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1.
Nat Immunol ; 21(5): 525-534, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32313246

RESUMEN

Multiple sclerosis (MS) is characterized by pathological inflammation that results from the recruitment of lymphoid and myeloid immune cells from the blood into the brain. Due to subset heterogeneity, defining the functional roles of the various cell subsets in acute and chronic stages of MS has been challenging. Here, we used index and transcriptional single-cell sorting to characterize the mononuclear phagocytes that infiltrate the central nervous system from the periphery in mice with experimentally induced autoimmune encephalomyelitis, a model of MS. We identified eight monocyte and three dendritic cell subsets at acute and chronic disease stages in which the defined transcriptional programs pointed toward distinct functions. Monocyte-specific cell ablation identified Cxcl10+ and Saa3+ monocytic subsets with a pathogenic potential. Transfer experiments with different monocyte and precursor subsets indicated that these Cxcl10+ and Saa3+ pathogenic cells were not derived from Ly6C+ monocytes but from early myeloid cell progenitors. These results suggest that blocking specific pathogenic monocytic subsets, including Cxcl10+ and Saa3+ monocytes, could be used for targeted therapeutic interventions.


Asunto(s)
Células Dendríticas/fisiología , Encefalomielitis Autoinmune Experimental/inmunología , Monocitos/fisiología , Esclerosis Múltiple/inmunología , Fagocitos/fisiología , Animales , Autoinmunidad , Diferenciación Celular , Células Cultivadas , Sistema Nervioso Central , Quimiocina CXCL10/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inflamación Neurogénica , Proteína Amiloide A Sérica/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/genética
4.
Nat Immunol ; 19(6): 636-644, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29777220

RESUMEN

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain's macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the 'RiboTag' method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.


Asunto(s)
Microglía , ARN Mensajero/análisis , Análisis de Secuencia de ARN/métodos , Animales , Inmunoprecipitación/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ribosomas
5.
Immunity ; 54(1): 176-190.e7, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33333014

RESUMEN

The developmental and molecular heterogeneity of tissue macrophages is unravelling, as are their diverse contributions to physiology and pathophysiology. Moreover, also given tissues harbor macrophages in discrete anatomic locations. Functional contributions of specific cell populations can in mice be dissected using Cre recombinase-mediated mutagenesis. However, single promoter-based Cre models show limited specificity for cell types. Focusing on macrophages in the brain, we establish here a binary transgenic system involving complementation-competent NCre and CCre fragments whose expression is driven by distinct promoters: Sall1ncre: Cx3cr1ccre mice specifically target parenchymal microglia and compound transgenic Lyve1ncre: Cx3cr1ccre animals target vasculature-associated macrophages, in the brain, as well as other tissues. We imaged the respective cell populations and retrieved their specific translatomes using the RiboTag in order to define them and analyze their differential responses to a challenge. Collectively, we establish the value of binary transgenesis to dissect tissue macrophage compartments and their functions.


Asunto(s)
Encéfalo/citología , Sistema Nervioso Central/fisiología , Integrasas/metabolismo , Macrófagos/fisiología , Microglía/fisiología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Especificidad de Órganos
6.
Immunity ; 53(5): 1033-1049.e7, 2020 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-33049219

RESUMEN

Microglia, the resident macrophages of the brain parenchyma, are key players in central nervous system (CNS) development, homeostasis, and disorders. Distinct brain pathologies seem associated with discrete microglia activation modules. How microglia regain quiescence following challenges remains less understood. Here, we explored the role of the interleukin-10 (IL-10) axis in restoring murine microglia homeostasis following a peripheral endotoxin challenge. Specifically, we show that lipopolysaccharide (LPS)-challenged mice harboring IL-10 receptor-deficient microglia displayed neuronal impairment and succumbed to fatal sickness. Addition of a microglial tumor necrosis factor (TNF) deficiency rescued these animals, suggesting a microglia-based circuit driving pathology. Single cell transcriptome analysis revealed various IL-10 producing immune cells in the CNS, including most prominently Ly49D+ NK cells and neutrophils, but not microglia. Collectively, we define kinetics of the microglia response to peripheral endotoxin challenge, including their activation and robust silencing, and highlight the critical role of non-microglial IL-10 in preventing deleterious microglia hyperactivation.


Asunto(s)
Endotoxinas/inmunología , Interleucina-10/metabolismo , Microglía/inmunología , Microglía/metabolismo , Animales , Biomarcadores , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Inmunofenotipificación , Interleucina-10/genética , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Lipopolisacáridos/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones
7.
Immunity ; 46(6): 1030-1044.e8, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28636953

RESUMEN

Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.


Asunto(s)
Hipocampo/metabolismo , MicroARNs/genética , Microglía/fisiología , Neuronas/fisiología , Ribonucleasa III/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Reparación del ADN , Femenino , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Humanos , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/metabolismo , Actividad Motora , Plasticidad Neuronal , Ribonucleasa III/genética
8.
Eur J Immunol ; 50(3): 353-362, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31762013

RESUMEN

Conditional mutagenesis and fate mapping have contributed considerably to our understanding of physiology and pathology. Specifically, Cre recombinase-based approaches allow the definition of cell type-specific contributions to disease development and of inter-cellular communication circuits in respective animal models. Here we compared Cx3 cr1CreER and Sall1CreER transgenic mice and their use to decipher the brain macrophage compartment as a showcase to discuss recent technological advances. Specifically, we highlight the need to define the accuracy of Cre recombinase expression, as well as strengths and pitfalls of these particular systems that should be taken into consideration when applying these models.


Asunto(s)
Encéfalo , Integrasas , Macrófagos , Ratones Transgénicos , Modelos Animales , Animales , Ratones , Factores de Transcripción
9.
Eur J Immunol ; 48(8): 1308-1318, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29697861

RESUMEN

Microglia are resident immune cells in the CNS, strategically positioned to clear dead cells and debris, and orchestrate CNS inflammation and immune defense. In steady state, these macrophages lack MHC class II (MHCII) expression, but microglia activation can be associated with MHCII induction. Whether microglial MHCII serves antigen presentation for critical local T-cell restimulation in CNS auto-immune disorders or modulates microglial signaling output remains under debate. To probe for such scenarios, we generated mice harboring an MHCII deficiency in microglia, but not peripheral myeloid cells. Using the CX3 CR1CreER -based approach we report that microglial antigen presentation is obsolete for the establishment of EAE, with disease onset, progression, and severity unaltered in mutant mice. Antigen presentation-independent roles of microglial MHCII were explored using a demyelination model induced by the copper chelator cuprizone. Absence of microglial I-Ab did not affect the extent of these chemically induced white matter alterations, nor did it affect microglial proliferation or gene expression associated with locally restricted de- and remyelination.


Asunto(s)
Presentación de Antígeno/inmunología , Enfermedades Desmielinizantes/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos/inmunología , Microglía/inmunología , Animales , Proliferación Celular , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología
10.
Trends Immunol ; 36(10): 614-624, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26431939

RESUMEN

Microglia are macrophages of the central nervous system (CNS) that continuously scrutinize their environment for damage. They colonize the cephalic mesenchyme during embryogenesis and actively shape the developing neuronal network by immune-mediated mechanisms. Upon CNS maturation, microglia drastically change phenotype and function. During health, adult microglia contribute to homeostasis, but also the establishment and resolution of inflammatory conditions. Fulfillment of these distinct tasks requires these long-lived cells to accurately adjust to their changing environment. Deciphering microglia responsiveness to divergent stimuli is central to understanding this cell type and for eventual microglia manipulation to potentially reduce disease burden. Here we discuss new aspects of myeloid cell biology in general with special emphasis on the shifting role of microglia during establishment and protection of CNS integrity.


Asunto(s)
Microglía/fisiología , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Enfermedades del Sistema Nervioso Central , Homeostasis , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Neuroinmunomodulación
11.
J Immunother Cancer ; 10(5)2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35580925

RESUMEN

BACKGROUND: Soluble human leucocyte antigen (sHLA) molecules, released into the plasma, carry their original peptide cargo and provide insight into the protein synthesis and degradation schemes of their source cells and tissues. Other body fluids, such as pleural effusions, may also contain sHLA-peptide complexes, and can potentially serve as a source of tumor antigens since these fluids are drained from the tumor microenvironment. We explored this possibility by developing a methodology for purifying and analyzing large pleural effusion sHLA class I peptidomes of patients with malignancies or benign diseases. METHODS: Cleared pleural fluids, cell pellets present in the pleural effusions, and the primary tumor cells cultured from cancer patients' effusions, were used for immunoaffinity purification of the HLA molecules. The recovered HLA peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and the resulting LC-MS/MS data were analyzed with the MaxQuant software tool. Selected tumor antigen peptides were tested for their immunogenicity potential with donor peripheral blood mononuclear cells (PBMCs) in an in vitro assay. RESULTS: Mass spectrometry analysis of the pleural effusions revealed 39,669 peptides attributable to 11,305 source proteins. The majority of peptides identified from the pleural effusions were defined as HLA ligands that fit the patients' HLA consensus sequence motifs. The membranal and soluble HLA peptidomes of each individual patient correlated to each other. Additionally, soluble HLA peptidomes from the same patient, obtained at different visits to the clinic, were highly similar. Compared with benign effusions, the soluble HLA peptidomes of malignant pleural effusions were larger and included HLA peptides derived from known tumor-associated antigens, including cancer/testis antigens, lung-related proteins, and vascular endothelial growth factor pathway proteins. Selected tumor-associated antigens that were identified by the immunopeptidomics were able to successfully prime CD8+ T cells. CONCLUSIONS: Pleural effusions contain sHLA-peptide complexes, and the pleural effusion HLA peptidome of patients with malignant tumors can serve as a rich source of biomarkers for tumor diagnosis and potential candidates for personalized immunotherapy.


Asunto(s)
Antígenos de Neoplasias , Derrame Pleural Maligno , Linfocitos T CD8-positivos , Cromatografía Liquida , Antígenos de Histocompatibilidad Clase I , Humanos , Leucocitos Mononucleares , Masculino , Péptidos , Espectrometría de Masas en Tándem , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular
12.
Elife ; 92020 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-31916932

RESUMEN

Monocytes are circulating short-lived macrophage precursors that are recruited on demand from the blood to sites of inflammation and challenge. In steady state, classical monocytes give rise to vasculature-resident cells that patrol the luminal side of the endothelium. In addition, classical monocytes feed macrophage compartments of selected organs, including barrier tissues, such as the skin and intestine, as well as the heart. Monocyte differentiation under conditions of inflammation has been studied in considerable detail. In contrast, monocyte differentiation under non-inflammatory conditions remains less well understood. Here we took advantage of a combination of cell ablation and precursor engraftment to investigate the generation of gut macrophages from monocytes. Collectively, we identify factors associated with the gradual adaptation of monocytes to tissue residency. Moreover, comparison of monocyte differentiation into the colon and ileum-resident macrophages revealed the graduated acquisition of gut segment-specific gene expression signatures.


Asunto(s)
Diferenciación Celular , Colon/fisiología , Íleon/fisiología , Macrófagos/metabolismo , Monocitos/citología , Animales , Ratones , Organismos Libres de Patógenos Específicos
13.
Nat Commun ; 9(1): 5206, 2018 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-30523248

RESUMEN

Microglia are yolk sac-derived macrophages residing in the parenchyma of brain and spinal cord, where they interact with neurons and other glial. After different conditioning paradigms and bone marrow (BM) or hematopoietic stem cell (HSC) transplantation, graft-derived cells seed the brain and persistently contribute to the parenchymal brain macrophage compartment. Here we establish that graft-derived macrophages acquire, over time, microglia characteristics, including ramified morphology, longevity, radio-resistance and clonal expansion. However, even after prolonged CNS residence, transcriptomes and chromatin accessibility landscapes of engrafted, BM-derived macrophages remain distinct from yolk sac-derived host microglia. Furthermore, engrafted BM-derived cells display discrete responses to peripheral endotoxin challenge, as compared to host microglia. In human HSC transplant recipients, engrafted cells also remain distinct from host microglia, extending our finding to clinical settings. Collectively, our data emphasize the molecular and functional heterogeneity of parenchymal brain macrophages and highlight potential clinical implications for HSC gene therapies aimed to ameliorate lysosomal storage disorders, microgliopathies or general monogenic immuno-deficiencies.


Asunto(s)
Encéfalo/metabolismo , Cromatina/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Macrófagos/metabolismo , Microglía/metabolismo , Transcriptoma , Animales , Encéfalo/citología , Proliferación Celular/genética , Cromatina/metabolismo , Femenino , Humanos , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/citología , Microglía/efectos de los fármacos
14.
J Exp Med ; 214(4): 905-917, 2017 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-28330904

RESUMEN

Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are up-regulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such as the spinal cord, during experimental autoimmune encephalomyelitis (EAE). In this study, using competitive in vitro and in vivo assays, we show that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte-autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes/macrophages, but not in microglia, delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6Chi effector monocytes. Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance, and function.


Asunto(s)
Inflamación/inmunología , Monocitos/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Supervivencia Celular , Encefalomielitis Autoinmune Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Microglía/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología
15.
Semin Immunopathol ; 37(6): 613-23, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26240063

RESUMEN

Macrophages can be of dual origin. Most tissue-resident macrophage compartments are generated before birth and subsequently maintain themselves independently from each other locally in healthy tissue. Under inflammatory conditions, these cells can however be complemented by macrophages derived from acute monocyte infiltrates. Due to the lack of suitable experimental systems, differential functional contributions of central nervous system (CNS)-resident microglia and monocyte-derived macrophages (MoMF) to CNS inflammation, such as experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis (MS), remain poorly understood. Here, we will review recent progress in this field that suggest distinct roles of microglia and MoMF in disease induction and progression, capitalizing on novel transgenic mouse models. The latter finding could have major implications for the rationale development of therapeutic approaches to the management of brain inflammation and MS therapy.


Asunto(s)
Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Macrófagos/inmunología , Microglía/inmunología , Monocitos/inmunología , Animales , Presentación de Antígeno/inmunología , Sistema Nervioso Central/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Inflamación/inmunología , Ratones , Fagocitosis/inmunología
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