RESUMEN
Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that mouse long-term ex vivo expanded AMs (exAMs) maintained a core AM gene expression program, but showed culture adaptations related to adhesion, metabolism and proliferation. Upon transplantation into the lung, exAMs reacquired full transcriptional and epigenetic AM identity, even after several months in culture and could self-maintain long-term in the alveolar niche. Changes in open chromatin regions observed in culture were fully reversible in transplanted exAMs and resulted in a gene expression profile indistinguishable from resident AMs. Our results indicate that long-term proliferation of AMs in culture did not compromise cellular identity in vivo. The robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of exAMs.
Asunto(s)
Pulmón , Macrófagos Alveolares , Animales , Cromatina/metabolismo , Epigénesis Genética , Epigenómica , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , RatonesRESUMEN
Migratory non-lymphoid tissue dendritic cells (NLT-DCs) transport antigens to lymph nodes (LNs) and are required for protective immune responses in the context of inflammation and to promote tolerance to self-antigens in steady-state. However, the molecular mechanisms that elicit steady-state NLT-DC maturation and migration are unknown. By comparing the transcriptome of NLT-DCs in the skin with their migratory counterparts in draining LNs, we have identified a novel NF-κB-regulated gene network specific to migratory DCs. We show that targeted deletion of IKKß in DCs, a major activator of NF-κB, prevents NLT-DC accumulation in LNs and compromises regulatory T cell conversion in vivo. This was associated with impaired tolerance and autoimmunity. NF-κB is generally considered the prototypical pro-inflammatory transcription factor, but this study describes a role for NF-κB signaling in DCs for immune homeostasis and tolerance that could have implications in autoimmune diseases and immunity.
Asunto(s)
Células Dendríticas/inmunología , Redes Reguladoras de Genes/inmunología , Homeostasis/inmunología , Tolerancia Inmunológica , FN-kappa B/inmunología , Transducción de Señal/inmunología , Animales , Autoantígenos/genética , Autoantígenos/inmunología , Autoinmunidad , Movimiento Celular , Células Dendríticas/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Quinasa I-kappa B/deficiencia , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Noqueados , Análisis por Micromatrices , FN-kappa B/genética , Piel/citología , Piel/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunologíaRESUMEN
Systemic mastocytosis (SM) is a KIT-driven hematopoietic neoplasm characterized by the excessive accumulation of neoplastic mast cells (MCs) in various organs and, mainly, the bone marrow (BM). Multiple genetic and epigenetic mechanisms contribute to the onset and severity of SM. However, little is known to date about the metabolic underpinnings underlying SM aggressiveness, which has thus far impeded the development of strategies to leverage metabolic dependencies when existing KIT-targeted treatments fail. Here, we show that plasma metabolomic profiles were able to discriminate indolent from advanced forms of the disease. We identified N-acetyl-d-glucosamine (GlcNAc) as the most predictive metabolite of SM severity. High plasma levels of GlcNAc in patients with advanced SM correlated with the activation of the GlcNAc-fed hexosamine biosynthesis pathway in patients BM aspirates and purified BM MCs. At the functional level, GlcNAc enhanced human neoplastic MCs proliferation and promoted rapid health deterioration in a humanized mouse model of SM. In addition, in the presence of GlcNAc, immunoglobulin E-stimulated MCs triggered enhanced release of proinflammatory cytokines and a stronger acute response in a mouse model of passive cutaneous anaphylaxis. Mechanistically, elevated GlcNAc levels promoted the transcriptional accessibility of chromatin regions that contain genes encoding mediators of receptor tyrosine kinases cascades and inflammatory responses, thus leading to a more aggressive phenotype. Therefore, GlcNAc is an oncometabolite driver of SM aggressiveness. This study suggests the therapeutic potential for targeting metabolic pathways in MC-related diseases to manipulate MCs effector functions.
Asunto(s)
Acetilglucosamina/análisis , Ensamble y Desensamble de Cromatina , Mastocitos/patología , Mastocitosis Sistémica/patología , Acetilglucosamina/metabolismo , Adulto , Animales , Progresión de la Enfermedad , Humanos , Mastocitos/metabolismo , Mastocitosis Sistémica/genética , Mastocitosis Sistémica/metabolismo , Metaboloma , Ratones SCID , Estudios ProspectivosRESUMEN
Mature differentiated macrophages can self-maintain by local proliferation in tissues and can be extensively expanded in culture under specific conditions, but the mechanisms of this phenomenon remain only partially defined. Here, we show that SIRT1, an evolutionary conserved regulator of life span, positively affects macrophage self-renewal ability in vitro and in vivo Overexpression of SIRT1 during bone marrow-derived macrophage differentiation increased their proliferative capacity. Conversely, decrease of SIRT1 expression by shRNA inactivation, CRISPR/Cas9 mediated deletion and pharmacological inhibition restricted macrophage self-renewal in culture. Furthermore, pharmacological SIRT1 inhibition in vivo reduced steady state and cytokine-induced proliferation of alveolar and peritoneal macrophages. Mechanistically, SIRT1 inhibition negatively regulated G1/S transition, cell cycle progression and a network of self-renewal genes. This included inhibition of E2F1 and Myc and concomitant activation of FoxO1, SIRT1 targets mediating cell cycle progression and stress response, respectively. Our findings indicate that SIRT1 is a key regulator of macrophage self-renewal that integrates cell cycle and longevity pathways. This suggests that macrophage self-renewal might be a relevant parameter of ageing.
Asunto(s)
Proliferación Celular , Autorrenovación de las Células , Macrófagos/fisiología , Sirtuina 1/metabolismo , Animales , Ciclo Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Ratones , Sirtuina 1/genéticaRESUMEN
T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed "exhausted" T cells. We compared the transcriptome of "exhausted" CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFß and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFß/IL-6-mediated induction of Maf.
Asunto(s)
Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Melanoma/metabolismo , Proteínas Proto-Oncogénicas c-maf/metabolismo , Microambiente Tumoral/fisiología , Animales , Linfocitos T CD8-positivos/metabolismo , Cartilla de ADN/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Interleucina-6/metabolismo , Luciferasas , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-maf/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-ß deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.
Asunto(s)
Linfocitos B/patología , Inflamación/inmunología , Inflamación/patología , Animales , Quimiocina CXCL13/metabolismo , Células Dendríticas/patología , Fibroblastos/patología , Ganglios Linfáticos/patología , Depleción Linfocítica , Linfocitos/patología , Ratones , Receptores CXCR5/deficiencia , Receptores CXCR5/metabolismo , Receptores de Complemento 3d/metabolismo , Células del Estroma/metabolismo , Células del Estroma/patología , Linfocitos TRESUMEN
As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFß exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFß signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
Asunto(s)
Caveolina 1 , Fibrosis Pulmonar Idiopática , Pulmón , MicroARNs , Factor de Crecimiento Transformador beta , Animales , Bleomicina/toxicidad , Caveolina 1/genética , Caveolina 1/metabolismo , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/fisiopatología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
In adoptive therapy, CD8 T cells expressing active STAT5 (STAT5CA) transcription factors were found to be superior to unmanipulated counterparts in long-term persistence, capacity to infiltrate autochthonous mouse melanomas, thrive in their microenvironment, and induce their regression. However, the molecular mechanisms sustaining these properties were undefined. In this study, we report that STAT5CA induced sustained expression of genes controlling tissue homing, cytolytic granule composition, type 1 CD8 cytotoxic T cell-associated effector molecules granzyme B(+), IFN-γ(+), TNF-α(+), and CCL3(+), but not IL-2, and transcription factors T-bet and eomesodermin (Eomes). Chromatin immunoprecipitation sequencing analyses identified the genes possessing regulatory regions to which STAT5 bound in long-term in vivo maintained STAT5CA-expressing CD8 T cells. This analysis identified 34% of the genes differentially expressed between STAT5CA-expressing and nonexpressing effector T cells as direct STAT5CA target genes, including those encoding T-bet, Eomes, and granzyme B. Additionally, genes encoding the IL-6R and TGFbRII subunits were stably repressed, resulting in dampened IL-17-producing CD8 T cell polarization in response to IL-6 and TGF-ß1. The absence of T-bet did not affect STAT5CA-driven accumulation of the T cells in tissue or their granzyme B expression but restored IL-2 secretion and IL-6R and TGFbRII expression and signaling, as illustrated by IL-17 induction. Therefore, concerted STAT5/T-bet/Eomes regulation controls homing, long-term maintenance, recall responses, and resistance to polarization towards IL-17-producing CD8 T cells while maintaining expression of an efficient type 1 CD8 cytotoxic T cell program (granzyme B(+), IFN-γ(+)).
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT5/metabolismo , Proteínas de Dominio T Box/genética , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Interleucina-17/metabolismo , Ratones , Ratones Transgénicos , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Factor de Transcripción STAT5/genética , Transducción de Señal , Proteínas de Dominio T Box/deficiencia , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Transcripción GenéticaRESUMEN
Hematopoietic stem cells (HSCs) maintain life-long production of immune cells and can directly respond to infection, but sustained effects on the immune response remain unclear. We show that acute immune stimulation with lipopolysaccharide (LPS) induced only transient changes in HSC abundance, composition, progeny, and gene expression, but persistent alterations in accessibility of specific myeloid lineage enhancers occurred, which increased responsiveness of associated immune genes to secondary stimulation. Functionally, this was associated with increased myelopoiesis of pre-exposed HSCs and improved innate immunity against the gram-negative bacterium P. aeruginosa. The accessible myeloid enhancers were enriched for C/EBPß targets, and C/EBPß deletion erased the long-term inscription of LPS-induced epigenetic marks and gene expression. Thus, short-term immune signaling can induce C/EBPß-dependent chromatin accessibility, resulting in HSC-trained immunity, during secondary infection. This establishes a mechanism for how infection history can be epigenetically inscribed in HSCs as an integral memory function of innate immunity.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT , Epigénesis Genética , Células Madre Hematopoyéticas/inmunología , Inmunidad Innata , Proteína beta Potenciadora de Unión a CCAAT/genética , Epigenómica , Humanos , MielopoyesisRESUMEN
Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Células Madre Embrionarias/citología , Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica , Macrófagos/citología , Animales , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Redes Reguladoras de Genes , Factor de Transcripción MafB/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-maf/metabolismo , Análisis de la Célula Individual , Activación TranscripcionalRESUMEN
Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.