye uptake and ATP release but did not promote Ca2+ influx, confirming that Panx1 Chs permeable to ATP are not permeable to Ca2+. These data strongly suggest that during antigen recognition, Panx1 Chs contribute to the sustained Ca2+ signal increase via release of ATP that activates P2 receptors, playing a critical role in the sequential events that leads to degranulation response during Type I hypersensitivity reactions.


Subject(s)
Cell Degranulation/physiology , Connexins/immunology , Hypersensitivity, Immediate/immunology , Mast Cells/immunology , Nerve Tissue Proteins/immunology , Animals , Connexins/metabolism , HeLa Cells , Humans , Hypersensitivity, Immediate/metabolism , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/metabolism
19.
Proc Natl Acad Sci U S A ; 116(51): 25839-25849, 2019 12 17.
Article in English | MEDLINE | ID: mdl-31776254

ABSTRACT

Naive CD4+ T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4+ T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 ß and γ isoforms (HP1ß/γ, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K-AKT-mTOR axis and switch to glycolysis. While differentiation of naive TRIM28-/- T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28-/- regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1ß/γ control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.


Subject(s)
Cell Differentiation/physiology , Cellular Reprogramming/physiology , Chromosomal Proteins, Non-Histone/metabolism , Epigenesis, Genetic/physiology , T-Lymphocytes/metabolism , Tripartite Motif-Containing Protein 28/metabolism , Animals , Autoimmunity/physiology , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation/genetics , Cell Plasticity/physiology , Cellular Reprogramming/genetics , Chromobox Protein Homolog 5 , Colon/pathology , Cytokines/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Gene Silencing , Histones/metabolism , Mice , Mice, Knockout , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transcriptome , Tripartite Motif-Containing Protein 28/genetics
20.
Placenta ; 86: 35-44, 2019 10.
Article in English | MEDLINE | ID: mdl-31345420

ABSTRACT

OBJECTIVE: Pregestational maternal obesity (PGMO) associates with foetoplacental vascular endothelial dysfunction and higher risk for insulin resistance in the neonate. We characterised the PGMO consequences on the insulin response of the human foetoplacental vasculature. METHODS: Umbilical veins were from pregnancies where the mother was with PGMO (body mass index 30-42.3 kg/m2, n = 33) or normal pregestational weight (PGMN) (body mass index 19.5-24.4 kg/m2, n = 21) with total gestational weight gain within the physiological range. Umbilical vein ring segments were mounted in a myograph for isometric force measurements. Primary cultures of human umbilical vein endothelial cells were used in passage 3. Vessel rings and cells were exposed to 1 nmol/L insulin (20 min) in the absence or presence of 100 µmol/L NG-nitro-l-arginine methyl ester (inhibitor of nitric oxide synthase, NOS). RESULTS: Vessel rings from PGMO showed reduced nitric oxide synthase-activity dependent dilation to insulin or calcitonin-gene related peptide compared with PGMN. PGMO associated with higher inhibitor phosphorylation of the insulin receptor substrate 1 (IRS-1) and lower activator phosphorylation of protein kinase B/Akt (Akt). Cells from PGMO also showed lower nitric oxide level and reduced activator serine1177 but increased inhibitor threonine495 phosphorylation of endothelial nitric oxide synthase (eNOS) and saturable transport of l-arginine. HUVECs from PGMO were not responsive to insulin. CONCLUSION: The lack of response to insulin by the foetoplacental endothelium may result from reduced IRS-1/Akt/eNOS signalling in PGMO. These findings may result in higher risk of insulin resistance in neonates to PGMO pregnancies.


Subject(s)
Endothelium, Vascular/physiopathology , Insulin , Obesity/physiopathology , Pregnancy Complications/physiopathology , Umbilical Veins/physiopathology , Adult , Arginine/metabolism , Case-Control Studies , Endothelial Cells/metabolism , Female , Human Umbilical Vein Endothelial Cells , Humans , Infant, Newborn , Insulin Receptor Substrate Proteins/metabolism , Myography , Pregnancy , Primary Cell Culture , Young Adult
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