RESUMEN
Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvß3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvß3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvß3 interaction enhances Src recruitment to integrin αvß3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvß3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvß3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvß3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Angiopoyetinas/metabolismo , Permeabilidad Capilar/fisiología , Integrina alfaVbeta3/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/deficiencia , Animales , Hipoxia de la Célula/fisiología , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/fisiopatología , Humanos , Ratones Noqueados , Fosforilación/fisiología , Retina/metabolismo , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. METHODS AND RESULTS: We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. CONCLUSIONS: These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.
Asunto(s)
Angiopoyetinas/uso terapéutico , Endotelio Vascular/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/prevención & control , Fenómeno de no Reflujo/metabolismo , Fenómeno de no Reflujo/prevención & control , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/deficiencia , Animales , Cardiotónicos/metabolismo , Cardiotónicos/uso terapéutico , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Infarto del Miocardio/patología , Miocitos Cardíacos/metabolismo , Conejos , Distribución AleatoriaRESUMEN
Proper vessel maturation, remodeling of endothelial junctions, and recruitment of perivascular cells is crucial for establishing and maintaining vessel functions. In proliferative retinopathies, hypoxia-induced angiogenesis is associated with disruption of the vascular barrier, edema, and vision loss. Therefore, identifying factors that regulate vascular maturation is critical to target pathological angiogenesis. Given the conflicting role of angiopoietin-like-4 (ANGPTL4) reported in the current literature using gain of function systems both in vitro and in vivo, the goal of this study was to characterize angiogenesis, focusing on perinatal retinal vascularization and pathological circumstances in angpl4-deficient mice. We report altered organization of endothelial junctions and pericyte coverage, both leading to impaired angiogenesis and increased vascular leakage that were eventually caught up, suggesting a delay in vessel maturation. In a model of oxygen-induced retinopathy, pathological neovascularization, which results from tissue hypoxia, was also strongly inhibited in angptl4-deficient mice. This study therefore shows that ANGPTL4 tunes endothelial cell junction organization and pericyte coverage and controls vascular permeability and angiogenesis, both during development and in pathological conditions.
Asunto(s)
Angiopoyetinas/metabolismo , Células Endoteliales/metabolismo , Uniones Intercelulares/metabolismo , Neovascularización Patológica/embriología , Pericitos/metabolismo , Retina/embriología , Neovascularización Retiniana/embriología , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/genética , Animales , Permeabilidad Capilar/genética , Células Endoteliales/patología , Hipoxia/inducido químicamente , Hipoxia/embriología , Hipoxia/genética , Hipoxia/patología , Uniones Intercelulares/genética , Uniones Intercelulares/patología , Ratones , Ratones Mutantes , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Oxígeno/toxicidad , Pericitos/patología , Retina/patología , Neovascularización Retiniana/inducido químicamente , Neovascularización Retiniana/genética , Neovascularización Retiniana/patologíaRESUMEN
CD4+ T follicular helper (Tfh) cells are essential for initiating and regulating efficient humoral responses in secondary lymphoid organs. Tfh polarization and differentiation is driven by multiple stimuli delivered by antigen presenting cells (APCs). APCs represent a complex population of immune cells, comprising several subpopulations (dendritic cells and macrophages) that are distinguished by their phenotype, ontogeny, and functions. In order to better identify and understand the role of the different APC subsets in human Tfh biology, we have used in vitro assays based on the co-culture of APCs and T cells. This chapter describes two complementary protocols. The first protocol describes an assay to study the capacity of APCs to drive Tfh polarization from naive CD4+ T cells. The second protocol is designed to address the role of APCs in modulating effector functions of mature Tfh cells.
Asunto(s)
Células Presentadoras de Antígenos , Diferenciación Celular , Humanos , Recuento de Linfocitos , Macrófagos , Subgrupos de Linfocitos T , Linfocitos T Colaboradores-InductoresRESUMEN
CD4+ T follicular helper (Tfh) cells are essential for inducing efficient humoral responses. T helper polarization is classically orientated by dendritic cells (DCs), which are composed of several subpopulations with distinct functions. Whether human DC subsets display functional specialization for Tfh polarization remains unclear. Here we find that tonsil cDC2 and CD14+ macrophages are the best inducers of Tfh polarization. This ability is intrinsic to the cDC2 lineage but tissue dependent for macrophages. We further show that human Tfh cells comprise two effector states producing either IL-21 or CXCL13. Distinct mechanisms drive the production of Tfh effector molecules, involving IL-12p70 for IL-21 and activin A and TGFß for CXCL13. Finally, using imaging mass cytometry, we find that tonsil CD14+ macrophages localize in situ in the B cell follicles, where they can interact with Tfh cells. Our results indicate that human lymphoid organ cDC2 and macrophages play complementary roles in the induction of Tfh responses.
Asunto(s)
Tejido Linfoide/inmunología , Macrófagos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Polaridad Celular , Quimiocina CXCL13/metabolismo , Células Dendríticas , Humanos , Interleucinas/metabolismo , Receptores de Lipopolisacáridos/inmunología , Tejido Linfoide/citología , Subgrupos de Linfocitos TRESUMEN
Presentation of exogenous antigens on MHC-I molecules, termed cross-presentation, is essential for cytotoxic CD8+ T cell responses. In mice, dendritic cells (DCs) that arise from monocytes (mo-DCs) during inflammation have a key function in these responses by cross-presenting antigens locally in peripheral tissues. Whether human naturally-occurring mo-DCs can cross-present is unknown. Here, we use human mo-DCs and macrophages directly purified from ascites to address this question. Single-cell RNA-seq data show that ascites CD1c+ DCs contain exclusively monocyte-derived cells. Both ascites mo-DCs and monocyte-derived macrophages cross-present efficiently, but are inefficient for transferring exogenous proteins into their cytosol. Inhibition of cysteine proteases, but not of proteasome, abolishes cross-presentation in these cells. We conclude that human monocyte-derived cells cross-present exclusively using a vacuolar pathway. Finally, only ascites mo-DCs provide co-stimulatory signals to induce effector cytotoxic CD8+ T cells. Our findings thus provide important insights on how to harness cross-presentation for therapeutic purposes.
Asunto(s)
Presentación de Antígeno , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Vacuolas/metabolismo , Antígenos CD1/metabolismo , Ascitis/etiología , Ascitis/inmunología , Capa Leucocitaria de la Sangre/citología , Técnicas de Cultivo de Célula , Células Cultivadas , Citosol/metabolismo , Células Dendríticas/metabolismo , Femenino , Perfilación de la Expresión Génica , Glicoproteínas/metabolismo , Voluntarios Sanos , Humanos , Macrófagos/metabolismo , Masculino , Monocitos/metabolismo , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/inmunología , Tonsila Palatina/citología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Vacuolas/inmunologíaRESUMEN
T follicular helper cells (Tfh) are important regulators of humoral responses. Human Tfh polarization pathways have been thus far associated with Th1 and Th17 polarization pathways. How human Tfh cells differentiate in Th2-skewed environments is unknown. We show that thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DCs) promote human Tfh differentiation from naive CD4 T cells. We identified a novel population, distinct from Th2 cells, expressing IL-21 and TNF, suggestive of inflammatory cells. TSLP-induced T cells expressed CXCR5, CXCL13, ICOS, PD1, BCL6, BTLA, and SAP, among other Tfh markers. Functionally, TSLP-DC-polarized T cells induced IgE secretion by memory B cells, and this depended on IL-4Rα. TSLP-activated DCs stimulated circulating memory Tfh cells to produce IL-21 and CXCL13. Mechanistically, TSLP-induced Tfh differentiation depended on OX40-ligand, but not on ICOS-ligand. Our results delineate a pathway of human Tfh differentiation in Th2 environments.
Asunto(s)
Citocinas/fisiología , Células Dendríticas/fisiología , Ligando OX40/fisiología , Células Th2/fisiología , Diferenciación Celular/fisiología , Quimiocina CXCL13/metabolismo , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Interleucinas/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Receptores CXCR5/metabolismo , Receptores Inmunológicos/metabolismo , Linfopoyetina del Estroma TímicoRESUMEN
Dendritic cells (DCs) are a rare population of antigen-presenting cells that initiate immune responses in secondary lymphoid organs. In order to better understand the properties of DC in humans, it is essential to analyze DC subsets directly purified from tissues. Here, we describe a protocol allowing the purification of DC subsets from human tonsils and human lymph nodes.
Asunto(s)
Células Dendríticas/citología , Ganglios Linfáticos/citología , Tonsila Palatina/citología , Separación Celular , Citometría de Flujo , HumanosRESUMEN
Dendritic cells (DCs) initiate and orient immune responses and comprise several subsets that display distinct phenotypes and properties. Most of our knowledge of DC subsets biology is based on mouse studies. In the past few years, the alignment of the human DC network with the mouse DC network has been the focus of much attention. Although comparative phenotypic and transcriptomic analysis have shown a high level of homology between mouse and human DC subsets, significant differences in phenotype and function have also been evidenced. Here, we review recent advances in our understanding of the human DC network and discuss some remaining gaps and future challenges of the human DC field.
RESUMEN
Dendritic cells (DCs) represent a heterogeneous population of antigen-presenting cells that initiate and orient immune responses in secondary lymphoid organs. In mice, lymphoid organ-resident CD8(+) DCs are specialized at cross-presentation and have developed specific adaptations of their endocytic pathway (high pH, low degradation, and high export to the cytosol). In humans, blood BDCA3(+) DCs were recently shown to be the homologues of mouse CD8(+) DCs. They were also proposed to cross-present antigens more efficiently than other blood DC subsets after in vitro activation, suggesting that in humans cross-presentation is restricted to certain DC subsets. The DCs that cross-present antigen physiologically, however, are the ones present in lymphoid organs. Here, we show that freshly isolated tonsil-resident BDCA1(+) DCs, BDCA3(+) DCs, and pDCs all cross-present soluble antigen efficiently, as compared to macrophages, in the absence of activation. In addition, BDCA1(+) and BDCA3(+) DCs display similar phagosomal pH and similar production of reactive oxygen species in their phagosomes. All three DC subsets, in contrast to macrophages, also efficiently export internalized proteins to the cytosol. We conclude that all freshly isolated lymphoid organ-resident human DCs, but not macrophages, display high intrinsic cross-presentation capacity.
Asunto(s)
Presentación de Antígeno/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Tejido Linfoide/citología , Fagocitos/inmunología , Animales , Antígenos CD1/inmunología , Separación Celular , Citosol/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Antígeno MART-1/inmunología , Ratones , Necrosis , Tonsila Palatina/citología , Fagosomas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Solubilidad , Receptores Toll-Like/inmunologíaRESUMEN
BACKGROUND: Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI. METHODS AND FINDINGS: Using a genetic model of tsp-1(-/-) mice subjected to femoral artery excision, we report that tsp-1(-/-) mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1(-/-) and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1(-/-) mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1(-/-) mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1(-/-) mice, thereby demonstrating that macrophages mediated tissue protection in these mice. CONCLUSION: This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.