RESUMEN
Brain arterioles are active, multicellular complexes whose diameters oscillate at â¼ 0.1 Hz. We assess the physiological impact and spatiotemporal dynamics of vaso-oscillations in the awake mouse. First, vaso-oscillations in penetrating arterioles, which source blood from pial arterioles to the capillary bed, profoundly impact perfusion throughout neocortex. The modulation in flux during resting-state activity exceeds that of stimulus-induced activity. Second, the change in perfusion through arterioles relative to the change in their diameter is weak. This implies that the capillary bed dominates the hydrodynamic resistance of brain vasculature. Lastly, the phase of vaso-oscillations evolves slowly along arterioles, with a wavelength that exceeds the span of the cortical mantle and sufficient variability to establish functional cortical areas as parcels of uniform phase. The phase-gradient supports traveling waves in either direction along both pial and penetrating arterioles. This implies that waves along penetrating arterioles can mix, but not directionally transport, interstitial fluids.
Asunto(s)
Circulación Cerebrovascular , Animales , Ratones , Arteriolas/fisiología , Circulación Cerebrovascular/fisiología , Masculino , Corteza Cerebral/fisiología , Corteza Cerebral/irrigación sanguínea , Ratones Endogámicos C57BL , Neocórtex/fisiología , Neocórtex/irrigación sanguíneaRESUMEN
How transient hyperglycemia contributes to cerebro-vascular disease has been a challenge to study under controlled physiological conditions. We use amplified, ultrashort laser-pulses to physically disrupt brain-venule endothelium at targeted locations. This vessel disruption is performed in conjunction with transient hyperglycemia from a single injection of metabolically active D-glucose into healthy mice. The observed real-time responses to laser-induced disruption include rapid serum extravasation, platelet aggregation, and neutrophil recruitment. Thrombo-inflammation is pharmacologically ameliorated by a platelet inhibitor, by a scavenger of reactive oxygen species, and by a nitric oxide donor. As a control, vessel thrombo-inflammation is significantly reduced in mice injected with metabolically inert L-glucose. Venules in mice with diabetes show a similar response to laser-induced disruption and damage is reduced by restoration of normo-glycemia. Our approach provides a controlled method to probe synergies between transient metabolic and physical vascular perturbations and can reveal new aspects of brain pathophysiology.
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Glucemia , Glucosa , Hiperglucemia , Animales , Ratones , Vénulas/metabolismo , Glucemia/metabolismo , Inflamación/metabolismo , Hiperglucemia/metabolismo , Plaquetas/metabolismo , Neutrófilos/metabolismo , Endotelio Vascular/metabolismoRESUMEN
Mosaic inactivation of CCM2 in humans causes cerebral cavernous malformations (CCMs) containing adjacent dilated blood-filled multi-cavernous lesions. We used CRISPR-Cas9 mutagenesis to induce mosaic inactivation of zebrafish ccm2 resulting in a novel lethal multi-cavernous lesion in the embryonic caudal venous plexus (CVP) caused by obstruction of blood flow by intraluminal pillars. These pillars mimic those that mediate intussusceptive angiogenesis; however, in contrast to the normal process, the pillars failed to fuse to split the pre-existing vessel in two. Abortive intussusceptive angiogenesis stemmed from mosaic inactivation of ccm2 leading to patchy klf2a overexpression and resultant aberrant flow signaling. Surviving adult fish manifested histologically typical hemorrhagic CCM. Formation of mammalian CCM requires the flow-regulated transcription factor KLF2; fish CCM and the embryonic CVP lesion failed to form in klf2a null fish indicating a common pathogenesis with the mammalian lesion. These studies describe a zebrafish CCM model and establish a mechanism that can explain the formation of characteristic multi-cavernous lesions.
Asunto(s)
Encéfalo/irrigación sanguínea , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Proteínas Musculares/genética , Neovascularización Patológica/genética , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Circulación Cerebrovascular , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Predisposición Genética a la Enfermedad , Hemangioma Cavernoso del Sistema Nervioso Central/embriología , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Mosaicismo , Proteínas Musculares/metabolismo , Fenotipo , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
De novo blood vessel formation occurs through coalescence of endothelial cells (ECs) into a cord-like structure, followed by lumenization either through cell-1-3 or cord-hollowing4-7. Vessels generated in this manner are restricted in diameter to one or two ECs, and these models fail to explain how vasculogenesis can form large-diameter vessels. Here, we describe a model for large vessel formation that does not require a cord-like structure or a hollowing step. In this model, ECs coalesce into a network of struts in the future lumen of the vessel, a process dependent upon bone morphogenetic protein signalling. The vessel wall forms around this network and consists initially of only a few patches of ECs. To withstand external forces and to maintain the shape of the vessel, strut formation traps erythrocytes into compartments to form a rigid structure. Struts gradually prune and ECs from struts migrate into and become part of the vessel wall. Experimental severing of struts resulted in vessel collapse, disturbed blood flow and remodelling defects, demonstrating that struts enable the patency of large vessels during their formation.
Asunto(s)
Vasos Sanguíneos/crecimiento & desarrollo , Células Endoteliales/fisiología , Morfogénesis/genética , Neovascularización Fisiológica/genética , Vasos Sanguíneos/metabolismo , Células Endoteliales/metabolismo , Eritrocitos/metabolismo , Eritrocitos/patología , HumanosRESUMEN
The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.
Asunto(s)
Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Sistema Nervioso Simpático/inmunología , Animales , Línea Celular , Células Cultivadas , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Expresión Génica/inmunología , Humanos , Inflamación/genética , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Norepinefrina/inmunología , Norepinefrina/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema Nervioso Simpático/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/inmunología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The immune system plays an important role in regulating tumor growth and metastasis. Classical monocytes promote tumorigenesis and cancer metastasis, but how nonclassical "patrolling" monocytes (PMo) interact with tumors is unknown. Here we show that PMo are enriched in the microvasculature of the lung and reduce tumor metastasis to lung in multiple mouse metastatic tumor models. Nr4a1-deficient mice, which specifically lack PMo, showed increased lung metastasis in vivo. Transfer of Nr4a1-proficient PMo into Nr4a1-deficient mice prevented tumor invasion in the lung. PMo established early interactions with metastasizing tumor cells, scavenged tumor material from the lung vasculature, and promoted natural killer cell recruitment and activation. Thus, PMo contribute to cancer immunosurveillance and may be targets for cancer immunotherapy.
Asunto(s)
Vigilancia Inmunológica/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Monocitos/inmunología , Animales , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/terapia , Ratones , Ratones Mutantes , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/secundario , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genéticaRESUMEN
BACKGROUND & AIMS: The lymphatic chemokine CCL21 is required for dendritic cell (DC) migration from tissues to lymph nodes, which helps establish tolerance to foreign yet harmless antigens. We demonstrate that CCL21 is almost completely absent from SAMP1/YitFc (SAMP) mice, which spontaneously develop chronic ileitis that resembles Crohn's disease, and that DC migration is severely impaired in these mice compared with AKR mice (controls). Toll-like receptor agonists like the Toll-like receptor 7 agonist R848 induce DC maturation and mobilization. METHODS: We collected intestinal and other tissues and mesenteric lymph nodes (MLN) from SAMP mice. Expression of CCL21 was measured by quantitative reverse transcription polymerase chain reaction and immunofluorescence analyses; spontaneous and induced migration of DCs were assessed by flow cytometry. We analyzed production of retinoic acid by DCs and their ability to induce development of regulatory T cells. Mice were fed R848 to determine its effects on migration of DCs and development of ileitis in SAMP mice. RESULTS: SAMP mice expressed almost no CCL21 in any tissue tested. Their CD11b(+)CD103(+) DCs were defective in migration from the ileal lamina propria to the MLN. DCs from SAMP mice also had a greatly reduced ability to produce retinoic acid and induce development of regulatory T cells compared with control mice. Young SAMP mice had reduced CCL21 expression and decreased DC migration before developing ileitis. Administration of R848 to adult SAMP mice increased migration of DC to the MLN and development of regulatory T cells there, and reduced the severity of ileitis. CONCLUSIONS: Loss of CCL21 signaling and DC migration is required for development of ileitis in SAMP mice. Reagents such as R848, which activate DC migration to the MLN, may be developed as treatments for patients with Crohn's disease.
Asunto(s)
Movimiento Celular/inmunología , Quimiocina CCL21/inmunología , Enfermedad de Crohn/inmunología , Células Dendríticas/inmunología , Ileítis/inmunología , Ganglios Linfáticos/inmunología , Animales , Movimiento Celular/efectos de los fármacos , Quimiocina CCL21/genética , Células Dendríticas/efectos de los fármacos , Modelos Animales de Enfermedad , Imidazoles/farmacología , Glicoproteínas de Membrana/agonistas , Ratones , Ratones Endogámicos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/inmunología , Receptor Toll-Like 7/agonistasRESUMEN
OBJECTIVE: Infection with hepatitis C virus (HCV) or human immunodeficiency virus (HIV) may be associated with atherosclerosis and vascular disease. Macrophages are a major component of atherosclerotic plaque, and classically activated (M1) macrophages contribute to plaque instability. Our goal was to identify plasma biomarkers that reflect macrophage inflammation and are associated with subclinical atherosclerosis. APPROACH AND RESULTS: We tested whether M1 macrophages produce galectin-3-binding protein in vitro. Then, we measured galectin-3-binding protein and the soluble macrophage biomarkers soluble cluster of differentiation (CD) 163 and soluble CD14 in 264 participants in the Women's Interagency HIV Study. Women were positive for HIV, HCV, both, or neither (66 in each group, matched for age, race/ethnicity, and smoking status). Carotid artery disease was assessed by ultrasound measurement of right distal common carotid artery intima-media thickness, distensibility, and presence of atherosclerotic lesions (intima-media thickness >1.5 mm). Plasma galectin-3-binding protein was higher in HCV+ than HCV- women (P<0.01) but did not differ by HIV status. The 3 inflammatory macrophage markers were significantly correlated with each other and negatively correlated with CD4+ counts in HIV-infected women. We defined a macrophage score as 1, 2, or 3 biomarkers elevated above the median. In models adjusted for traditional risk factors, higher macrophage scores were significantly associated with increased atherosclerotic lesions and lower carotid distensibility. Receiver-operator curve analysis of lesions revealed that the markers added predictive value beyond traditional risk factors and C-reactive protein. CONCLUSIONS: The macrophage inflammatory markers galectin-3-binding protein, soluble CD163, and soluble CD14 are significantly associated with carotid artery disease in the setting of HIV and HCV infection.
Asunto(s)
Enfermedades de las Arterias Carótidas/sangre , Coinfección , Infecciones por VIH/sangre , Hepatitis C/sangre , Mediadores de Inflamación/sangre , Macrófagos/metabolismo , Adulto , Antígenos CD/sangre , Antígenos de Diferenciación Mielomonocítica/sangre , Antígenos de Neoplasias/sangre , Enfermedades Asintomáticas , Biomarcadores/sangre , Biomarcadores de Tumor/sangre , Enfermedades de las Arterias Carótidas/diagnóstico , Enfermedades de las Arterias Carótidas/inmunología , Grosor Intima-Media Carotídeo , Proteínas Portadoras/sangre , Estudios Transversales , Femenino , Glicoproteínas/sangre , Infecciones por VIH/diagnóstico , Infecciones por VIH/inmunología , Hepatitis C/diagnóstico , Hepatitis C/inmunología , Humanos , Receptores de Lipopolisacáridos/sangre , Macrófagos/inmunología , Persona de Mediana Edad , Receptores de Superficie Celular/sangre , Factores de Riesgo , Factores Sexuales , Fumar/sangre , Fumar/inmunología , Rigidez VascularRESUMEN
Immune cells sense microbial products through Toll-like receptors (TLR), which trigger host defense responses including type 1 interferons (IFNs) secretion. A coding polymorphism in the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is a susceptibility allele for human autoimmune and infectious disease. We report that Ptpn22 selectively regulated type 1 IFN production after TLR engagement in myeloid cells. Ptpn22 promoted host antiviral responses and was critical for TLR agonist-induced, type 1 IFN-dependent suppression of inflammation in colitis and arthritis. PTPN22 directly associated with TNF receptor-associated factor 3 (TRAF3) and promotes TRAF3 lysine 63-linked ubiquitination. The disease-associated PTPN22W variant failed to promote TRAF3 ubiquitination, type 1 IFN upregulation, and type 1 IFN-dependent suppression of arthritis. The findings establish a candidate innate immune mechanism of action for a human autoimmunity "risk" gene in the regulation of host defense and inflammation.
Asunto(s)
Autoinmunidad/inmunología , Inmunidad/inmunología , Interferón Tipo I/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 22/inmunología , Receptores Toll-Like/inmunología , Animales , Artritis/genética , Artritis/inmunología , Autoinmunidad/genética , Línea Celular , Células Cultivadas , Colitis/inducido químicamente , Colitis/genética , Colitis/inmunología , Sulfato de Dextran/inmunología , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad/genética , Immunoblotting , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Mieloides/inmunología , Células Mieloides/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 22/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Ubiquitinación/inmunologíaRESUMEN
Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.
Asunto(s)
Aterosclerosis/inmunología , Colesterol/biosíntesis , Desmosterol/metabolismo , Células Espumosas/metabolismo , Metabolismo de los Lípidos , Transcriptoma , Animales , Aterosclerosis/metabolismo , Colesterol/análogos & derivados , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Células Espumosas/inmunología , Técnicas de Silenciamiento del Gen , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
RATIONALE: NR4A1 (Nur77) is a nuclear receptor that is expressed in macrophages and within atherosclerotic lesions, yet its function in atherosclerosis is unknown. OBJECTIVE: Nur77 regulates the development of monocytes, particularly patrolling Ly6C(-) monocytes that may be involved in resolution of inflammation. We sought to determine how absence of nuclear receptor subfamily 4, group A, member 1 (NR4A1) in hematopoietic cells affected atherosclerosis development. METHODS AND RESULTS: Nur77(-/-) chimeric mice on a Ldlr(-/-) background showed a 3-fold increase in atherosclerosis development when fed a Western diet for 20 weeks, despite having a drastic reduction in Ly6C(-) patrolling monocytes. In a second model, mice deficient in both Nur77 and ApoE (ApoE(-/-)Nur77(-/-)) also showed increased atherosclerosis after 11 weeks of Western diet. Atherosclerosis was associated with a significant change in macrophage polarization toward a proinflammatory phenotype, with high expression of tumor necrosis factor-α and nitric oxide and low expression of Arginase-I. Moreover, we found increased expression of toll-like receptor 4 mRNA and protein in Nur77(-/-) macrophages as well as increased phosphorylation of the p65 subunit of NFκB. Inhibition of NFκB activity blocked excess activation of Nur77(-/-) macrophages. CONCLUSIONS: We conclude that the absence of Nur77 in monocytes and macrophages results in enhanced toll-like receptor signaling and polarization of macrophages toward a proinflammatory M1 phenotype. Despite having fewer monocytes, Nur77(-/-) mice developed significant atherosclerosis when fed a Western diet. These studies indicate that Nur77 is a novel target for modulating the inflammatory phenotype of monocytes and macrophages and may be important for regulation of atherogenesis.
Asunto(s)
Aterosclerosis/patología , Eliminación de Gen , Inflamación/patología , Macrófagos/patología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/deficiencia , Fenotipo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apolipoproteínas E/fisiología , Aterosclerosis/etiología , Aterosclerosis/fisiopatología , Dieta/efectos adversos , Modelos Animales de Enfermedad , Humanos , Inflamación/fisiopatología , Metabolismo de los Lípidos/fisiología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/fisiología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/fisiología , Receptores Toll-Like/fisiologíaRESUMEN
In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors, such as macrophage colony-stimulation factor (M-CSF), and chemokines, such as platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with M-CSF-induced macrophages or macrophages polarized with IFN-gamma/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for 6 d. mRNA expression was measured by Affymetrix gene chips, and differences were analyzed by local pooled error test, profile of complex functionality, and gene set enrichment analysis. Three hundred seventy-five genes were differentially expressed between M-CSF- and CXCL4-induced macrophages; 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, Ag processing and presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level; however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently upregulated or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters showed higher expression in CXCL4- than M-CSF-induced macrophages, resulting in lower low-density lipoprotein content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4.
Asunto(s)
Diferenciación Celular/inmunología , Perfilación de la Expresión Génica , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Factor Plaquetario 4/fisiología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Biomarcadores/sangre , Diferenciación Celular/genética , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Polaridad Celular/genética , Polaridad Celular/inmunología , Células Cultivadas , Progresión de la Enfermedad , Humanos , Factor Estimulante de Colonias de Macrófagos/sangre , Factor Estimulante de Colonias de Macrófagos/fisiología , Macrófagos/citología , Monocitos/citología , Factor Plaquetario 4/sangreRESUMEN
RATIONALE: CXCL4 is a platelet-derived chemokine that promotes macrophage differentiation from monocytes. Deletion of the PF4 gene that encodes CXCL4 reduces atherosclerotic lesions in ApoE(-/-) mice. OBJECTIVE: We sought to study effects of CXCL4 on macrophage differentiation with possible relevance for atherogenesis. METHODS AND RESULTS: Flow cytometry for expression of surface markers in macrophage colony-stimulating factor (M-CSF)- and CXCL4-induced macrophages demonstrated virtually complete absence of the hemoglobin scavenger receptor CD163 in CXCL4-induced macrophages. mRNA for CD163 was downregulated as early as 2 hours after CXCL4. CD163 protein reached a minimum after 3 days, which was not reversed by treatment of cells with M-CSF. The CXCL4 effect was entirely neutralized by heparin, which bound CXCL4 and prevented CXCL4 surface binding to monocytes. Pretreatment of cells with chlorate, which inhibits glycosaminoglycan synthesis, strongly inhibited CXCL4-dependent downregulation of CD163. Similar to recombinant CXCL4, releasate from human platelets also reduced CD163 expression. CXCL4-differentiated macrophages were unable to upregulate the atheroprotective enzyme heme oxygenase-1 at the RNA and protein level in response to hemoglobin-haptoglobin complexes. Immunofluorescence of human atherosclerotic plaques demonstrated presence of both CD68+CD163+ and CD68+CD163- macrophages. PF4 and CD163 gene expression within human atherosclerotic lesions were inversely correlated, supporting the in vivo relevance of CXCL4-induced downregulation of CD163. CONCLUSIONS: CXCL4 may promote atherogenesis by suppressing CD163 in macrophages, which are then unable to upregulate the atheroprotective enzyme heme oxygenase-1 in response to hemoglobin.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Aterosclerosis/metabolismo , Macrófagos/metabolismo , Factor Plaquetario 4/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptores Depuradores/biosíntesis , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Hemoglobinas/genética , Hemoglobinas/metabolismo , Hemoglobinas/farmacología , Humanos , Ratones , Ratones Noqueados , Monocitos/metabolismo , Factor Plaquetario 4/genética , Factor Plaquetario 4/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Depuradores/agonistas , Receptores Depuradores/genéticaRESUMEN
MicroRNAs (miRNAs) contribute to both neuronal and immune cell fate, but their involvement in intertissue communication remained unexplored. The brain, via vagal secretion of acetylcholine (ACh), suppresses peripheral inflammation by intercepting cytokine production; therefore, we predicted that microRNAs targeting acetylcholinesterase (AChE) can attenuate inflammation. Here, we report that inflammatory stimuli induced leukocyte overexpression of the AChE-targeting miR-132. Injected locked nucleic acid (LNA)-modified anti-miR-132 oligonucleotide depleted miR-132 amounts while elevating AChE in mouse circulation and tissues. In transfected cells, a mutated 3'UTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice overexpressing 3'UTR null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 upregulation in brain and bone marrow. Our findings identify the AChE mRNA-targeting miR-132 as a functional regulator of the brain-to-body resolution of inflammation, opening avenues for study and therapeutic manipulations of the neuro-immune dialog.
Asunto(s)
Acetilcolinesterasa/genética , Inflamación/enzimología , MicroARNs/metabolismo , Acetilcolina/metabolismo , Animales , Secuencia de Bases , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Línea Celular , Regulación hacia Abajo , Femenino , Humanos , Inflamación/inmunología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/inmunología , Lipopolisacáridos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Transgénicos , MicroARNs/agonistas , MicroARNs/genética , Datos de Secuencia Molecular , Oligonucleótidos/farmacología , Eliminación de Secuencia , Transducción de Señal , Regulación hacia ArribaRESUMEN
Mammalian stress responses present a case study for investigating alternative splicing reactions in general, and changes in acetylcholinesterase (AChE) gene expression in particular, under endangered homeostasis. Acetylcholine (ACh) is a major regulator of stress responses, which was recently found to function as an essential route by which neurons can "talk" to immune cells. Therefore, chemical, physical, or psychological insults to the brain might all be traced in peripheral immune cells, which serve as key determinants in the physiological reactions to stress. Stress-induced changes in the alternative splicing patterns of AChE pre-mRNA give this gene and its different protein products diverse stress responsive functions that are associated with both the enzymatic and noncatalytic properties of AChE variants. Transgenic manipulations of AChE gene expression uncovered previously nonperceived aspects of stress responses, including brain-to-blood as well as immune-to-neuronal communication. Herein we discuss the newly gained understanding achieved by using genomic manipulations of AChE gene expression as tools for approaching the alternative splicing features of mammalian stress responses.
Asunto(s)
Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Empalme Alternativo , Encéfalo/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrés Psicológico , Acetilcolinesterasa/química , Animales , Conducta Animal/fisiología , Cognición/fisiología , Regulación Enzimológica de la Expresión Génica , Homeostasis , Humanos , Modelos Moleculares , Conformación Proteica , Isoformas de Proteínas/químicaRESUMEN
Cholinergic signaling and acetylcholinesterase (AChE) influence immune response and inflammation. Autoimmune myasthenia gravis (MG) is mediated by antibodies to the acetylcholine receptor and current therapy is based on anti-AChE drugs. MG is associated with thymic hyperplasia, showing signs of inflammation. The objectives of this study were to analyze the involvement of AChE variants in thymic hyperplasia. We found lower hydrolytic activities in the MG thymus compared with adult controls, accompanied by translocation of AChE-R from the cytoplasm to the membrane and increased expression of the signaling protein kinase PKC-betaII. To explore possible causal association of AChE-R changes with thymic composition and function, we used an AChE-R transgenic model and showed smaller thymic medulla compared with strain-matched controls, indicating that AChE-R overexpression interferes with thymic differentiation mechanisms. Interestingly, AChE-R transgenic mice showed increased numbers of CD4(+)CD8(+) cells that were considerably more resistant in vitro to apoptosis than normal thymocytes, suggesting possibly altered positive selection. We further analyzed microarray data of MG thymic hyperplasia compared with healthy controls and found continuous and discrete changes in AChE-annotated GO categories. Together, these findings show that modified AChE gene expression and properties are causally involved in thymic function and development.
Asunto(s)
Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Empalme Alternativo , Miastenia Gravis/genética , Miastenia Gravis/metabolismo , Timo/enzimología , Adolescente , Adulto , Anciano , Animales , Células Cultivadas , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Lactante , Recién Nacido , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Timo/metabolismoRESUMEN
MicroRNAs (miRNAs) are abundant small regulatory RNAs with multiple roles in cell fate determination. The processes regulating cellular miRNA levels are still unclear and experimental oligonucleotide tools to readily mimic their effects are not yet available. Here, we report that thapsigargin-induced intracellular Ca(++) release suppressed pre-miR-181a levels in human promegakaryotic Meg-01 cells, induced differentiation-associated nuclear endoreduplication and caspase-3 activation and replaced the acetylcholinesterase 3' splice variant AChE-S with AChE-R. AChE, PKC and PKA inhibitors all attenuated the pre-miR-181a decline and the induced differentiation. AChmiON, a synthetic 23-mer 2'-oxymethylated oligonucleotide mimicking the miR-181a sequence, blocked the calcium-induced differentiation while elevating cellular pre-miR-181a levels and inducing DNA fragmentation and cell death. Moreover, when added to RW 264.7 macrophages, AChmiON at 100 nM induced nitric oxide production with efficiency close to that of bacterial endotoxin, demonstrating physiologically relevant activities also in blood-born monocytes/macrophages. The stress-induced modulation of hematopoietic miR-181a levels through AChE, PKC and PKA cascade(s) suggests using miRNA mimics for diverting the fate of hematopoietic tumor cells towards differentiation and/or apoptosis.
Asunto(s)
Acetilcolinesterasa/metabolismo , Megacariocitos/metabolismo , MicroARNs/metabolismo , Oligonucleótidos/farmacología , Acetilcolinesterasa/efectos de los fármacos , Acetilcolinesterasa/genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Calcio/metabolismo , Calcio/farmacología , Caspasa 3 , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Macrófagos/efectos de los fármacos , Megacariocitos/efectos de los fármacos , Ratones , MicroARNs/efectos de los fármacos , MicroARNs/genética , Óxido Nítrico/biosíntesis , Oligonucleótidos/síntesis química , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tapsigargina/antagonistas & inhibidores , Tapsigargina/farmacologíaRESUMEN
Acute or chronic glaucoma is often associated with an increase in intraocular pressure (IOP). In many patients, however, therapeutic pressure reduction does not halt disease progression. Neuroprotection has been proposed as a complementary therapeutic approach. We previously demonstrated effective T-cell-based neuroprotection in experimental animals vaccinated with the synthetic copolymer glatiramer acetate (copolymer-1, Cop-1), a weak agonist of self-antigens. This study was undertaken to test different routes and modes of vaccination with Cop-1 as treatment modalities for protection against retinal ganglion cell (RGC) death caused by chronic elevation of IOP in rats, and to determine whether anatomical neuroprotection is accompanied by functional neuroprotection. In a chronic model of unilaterally high IOP, Cop-1 vaccination, with or without an adjuvant, protected rats against IOP-induced loss of RGCs by eliciting a systemic T-cell-mediated response capable of cross-reacting with self-antigens residing in the eye. In rats deprived of T cells, Cop-1 (unlike treatment with alpha2-adrenoreceptor agonists) was not protective of RGCs, substantiating the contention that its beneficial effect is not conferred directly but is T-cell-mediated. Pattern electroretinography provided evidence of functional protection. Thus, vaccination with adjuvant-free Cop-1 can protect RGCs from the consequences of elevated IOP in rats. This protection is manifested both morphologically and functionally. These findings can be readily implemented for the development of a therapeutic vaccination to arrest the progression of glaucoma.
Asunto(s)
Modelos Animales de Enfermedad , Presión Intraocular/efectos de los fármacos , Proteínas Represoras/uso terapéutico , Células Ganglionares de la Retina/efectos de los fármacos , Linfocitos T/inmunología , Vacunación , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/uso terapéutico , Animales , Apoptosis , Proteínas CCN de Señalización Intercelular , Citoprotección/inmunología , Electrorretinografía , Presión Intraocular/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Soluciones Oftálmicas/administración & dosificación , Soluciones Oftálmicas/uso terapéutico , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Proteínas Represoras/administración & dosificación , Células Ganglionares de la Retina/inmunología , Células Ganglionares de la Retina/patología , Factores de TiempoRESUMEN
After an injury to the central nervous system (CNS), activated microglia have been shown to contribute to the ongoing destructive processes leading to secondary neuronal degeneration. They can, however, also express neuroprotective activity. Studies from our laboratory point to the existence of a physiological T cell-mediated neuroprotective mechanism (adaptive immunity) that is amenable to boosting. We postulate that the beneficial or destructive outcome of the local microglial (innate) response is determined by a well-controlled dialog between the innate and the adaptive immune players. Here, we show that spontaneous or exogenously boosted T cell-mediated neuroprotection is correlated with early activation of microglia as antigen-presenting cells. We suggest that such microglial activity, if well controlled, is a crucial step in determining the fate of the neurons in a hostile environment.