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Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. In this study, we addressed the role of Met signaling in distinct steps of LC/dermal DC emigration from the skin by employing a conditionally Met-deficient mouse model (Metflox/flox). We found that Met deficiency severely impaired podosome formation in DCs and concomitantly decreased the proteolytic degradation of gelatin. Accordingly, Met-deficient LCs failed to efficiently cross the extracellular matrix-rich basement membrane between the epidermis and the dermis. We further observed that HGF-dependent Met activation reduced the adhesion of bone marrow-derived LCs to various extracellular matrix factors and enhanced the motility of DCs in three-dimensional collagen matrices, which was not the case for Met-deficient LCs/DCs. We found no impact of Met signaling on the integrin-independent amoeboid migration of DCs in response to the CCR7 ligand CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.
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Podosomas , Ratones , Animales , Movimiento Celular , Piel , Células de Langerhans/metabolismo , Transducción de Señal , Células Dendríticas/metabolismo , Ganglios LinfáticosRESUMEN
Engineered magnetic nanoparticles (MNPs) are emerging as advanced tools for medical applications. The coating of MNPs using polyelectrolytes (PEs) is a versatile means to tailor MNP properties and is used to optimize MNP functionality. Dendritic cells (DCs) are critical regulators of adaptive immune responses. Functionally distinct DC subsets exist, either under steady-state or inflammatory conditions, which are explored for the specific treatment of various diseases, such as cancer, autoimmunity, and transplant rejection. Here, the impact of the PE coating of ferumoxytol for uptake into both inflammatory and steady-state DCs and the cellular responses to MNP labeling is addressed. Labeling efficiency by uncoated and PE-coated ferumoxytol is highly variable in different DC subsets, and PE coating significantly improves the labeling of steady-state DCs. Uncoated ferumoxytol results in increased cytotoxicity of steady-state DCs after labeling, which is abolished by the PE coating, while no increased cell death is observed in inflammatory DCs. Furthermore, uncoated and PE-coated ferumoxytol appear immunologically inert in inflammatory DCs, but they induce activation of steady-state DCs. These results show that the PE coating of MNPs can be applied to endow particles with desired properties for enhanced uptake and cell type-specific responses in distinct target DC populations.
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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.
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The spleen contains phenotypically and functionally distinct conventional dendritic cell (cDC) subpopulations, termed cDC1 and cDC2, which each can be divided into several smaller and less well-characterized subsets. Despite advances in understanding the complexity of cDC ontogeny by transcriptional programming, the significance of posttranslational modifications in controlling tissue-specific cDC subset immunobiology remains elusive. Here, we identified the cell-surface-expressed A-disintegrin-and-metalloproteinase 10 (ADAM10) as an essential regulator of cDC1 and cDC2 homeostasis in the splenic marginal zone (MZ). Mice with a CD11c-specific deletion of ADAM10 (ADAM10ΔCD11c) exhibited a complete loss of splenic ESAMhi cDC2A because ADAM10 regulated the commitment, differentiation, and survival of these cells. The major pathways controlled by ADAM10 in ESAMhi cDC2A are Notch, signaling pathways involved in cell proliferation and survival (e.g., mTOR, PI3K/AKT, and EIF2 signaling), and EBI2-mediated localization within the MZ. In addition, we discovered that ADAM10 is a molecular switch regulating cDC2 subset heterogeneity in the spleen, as the disappearance of ESAMhi cDC2A in ADAM10ΔCD11c mice was compensated for by the emergence of a Clec12a+ cDC2B subset closely resembling cDC2 generally found in peripheral lymph nodes. Moreover, in ADAM10ΔCD11c mice, terminal differentiation of cDC1 was abrogated, resulting in severely reduced splenic Langerin+ cDC1 numbers. Next to the disturbed splenic cDC compartment, ADAM10 deficiency on CD11c+ cells led to an increase in marginal metallophilic macrophage (MMM) numbers. In conclusion, our data identify ADAM10 as a molecular hub on both cDC and MMM regulating their transcriptional programming, turnover, homeostasis, and ability to shape the anatomical niche of the MZ.
Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Células Dendríticas/metabolismo , Proteínas de la Membrana/metabolismo , Proteína ADAM10/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Animales , Células Presentadoras de Antígenos/metabolismo , Antígeno CD11c/metabolismo , Diferenciación Celular , Proliferación Celular , Femenino , Homeostasis , Tejido Linfoide/metabolismo , Macrófagos/metabolismo , Masculino , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Transducción de Señal , Bazo/citología , Bazo/metabolismoRESUMEN
Nonalcoholic steatohepatitis (NASH) is the most common chronic, progressive liver disease in Western countries. The significance of cellular interactions of the HGF/c-Met axis in different liver cell subtypes and its relation to the oxidative stress response remains unclear so far. Hence, the present study is aimed at investigating the role of c-Met and the interaction with the oxidative stress response during NASH development in mice and humans. Conditional c-Met knockout (KO) lines (LysCre for Kupffer cells/macrophages, GFAPCre for α-SMA+ and CK19+ cells and MxCre for bone marrow-derived immune cells) were fed chow and either methionine-choline-deficient diet (MCD) for 4 weeks or high-fat diet (HFD) for 24 weeks. Mice lacking c-Met either in Kupffer cells, α-SMA+ and CK19+ cells, or bone marrow-derived immune cells displayed earlier and faster progressing steatohepatitis during dietary treatments. Severe fatty liver degeneration and histomorphological changes were accompanied by an increased infiltration of immune cells and a significant upregulation of inflammatory cytokine expression reflecting an earlier initiation of steatohepatitis development. In addition, animals with a cell-type-specific deletion of c-Met exhibited a strong generation of reactive oxygen species (ROS) by dihydroethidium (hydroethidine) (DHE) staining showing a significant increase in the oxidative stress response especially in LysCre/c-Metmut and MxCre/c-Metmut animals. All these changes finally lead to earlier and stronger fibrosis progression with strong accumulation of collagen within liver tissue of mice deficient for c-Met in different liver cell types. The HGF/c-Met signaling pathway prevents from steatosis development and has a protective function in the progression to steatohepatitis and fibrosis. It conveys an antifibrotic role independent on which cell type c-Met is missing (Kupffer cells/macrophages, α-SMA+ and CK19+ cells, or bone marrow-derived immune cells). These results highlight a global protective capacity of c-Met in NASH development and progression.
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Cirrosis Hepática/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Progresión de la Enfermedad , Técnicas de Inactivación de Genes , Hepatocitos/metabolismo , Humanos , Macrófagos del Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/patología , Transducción de Señal/fisiologíaRESUMEN
Langerhans cells (LCs), the epidermal dendritic cell (DC) subset, express the transmembrane tyrosine kinase receptor Met also known as hepatocyte growth factor (HGF) receptor. HGF is the exclusive ligand of Met and upon binding executes mitogenic, morphogenic, and motogenic activities to various cells. HGF exerts anti-inflammatory activities via Met signaling and was found to regulate various functions of immune cells, including differentiation and maturation, cytokine production, cellular migration and adhesion, and T cell effector function. It has only recently become evident that a number of HGF-regulated functions in inflammatory processes and immune responses are imparted via DCs. However, the mechanisms by which Met signaling in DCs conveys its immunoregulatory effects have not yet been fully understood. In this review, we focus on the current knowledge of Met signaling in DCs with particular attention on the morphogenic and motogenic activities. Met signaling was shown to promote DC mobility by regulating matrix metalloproteinase activities and adhesion. This is a striking resemblance to the role of Met in regulating a cell fate program during embryonic development, wound healing, and in tumor invasion known as epithelial-mesenchymal transition (EMT). Hence, we propose the concept that an EMT program is executed by Met signaling in LCs.
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Células de Langerhans/fisiología , Proteínas Proto-Oncogénicas c-met/fisiología , Piel/inmunología , Animales , Movimiento Celular , Transición Epitelial-Mesenquimal , Factor de Crecimiento de Hepatocito/fisiología , Humanos , Piel/lesiones , Piel/metabolismoRESUMEN
Mucosal Langerhans cells (LCs) originate from pre-dendritic cells and monocytes. However, the mechanisms involved in their in situ development remain unclear. Here, we demonstrate that the differentiation of murine mucosal LCs is a two-step process. In the lamina propria, signaling via BMP7-ALK3 promotes translocation of LC precursors to the epithelium. Within the epithelium, TGF-ß1 finalizes LC differentiation, and ALK5 is crucial to this process. Moreover, the local microbiota has a major impact on the development of mucosal LCs, whereas LCs in turn maintain mucosal homeostasis and prevent tissue destruction. These results reveal the differential and sequential role of TGF-ß1 and BMP7 in LC differentiation and highlight the intimate interplay of LCs with the microbiota.
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Proteína Morfogenética Ósea 7/inmunología , Células de Langerhans/inmunología , Microbiota/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Proteína Morfogenética Ósea 7/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Humanos , Inmunidad Mucosa , Células de Langerhans/citología , Células de Langerhans/metabolismo , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Lectinas de Unión a Manosa/deficiencia , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucosa Bucal/citología , Mucosa Bucal/inmunología , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal/inmunología , Células Madre/citología , Células Madre/inmunología , Transcriptoma , Factor de Crecimiento Transformador beta1/genética , Regulación hacia ArribaRESUMEN
Langerhans cells (LCs) populate the mucosal epithelium, a major entry portal for pathogens, yet their ontogeny remains unclear. We found that, in contrast to skin LCs originating from self-renewing radioresistant embryonic precursors, oral mucosal LCs derive from circulating radiosensitive precursors. Mucosal LCs can be segregated into CD103(+)CD11b(lo) (CD103(+)) and CD11b(+)CD103(-) (CD11b(+)) subsets. We further demonstrated that similar to non-lymphoid dendritic cells (DCs), CD103(+) LCs originate from pre-DCs, whereas CD11b(+) LCs differentiate from both pre-DCs and monocytic precursors. Despite this ontogenetic discrepancy between skin and mucosal LCs, the transcriptomic signature and immunological function of oral LCs highly resemble those of skin LCs but not DCs. These findings, along with the epithelial position, morphology, and expression of the LC-associated phenotype strongly suggest that oral mucosal LCs are genuine LCs. Collectively, in a tissue-dependent manner, murine LCs differentiate from at least three distinct precursors (embryonic, pre-DC, and monocytic) in steady state.
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Diferenciación Celular , Células Dendríticas/inmunología , Células de Langerhans/inmunología , Monocitos/inmunología , Mucosa Bucal/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Circulación Sanguínea , Antígeno CD11b/metabolismo , Células Cultivadas , Epitelio/inmunología , Cadenas alfa de Integrinas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Piel/inmunología , Transcriptoma/inmunologíaRESUMEN
Hepatocyte growth factor (HGF)-signaling via Met can induce mitogenic, morphogenic, and motogenic activity in various cell types. Met expression in the immune system is limited to cells with antigen-presenting capacities, including dendritic cells (DCs). Thus, it appears highly conceivable that Met-signaling impacts on adaptive immune responses. However, the mechanisms by which HGF imparts its effects on immunological responses are not yet fully understood. DCs possess unique functionalities that are critically involved in controlling both tolerance and immunity. HGF conveys immunoregulatory functions, which strongly correlate with that of DCs orchestrating the apt immune response in inflammation. Therefore, this review focuses on the current knowledge of Met-signaling in DCs with specific emphasis on the morphogenic and motogenic activities. HGF has been identified to play a role in peripheral immune tolerance by directing DC differentiation towards a tolerogenic phenotype. In skin immunity, Met-signaling was shown to drive mobilization of DCs by regulating matrix metalloproteinase activities. This is strikingly reminiscent of the role of Met for regulating a cell fate program during embryonic development, wound healing, and in tumor invasion known as epithelial-mesenchymal transition (EMT). Thus, the concept emerges that an EMT program is executed by Met-signaling in DCs, which will be also discussed.
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Langerhans cells (LC), the skin epidermal contingent of dendritic cells (DC), possess an exceptional life cycle and developmental origin. LC, like all mature blood cells, develop from haematopoietic stem cells (HSC) through successive steps of lineage commitment and differentiation. However, LC development is different to that of other DC subsets and not yet fully understood. Haematopoietic cell fate decisions are instructed by specific growth factors and cytokines produced in specialized microenvironments or niches. Upon ligand binding the cognate surface receptors on HSC and further restricted progenitor cells regulate the signalling pathways that eventually leads to the execution of lineage-determining genetic programs. In this review we focus on a specific set of surface receptor kinases that have been identified as critical regulators of LC development using genetically modified mice. Recent studies suggest for some of these kinases to impact on LC/LC progenitor interaction with the local niche by regulating adhesion and/or migration. During embryonic development, in wound healing and aberrantly in tumour invasion the same kinase receptors control a genetic program known as epithelial-to-mesenchymal-transition (EMT). We will discuss how EMT and its reverse program of mesenchymal-to-epithelial-transition (MET) can serve as universal concepts operating also in LC development.
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Movimiento Celular/inmunología , Homeostasis/inmunología , Células de Langerhans/inmunología , Piel/inmunología , Adhesión Celular/inmunología , Diferenciación Celular/inmunología , Transición Epitelial-Mesenquimal/inmunología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células de Langerhans/metabolismo , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Piel/citología , Piel/metabolismoRESUMEN
We have recently described two types of Langerhans cells (LCs), which develop via separate pathways in steady-state conditions and during inflammation. Here, we propose that these two types of LCs differ in their requirement for transforming growth factor ß1 (TGFß1), and we discuss how TGFß1 impacts on the development of other dendritic cell subtypes.
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Cord blood-derived haematopoietic stem cells (CB-HSCs) are an attractive source for transplantation in haematopoietic disorders. However, the yield of CB-HSCs per graft is limited and often insufficient, particularly for the treatment of adult patients. Here we compare the capacity of three cytokine cocktails to expand CB-CD34(+) cells. Cells were cultured for 5 or 14 days in media supplemented with: (a) SCF, FL, IL-3 and IL-6 (SFLIL3/6); (b) SCF, TPO, FGF-1 and IL-6 (STFIL6); and (c) SCF, TPO, FGF-1, IGFBP2 and Angptl-5 (STFAI). We observed that STFAI-culture expansion sustained the most vigorous cell proliferation, maintenance of CD34(+) phenotype and colony-forming unit counts. In addition, STFAI-cultured cells had a potent ex vivo migration activity. STFAI-expanded cells were able to engraft NSG mice. However, no significant difference in overall engraftment was observed among the expansion cocktails. Assessment of short-term reconstitution using multilineage markers demonstrated that the STFAI cocktail for HSCs expansion greatly improved total cell expansion but may impair short-term lymphoid repopulation.
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Angiopoyetinas/farmacología , Antígenos CD34/metabolismo , Sangre Fetal/citología , Sangre Fetal/efectos de los fármacos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Linfocitos/citología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Clonales , Ensayo de Unidades Formadoras de Colonias , Citocinas/farmacología , Sangre Fetal/metabolismo , Humanos , Antígenos Comunes de Leucocito/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Bazo/citología , Factor de Células Madre/farmacología , Trombopoyetina/farmacologíaRESUMEN
Currently, ex vivo expansion of hematopoietic stem cells (HSC) is still insufficient. Traditional approaches for HSC expansion include the use of stromal cultures, growth factors, and/or bioreactors. Biomaterial-based strategies provide new perspectives. We focus on identifying promising two-dimensional (2D) polymer candidates for HSC expansion. After a 7-day culture period with cytokine supplementation, 2D fibrin, poly(D,L-lactic-co-glycolic acid; Resomer® RG503), and Poly(É-caprolactone; PCL) substrates supported expansion of cord blood (CB)-derived CD34⺠cells ex vivo. Fibrin cultures achieved the highest proliferation rates (>8700-fold increase of total nuclear cells, p<0.001), high total colony-forming units (3.6-fold increase, p<0.001), and highest engraftment in NSG mice (7.69-fold more donor cells compared with tissue culture polysterene, p<0.001). In addition, the presence of multiple human hematopoietic lineages such as myeloid (CD13âº), erythroid (GypCâº), and lymphoid (CD20âº/CD56âº) in murine transplant recipients confirmed the multilineage engraftment potential of fibrin-based cultures. Filopodia development in fibrin-expanded cells was a further indicator for superior cell adhesion capacities. We propose application of fibrin, Resomer® RG503, and PCL for future strategies of CB-CD34⺠cell expansion. Suitable polymers for HSC expansion might also be appropriate for future drug discovery applications or for studies aimed to develop hematological therapies.
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Técnicas de Cultivo de Célula/métodos , Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Polímeros/farmacología , Animales , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula , Supervivencia Celular , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/ultraestructura , Humanos , Inmunohistoquímica , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Especificidad de Órganos/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
Langerhans cells (LCs), the dendritic cells (DCs) in skin epidermis, possess an exceptional life cycle and developmental origin. Here we identified two types of LCs, short-term and long-term LCs, which transiently or stably reconstitute the LC compartment, respectively. Short-term LCs developed from Gr-1(hi) monocytes under inflammatory conditions and occurred independently of the transcription factor Id2. Long-term LCs arose from bone marrow in steady state and were critically dependent on Id2. Surface marker and gene expression analysis positioned short-term LCs close to Gr-1(hi) monocytes, which is indicative of their monocytic origin. We also show that LC reconstitution after UV light exposure occurs in two waves: an initial fast and transient wave of Gr-1(hi) monocyte-derived short-term LCs is followed by a second wave of steady-state precursor-derived long-term LCs. Our data demonstrate the presence of two types of LCs that develop through different pathways in inflammation and steady state.
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Inflamación/metabolismo , Inflamación/patología , Células de Langerhans/metabolismo , Células de Langerhans/patología , Piel/citología , Piel/metabolismo , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Médula Ósea/inmunología , Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Epidérmicas , Epidermis/inmunología , Epidermis/metabolismo , Epidermis/patología , Expresión Génica , Inflamación/genética , Inflamación/inmunología , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Células de Langerhans/inmunología , Ratones , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/patología , Piel/inmunología , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Rayos UltravioletaRESUMEN
This work deals with the production and characterization of water-compatible, iron oxide based nanoparticles covered with functional poly(ethylene glycol) (PEG)-biotin surface groups (SPIO-PEG-biotin). Synthesis of the functionalized colloids occurred by incubating the oleate coated particles used as precursor magnetic fluid with anionic liposomes containing 14 mol% of a phospholipid-PEG-biotin conjugate. The latter was prepared by coupling dimyristoylphosphatidylethanolamine (DC(14:0)PE) to activated α-biotinylamido-ω -N-hydroxy-succinimidcarbonyl-PEG (NHS-PEG-biotin). Physical characterization of the oleate and PEG-biotin iron oxide nanocolloids revealed that they appear as colloidal stable clusters with a hydrodynamic diameter of 160 nm and zeta potentials of - 39 mV (oleate coated particles) and - 14 mV (PEG-biotin covered particles), respectively, as measured by light scattering techniques. Superconducting quantum interference device (SQUID) measurements revealed specific saturation magnetizations of 62-73 emu g(-1) Fe(3)O(4) and no hysteresis was observed at 300 K. MR relaxometry at 3 T revealed very high r(2) relaxivities and moderately high r(1) values. Thus, both nanocolloids can be classified as small, superparamagnetic, negative MR contrast agents. The capacity to functionalize the particles was illustrated by binding streptavidin alkaline phosphatase (SAP). It was found, however, that these complexes become highly aggregated after capturing them on the magnetic filter device during high-gradient magnetophoresis, thereby reducing the accessibility of the SAP.
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Fosfatasa Alcalina/química , Biotina/química , Nanopartículas de Magnetita/química , Estreptavidina/química , Fosfatasa Alcalina/metabolismo , Glicerofosfolípidos/química , Ácido Oléico/química , Tamaño de la Partícula , Polietilenglicoles/química , Unión ProteicaRESUMEN
The Met tyrosine kinase has a pivotal role in embryonic development and tissue regeneration, and deregulated Met signaling contributes to tumorigenesis. After binding of its cognate ligand hepatocyte growth factor, Met signaling confers mitogenic, morphogenic, and motogenic activity to various cells. Met expression in the hematopoietic compartment is limited to progenitor cells and their Ag-presenting progeny, including dendritic cells (DCs). In this study, we demonstrate that Met signaling in skin-resident DCs is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. By using a conditional Met-deficient mouse model (Met(flox/flox)), we show that Met acts on the initial step of DC release from skin tissue. Met-deficient DCs fail to reach skin-draining lymph nodes upon activation while exhibiting an activated phenotype. Contact hypersensitivity reactions in response to various contact allergens is strongly impaired in Met-deficient mice. Inhibition of Met signaling by single-dose epicutaneous administration of the Met kinase-specific inhibitor SU11274 also suppressed contact hypersensitivity in wild-type mice. Additionally, we found that Met signaling regulates matrix metalloproteinase MMP2 and MMP9 activity, which is important for DC migration through extracellular matrix. These data unveil Met signaling in DCs as a critical determinant for the maintenance of normal immune function and suggest Met as a potential target for treatment of autoimmune skin diseases.
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Movimiento Celular/inmunología , Células Dendríticas/inmunología , Proteínas Proto-Oncogénicas c-met/inmunología , Piel/inmunología , Animales , Células Dendríticas/enzimología , Citometría de Flujo , Immunoblotting , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Proteínas Proto-Oncogénicas c-met/deficiencia , Proteínas Proto-Oncogénicas c-met/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Piel/citologíaRESUMEN
Expansion of multipotent, undifferentiated and proliferating cord blood (CB)-hematopoietic stem cells (HSC) in vitro is limited and insufficient. Bone marrow (BM) engineering in vitro allows mimicking the main components of the hematopoietic niche compared to conventional expansion strategies. In this study, four different 3D biomaterial scaffolds (PCL, PLGA, fibrin and collagen) were tested for freshly isolated cord blood (CB)-CD34(+) cell expansion in presence of (i) efficient exogenous cytokine supplementation and (ii) umbilical cord (UC)-mesenchymal stem cells (MSC). Cell morphology, growth and proliferation were analyzed in vitro as well as multi-organ engraftment and multilineage differentiation in a murine transplantation model. All scaffolds, except 3D PLGA meshes, supported CB-CD34(+) cell expansion, which was additionally stimulated by UC-MSC support. CB-CD34(+) cells cultured on human-derived 3D fibrin scaffolds with UC-MSC support i) reached the highest overall growth (5 × 10(8)-fold expansion of total nuclear cells after fourteen days and 3 × 10(7)-fold expansion of CD34(+) cells after seven days, p < 0.001), ii) maintained a more primitive immunophenotype for more cell divisions, iii) exhibited superior morphological, migratory and adhesive properties, and iv) showed the significantly highest numbers of engraftment and multilineage differentiation (CD45, CD34, CD13, CD3 and CD19) in BM, spleen and peripheral blood in long-term transplanted NSG mice compared to the other 3D biomaterial scaffolds. Thus, the 3D fibrin scaffold based BM-mimicry strategy reveals optimal requirements for translation into clinical protocols for CB expansion and transplantation.
Asunto(s)
Sangre Fetal/citología , Fibrina/química , Células Madre Hematopoyéticas/citología , Andamios del Tejido/química , Animales , Antígenos CD34/biosíntesis , Materiales Biocompatibles , Células de la Médula Ósea/citología , Proliferación Celular , Trasplante de Células/métodos , Células Cultivadas/citología , Citocinas/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía Electrónica de Rastreo/métodos , Fenotipo , Ingeniería de Tejidos/métodos , Cordón Umbilical/citologíaRESUMEN
OBJECTIVE: Inflammatory leukocyte accumulation drives atherosclerosis. Although monocytes/macrophages and polymorphonuclear neutrophilic leukocytes (PMN) contribute to lesion formation, sequelae of myeloproliferative disease remain to be elucidated. METHODS AND RESULTS: We used mice deficient in interferon regulatory factor 8 (IRF8(-/-)) in hematopoietic cells that develop a chronic myelogenous leukemia-like phenotype. Apolipoprotein E-deficient mice reconstituted with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow displayed an exacerbated atherosclerotic lesion formation compared with controls. The chronic myelogenous leukemia-like phenotype in mice with IRF8(-/-) bone marrow, reflected by an expansion of PMN in the circulation, was associated with an increased lesional accumulation and apoptosis of PMN, and enlarged necrotic cores. IRF8(-/-) compared with IRF8(+/+) PMN displayed unaffected reactive oxygen species formation and discharge of PMN granule components. In contrast, accumulating in equal numbers at sites of inflammation, IRF8(-/-) macrophages were defective in efferocytosis, lipid uptake, and interleukin-10 cytokine production. Importantly, depletion of PMN in low-density lipoprotein receptor or apolipoprotein E-deficient mice with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow abrogated increased lesion formation. CONCLUSIONS: These findings indicate that a chronic myelogenous leukemia-like phenotype contributes to accelerated atherosclerosis in mice. Among proatherosclerotic effects of other cell types, this, in part, is linked to an expansion of functionally intact PMN.
Asunto(s)
Aterosclerosis/etiología , Factores Reguladores del Interferón/fisiología , Animales , Apolipoproteínas E/fisiología , Apoptosis , Trasplante de Médula Ósea , Permeabilidad Capilar , Femenino , Interleucina-10/biosíntesis , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/fisiología , Peroxidasa/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptores de LDL/fisiologíaRESUMEN
Iron oxide-based magnetic nanoparticles (MNPs) offer unique properties for cell tracking by magnetic resonance imaging (MRI) in cellular immunotherapy. In this study, we investigated the uptake of chemically engineered NPs into antigen-presenting dendritic cells (DCs). DCs are expected to perceive MNPs as foreign antigens, thus exhibiting the capability to immunologically sense MNP surface chemistry. To systematically evaluate cellular uptake and T2/T2(â) MR imaging properties of MNPs, we synthesized polymer-based MNPs by employing layer-by-layer (LbL) technology. Thereby, we achieved modification of particle shell parameters, such as size, surface charge, and chemistry. We found that subcellular packaging of MNPs rather than MNP content in DCs influences MR imaging quality. Increased local intracellular electron density as inferred from transmission electron microscopy (TEM) strongly correlated with enhanced contrast in MRI. Thus, LbL-tailoring of MNP shells using polyelectrolytes that impact on uptake and subcellular localization can be used for modulating MR imaging properties. FROM THE CLINICAL EDITOR: In this study, layer-by-layer tailoring of magnetic NP shells was performed using polyelectrolytes to improve uptake by dendritic cells for cell-specific MR imaging. The authors conclude that polyelectrolyte modified NP-s can be used for modulating improving MR imaging quality by increasing subcellular localization.
Asunto(s)
Células Dendríticas/citología , Compuestos Férricos/química , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita , Animales , Supervivencia Celular/efectos de los fármacos , Rastreo Celular , Células Cultivadas , Células Madre Hematopoyéticas/citología , Nanopartículas de Magnetita/administración & dosificación , Nanopartículas de Magnetita/química , Ratones , Microscopía Electrónica de Transmisión , Coloración y EtiquetadoRESUMEN
The low yield of hematopoietic progenitor cells (HPC) present in cord blood grafts limits their application in clinics. A reliable strategy for ex vivo expansion of functional HPC is a present goal in regenerative medicine. Here we evaluate the capacity of several two-dimensional polymers to support HPC proliferation. Basic compatibility was tested by measuring cell viability, cytotoxicity and apoptosis of CD34(+) progenitors that were short and long-term exposed to sixteen bio and synthetic polymers. Resomer(®) RG503, PCL and Fibrin might be good alternatives to tissue culture plastic for culture of CB-derived CD34(+) progenitors. Further, these polymers will be produced in three-dimensional structures and tested for their cytocompatibility.