RESUMEN
Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.
Asunto(s)
Infecciones Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Hidroxicolesteroles/metabolismo , Interferones/aislamiento & purificación , Fagocitos/inmunología , Estreptolisinas/inmunología , Animales , Bacterias/inmunología , Bacterias/metabolismo , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Femenino , Interacciones Microbiota-Huesped/inmunología , Humanos , Microscopía Intravital , Masculino , Ratones , Ratones Transgénicos , Fagocitos/citología , Fagocitos/metabolismo , Cultivo Primario de Células , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Estreptolisinas/administración & dosificación , Estreptolisinas/metabolismoRESUMEN
Foxo transcription factors play an essential role in regulating specialized lymphocyte functions and in maintaining T cell quiescence. Here, we used a system in which Foxo1 transcription-factor activity, which is normally terminated upon cell activation, cannot be silenced, and we show that enforcing Foxo1 activity disrupts homeostasis of CD4 conventional and regulatory T cells. Despite limiting cell metabolism, continued Foxo1 activity is associated with increased activation of the kinase Akt and a cell-intrinsic proliferative advantage; however, survival and cell division are decreased in a competitive setting or growth-factor-limiting conditions. Via control of expression of the transcription factor Myc and the IL-2 receptor ß-chain, termination of Foxo1 signaling couples the increase in cellular cholesterol to biomass accumulation after activation, thereby facilitating immunological synapse formation and mTORC1 activity. These data reveal that Foxo1 regulates the integration of metabolic and mitogenic signals essential for T cell competitive fitness and the coordination of cell growth with cell division.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Proteína Forkhead Box O1/metabolismo , Linfocitos T Reguladores/fisiología , Animales , Proliferación Celular , Células Cultivadas , Colesterol/metabolismo , Proteína Forkhead Box O1/genética , Perfilación de la Expresión Génica , Homeostasis , Sinapsis Inmunológicas/metabolismo , Subunidad beta del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de SeñalRESUMEN
Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.
Asunto(s)
Colesterol/metabolismo , Inmunidad Innata , Interferón gamma/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Humanos , Interferon beta-1b , Proteínas de la Membrana/metabolismo , Ácido Mevalónico/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.
Asunto(s)
Inflamación , Interleucina-10 , Esfingolípidos , Animales , Humanos , Ratones , Ceramidas/química , Ceramidas/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/metabolismo , Homeostasis , Inmunidad Innata , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-10/deficiencia , Interleucina-10/genética , Interleucina-10/metabolismo , Proteínas Proto-Oncogénicas c-rel , Esfingolípidos/metabolismoRESUMEN
Type I interferons (IFNs) can reprogram the cholesterol biosynthetic pathway to facilitate innate immune responses. In this issue of Immunity, Xiao et al. (2020) reveal that type I IFN signaling and 7-dehydrocholesterol (7-DHC) accumulation form a positive feedback loop to amplify innate immune responses to control viral infections by activating AKT3.
Asunto(s)
Inmunidad Innata , Fosfatidilinositol 3-Quinasas , Colesterol , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , EsterolesRESUMEN
Tissue resident memory CD8+ T (TRM) cells offer rapid and long-term protection at sites of reinfection1. Tumour-infiltrating lymphocytes with characteristics of TRM cells maintain enhanced effector functions, predict responses to immunotherapy and accompany better prognoses2,3. Thus, an improved understanding of the metabolic strategies that enable tissue residency by T cells could inform new approaches to empower immune responses in tissues and solid tumours. Here, to systematically define the basis for the metabolic reprogramming supporting TRM cell differentiation, survival and function, we leveraged in vivo functional genomics, untargeted metabolomics and transcriptomics of virus-specific memory CD8+ T cell populations. We found that memory CD8+ T cells deployed a range of adaptations to tissue residency, including reliance on non-steroidal products of the mevalonate-cholesterol pathway, such as coenzyme Q, driven by increased activity of the transcription factor SREBP2. This metabolic adaptation was most pronounced in the small intestine, where TRM cells interface with dietary cholesterol and maintain a heightened state of activation4, and was shared by functional tumour-infiltrating lymphocytes in diverse tumour types in mice and humans. Enforcing synthesis of coenzyme Q through deletion of Fdft1 or overexpression of PDSS2 promoted mitochondrial respiration, memory T cell formation following viral infection and enhanced antitumour immunity. In sum, through a systematic exploration of TRM cell metabolism, we reveal how these programs can be leveraged to fuel memory CD8+ T cell formation in the context of acute infections and enhance antitumour immunity.
Asunto(s)
Linfocitos T CD8-positivos , Linfocitos Infiltrantes de Tumor , Neoplasias , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Respiración de la Célula , Colesterol/metabolismo , Colesterol/farmacología , Memoria Inmunológica , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Metabolómica , Ácido Mevalónico/metabolismo , Neoplasias/inmunología , Ubiquinona/metabolismo , Virosis/inmunología , Virus/inmunología , Mitocondrias/metabolismoRESUMEN
The mevalonate pathway is an essential metabolic pathway in T cells regulating development, proliferation, survival, differentiation, and effector functions. The mevalonate pathway is a complex, branched pathway composed of many enzymes that ultimately generate cholesterol and nonsterol isoprenoids. T cells must tightly control metabolic flux through the branches of the mevalonate pathway to ensure sufficient isoprenoids and cholesterol are available to meet cellular demands. Unbalanced metabolite flux through the sterol or the nonsterol isoprenoid branch is metabolically inefficient and can have deleterious consequences for T cell fate and function. Accordingly, there is tight regulatory control over metabolic flux through the branches of this essential lipid synthetic pathway. In this review we provide an overview of how the branches of the mevalonate pathway are regulated in T cells and discuss our current understanding of the relationship between mevalonate metabolism, cholesterol homeostasis and T cell function.
Asunto(s)
Ácido Mevalónico , Linfocitos T , Humanos , Ácido Mevalónico/metabolismo , Linfocitos T/metabolismo , Colesterol/metabolismo , Redes y Vías Metabólicas , Terpenos/metabolismoRESUMEN
Newly activated CD8(+) T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals that mediate metabolic reprogramming remain poorly defined. Here we demonstrate an essential role for sterol regulatory element-binding proteins (SREBPs) in the acquisition of effector-cell metabolism. Without SREBP signaling, CD8(+) T cells were unable to blast, which resulted in attenuated clonal expansion during viral infection. Mechanistic studies indicated that SREBPs were essential for meeting the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs were dispensable for homeostatic proliferation, which indicated a context-specific requirement for SREBPs in effector responses. Our studies provide insights into the molecular signals that underlie the metabolic reprogramming of CD8(+) T cells during the transition from quiescence to activation.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Inmunidad Adaptativa/genética , Animales , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Transgenes/genéticaRESUMEN
C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.
Asunto(s)
Transformación Celular Neoplásica/genética , Regulación de la Expresión Génica , Factor de Transcripción Ikaros/genética , Leucemia/genética , Linfopoyesis/genética , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Secuencia de Bases , Sitios de Unión , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Perfilación de la Expresión Génica , Mutación de Línea Germinal , Secuenciación de Nucleótidos de Alto Rendimiento , Factor de Transcripción Ikaros/metabolismo , Inmunofenotipificación , Leucemia/metabolismo , Leucemia/mortalidad , Linfoma/genética , Linfoma/metabolismo , Linfoma/mortalidad , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Motivos de Nucleótidos , Fenotipo , Posición Específica de Matrices de Puntuación , Unión Proteica , Timocitos/metabolismoRESUMEN
Liver X receptors (LXRs) are regulators of cholesterol metabolism that also modulate immune responses. Inactivation of LXR α and ß in mice leads to autoimmunity; however, how the regulation of cholesterol metabolism contributes to autoimmunity is unclear. Here we found that cholesterol loading of CD11c+ cells triggered the development of autoimmunity, whereas preventing excess lipid accumulation by promoting cholesterol efflux was therapeutic. LXRß-deficient mice crossed to the hyperlipidemic ApoE-deficient background or challenged with a high-cholesterol diet developed autoantibodies. Cholesterol accumulation in lymphoid organs promoted T cell priming and stimulated the production of the B cell growth factors Baff and April. Conversely, B cell expansion and the development of autoantibodies in ApoE/LXR-ß-deficient mice was reversed by ApoA-I expression. These findings implicate cholesterol imbalance as a contributor to immune dysfunction and suggest that stimulating HDL-dependent reverse cholesterol transport could be beneficial in the setting of autoimmune disease.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Enfermedades Autoinmunes/inmunología , Colesterol/metabolismo , Hipercolesterolemia/metabolismo , Animales , Enfermedades Autoinmunes/metabolismo , Antígeno CD11c/inmunología , Colesterol/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , Hipercolesterolemia/inmunología , Receptores X del Hígado/inmunología , Receptores X del Hígado/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , TranscriptomaRESUMEN
Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.
Asunto(s)
Smegmamorpha , Animales , Smegmamorpha/metabolismo , Mitocondrias/metabolismo , Metabolismo Energético , Glucólisis , Fosforilación Oxidativa , Adenosina Trifosfato/metabolismo , Mamíferos/metabolismoRESUMEN
Mutations in the gene encoding the orphan nuclear receptor Nurr1 are linked to a rare familial form of Parkinson's disease. By examining the function of its mouse homolog, Saijo et al. (2009) provide evidence that Nurr1 protects dopaminergic neurons by suppressing inflammatory gene expression in astrocytes and microglia.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enfermedad de Parkinson/metabolismo , Factores de Transcripción/metabolismo , Animales , Astrocitos/metabolismo , Ratones , Microglía/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Transducción de SeñalRESUMEN
Cholesterol is essential for membrane synthesis; however, the mechanisms that link cellular lipid metabolism to proliferation are incompletely understood. We demonstrate here that cellular cholesterol levels in dividing T cells are maintained in part through reciprocal regulation of the LXR and SREBP transcriptional programs. T cell activation triggers induction of the oxysterol-metabolizing enzyme SULT2B1, consequent suppression of the LXR pathway for cholesterol transport, and promotion of the SREBP pathway for cholesterol synthesis. Ligation of LXR during T cell activation inhibits mitogen-driven expansion, whereas loss of LXRbeta confers a proliferative advantage. Inactivation of the sterol transporter ABCG1 uncouples LXR signaling from proliferation, directly linking sterol homeostasis to the antiproliferative action of LXR. Mice lacking LXRbeta exhibit lymphoid hyperplasia and enhanced responses to antigenic challenge, indicating that proper regulation of LXR-dependent sterol metabolism is important for immune responses. These results implicate LXR signaling in a metabolic checkpoint that modulates cell proliferation and immunity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Esteroles/metabolismo , Linfocitos T/inmunología , Envejecimiento , Animales , Proliferación Celular , Proteínas de Unión al ADN/genética , Humanos , Receptores X del Hígado , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease and cirrhosis. NAFLD is mediated by changes in lipid metabolism and known risk factors include obesity, metabolic syndrome, and diabetes. The aim of this study was to better understand differences in the lipid composition of individuals with NAFLD compared to controls, by performing direct infusion lipidomics on serum biospecimens from a cohort study of adults in Mexico. METHODS: A nested case-control study was conducted with a sample of 98 NAFLD cases and 100 healthy controls who are participating in an on-going, longitudinal study in Mexico. NAFLD cases were clinically confirmed using elevated liver enzyme tests and liver ultrasound or liver ultrasound elastography, after excluding alcohol abuse, and 100 controls were identified as having at least two consecutive normal alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (< 40 U/L) results in a 6-month period, and a normal liver ultrasound elastography result in January 2018. Samples were analyzed on the Sciex Lipidyzer Platform and quantified with normalization to serum volume. As many as 1100 lipid species can be identified using the Lipidyzer targeted multiple-reaction monitoring list. The association between serum lipids and NAFLD was investigated using analysis of covariance, random forest analysis, and by generating receiver operator characteristic (ROC) curves. RESULTS: NAFLD cases had differences in total amounts of serum cholesterol esters, lysophosphatidylcholines, sphingomyelins, and triacylglycerols (TAGs), however, other lipid subclasses were similar to controls. Analysis of individual TAG species revealed increased incorporation of saturated fatty acyl tails in serum of NAFLD cases. After adjusting for age, sex, body mass index, and PNPLA3 genotype, a combined panel of ten lipids predicted case or control status better than an area under the ROC curve of 0.83. CONCLUSIONS: These preliminary results indicate that the serum lipidome differs in patients with NAFLD, compared to healthy controls, and suggest that assessing the desaturation state of TAGs or a specific lipid panel may be useful clinical tools for the diagnosis of NAFLD.
Asunto(s)
Colesterol/sangre , Lisofosfatidilcolinas/sangre , Enfermedad del Hígado Graso no Alcohólico/sangre , Esfingomielinas/sangre , Triglicéridos/sangre , Adulto , Anciano , Biomarcadores/sangre , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Lipidómica , Masculino , México , Persona de Mediana Edad , Curva ROCRESUMEN
Macrophages are generally assumed to unload surplus cholesterol through direct interactions between ABC transporters on the plasma membrane and HDLs, but they have also been reported to release cholesterol-containing particles. How macrophage-derived particles are formed and released has not been clear. To understand the genesis of macrophage-derived particles, we imaged mouse macrophages by EM and nanoscale secondary ion mass spectrometry (nanoSIMS). By scanning EM, we found that large numbers of 20- to 120-nm particles are released from the fingerlike projections (filopodia) of macrophages. These particles attach to the substrate, forming a "lawn" of particles surrounding macrophages. By nanoSIMS imaging we showed that these particles are enriched in the mobile and metabolically active accessible pool of cholesterol (detectable by ALO-D4, a modified version of a cholesterol-binding cytolysin). The cholesterol content of macrophage-derived particles was increased by loading the cells with cholesterol or by adding LXR and RXR agonists to the cell-culture medium. Incubating macrophages with HDL reduced the cholesterol content of macrophage-derived particles. We propose that release of accessible cholesterol-rich particles from the macrophage plasma membrane could assist in disposing of surplus cholesterol and increase the efficiency of cholesterol movement to HDL.
Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Animales , Micropartículas Derivadas de Células/ultraestructura , Lipoproteínas HDL/ultraestructura , Macrófagos/ultraestructura , Ratones , Ratones Noqueados , Microscopía Electrónica , Células RAW 264.7 , Espectrometría de Masa de Ion SecundarioRESUMEN
Objective- The objective of this study was to determine the basis of resistance to atherosclerosis of inbred mouse strain BALB/cJ. Approach and Results- BALB/cJ mice carry a naturally occurring null mutation of the gene encoding the transcription factor Zhx2, and genetic analyses suggested that this may confer resistance to atherosclerosis. On a hyperlipidemic low-density lipoprotein receptor null background, BALB/cJ mice carrying the mutant allele for Zhx2 exhibited up to a 10-fold reduction in lesion size as compared with an isogenic strain carrying the wild-type allele. Several lines of evidence, including bone marrow transplantation studies, indicate that this effect of Zhx2 is mediated, in part, by monocytes/macrophages although nonbone marrow-derived pathways are clearly involved as well. Both in culture and in atherosclerotic lesions, macrophages from Zhx2 null mice exhibited substantially increased apoptosis. Zhx2 null macrophages were also enriched for M2 markers. Effects of Zhx2 on proliferation and other bone marrow-derived cells, such as lymphocytes, were at most modest. Expression microarray analyses identified >1000 differentially expressed transcripts between Zhx2 wild-type and null macrophages. To identify the global targets of Zhx2, we performed ChIP-seq (chromatin immunoprecipitation sequencing) studies with the macrophage cell line RAW264.7. The ChIP-seq peaks overlapped significantly with gene expression and together suggested roles for transcriptional repression and apoptosis. Conclusions- A mutation of Zhx2 carried in BALB/cJ mice is responsible in large part for its relative resistance to atherosclerosis. Our results indicate that Zhx2 promotes macrophage survival and proinflammatory functions in atherosclerotic lesions, thereby contributing to lesion growth.
Asunto(s)
Apoptosis , Aterosclerosis/fisiopatología , Proteínas de Homeodominio/fisiología , Macrófagos/fisiología , Factores de Transcripción/fisiología , Dedos de Zinc/fisiología , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Expresión Génica , Proteínas de Homeodominio/genética , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Factores de Transcripción/genética , Dedos de Zinc/genéticaRESUMEN
Persistent viral infections are simultaneously associated with chronic inflammation and highly potent immunosuppressive programs mediated by IL-10 and PDL1 that attenuate antiviral T cell responses. Inhibiting these suppressive signals enhances T cell function to control persistent infection; yet, the underlying signals and mechanisms that program immunosuppressive cell fates and functions are not well understood. Herein, we use lymphocytic choriomeningitis virus infection (LCMV) to demonstrate that the induction and functional programming of immunosuppressive dendritic cells (DCs) during viral persistence are separable mechanisms programmed by factors primarily considered pro-inflammatory. IFNγ first induces the de novo development of naive monocytes into DCs with immunosuppressive potential. Type I interferon (IFN-I) then directly targets these newly generated DCs to program their potent T cell immunosuppressive functions while simultaneously inhibiting conventional DCs with T cell stimulating capacity. These mechanisms of monocyte conversion are constant throughout persistent infection, establishing a system to continuously interpret and shape the immunologic environment. MyD88 signaling was required for the differentiation of suppressive DCs, whereas inhibition of stimulatory DCs was dependent on MAVS signaling, demonstrating a bifurcation in the pathogen recognition pathways that promote distinct elements of IFN-I mediated immunosuppression. Further, a similar suppressive DC origin and differentiation was also observed in Mycobacterium tuberculosis infection, HIV infection and cancer. Ultimately, targeting the underlying mechanisms that induce immunosuppression could simultaneously prevent multiple suppressive signals to further restore T cell function and control persistent infections.
Asunto(s)
Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Interferones/inmunología , Virosis/inmunología , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , VIH , Infecciones por VIH/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T/inmunología , Tuberculosis/inmunologíaRESUMEN
Effective clearance of apoptotic cells by macrophages is essential for immune homeostasis. The transcriptional pathways that allow macrophages to sense and respond to apoptotic cells are poorly defined. We found that liver X receptor (LXR) signaling was important for both apoptotic cell clearance and the maintenance of immune tolerance. Apoptotic cell engulfment activated LXR and thereby induced the expression of Mer, a receptor tyrosine kinase critical for phagocytosis. LXR-deficient macrophages exhibited a selective defect in phagocytosis of apoptotic cells and an aberrant proinflammatory response to them. As a consequence of these defects, mice lacking LXRs manifested a breakdown in self-tolerance and developed autoantibodies and autoimmune glomerulonephritis. Treatment with an LXR agonist ameliorated disease progression in a mouse model of lupus-like autoimmunity. Thus, activation of LXR by apoptotic cells engages a virtuous cycle that promotes their own clearance and couples engulfment to the suppression of inflammatory pathways.
Asunto(s)
Apoptosis/inmunología , Enfermedades Autoinmunes/inmunología , Proteínas de Unión al ADN/agonistas , Macrófagos/inmunología , Receptores Citoplasmáticos y Nucleares/agonistas , Bazo/inmunología , Animales , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Autoinmunidad/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Tolerancia Inmunológica/inmunología , Receptores X del Hígado , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Nucleares Huérfanos , Fagocitosis/inmunología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/inmunología , Transducción de Señal/inmunología , Bazo/citología , Bazo/metabolismo , Tirosina Quinasa c-MerRESUMEN
Reelin is a regulator of cell migration in the nervous system, and has other functions in the development of a number of non-neuronal tissues. In addition, alterations in reelin expression levels have been reported in breast, pancreatic, liver, gastric, and other cancers. Reelin is normally expressed in mammary gland stromal cells, but whether stromal reelin contributes to breast cancer progression is unknown. Herein, we used a syngeneic mouse mammary tumor transplantation model to examine the impact of host-derived reelin on breast cancer progression. We found that transplanted syngeneic tumors grew more slowly in reelin-deficient (rl Orl -/- ) mice and had delayed metastatic colonization of the lungs. Immunohistochemistry of primary tumors revealed that tumors grown in rl Orl -/- animals had fewer blood vessels and increased macrophage infiltration. Gene expression studies from tumor tissues indicate that loss of host-derived reelin alters the balance of M1- and M2-associated macrophage markers, suggesting that reelin may influence the polarization of these cells. Consistent with this, rl Orl -/- M1-polarized bone marrow-derived macrophages have heightened levels of the M1-associated cytokines iNOS and IL-6. Based on these observations, we propose a novel function for the reelin protein in breast cancer progression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular Neuronal/metabolismo , Proliferación Celular/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Mama/metabolismo , Mama/patología , Línea Celular , Línea Celular Tumoral , Citocinas/metabolismo , Progresión de la Enfermedad , Femenino , Expresión Génica/fisiología , Células HEK293 , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Proteína ReelinaRESUMEN
Stable isotope labeling has become an important methodology for determining lipid metabolic parameters of normal and neoplastic cells. Conventional methods for fatty acid and cholesterol analysis have one or more issues that limit their utility for in vitro stable isotope-labeling studies. To address this, we developed a method optimized for measuring both fatty acids and cholesterol from small numbers of stable isotope-labeled cultured cells. We demonstrate quantitative derivatization and extraction of fatty acids from a wide range of lipid classes using this approach. Importantly, cholesterol is also recovered, albeit at a modestly lower yield, affording the opportunity to quantitate both cholesterol and fatty acids from the same sample. Although we find that background contamination can interfere with quantitation of certain fatty acids in low amounts of starting material, our data indicate that this optimized method can be used to accurately measure mass isotopomer distributions for cholesterol and many fatty acids isolated from small numbers of cultured cells. Application of this method will facilitate acquisition of lipid parameters required for quantifying flux and provide a better understanding of how lipid metabolism influences cellular function.