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Macrophages fight infection and ensure tissue repair, often operating at nutrient-poor wound sites. We investigated the ability of human macrophages to metabolize glycogen. We observed that the cytokines GM-CSF and M-CSF plus IL-4 induced glycogenesis and the accumulation of glycogen by monocyte-derived macrophages. Glyconeogenesis occurs in cells cultured in the presence of the inflammatory cytokines GM-CSF and IFNγ (M1 cells), via phosphoenolpyruvate carboxykinase 2 (PCK2) and fructose-1,6-bisphosphatase 1 (FBP1). Enzyme inhibition with drugs or gene silencing techniques and 13C-tracing demonstrate that glutamine (metabolized by the TCA cycle), lactic acid, and glycerol were substrates of glyconeogenesis only in M1 cells. Tumor-associated macrophages (TAMs) also store glycogen and can perform glyconeogenesis. Finally, macrophage glycogenolysis and the pentose phosphate pathway (PPP) support cytokine secretion and phagocytosis regardless of the availability of extracellular glucose. Thus, glycogen metabolism supports the functions of human M1 and M2 cells, with inflammatory M1 cells displaying a possible dependence on glyconeogenesis.
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Rationale: Extracellular histones, released into the surrounding environment during extensive cell death, promote inflammation and cell death, and these deleterious roles have been well documented in sepsis. Clusterin (CLU) is a ubiquitous extracellular protein that chaperones misfolded proteins and promotes their removal. Objectives: We investigated whether CLU could protect against the deleterious properties of histones. Methods: We assessed CLU and histone expression in patients with sepsis and evaluated the protective role of CLU against histones in in vitro assays and in vivo models of experimental sepsis. Measurements and Main Results: We show that CLU binds to circulating histones and reduces their inflammatory, thrombotic, and cytotoxic properties. We observed that plasma CLU levels decreased in patients with sepsis and that the decrease was greater and more durable in nonsurvivors than in survivors. Accordingly, CLU deficiency was associated with increased mortality in mouse models of sepsis and endotoxemia. Finally, CLU supplementation improved mouse survival in a sepsis model. Conclusions: This study identifies CLU as a central endogenous histone-neutralizing molecule and suggests that, in pathologies with extensive cell death, CLU supplementation may improve disease tolerance and host survival.
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Antineoplásicos , Sepsis , Animales , Ratones , Histonas/metabolismo , Clusterina/metabolismo , Inflamación , Muerte Celular , Sepsis/tratamiento farmacológicoRESUMEN
Microglia are the major myeloid-immune cells of the brain parenchyma. In a steady state, microglia monitor their environment for pathogens or damaged cells. In response to neural injury or inflammation, microglia become competent APCs able to prime CD4(+) and CD8(+) T lymphocytes. We previously demonstrated that neonatal and adult microglia cross-present exogenous soluble Ags in vitro. However, whether microglia are able to cross-present Ag to naive CD8(+) T cells in vivo, within the brain microenvironment, remains undetermined. Here, we have designed an original protocol in order to exclude the involvement in cross-presentation activity of peripheral migrating APCs and of CNS-associated APCs. In C57Bl/6 mice, in which the body but not the head has been properly irradiated, we analyzed the ability of resident microglia to stimulate intracerebrally injected CD8(+) T cells in vivo. This study demonstrates for the first time that adult microglia cross-present Ag to naive CD8(+) T cells in vivo and that full microglia activation is required to overcome the inhibitory constrains of the brain and to render microglia able to cross-prime naive CD8(+) T cells injected in the brain. These observations offer new insights in brain-tumor immunotherapy based on the induction of cytotoxic antitumoral T cells.
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Células Presentadoras de Antígenos/inmunología , Encéfalo/inmunología , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada , Microglía/inmunología , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/citología , Antígenos/inmunología , Encéfalo/citología , Linfocitos T CD8-positivos/trasplante , Rayos gamma , Inyecciones Intraventriculares , Ratones , Ratones Endogámicos C57BL , Microglía/citología , SolubilidadRESUMEN
Brain-gut axis refers to the bidirectional functional connection between the brain and the gut, which sustains vital functions for vertebrates. This connection also underlies the gastrointestinal (GI) comorbidities associated with brain disorders. Using a mouse model of glioma, based on the orthotopic injection of GL261 cell line in syngeneic C57BL6 mice, we show that late-stage glioma is associated with GI functional alteration and with a shift in the level of some bacterial metabolites in the cecum. By performing cecal content transfer experiments, we further show that cancer-associated alteration in cecal metabolites is involved in end-stage disease progression. Antibiotic treatment results in a slight but significant delay in mice death and a shift in the proportion of myeloid cells in the brain tumor environment. This work rationally considers microbiota modulating strategies in the clinical management of patients with late-stage glioma.
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SARS-CoV-2 coronavirus infection induces heterogeneous symptoms, ranging from asymptomatic to lethal forms. Severe forms usually occur in the elderly and/or individuals with comorbidities. Children generally remain asymptomatic to primary infection, suggesting that they may have an effective local innate immune response. IFN-I and -III have non-redundant protective roles against SARS-CoV-2, although sometimes damaging the host. The expression and role of anti-viral peptides during SARS-CoV-2 infection have thus far been little studied. We aimed to identify the innate immune molecules present at the SARS-CoV-2 entry point. We analyzed the mRNA levels of type I (IFN-α and -ß) and type III (IFN-λ1-3) interferons and selected antiviral peptides (i.e., ß-defensins 1-3, α-defensins [HNP1-3, HD5] pentraxin-3, surfactant protein D, the cathelicidin LL-37 and interleukin-26) in nasopharyngeal swabs from 226 individuals of various ages, either infected with SARS-CoV-2 (symptomatic or asymptomatic) or negative for the virus. We observed that infection induced selective upregulation of IFN-λ1 expression in pediatric subjects (≤15 years), whereas IFN-α, IFN-ß, IFN-λ2/λ3, and ß-defensin 1-3 expression was unaffected. Conversely, infection triggered upregulation of IFN-α, IFN-ß, IFN-λ2/λ3, and ß-defensin 1-3 mRNA expression in adults (15-65 years) and the elderly (≥ 65 years), but without modulation of IFN-λ1. The expression of these innate molecules was not associated with gender or symptoms. Expression of the interferon-stimulated genes IFITM1 and IFITM3 was upregulated in SARS-CoV-2-positive subjects and reached similar levels in the three age groups. Finally, age-related differences in nasopharyngeal innate immunity were also observed in SARS-CoV-2-negative subjects. This study shows that the expression patterns of IFN-I/-III and certain anti-viral molecules in the nasopharyngeal mucosa of SARS-CoV-2-infected subjects differ with age and suggests that susceptibility to SARS-CoV-2 may be related to intrinsic differences in the nature of mucosal anti-viral innate immunity.
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Factores de Restricción Antivirales/análisis , Interferón Tipo I/biosíntesis , Interferón gamma/biosíntesis , Mucosa Nasal/inmunología , SARS-CoV-2/inmunología , beta-Defensinas/biosíntesis , Adolescente , Adulto , Factores de Edad , Anciano , COVID-19/inmunología , Células Cultivadas , Femenino , Humanos , Inmunidad Innata/inmunología , Interferón Tipo I/inmunología , Interferón gamma/inmunología , Interferones/biosíntesis , Interferones/inmunología , Interleucinas/biosíntesis , Interleucinas/inmunología , Masculino , Persona de Mediana Edad , Nasofaringe/inmunología , Adulto Joven , beta-Defensinas/inmunología , Interferón lambdaRESUMEN
A controversial issue in neurobiology concerns the respective functions of central nervous system (CNS)-resident macrophages and systemic infiltrating macrophages morphologically and phenotypically similar during most of CNS injury processes. In a previous work, we isolated sixteen mRNAs differentially expressed between two microglial EOC clones. By studying their pattern of expression, we found that three of them were not expressed in peripheral macrophages, even after stimulation with IFNgamma, TNFalpha or IL10. These three molecules are physiologically expressed by murine adult microglia and could be used to evaluate in vivo their discriminative potential toward CNS-infiltrating macrophages during inflammatory events.
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Biomarcadores/metabolismo , Sistema Nervioso Central/citología , Activación de Macrófagos/fisiología , Macrófagos/metabolismo , Animales , Antígenos CD/clasificación , Antígenos CD/metabolismo , Northern Blotting/métodos , Células Cultivadas , ADN Complementario/metabolismo , Citometría de Flujo/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Interferón-alfa/farmacología , Interferón gamma/farmacología , Interleucina-10/farmacología , Ratones , Ratones Endogámicos C3H , Microglía/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
In this work, multifunctional lipid nanocapsules (M-LNC) were designed to combine the activity of the cytotoxic drug paclitaxel (PTX) with the immunostimulant CpG. This nanosystem, consisting of modified lipid nanocapsules coated with a cationic polymeric shell composed of chitosan (CS), was able to allocate the hydrophobic drug PTX in the inner oily core, and to associate onto the surface the genetic material CpG. The CS-coated LNC (CS-LNC), showed a narrow size distribution with an average size of 70 nm and a positive zeta potential (+25 mV). They encapsulated PTX in a high amount (98%), and, due to the cationic surface charge, were able to adsorb CpG without losing stability. As a preliminary in vitro study, the apoptotic effect on GL261 glioma cells was investigated. The drug-loaded CS-LNC exhibited the ability to interact with glioma cells and induce an important apoptotic effect in comparison with blank systems. Finally, the M-LNC made of CS-LNC loaded with both CpG and PTX were tested in vivo, injected via convention enhanced delivery (CED) in GL261-glioma-bearing mice. The results showed that the overall survival of mice treated with the M-LNC was significantly increased in comparison with the control, Taxol(®), or the separated injection of PTX-loaded LNC and CpG. This effect was also confirmed by magnetic resonance imaging (MRI) which revealed the reduction of tumor growth in the animals treated with CpG and PTX-loaded M-LNC. All these findings suggested that the developed M-LNC could potentiate both CpG immunopotency and PTX antitumor activity by enhancing its delivery into the tumor microenvironment.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Glioblastoma/tratamiento farmacológico , Lípidos/administración & dosificación , Nanocápsulas/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/farmacología , Paclitaxel/administración & dosificación , Paclitaxel/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Quitosano/química , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Lípidos/química , Imagen por Resonancia Magnética , Ratones , Nanocápsulas/químicaRESUMEN
Using brain lymphoma model, we demonstrate that immunotherapy combining Treg depletion (using anti-CD25 mAb PC61) followed by intracranial CpG-ODN administration induced tumor rejection in all treated mice and led to the establishment of a memory antitumor immune response in 60% of them. This protective effect was associated with a recruitment of NK cells and, to a lesser extent, of dendritic cells, B cells and T lymphocytes. NK cell depletion abolished the protective effect of the treatment, confirming a major role of NK cells in brain tumor elimination. Each treatment used alone failed to protect brain tumor bearing mice, revealing the therapeutic benefit of combining Treg depletion and local CpG-ODN injection.
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Adyuvantes Inmunológicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Oligodesoxirribonucleótidos/uso terapéutico , Linfocitos T Reguladores/fisiología , Animales , Anticuerpos/toxicidad , Antígenos Ly/inmunología , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Etopósido/farmacología , Etopósido/uso terapéutico , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/fisiología , Linfoma/complicaciones , Linfoma/metabolismo , Linfoma/patología , Trastornos de la Memoria/etiología , Trastornos de la Memoria/prevención & control , Ratones , Ratones Endogámicos C57BL , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Trasplante de Neoplasias , Linfocitos T Reguladores/efectos de los fármacos , Factores de Tiempo , Receptor Toll-Like 9/metabolismoRESUMEN
Some observations have suggested that cells from the central nervous system (CNS) could present exogenous antigens on major histocompatibility complex (MHC) class I molecules to CD8(+) T cells (a process called cross-presentation). Microglia are the major myeloid immunocompetent cells of the CNS. When activated, following the injury of the nervous parenchyma, they become fully competent antigen-presenting cells (APC) that prime CD4(+) T lymphocytes. We therefore tested the cross-presentation capacity of murine microglia. We report that a microglial cell line (C8-B4), neonatal microglia, and interestingly adult microglia cross-present soluble exogenous antigen (ovalbumin) to a OVA-specific CD8(+) T-cell hybridoma and cross-prime OVA-specific naive OT-1 CD8(+) T cells. In both these cases, C8-B4 and neonatal microglia cross-present OVA as well as peritoneal macrophages. Although cross-presentation by adult microglia is less efficient, it is increased by GM-CSF and CpG oligodeoxynucleotide (ODN) stimulation. Using microglial cells either exposed to an inhibitor of proteasome, lactacystin, or purified from TAP(-/-) mice, we demonstrate that the microglia cross-present antigen in proteasome- and TAP-dependant pathways, respectively. Last, microglia purified from adult mice injected intracerebrally with OVA efficiently stimulate OVA-specific CD8(+) T cells, thereby showing that microglia take up and process exogenous antigen into MHC class I in vivo. This first demonstration of the cross-presentation property of microglia offers novel therapeutic approaches to modulate CD8 T-cell responses in the brain.
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Envejecimiento/inmunología , Animales Recién Nacidos/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos/inmunología , Microglía/inmunología , Animales , Antígenos CD8/inmunología , Línea Celular , Células Cultivadas , Citometría de Flujo , Genes MHC Clase I/genética , Genes MHC Clase I/inmunología , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/inmunología , Fenotipo , Complejo de la Endopetidasa Proteasomal , Linfocitos T/inmunologíaRESUMEN
In recent years, a neuroimmunomodulatory role for 1,25-dihydroxyvitamine D(3) [1,25(OH)(2)D(3)] has emerged. Microglial cells present a potential target for the effects of this hormone in the brain. This study focuses on the effect of 1,25(OH)(2)D(3) on the expression and production of inflammatory cytokines and nitric oxide (NO) by the EOC13 microglial cell line. The presence of the vitamin D3 receptor in microglia was demonstrated by RT-PCR. 1,25(OH)(2)D(3) inhibited the production of tumor necrosis factor-alpha, interleukin-6, and NO by stimulated microglia in a concentration-related fashion. The production of transforming growth factor-beta1 (TGF-beta1), an anti-inflammatory cytokine, was not modified in the presence of 1,25(OH)(2)D(3), indicating that the effects of 1,25(OH)(2)D(3) may not involve TGF-beta1 regulation. These results show that 1,25(OH)(2)D(3) has direct anti-inflammatory properties on microglia. It further supports the hypothesis that 1,25(OH)(2)D(3) could be involved in the maintenance of the brain homeostasis and may have a therapeutic potential in inflammatory pathologies of the central nervous system.