RESUMEN
The cytokine transforming growth factor-ß (TGF-ß) regulates the development and homeostasis of several tissue-resident macrophage populations, including microglia. TGF-ß is not critical for microglia survival but is required for the maintenance of the microglia-specific homeostatic gene signature1,2. Under defined host conditions, circulating monocytes can compete for the microglial niche and give rise to long-lived monocyte-derived macrophages residing in the central nervous system (CNS)3-5. Whether monocytes require TGF-ß for colonization of the microglial niche and maintenance of CNS integrity is unknown. We found that abrogation of TGF-ß signaling in CX3CR1+ monocyte-derived macrophages led to rapid onset of a progressive and fatal demyelinating motor disease characterized by myelin-laden giant macrophages throughout the spinal cord. Tgfbr2-deficient macrophages were characterized by high expression of genes encoding proteins involved in antigen presentation, inflammation and phagocytosis. TGF-ß is thus crucial for the functional integration of monocytes into the CNS microenvironment.
Asunto(s)
Encéfalo/inmunología , Enfermedades Desmielinizantes/inmunología , Macrófagos/patología , Médula Espinal/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Transducción de Señal , Médula Espinal/metabolismo , Médula Espinal/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Our in silico drug screening reveals topoisomerase 1 (TOP1) inhibitors as promising drug candidates for microglial modulation. We show that TOP1 is highly expressed in neuroinflammatory conditions, and TOP1 inhibition using camptothecin (CPT) and its FDA-approved analog topotecan (TPT) reduces inflammatory responses in microglia/macrophages and ameliorates neuroinflammation in vivo. Transcriptomic analyses of sorted microglia from LPS-challenged mice reveal an altered transcriptional phenotype following TPT treatment. To target myeloid cells, we design a nanosystem using ß-glucan-coated DNA origami (MyloGami) loaded with TPT (TopoGami). MyloGami shows enhanced specificity to myeloid cells while preventing the degradation of the DNA origami scaffold. Myeloid-specific TOP1 inhibition using TopoGami significantly suppresses the inflammatory response in microglia and mitigates MS-like disease progression. Our findings suggest that TOP1 inhibition in myeloid cells represents a therapeutic strategy for neuroinflammatory diseases and that the myeloid-specific nanosystems we designed may also benefit the treatment of other diseases with dysfunctional myeloid cells.
Asunto(s)
Enfermedades Neuroinflamatorias , Inhibidores de Topoisomerasa I , Animales , ADN , Macrófagos , Ratones , Inhibidores de Topoisomerasa I/farmacología , Topotecan/farmacologíaRESUMEN
BACKGROUND: Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia. METHODS: Immunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies. RESULTS: We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre-Eyfp- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre-Eyfp- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1highCre-Eyfp- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre-Eyfp- microglia are Cx3cr1wt/wtCre-Eyfp-. Finally, we demonstrated that CX3CL1-CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro. CONCLUSIONS: Our results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.
Asunto(s)
Microglía , Transducción de Señal , Animales , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Ratones , Ratones Transgénicos , Microglía/metabolismoRESUMEN
Microglia are implicated in the pathophysiology of several neurodegenerative disorders, including Alzheimer's disease. While the role of microglia and peripheral macrophages in regulating amyloid beta pathology has been well characterized, the impact of these distinct cell subsets on tau pathology remains poorly understood. We and others have recently demonstrated that monocytes can engraft the brain and give rise to long-lived parenchymal macrophages, even under nonpathological conditions. We undertook the current study to investigate the regulation of tau pathology by microglia and peripheral macrophages using hTau transgenic mice, which do not exhibit microglial activation/pathology or macrophage engraftment. To assess the direct impact of microglia on tau pathology we developed a protocol for long-term microglial depletion in Cx3cr1CreER R26DTA mice and crossed them with hTau mice. We then depleted microglia up to 3 months in both young and old mice, but no net change in forebrain soluble oligomeric tau or total or phosphorylated levels of aggregated tau was recorded. To investigate the consequence of peripherally-derived parenchymal macrophages on tau aggregation we partially repopulated the hTau microglial pool with peripheral macrophages, but this also did not affect levels of tau oligomers or insoluble aggregates. Our study questions the direct involvement of microglia or peripheral macrophages in the development of tau pathology in the hTau model.
Asunto(s)
Enfermedad de Alzheimer/patología , Macrófagos/metabolismo , Microglía/metabolismo , Tauopatías/patología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Ratones Transgénicos , Microglía/patología , Monocitos/metabolismoRESUMEN
Microglia, predominant parenchymal resident macrophages in the central nervous system (CNS), are crucial players in neurodevelopment and CNS homeostasis. In disease conditions, pro-inflammatory microglia predominate over their regulatory counterparts, and are thus a potential immunotherapeutic target. It has been well documented that microglia can be effectively depleted using both conditional genetic Cx3cr1Cre-diphtheria toxin receptor (DTR)/diphtheria toxin subunit A (DTA) animal models and pharmacological colony-stimulating factor 1 receptor (CSF1R) inhibitors. Recent advances using these approaches have expanded our knowledge of the multitude of tasks conducted by microglia in both homeostasis and diseases. Importantly, experimental microglial depletion has been proven to exert neuroprotective effects in an increasing number of disease models, mostly explained by reduced neuroinflammation. However, the comprehensive effects of additional targets such as circulating monocytes and peripheral tissue macrophages during microglial depletion periods have not been investigated widely, and for those studies addressing the issue the conclusions are mixed. In this study, we demonstrate that experimental microglial depletion using both Cx3cr1CreER/+Rosa26DTA/+ mice and different doses of CSF1R inhibitor PLX3397 exert crucial influences on circulating monocytes and peripheral tissue macrophages. Our results suggest that effects on peripheral immunity should be considered both in interpretation of microglial depletion studies, and especially in the potential translation of microglial depletion and replacement therapies.
Asunto(s)
Macrófagos/metabolismo , Microglía/metabolismo , Fármacos Neuroprotectores/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Aminopiridinas/farmacología , Animales , Femenino , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Ratones , Ratones Transgénicos , Pirroles/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genéticaRESUMEN
Multiple sclerosis (MS) is a chronic neuroinflammatory disorder of the central nervous system (CNS) that usually presents in young adults and predominantly in females. Microglia, a major resident immune cell in the CNS, are critical players in both CNS homeostasis and disease. We have previously demonstrated that microglia can be efficiently depleted by the administration of tamoxifen in Cx3cr1CreER/+Rosa26DTA/+ mice, with ensuing repopulation deriving from both the proliferation of residual CNS resident microglia and the engraftment of peripheral monocyte-derived microglia-like cells. In this study, tamoxifen was administered to Cx3cr1CreER/+Rosa26DTA/+ and Cx3cr1CreER/+ female and male mice. Experimental autoimmune encephalomyelitis (EAE), a widely used animal model of MS, was induced by active immunization with myelin oligodendrocyte glycoprotein (MOG) one month after tamoxifen injections in Cx3cr1CreER/+Rosa26DTA/+ mice and Cx3cr1CreER/+ mice, a time point when the CNS niche was colonized by microglia derived from both CNS microglia and peripherally-derived macrophages. We demonstrate that engraftment of microglia-like cells following microglial depletion exacerbated EAE in Cx3cr1CreER/+Rosa26DTA/+ female mice as assessed by clinical symptoms and the expression of CNS inflammatory factors, but these findings were not evident in male mice. Higher major histocompatibility complex class II expression and cytokine production in the female CNS contributed to the sex-dependent EAE severity in mice following engraftment of microglia-like cells. An underestimated yet marked sex-dependent microglial activation pattern may exist in the injured CNS during EAE.
Asunto(s)
Sistema Nervioso Central/citología , Encefalomielitis Autoinmune Experimental/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Macrófagos/metabolismo , Microglía/citología , Monocitos/metabolismo , Esclerosis Múltiple/metabolismo , Glicoproteína Mielina-Oligodendrócito/inmunología , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/inmunología , Microglía/metabolismo , Monocitos/inmunología , Tamoxifeno/administración & dosificaciónRESUMEN
BACKGROUND: The advent of array-based genome-wide DNA methylation methods has enabled quantitative measurement of single CpG methylation status at relatively low cost and sample input. Whereas the use of Infinium Human Methylation BeadChips has shown great utility in clinical studies, no equivalent tool is available for rodent animal samples. We examined the feasibility of using the new Infinium MethylationEPIC BeadChip for studying DNA methylation in mouse. RESULTS: In silico, we identified 19,420 EPIC probes (referred as mEPIC probes), which align with a unique best alignment score to the bisulfite converted reference mouse genome mm10. Further annotation revealed that 85% of mEPIC probes overlapped with mm10.refSeq genes at different genomic features including promoters (TSS1500 and TSS200), 1st exons, 5'UTRs, 3'UTRs, CpG islands, shores, shelves, open seas and FANTOM5 enhancers. Hybridization of mouse samples to Infinium Human MethylationEPIC BeadChips showed successful measurement of mEPIC probes and reproducibility between inter-array biological replicates. Finally, we demonstrated the utility of mEPIC probes for data exploration such as hierarchical clustering. CONCLUSIONS: Given the absence of cost and labor convenient genome-wide technologies in the murine system, our findings show that the Infinium MethylationEPIC BeadChip platform is suitable for investigation of the mouse methylome. Furthermore, we provide the "mEPICmanifest" with genomic features, available to users of Infinium Human MethylationEPIC arrays for mouse samples.
Asunto(s)
Metilación de ADN , Ratones/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Islas de CpG , Genómica , Humanos , Reproducibilidad de los ResultadosRESUMEN
Intracerebral levels of Transforming Growth Factor beta (TGFß) rise rapidly during the onset of experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis (MS). We addressed the role of TGFß responsiveness in EAE by targeting the TGFß receptor in myeloid cells, determining that Tgfbr2 was specifically targeted in monocyte-derived dendritic cells (moDCs) but not in CNS resident microglia by using bone-marrow chimeric mice. TGFß responsiveness in moDCs was necessary for the remission phase since LysM(Cre) Tgfbr2(fl/fl) mice developed a chronic form of EAE characterized by severe demyelination and extensive infiltration of activated moDCs in the CNS. Tgfbr2 deficiency resulted in increased moDC IL-12 secretion that skewed T cells to produce IFN-γ, which in turn enhanced the production of moDC-derived reactive oxygen species that promote oxidative damage and demyelination. We identified SNPs in the human NOX2 (CYBB) gene that associated with the severity of MS, and significantly increased CYBB expression was recorded in PBMCs from both MS patients and from MS severity risk allele rs72619425-A carrying individuals. We thus identify a novel myeloid cell-T cell activation loop active in the CNS during chronic disease that could be therapeutically targeted. GLIA 2016;64:1925-1937.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Polaridad Celular/fisiología , Citocinas/metabolismo , Células Dendríticas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Células TH1/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Polaridad Celular/genética , Estudios de Cohortes , Citocinas/genética , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Regulación de la Expresión Génica/genética , Genotipo , Humanos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/citología , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/toxicidad , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/metabolismo , Polimorfismo de Nucleótido Simple/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Microglia are resident antigen-presenting cells in the central nervous system (CNS) that either suppress or promote disease depending on their activation phenotype and the microenvironment. Multiple sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the CNS, and experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate pathogenic mechanisms and therapeutic effects. We isolated and cultured microglia from adult mouse brains and exposed them to specific combinations of stimulatory molecules and cytokines, the combination of IL-4, IL-10, and TGF-ß yielding the optimal regime for induction of an immunosuppressive phenotype (M2). M2 microglia were characterized by decreased expression or production of CD86, PD-L1, nitric oxide, and IL-6, increased expression of PD-L2, and having a potent capacity to retain their phenotype on secondary proinflammatory stimulation. M2 microglia induced regulatory T cells, suppressed T-cell proliferation, and downmodulated M1-associated receptor expression in M1 macrophages. Myelin oligodendrocyte glycoprotein (MOG)-induced EAE was induced in DBA/1 mice and at different time points (0, 5, 12, or 15 days postimmunization) 3 × 105 M2 microglia were transferred intranasally. A single transfer of M2 microglia attenuated the severity of established EAE, which was particularly obvious when the cells were injected at 15 days postimmunization. M2 microglia-treated mice had reduced inflammatory responses and less demyelination in the CNS. Our findings demonstrate that adult M2 microglia therapy represents a novel intervention that alleviated established EAE and that this therapeutic principle may have relevance for treatment of MS patients.
Asunto(s)
Traslado Adoptivo/métodos , Citocinas/farmacología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Microglía/inmunología , Microglía/trasplante , Animales , Técnicas de Cocultivo , Femenino , Humanos , Inmunomodulación/inmunología , Ratones , Ratones Endogámicos DBA , Microglía/efectos de los fármacosRESUMEN
In dorsal root ganglia (DRG), macrophages reside close to sensory neurons and have largely been explored in the context of pain, nerve injury, and repair. However, we discovered that most DRG macrophages interact with and monitor the vasculature by sampling macromolecules from the blood. Characterization of the DRG vasculature revealed a specialized endothelial bed that transformed in molecular, structural, and permeability properties along the arteriovenous axis and was covered by macrophage-interacting pericytes and fibroblasts. Macrophage phagocytosis spatially aligned with peak endothelial permeability, a process regulated by enhanced caveolar transcytosis in endothelial cells. Profiling the DRG immune landscape revealed two subsets of perivascular macrophages with distinct transcriptome, turnover, and function. CD163+ macrophages self-maintained locally, specifically participated in vasculature monitoring, displayed distinct responses during peripheral inflammation, and were conserved in mouse and man. Our work provides a molecular explanation for the permeability of the blood-DRG barrier and identifies an unappreciated role of macrophages as integral components of the DRG-neurovascular unit.
Asunto(s)
Células Endoteliales , Ganglios Espinales , Humanos , Macrófagos , Pericitos , PermeabilidadRESUMEN
BACKGROUND: The dorsal root ganglion (DRG) is structurally complex and pivotal to systems processing nociception. Whole mount analysis allows examination of intricate microarchitectural and cellular relationships of the DRG in three-dimensional (3D) space. NEW METHOD: We present DRGquant a set of tools and techniques optimized as a pipeline for automated image analysis and reconstruction of cells/structures within the DRG. We have developed an open source software pipeline that utilizes machine learning to identify substructures within the DRG and reliably classify and quantify them. RESULTS: Our methods were sufficiently sensitive to isolate, analyze, and classify individual DRG substructures including macrophages. The activation of macrophages was visualized and quantified in the DRG following intrathecal injection of lipopolysaccharide, and in a model of chemotherapy induced peripheral neuropathy. The percent volume of infiltrating macrophages was similar to a commercial source in quantification. Circulating fluorescent dextran was visualized within DRG macrophages using whole mount preparations, which enabled 3D reconstruction of the DRG and DRGquant demonstrated subcellular spatial resolution within individual macrophages. COMPARISON WITH EXISTING METHOD(S): Here we describe a reliable and efficient methodologic pipeline to prepare cleared and whole mount DRG tissue. DRGquant allows automated image analysis without tedious manual gating to reduce bias. The quantitation of DRG macrophages was superior to commercial solutions. CONCLUSIONS: Using machine learning to separate signal from noise and identify individual cells, DRGquant enabled us to isolate individual structures or areas of interest within the DRG for a more granular and fine-tuned analysis. Using these 3D techniques, we are better able to appreciate the biology of the DRG under experimental inflammatory conditions.
Asunto(s)
Ganglios Espinales , Macrófagos , Procesamiento de Imagen Asistido por Computador/métodos , Lipopolisacáridos , Aprendizaje AutomáticoRESUMEN
Hippuric acid is synthesized and produced primarily by the gastrointestinal (GI) microflora. However, there is no known health benefit for hippuric acid except its catabolic conjugation of benzene-type compounds via glycine and subsequent excretion in the urine. For years the GI tract microflora were known to metabolize quinic acid to hippuric acid. Recently it was also proposed that DNA repair was strongly enhanced by quinic acid. In order to explain these quinic acid effects, Pero and colleagues have examined whether tryptophan and nicotinamide were also enhanced by quinic acid levels in urine. They were indeed, and so another study was designed using a natural supplement source of quinic acid called AIO + AC-11®, and then the effects of intervention were measured after only 21 days. It was possible to show profound increases in quinic acid that were again paralleled by increases in tryptophan and nicotinamide urinary levels. Because the high pressure liquid chromatography (HPLC) methods differed greatly between the two studies, differences in chemical analyses probably did not contribute to the data base.
Asunto(s)
Niacinamida/orina , Ácido Quínico/administración & dosificación , Ácido Quínico/orina , Triptófano/orina , Adolescente , Adulto , Anciano , Antioxidantes/administración & dosificación , Antioxidantes/farmacocinética , Suplementos Dietéticos , Femenino , Hipuratos/metabolismo , Humanos , Estilo de Vida , Masculino , Persona de Mediana Edad , Niacinamida/biosíntesis , Extractos Vegetales/administración & dosificación , Extractos Vegetales/farmacocinética , Ácido Quínico/farmacocinética , Triptófano/biosíntesis , Adulto JovenRESUMEN
Multiple sclerosis (MS) is a leading cause of incurable progressive disability in young adults caused by inflammation and neurodegeneration in the central nervous system (CNS). The capacity of microglia to clear tissue debris is essential for maintaining and restoring CNS homeostasis. This capacity diminishes with age, and age strongly associates with MS disease progression, although the underlying mechanisms are still largely elusive. Here, we demonstrate that the recovery from CNS inflammation in a murine model of MS is dependent on the ability of microglia to clear tissue debris. Microglia-specific deletion of the autophagy regulator Atg7, but not the canonical macroautophagy protein Ulk1, led to increased intracellular accumulation of phagocytosed myelin and progressive MS-like disease. This impairment correlated with a microglial phenotype previously associated with neurodegenerative pathologies. Moreover, Atg7-deficient microglia showed notable transcriptional and functional similarities to microglia from aged wild-type mice that were also unable to clear myelin and recover from disease. In contrast, induction of autophagy in aged mice using the disaccharide trehalose found in plants and fungi led to functional myelin clearance and disease remission. Our results demonstrate that a noncanonical form of autophagy in microglia is responsible for myelin degradation and clearance leading to recovery from MS-like disease and that boosting this process has a therapeutic potential for age-related neuroinflammatory conditions.
Asunto(s)
Proteína 7 Relacionada con la Autofagia/deficiencia , Encefalomielitis Autoinmune Experimental/inmunología , Microglía/inmunología , Esclerosis Múltiple/inmunología , Fagocitosis/inmunología , Animales , Autofagia/inmunología , Proteína 7 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/deficiencia , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Encéfalo/citología , Encéfalo/inmunología , Encéfalo/patología , Células Cultivadas , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Microglía/metabolismo , Esclerosis Múltiple/patología , Vaina de Mielina/metabolismo , Cultivo Primario de Células , Médula Espinal/citología , Médula Espinal/inmunología , Médula Espinal/patologíaRESUMEN
For over 50 years, hippuric/quinic acids were believed to have no biological efficacy. Here data are presented to support the hypothesis that quinic acid is not responsible for any efficacy, but rather that quinic acid nutritionally supports the synthesis of tryptophan and nicotinamide in the gastrointestinal (GI) tract, and that this in turn leads to DNA repair enhancement and NF-kB inhibition via increased nicotinamide and tryptophan production.Moreover, it is shown that quinic acid is a normal constituent of our diet, capable of conversion to tryptophan and nicotinamide via the GI tract microflora, thus providing an in situ physiological source of these essential metabolic ingredients to humans. The concentrations of quinic and hippuric acids in the diet were dependent on each other when analysed in urine, as was evidenced by a significant linear regression analysis that included unsupplemented control subjects (n = 45, p < 0.001). Thus, these ingredients were identified as major dietary components, and not simply originating from environmental pollution as previously had been thought.
Asunto(s)
Antioxidantes/metabolismo , Hipuratos/farmacocinética , Niacinamida/orina , Ácido Quínico/farmacocinética , Triptófano/orina , Dieta , Hipuratos/sangre , Hipuratos/orina , Humanos , Modelos Lineales , Masculino , Niacinamida/biosíntesis , Ácido Quínico/sangre , Ácido Quínico/orina , Compuestos de Sulfhidrilo/sangre , Triptófano/biosíntesisRESUMEN
Circulating monocytes can compete for virtually any tissue macrophage niche and become long-lived replacements that are phenotypically indistinguishable from their embryonic counterparts. As the factors regulating this process are incompletely understood, we studied niche competition in the brain by depleting microglia with >95% efficiency using Cx3cr1CreER/+R26DTA/+ mice and monitored long-term repopulation. Here we show that the microglial niche is repopulated within weeks by a combination of local proliferation of CX3CR1+F4/80lowClec12a- microglia and infiltration of CX3CR1+F4/80hiClec12a+ macrophages that arise directly from Ly6Chi monocytes. This colonization is independent of blood brain barrier breakdown, paralleled by vascular activation, and regulated by type I interferon. Ly6Chi monocytes upregulate microglia gene expression and adopt microglia DNA methylation signatures, but retain a distinct gene signature from proliferating microglia, displaying altered surface marker expression, phagocytic capacity and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct.
Asunto(s)
Encéfalo/inmunología , Linaje de la Célula/inmunología , Regulación de la Expresión Génica/inmunología , Microglía/inmunología , Monocitos/inmunología , Traslado Adoptivo , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Antígenos Ly/genética , Antígenos Ly/inmunología , Proteínas Bacterianas/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Encéfalo/citología , Encéfalo/efectos de la radiación , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/inmunología , Linaje de la Célula/efectos de la radiación , Proliferación Celular , Metilación de ADN , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Proteínas Luminiscentes/inmunología , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/efectos de la radiación , Ratones , Ratones Transgénicos , Microglía/citología , Microglía/efectos de la radiación , Monocitos/citología , Monocitos/efectos de la radiación , Monocitos/trasplante , Fagocitosis , Receptores Mitogénicos/genética , Receptores Mitogénicos/inmunología , Transducción de Señal , Quimera por Trasplante , Irradiación Corporal TotalRESUMEN
While bone marrow-derived Ly6Chi monocytes can infiltrate the central nervous system (CNS) they are developmentally and functionally distinct from resident microglia. Our understanding of the relative importance of these two populations in the distinct processes of pathogenesis and resolution of inflammation during neurodegenerative disorders was limited by a lack of tools to specifically manipulate each cell type. During recent years, the development of experimental cell-specific depletion models has enabled this issue to be addressed. Herein we compare and contrast the different depletion approaches that have been used, focusing on the respective functionalities of microglia and monocyte-derived macrophages in a range of neurodegenerative disease states, and discuss their prospects for immunotherapy.
RESUMEN
Neuroinflammation plays an influential role in Alzheimer's disease (AD), although the mechanisms underlying this phenomenon remain largely unknown. Microglia are thought to be responsible for the majority of these effects and can be characterized into resting (M0), proinflammatory (M1), or anti-inflammatory (M2) functional phenotypes. We investigated the effects of conditioned macrophage media, as an analogue to microglia, on the transfer of oligomeric amyloid beta (oAß) between differentiated SH-SY5Y cells. We also investigated how the different inflammatory environments related to intercellular and intracellular changes. We demonstrate that M2 products decrease interneuronal transfer of oAß, while recombinant interleukin (IL)-4, IL-10, and IL-13 increase transfer. There were no alterations to the mRNA of a number of AD-related genes in response to the combination of oAß and M0, M1, or M2, but several intracellular proteins, some relating to protein trafficking and the endosomal/lysosomal system, were altered. Stimulating microglia to an M2 phenotype may thus slow down the progression of AD and could be a target for future therapies.
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Péptidos beta-Amiloides/metabolismo , Macrófagos/fisiología , Microglía/fisiología , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Línea Celular , Endosomas/metabolismo , Humanos , Interleucina-10/fisiología , Interleucina-13/fisiología , Interleucina-4/fisiología , Lisosomas/metabolismo , Transporte de Proteínas , Proteínas RecombinantesRESUMEN
Prolonged infections or adjuvant usage can trigger emergency granulopoiesis (EG), leading to dysregulation in neutrophil blood counts. However, the impact of EG on T and B cell function remains largely unknown. In this study, to address this question, we used a mouse model of neutropenia and studied immune activation after adjuvant administration. The initial neutropenic state fostered an environment of increased dendritic cell activation and T cell-derived IL-17 production. Interestingly, neutropenic lysozyme 2-diphtheria toxin A mice exhibited striking EG and amplified neutrophil recruitment to the lymph nodes (LNs) that was dependent on IL-17-induced prostaglandin activity. The recruited neutrophils secreted a B cell-activating factor that highly accelerated plasma cell generation and antigen-specific antibody production. Reduction of neutrophil functions via granulocyte colony-stimulating factor neutralization significantly diminished plasma cell formation, directly linking EG with the humoral immune response. We conclude that neutrophils are capable of directly regulating T cell-dependent B cell responses in the LN.
Asunto(s)
Formación de Anticuerpos/fisiología , Factor Activador de Células B/metabolismo , Inmunidad Humoral/fisiología , Mielopoyesis/fisiología , Neutrófilos/metabolismo , Células Plasmáticas/metabolismo , Animales , Factor Activador de Células B/genética , Factor Activador de Células B/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Ratones , Ratones Noqueados , Neutropenia/genética , Neutropenia/inmunología , Neutropenia/metabolismo , Neutrófilos/citología , Neutrófilos/inmunología , Células Plasmáticas/inmunologíaRESUMEN
BACKGROUND: The progression of Alzheimer's disease (AD) is associated with an increase of phosphorylated tau in the brain. One of the earliest phosphorylated sites on tau is Ser262 that is preferentially phosphorylated by microtubule affinity regulating kinase (MARK), of which four isoforms exist. Herein we investigated the expression of MARK1-4 in the hippocampus of non-demented elderly (NDE) and AD cases. RESULTS: In situ hybridization revealed a uniform, neuronal distribution of all four isoform mRNAs in NDE and AD. Immunohistochemical analyses using isoform-selective antibodies demonstrated that MARK4 in a phosphorylated form colocalizes with p-tau Ser262 in granulovacuolar degeneration bodies (GVDs) that progressively accumulate in AD. In contrast MARK4 is largely absent in the neuronal cytoplasm. MARK3 was localized to a subset of the GVD-containing neurons and also had a weak general cytoplasmic neuronal staining in both NDE and AD. These results suggest that in AD, phosphorylated MARK3 and MARK4 are sequestered and proteolysed in GVDs. MARK1 and MARK2 were absent in GVDs and exhibited relatively uniform neuronal expressions with no apparent differences between NDE and AD. CONCLUSION: We found that the phosphorylated and fragmented forms of MARK4 and to some extent MARK3 are present in GVDs in AD, and that this expression is highly correlated with phosphorylation of tau at Ser262. This may represent a cellular defense mechanism to remove activated MARK and p-tau Ser262 from the cytosol, thereby reducing the phosphorylating effect on tau Ser262 that appears to be a critical step for subsequent neurodegeneration.
Asunto(s)
Enfermedad de Alzheimer/patología , Gránulos Citoplasmáticos/patología , Hipocampo/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Vacuolas/patología , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Gránulos Citoplasmáticos/metabolismo , Femenino , Células HEK293 , Hipocampo/patología , Humanos , Masculino , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Serina/metabolismo , Transfección , Vacuolas/metabolismoRESUMEN
The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimer's disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser²6² mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4-tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4-tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy.