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1.
Blood Adv ; 7(23): 7169-7183, 2023 12 12.
Article in English | MEDLINE | ID: mdl-37792794

ABSTRACT

E-cadherin is a crucial regulator of epithelial cell-to-cell adhesion and an established tumor suppressor. Aside epithelia, E-cadherin expression marks the erythroid cell lineage during human but not mouse hematopoiesis. However, the role of E-cadherin in human erythropoiesis remains unknown. Because rat erythropoiesis was postulated to reflect human erythropoiesis more closely than mouse erythropoiesis, we investigated E-cadherin expression in rat erythroid progenitors. E-cadherin expression is conserved within the erythroid lineage between rat and human. In response to anemia, erythroblasts in rat bone marrow (BM) upregulate E-cadherin as well as its binding partner ß-catenin. CRISPR/Cas9-mediated knock out of E-cadherin revealed that E-cadherin expression is required to stabilize ß-catenin in human and rat erythroblasts. Suppression of ß-catenin degradation by glycogen synthase kinase 3ß (GSK3ß) inhibitor CHIR99021 also enhances ß-catenin stability in human erythroblasts but hampers erythroblast differentiation and survival. In contrast, direct activation of ß-catenin signaling, using an inducible, stable ß-catenin variant, does not perturb maturation or survival of human erythroblasts but rather enhances their differentiation. Although human erythroblasts do not respond to Wnt ligands and direct GSK3ß inhibition even reduces their survival, we postulate that ß-catenin stability and signaling is mostly controlled by E-cadherin in human and rat erythroblasts. In response to anemia, E-cadherin-driven upregulation and subsequent activation of ß-catenin signaling may stimulate erythroblast differentiation to support stress erythropoiesis in the BM. Overall, we uncover E-cadherin/ß-catenin expression to mark stress erythropoiesis in rat BM. This may provide further understanding of the underlying molecular regulation of stress erythropoiesis in the BM, which is currently poorly understood.


Subject(s)
Anemia , beta Catenin , Humans , Rats , Mice , Animals , beta Catenin/metabolism , Erythropoiesis/physiology , Glycogen Synthase Kinase 3 beta , Cadherins/genetics , Cadherins/metabolism
2.
Blood Adv ; 7(19): 5868-5876, 2023 10 10.
Article in English | MEDLINE | ID: mdl-37428870

ABSTRACT

Activated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell's DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.


Subject(s)
Extracellular Traps , Humans , Extracellular Traps/metabolism , Neutrophils/metabolism , Histones/metabolism , DNA/metabolism , Cell Membrane/metabolism
3.
Front Immunol ; 14: 1183180, 2023.
Article in English | MEDLINE | ID: mdl-37261342

ABSTRACT

Cancer is one of the leading causes of death worldwide. Treatment outcome is largely dictated by the tumor type, disease stage, and treatment success rates, but also by the variation among patients in endogenous anti-tumor responses. Studies indicate that the presence of neutrophils in the tumor microenvironment is associated with a worse patient outcome due to their ability to suppress local anti-tumor T cell activity. Our previous studies investigated the mechanisms by which neutrophils suppress and damage T cells to become smaller in size (small T cells), debilitating their effector activities. Several studies indicate a role for tumor-associated macrophages in scavenging damaged or dead cells. We hypothesized that the observed lack of small T cells in the TME by confocal microscopy is due to immediate uptake by macrophages. In this study, we confirmed that indeed only the smaller, damaged T cells are taken up by macrophages, once serum-opsonized. Damaged T cells opsonized with complement factor C3 fragments were phagocytosed by macrophages, resulting in almost instantaneous and highly efficient uptake of these small T cells. Inhibition of the complement receptors CR1, CR3 and CR4 expressed by macrophages completely blocked phagocytosis. By contrast, actively proliferating T cells (large T cells) were neither impaired in neutrophil-MDSC activity nor opsonized for phagocytosis by macrophages. Rapid removal of damaged T cells suggests a role of complement and macrophages within the tumor microenvironment to clear suppressed T cells in cancer patients.


Subject(s)
Macrophages , T-Lymphocytes , Humans , Receptors, Complement 3b , Receptors, Complement/physiology , Complement C3
4.
Front Immunol ; 14: 1105103, 2023.
Article in English | MEDLINE | ID: mdl-36969261

ABSTRACT

Introduction: MISTRG mice have been genetically modified to allow development of a human myeloid compartment from engrafted human CD34+ haemopoietic stem cells, making them particularly suited to study the human innate immune system in vivo. Here, we characterized the human neutrophil population in these mice to establish a model that can be used to study the biology and contribution in immune processes of these cells in vivo. Methods and results: We could isolate human bone marrow neutrophils from humanized MISTRG mice and confirmed that all neutrophil maturation stages from promyelocytes (CD11b-CD16-) to end-stage segmented cells (CD11b+CD16+) were present. We documented that these cells possessed normal functional properties, including degranulation, reactive oxygen species production, adhesion, and antibody-dependent cellular cytotoxicity towards antibody-opsonized tumor cells ex vivo. The acquisition of functional capacities positively correlated with the maturation state of the cell. We found that human neutrophils were retained in the bone marrow of humanized MISTRG mice during steady state. However, the mature segmented CD11b+CD16+ human neutrophils were released from the bone marrow in response to two well-established neutrophil-mobilizing agents (i.e., G-CSF and/or CXCR4 antagonist Plerixafor). Moreover, the neutrophil population in the humanized MISTRG mice actively reacted to thioglycolate-induced peritonitis and could infiltrate implanted human tumors, as shown by flow cytometry and fluorescent microscopy. Discussion: These results show that functional human neutrophils are generated and can be studied in vivo using the humanized MISTRG mice, providing a model to study the various functions of neutrophils in inflammation and in tumors.


Subject(s)
Heterocyclic Compounds , Neutrophils , Humans , Mice , Animals , Hematopoietic Stem Cell Mobilization , Bone Marrow , Immunity
5.
Nat Protoc ; 16(2): 791-811, 2021 02.
Article in English | MEDLINE | ID: mdl-33349704

ABSTRACT

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.


Subject(s)
Skin/immunology , Skin/pathology , Staining and Labeling/methods , Animals , Biopsy/methods , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Culture Techniques/methods , Humans , Mice , Skin/cytology
6.
Haematologica ; 105(12): 2746-2756, 2020 12 01.
Article in English | MEDLINE | ID: mdl-33256374

ABSTRACT

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.


Subject(s)
Hematopoietic Stem Cell Transplantation , Podosomes , Animals , Bone Marrow , Bone Marrow Cells , Cell Movement , Endothelial Cells , Endothelium , Hematopoietic Stem Cells , Mice , Mice, Inbred C57BL
7.
Front Immunol ; 11: 570963, 2020.
Article in English | MEDLINE | ID: mdl-33162986

ABSTRACT

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPαΔCYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPαΔCYT>LDLR-/-) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.


Subject(s)
Atherosclerosis/immunology , B-Lymphocytes/immunology , CD47 Antigen/metabolism , Lymphoid Tissue/immunology , Receptors, Immunologic/metabolism , Animals , Antibody Formation , Autoantibodies/metabolism , Cell Movement , Cells, Cultured , Cytokines/metabolism , Immunomodulation , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Immunologic/genetics , Receptors, LDL/genetics , Th1 Cells/immunology , Transplantation Chimera
8.
Mol Cell Proteomics ; 19(7): 1179-1192, 2020 07.
Article in English | MEDLINE | ID: mdl-32332107

ABSTRACT

The vessel wall is continuously exposed to hemodynamic forces generated by blood flow. Endothelial mechanosensors perceive and translate mechanical signals via cellular signaling pathways into biological processes that control endothelial development, phenotype and function. To assess the hemodynamic effects on the endothelium on a system-wide level, we applied a quantitative mass spectrometry approach combined with cell surface chemical footprinting. SILAC-labeled endothelial cells were subjected to flow-induced shear stress for 0, 24 or 48 h, followed by chemical labeling of surface proteins using a non-membrane permeable biotin label, and analysis of the whole proteome and the cell surface proteome by LC-MS/MS analysis. These studies revealed that of the >5000 quantified proteins 104 were altered, which were highly enriched for extracellular matrix proteins and proteins involved in cell-matrix adhesion. Cell surface proteomics indicated that LAMA4 was proteolytically processed upon flow-exposure, which corresponded to the decreased LAMA4 mass observed on immunoblot. Immunofluorescence microscopy studies highlighted that the endothelial basement membrane was drastically remodeled upon flow exposure. We observed a network-like pattern of LAMA4 and LAMA5, which corresponded to the localization of laminin-adhesion molecules ITGA6 and ITGB4. Furthermore, the adaptation to flow-exposure did not affect the inflammatory response to tumor necrosis factor α, indicating that inflammation and flow trigger fundamentally distinct endothelial signaling pathways with limited reciprocity and synergy. Taken together, this study uncovers the blood flow-induced remodeling of the basement membrane and stresses the importance of the subendothelial basement membrane in vascular homeostasis.


Subject(s)
Basement Membrane/metabolism , Blood Circulation , Endothelial Cells/metabolism , Integrins/metabolism , Laminin/metabolism , Blood Circulation/physiology , Cells, Cultured , Chromatography, Liquid , Endothelial Cells/cytology , Endothelial Cells/drug effects , Extracellular Matrix/metabolism , Fluorescent Antibody Technique , Gene Expression Profiling , Gene Ontology , Hemodynamics , Humans , Integrin alpha Chains/metabolism , Integrin alpha6/metabolism , Integrin beta Chains/metabolism , Integrin beta4/metabolism , Protein Interaction Maps/physiology , Proteomics , Tandem Mass Spectrometry , Tumor Necrosis Factor-alpha/pharmacology
9.
Nat Immunol ; 21(6): 696, 2020 06.
Article in English | MEDLINE | ID: mdl-32210390

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

10.
Haematologica ; 105(6): 1677-1685, 2020 06.
Article in English | MEDLINE | ID: mdl-31467128

ABSTRACT

Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate TF gene expression and activity in these vascular cells. The LIM-only protein FHL2 is a scaffolding protein that modulates signal transduction pathways with crucial functions in endothelial and smooth muscle cells. However, the role of FHL2 in TF regulation and thrombosis remains unexplored. Using a murine model of venous thrombosis in mesenteric vessels, we demonstrated that FHL2 deficiency results in exacerbated thrombus formation. Gain- and loss-of-function experiments revealed that FHL2 represses TF expression in endothelial and smooth muscle cells through inhibition of the transcription factors nuclear factor κB and activating protein-1. Furthermore, we observed that FHL2 interacts with the cytoplasmic tail of TF. In line with our in vivo observations, FHL2 decreases TF activity in endothelial and smooth muscle cells whereas FHL2 knockdown or deficiency results in enhanced TF activity. Finally, the FHL2 single nucleotide polymorphism rs4851770 was associated with the risk of venous thrombosis in a large population of venous thrombosis cases and control subjects from 12 studies (INVENT consortium). Altogether, our results highlight functional involvement of FHL2 in TF-mediated coagulation and identify FHL2 as a novel gene associated with venous thrombosis in humans.


Subject(s)
Thromboplastin , Venous Thrombosis , Animals , Genetic Variation , Humans , LIM-Homeodomain Proteins/genetics , Mice , Muscle Proteins/genetics , Thromboplastin/genetics , Transcription Factors/genetics , Venous Thrombosis/genetics
11.
Sci Rep ; 9(1): 14401, 2019 10 07.
Article in English | MEDLINE | ID: mdl-31591420

ABSTRACT

Culture expanded mesenchymal stromal cells (MSCs) are being extensively studied for therapeutic applications, including treatment of graft-versus-host disease, osteogenesis imperfecta and for enhancing engraftment of hematopoietic stem cells after transplantation. Thus far, clinical trials have shown that the therapeutic efficiency of MSCs is variable, which may in part be due to inefficient cell migration. Here we demonstrate that human MSCs display remarkable low migratory behaviour compared to other mesodermal-derived primary human cell types. We reveal that specifically in MSCs the nucleus is irregularly shaped and nuclear lamina are prone to wrinkling. In addition, we show that expression of Lamin A/C is relatively high in MSCs. We further demonstrate that in vitro MSC migration through confined pores is limited by their nuclei, a property that might correlate to the therapeutic inefficiency of administered MSC in vivo. Silencing expression of Lamin A/C in MSCs improves nuclear envelope morphology, promotes the protrusive activity of MSCs through confined pores and enhances their retention in the lung after intravenous administration in vivo. Our findings suggest that the intrinsic nuclear lamina properties of MSCs underlie their limited capacity to migrate, and that strategies that target the nuclear lamina might alter MSC-based cellular therapies.


Subject(s)
Cell Nucleus Shape , Gene Expression Regulation , Lamin Type A/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Adult , Cell Movement , Humans , Nuclear Envelope/metabolism , Porosity
12.
Nat Immunol ; 20(6): 756-764, 2019 06.
Article in English | MEDLINE | ID: mdl-31110315

ABSTRACT

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Immunologic Memory , Skin/immunology , Animals , Antigens/immunology , Cell Line, Tumor , Cell Movement/immunology , Epidermis/immunology , Epidermis/metabolism , Fluorescent Antibody Technique , Humans , Mice , Organ Specificity/immunology , Single-Domain Antibodies/immunology , Skin/metabolism , Vaccines, DNA/genetics , Vaccines, DNA/immunology
13.
Eur J Immunol ; 49(4): 576-589, 2019 04.
Article in English | MEDLINE | ID: mdl-30707456

ABSTRACT

The BM serves as a blood-forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T-cell migration to and localization inside the BM. As BM accumulates many CXCR3-expressing memory CD8+ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8+ T cells in BM, is critically important for homing of all CD8+ T-cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8+ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM-1+VCAM-1+BP-1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL-7 and IL-15. We therefore conclude that CXCR4 is not only crucial for entry of CD8+ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.


Subject(s)
Bone Marrow/immunology , Bone Marrow/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Movement/immunology , Cellular Microenvironment , Receptors, CXCR4/metabolism , Animals , Bone Marrow/blood supply , Bone Marrow/pathology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Communication/immunology , Cellular Microenvironment/genetics , Cellular Microenvironment/immunology , Cytokines/biosynthesis , Immunologic Memory , Mice , Receptors, CXCR3 , Stromal Cells/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
14.
Cell Rep ; 24(12): 3115-3124, 2018 09 18.
Article in English | MEDLINE | ID: mdl-30231995

ABSTRACT

Leukocytes follow the well-defined steps of rolling, spreading, and crawling prior to diapedesis through endothelial cells (ECs). We found increased expression of DLC-1 in stiffness-associated diseases like atherosclerosis and pulmonary arterial hypertension. Depletion of DLC-1 in ECs cultured on stiff substrates drastically reduced cell stiffness and mimicked leukocyte transmigration kinetics observed for ECs cultured on soft substrates. Mechanistic studies revealed that DLC-1-depleted ECs or ECs cultured on soft substrates failed to recruit the actin-adaptor proteins filamin B, α-actinin-4, and cortactin to clustered ICAM-1, thereby preventing the ICAM-1 adhesome formation and impairing leukocyte spreading. This was rescued by overexpressing DLC-1, resulting in ICAM-1 adhesome stabilization and leukocyte spreading. Our results reveal an essential role for substrate stiffness-regulated endothelial DLC-1, independent of its GAP domain, in locally stabilizing the ICAM-1 adhesome to promote leukocyte spreading, essential for efficient leukocyte transendothelial migration.


Subject(s)
GTPase-Activating Proteins/genetics , Leukocytes/physiology , Transendothelial and Transepithelial Migration , Tumor Suppressor Proteins/genetics , Vascular Stiffness , Cells, Cultured , GTPase-Activating Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/physiology , Humans , Intercellular Adhesion Molecule-1/metabolism , Leukocytes/metabolism , Microfilament Proteins/metabolism , Tumor Suppressor Proteins/metabolism
15.
J Immunol ; 200(5): 1790-1801, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29386254

ABSTRACT

Leukocyte transendothelial migration is key to inflammation. Leukocytes first start rolling over the inflamed endothelium, followed by firmly adhering to it. Under inflammatory conditions, endothelial cells express small finger-like protrusions that stick out into the lumen. The function and regulation of these structures are unclear. We present evidence that these ICAM-1- and F-actin-rich endothelial finger-like protrusions are filopodia and function as adhesive structures for leukocytes to transit from rolling to crawling but are dispensable for diapedesis. Mechanistically, these structures require the motor function of myosin-X, activity of the small GTPase Cdc42, and p21-activated kinase 4. Moreover, myosin-X expression is under control of TNF-α-mediated c-Jun N-terminal kinase activity and is upregulated in human atherosclerotic regions. To our knowledge, this is the first study to identify that regulation of endothelial filopodia is crucial for leukocyte extravasation, in particular for the initiation of leukocyte adhesion under flow conditions.


Subject(s)
Endothelial Cells/metabolism , GTP Phosphohydrolases/metabolism , Inflammation/metabolism , Intercellular Adhesion Molecule-1/metabolism , Leukocytes/metabolism , Myosins/metabolism , Pseudopodia/metabolism , Actins/metabolism , Cell Adhesion/physiology , Cell Line , Cell Line, Tumor , Endothelium, Vascular/metabolism , HL-60 Cells , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Signal Transduction/physiology , Transendothelial and Transepithelial Migration/physiology , Up-Regulation/physiology
17.
J Immunol ; 197(11): 4312-4324, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27815426

ABSTRACT

Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KODC), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KODC mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KODC DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.


Subject(s)
Cell Movement/immunology , Chemokine CCL21/immunology , Dendritic Cells/immunology , GATA1 Transcription Factor/immunology , Lymph Nodes/immunology , Sialic Acids/immunology , Animals , Cell Movement/genetics , Chemokine CCL19/genetics , Chemokine CCL19/immunology , Chemokine CCL21/genetics , Dendritic Cells/cytology , GATA1 Transcription Factor/deficiency , Lymph Nodes/cytology , Mice , Mice, Knockout , Sialic Acids/genetics
18.
PLoS One ; 11(5): e0154342, 2016.
Article in English | MEDLINE | ID: mdl-27152938

ABSTRACT

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cKOMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches.


Subject(s)
Cell Compartmentation , GATA1 Transcription Factor/genetics , Megakaryocytes/cytology , Animals , Enzyme-Linked Immunosorbent Assay , Megakaryocytes/metabolism , Mice , Mice, Knockout , Transcription, Genetic
19.
J Immunol ; 196(3): 1272-83, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26718340

ABSTRACT

Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response.


Subject(s)
Aspergillosis/immunology , Aspergillus fumigatus/immunology , Hyphae/immunology , Neutrophils/immunology , Spores, Fungal/immunology , Cytotoxicity, Immunologic/immunology , Extracellular Traps/immunology , Humans , Immunity, Innate , Immunologic Deficiency Syndromes/immunology , Microscopy, Confocal , Phagocytes/immunology
20.
J Immunol ; 196(1): 72-9, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26597010

ABSTRACT

Multiple sclerosis (MS) is a chronic demyelinating disorder of the CNS characterized by immune cell infiltration across the brain vasculature into the brain, a process not yet fully understood. We previously demonstrated that the sphingolipid metabolism is altered in MS lesions. In particular, acid sphingomyelinase (ASM), a critical enzyme in the production of the bioactive lipid ceramide, is involved in the pathogenesis of MS; however, its role in the brain vasculature remains unknown. Transmigration of T lymphocytes is highly dependent on adhesion molecules in the vasculature such as intercellular adhesion molecule-1 (ICAM-1). In this article, we hypothesize that ASM controls T cell migration by regulating ICAM-1 function. To study the role of endothelial ASM in transmigration, we generated brain endothelial cells lacking ASM activity using a lentiviral shRNA approach. Interestingly, although ICAM-1 expression was increased in cells lacking ASM activity, we measured a significant decrease in T lymphocyte adhesion and consequently transmigration both in static and under flow conditions. As an underlying mechanism, we revealed that upon lack of endothelial ASM activity, the phosphorylation of ezrin was perturbed as well as the interaction between filamin and ICAM-1 upon ICAM-1 clustering. Functionally this resulted in reduced microvilli formation and impaired transendothelial migration of T cells. In conclusion, in this article, we show that ASM coordinates ICAM-1 function in brain endothelial cells by regulating its interaction with filamin and phosphorylation of ezrin. The understanding of these underlying mechanisms of T lymphocyte transmigration is of great value to develop new strategies against MS lesion formation.


Subject(s)
Brain/metabolism , Intercellular Adhesion Molecule-1/metabolism , Sphingomyelin Phosphodiesterase/metabolism , T-Lymphocytes/immunology , Transendothelial and Transepithelial Migration/immunology , Adult , Aged , Aged, 80 and over , Brain/cytology , Brain/immunology , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Line , Ceramides/metabolism , Cytoskeletal Proteins/metabolism , Endothelial Cells/immunology , Endothelial Cells/metabolism , Female , Filamins/metabolism , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Intercellular Adhesion Molecule-1/immunology , Male , Middle Aged , Multiple Sclerosis/immunology , Phosphorylation/genetics , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/immunology
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