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Lymph node (LN) stromal cells play a crucial role in LN development and in supporting adaptive immune responses. However, their origin, differentiation pathways, and transcriptional programs are still elusive. Here, we used lineage-tracing approaches and single-cell transcriptome analyses to determine origin, transcriptional profile, and composition of LN stromal and endothelial progenitors. Our results showed that all major stromal cell subsets and a large proportion of blood endothelial cells originate from embryonic Hoxb6+ progenitors of the lateral plate mesoderm (LPM), whereas lymphatic endothelial cells arise from Pax3+ progenitors of the paraxial mesoderm (PXM). Single-cell RNA sequencing revealed the existence of different Cd34+ and Cxcl13+ stromal cell subsets and showed that embryonic LNs contain proliferating progenitors possibly representing the amplifying populations for terminally differentiated cells. Taken together, our work identifies the earliest embryonic sources of LN stromal and endothelial cells and demonstrates that stromal diversity begins already during LN development.
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Células Endoteliales , Células Endoteliales/metabolismo , Ganglios Linfáticos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Células del Estroma , Factores de Transcripción/metabolismoRESUMEN
Eukaryotic DNA is organized in nucleosomes, which package DNA and regulate its accessibility to transcription, replication, recombination and repair. Here, we show that in living cells nucleosomes protect DNA from high-energy radiation and reactive oxygen species. We combined sequence-based methods (ATAC-seq and BLISS) to determine the position of both nucleosomes and double strand breaks (DSBs) in the genome of nucleosome-rich malignant mesothelioma cells, and of the same cells partially depleted of nucleosomes. The results were replicated in the human MCF-7 breast carcinoma cell line. We found that, for each genomic sequence, the probability of DSB formation is directly proportional to the fraction of time it is nucleosome-free; DSBs accumulate distal from the nucleosome dyad axis. Nucleosome free regions and promoters of actively transcribed genes are more sensitive to DSB formation, and consequently to mutation. We argue that this may be true for a variety of chemical and physical DNA damaging agents.
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Roturas del ADN de Doble Cadena/efectos de la radiación , ADN/efectos de la radiación , Nucleosomas/metabolismo , Animales , Línea Celular , Humanos , Células MCF-7 , RatonesRESUMEN
Invariant natural killer T cells (iNKT) cells are T lymphocytes displaying innate effector functions, acquired through a distinct thymic developmental program regulated by microRNAs (miRNAs). Deleting miRNAs by Dicer ablation (Dicer KO) in thymocytes selectively impairs iNKT cell survival and functional differentiation. To unravel this miRNA-dependent program, we systemically identified transcripts that were differentially expressed between WT and Dicer KO iNKT cells at different differentiation stages and predicted to be targeted by the iNKT cell-specific miRNAs. TGF-ß receptor II (TGF-ßRII), critically implicated in iNKT cell differentiation, was found up-regulated in iNKT Dicer KO cells together with enhanced TGF-ß signaling. miRNA members of the miR-17â¼92 family clusters were predicted to target Tgfbr2 mRNA upon iNKT cell development. iNKT cells lacking all three miR-17â¼92 family clusters (miR-17â¼92, miR-106aâ¼363, miR-106bâ¼25) phenocopied both increased TGF-ßRII expression and signaling, and defective effector differentiation, displayed by iNKT Dicer KO cells. Consistently, genetic ablation of TGF-ß signaling in the absence of miRNAs rescued iNKT cell differentiation. These results elucidate the global impact of miRNAs on the iNKT cell developmental program and uncover the targeting of a lineage-specific cytokine signaling by miRNAs as a mechanism regulating innate-like T-cell development and effector differentiation.
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MicroARNs/genética , Células T Asesinas Naturales/citología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Antígenos CD1d/metabolismo , Diferenciación Celular , Citocinas/metabolismo , ARN Helicasas DEAD-box/genética , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Familia de Multigenes , Fenotipo , ARN Mensajero/metabolismo , Ribonucleasa III/genética , Transducción de Señal , Timo/metabolismoRESUMEN
Vertebrate organogenesis is critically sensitive to gene dosage and even subtle variations in the expression levels of key genes may result in a variety of tissue anomalies. MicroRNAs (miRNAs) are fundamental regulators of gene expression and their role in vertebrate tissue patterning is just beginning to be elucidated. To gain further insight into this issue, we analysed the transcriptomic consequences of manipulating the expression of miR-204 in the Medaka fish model system. We used RNA-Seq and an innovative bioinformatics approach, which combines conventional differential expression analysis with the behavior expected by miR-204 targets after its overexpression and knockdown. With this approach combined with a correlative analysis of the putative targets, we identified a wider set of miR-204 target genes belonging to different pathways. Together, these approaches confirmed that miR-204 has a key role in eye development and further highlighted its putative function in neural differentiation processes, including axon guidance as supported by in vivo functional studies. Together, our results demonstrate the advantage of integrating next-generation sequencing and bioinformatics approaches to investigate miRNA biology and provide new important information on the role of miRNAs in the control of axon guidance and more broadly in nervous system development.
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Axones/fisiología , Perfilación de la Expresión Génica , MicroARNs/metabolismo , Neurogénesis/genética , Oryzias/genética , Animales , Axones/ultraestructura , Biología Computacional , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos Animales , Oryzias/embriología , Oryzias/metabolismo , Retina/embriología , Retina/metabolismo , Retina/ultraestructura , Análisis de Secuencia de ARNRESUMEN
Multiple myeloma (MM) is a malignancy of plasma cells characterized by multifocal osteolytic bone lesions. Macroscopic and genetic heterogeneity has been documented within MM lesions. Understanding the bases of such heterogeneity may unveil relevant features of MM pathobiology. To this aim, we deployed unbiased ¹H high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) metabolomics to analyze multiple biopsy specimens of osteolytic lesions from one case of pathological fracture caused by MM. Multivariate analyses on normalized metabolite peak integrals allowed clusterization of samples in accordance with a posteriori histological findings. We investigated the relationship between morphological and NMR features by merging morphological data and metabolite profiling into a single correlation matrix. Data-merging addressed tissue heterogeneity, and greatly facilitated the mapping of lesions and nearby healthy tissues. Our proof-of-principle study reveals integrated metabolomics and histomorphology as a promising approach for the targeted study of osteolytic lesions.
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Espectroscopía de Resonancia Magnética , Metabolómica/métodos , Mieloma Múltiple/metabolismo , Osteólisis/metabolismo , Anciano , Biopsia , Femenino , Humanos , Mieloma Múltiple/complicaciones , Mieloma Múltiple/patología , Análisis Multivariante , Osteólisis/complicaciones , Osteólisis/patologíaRESUMEN
The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4(+) T-cell lineages.
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Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/fisiología , Células TH1/fisiología , Células Th17/fisiología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Linaje de la Célula/inmunología , Proliferación Celular , Células Clonales/citología , Células Clonales/fisiología , Femenino , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Masculino , ARN Interferente Pequeño , Receptores CCR4/genética , Receptores CCR4/metabolismo , Células TH1/metabolismo , Células Th17/metabolismo , TranscriptomaRESUMEN
Transcription factor dynamics is fundamental to determine the activation of accurate transcriptional programs and yet is heterogeneous at a single-cell level, even within homogeneous populations. We asked how such heterogeneity emerges for the nuclear factor κB (NF-κB). We found that clonal populations of immortalized fibroblasts derived from a single mouse embryo display robustly distinct NF-κB dynamics upon tumor necrosis factor É (TNF-É) stimulation including persistent, oscillatory, and weak activation, giving rise to differences in the transcription of its targets. By combining transcriptomics and simulations we show how less than two-fold differences in the expression levels of genes coding for key proteins of the signaling cascade and feedback system are predictive of the differences of the NF-κB dynamic response of the clones to TNF-É and IL-1ß. We propose that small transcriptional differences in the regulatory circuit of a transcription factor can lead to distinct signaling dynamics in cells within homogeneous cell populations and among different cell types.
RESUMEN
Noncoding RNAs (ncRNAs), which include circular RNAs (circRNAs) and microRNAs (miRNAs), regulate the development of cardiovascular diseases (CVD). Notably, circRNAs can interact with miRNAs, influencing their specific mRNA targets' levels and shaping a competing endogenous RNAs (ceRNA) network. However, these interactions and their respective functions remain largely unexplored in ischemic heart failure (IHF). This study is aimed at identifying circRNA-centered ceRNA networks in non-end-stage IHF. Approximately 662 circRNA-miRNA-mRNA interactions were identified in the heart by combining state-of-the-art bioinformatics tools with experimental data. Importantly, KEGG terms of the enriched mRNA indicated CVD-related signaling pathways. A specific network centered on circBPTF was validated experimentally. The levels of let-7a-5p, miR-18a-3p, miR-146b-5p, and miR-196b-5p were enriched in circBPTF pull-down experiments, and circBPTF silencing inhibited the expression of HDAC9 and LRRC17, which are targets of miR-196b-5p. Furthermore, as suggested by the enriched pathway terms of the circBPTF ceRNA network, circBPTF inhibition elicited endothelial cell cycle arrest. circBPTF expression increased in endothelial cells exposed to hypoxia, and its upregulation was confirmed in cardiac samples of 36 end-stage IHF patients compared to healthy controls. In conclusion, circRNAs act as miRNA sponges, regulating the functions of multiple mRNA targets, thus providing a novel vision of HF pathogenesis and laying the theoretical foundation for further experimental studies.
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Enfermedades Cardiovasculares , Insuficiencia Cardíaca , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Endoteliales/metabolismo , Insuficiencia Cardíaca/genéticaRESUMEN
CRISPR/Cas9-mediated therapeutic gene editing is a promising technology for durable treatment of incurable monogenic diseases such as myotonic dystrophies. Gene-editing approaches have been recently applied to in vitro and in vivo models of myotonic dystrophy type 1 (DM1) to delete the pathogenic CTG-repeat expansion located in the 3' untranslated region of the DMPK gene. In DM1-patient-derived cells removal of the expanded repeats induced beneficial effects on major hallmarks of the disease with reduction in DMPK transcript-containing ribonuclear foci and reversal of aberrant splicing patterns. Here, we set out to excise the triplet expansion in a time-restricted and cell-specific fashion to minimize the potential occurrence of unintended events in off-target genomic loci and select for the target cell type. To this aim, we employed either a ubiquitous promoter-driven or a muscle-specific promoter-driven Cas9 nuclease and tetracycline repressor-based guide RNAs. A dual-vector approach was used to deliver the CRISPR/Cas9 components into DM1 patient-derived cells and in skeletal muscle of a DM1 mouse model. In this way, we obtained efficient and inducible gene editing both in proliferating cells and differentiated post-mitotic myocytes in vitro as well as in skeletal muscle tissue in vivo.
RESUMEN
When small B lymphocytes bind antigen in the context of suitable signals, a profound geno-proteomic metamorphosis is activated that generates antibody-secreting cells. To study the metabolic changes associated with this differentiation program, we compared the exometabolome of differentiating murine B lymphoma cells and primary B cells by monodimensional proton nuclear magnetic resonance spectroscopy and mass spectrometry coupled to liquid chromatography. Principal component analysis, a multivariate statistical analysis, highlighted metabolic hallmarks of the sequential differentiation phases discriminating between the proliferation and antibody secreting phases and revealing novel metabolic pathways. During proliferation, lactate production increased together with consumption of essential amino acids; massive Ig secretion was paralleled by alanine and glutamate production, glutamine being used as carbon and energy sources. Notably, ethanol and 5'-methylthioadenosine were produced during the last phase of protein secretion and the proliferative burst, respectively. Our metabolomics results are in agreement with previous genoproteomics studies. Thus, metabolic profiling of extracellular medium is a useful tool to characterize the functional state of differentiating B cells and to identify novel underlying metabolic pathways.
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Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular/fisiología , Metaboloma/fisiología , Metabolómica/métodos , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , Animales , Línea Celular Tumoral , Linfoma de Células B/patología , Espectrometría de Masas , Ratones , Resonancia Magnética Nuclear Biomolecular , Análisis de Componente Principal , Huella de ProteínaRESUMEN
Molecular mechanisms associated with human germ cell aplasia in infertile men remain undefined. Here we perform single-cell transcriptome profiling to highlight differentially expressed genes and pathways in each somatic cell type in testes of men with idiopathic germ cell aplasia. We identify immaturity of Leydig cells, chronic tissue inflammation, fibrosis, and senescence phenotype of the somatic cells, as well markers of chronic inflammation in the blood. We find that deregulated expression of parentally imprinted genes in myoid and immature Leydig cells, with relevant changes in the ratio of Lamin A/C transcripts and an active DNA damage response in Leydig and peritubular myoid cells are also indicative of senescence of the testicular niche. This study offers molecular insights into the pathogenesis of idiopathic germ cell aplasia.
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Envejecimiento/fisiología , Daño del ADN , Inflamación , Testículo/metabolismo , Envejecimiento/genética , Comunicación Celular , Quimiocinas , Perfilación de la Expresión Génica , Células Germinativas , Humanos , Inflamación/patología , Células Intersticiales del Testículo , Masculino , Fenotipo , Alineación de Secuencia , Espermatogénesis/genética , Espermatogénesis/fisiología , Espermatogonias/metabolismo , TranscriptomaRESUMEN
Bone marrow (BM) angiogenesis significantly influences disease progression in multiple myeloma (MM) patients and correlates with adverse prognosis. The present study shows a statistically significant correlation of the AP-1 family member JunB with VEGF, VEGFB, and IGF1 expression levels in MM. In contrast to the angiogenic master regulator Hif-1α, JunB protein levels were independent of hypoxia. Results in tumor-cell models that allow the induction of JunB knockdown or JunB activation, respectively, corroborated the functional role of JunB in the production and secretion of these angiogenic factors (AFs). Consequently, conditioned media derived from MM cells after JunB knockdown or JunB activation either inhibited or stimulated in vitro angiogenesis. The impact of JunB on MM BM angiogenesis was finally confirmed in a dynamic 3D model of the BM microenvironment, a xenograft mouse model as well as in patient-derived BM sections. In summary, in continuation of our previous study (Fan et al., 2017), the present report reveals for the first time that JunB is not only a mediator of MM cell survival, proliferation, and drug resistance, but also a promoter of AF transcription and consequently of MM BM angiogenesis. Our results thereby underscore worldwide efforts to target AP-1 transcription factors such as JunB as a promising strategy in MM therapy.
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Médula Ósea/irrigación sanguínea , Mieloma Múltiple/irrigación sanguínea , Factores de Transcripción/genética , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular Tumoral , Femenino , Xenoinjertos , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Cultivo Primario de Células , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor B de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Obesity and its associated metabolic abnormalities have become a global emergency with considerable morbidity and mortality. Epidemiologic and animal model data suggest an epigenetic contribution to obesity. Nevertheless, the cellular and molecular mechanisms through which epigenetics contributes to the development of obesity remain to be elucidated. Suv420h1 and Suv420h2 are histone methyltransferases responsible for chromatin compaction and gene repression. Through in vivo, ex vivo, and in vitro studies, we found that Suv420h1 and Suv420h2 respond to environmental stimuli and regulate metabolism by down-regulating peroxisome proliferator-activated receptor gamma (PPAR-γ), a master transcriptional regulator of lipid storage and glucose metabolism. Accordingly, mice lacking Suv420h proteins activate PPAR-γ target genes in brown adipose tissue to increase mitochondria respiration, improve glucose tolerance, and reduce adipose tissue to fight obesity. We conclude that Suv420h proteins are key epigenetic regulators of PPAR-γ and the pathways controlling metabolism and weight balance in response to environmental stimuli.
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Metabolismo Energético , N-Metiltransferasa de Histona-Lisina/metabolismo , PPAR gamma/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Animales , Cromatina/metabolismo , Frío , Dieta Alta en Grasa , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Obesidad/patología , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
Myoblast fusion (MF) is required for muscle growth and repair, and its alteration contributes to muscle diseases. The mechanisms governing this process are incompletely understood, and no epigenetic regulator has been previously described. Ash1L is an epigenetic activator belonging to the Trithorax group of proteins and is involved in FSHD muscular dystrophy, autism and cancer. Its physiological role in skeletal muscle is unknown. Here we report that Ash1L expression is positively correlated with MF and reduced in Duchenne muscular dystrophy. In vivo, ex vivo and in vitro experiments support a selective and evolutionary conserved requirement for Ash1L in MF. RNA- and ChIP-sequencing indicate that Ash1L is required to counteract Polycomb repressive activity to allow activation of selected myogenesis genes, in particular the key MF gene Cdon. Our results promote Ash1L as an important epigenetic regulator of MF and suggest that its activity could be targeted to improve cell therapy for muscle diseases.
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Moléculas de Adhesión Celular/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Línea Celular , Proteínas de Unión al ADN , N-Metiltransferasa de Histona-Lisina/genética , Ratones , Ratones Endogámicos C57BL , Distrofias MuscularesRESUMEN
Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2.
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Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/genética , Distrofia Miotónica/genética , Complejo Silenciador Inducido por ARN/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/genética , Humanos , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Microglia activation is a commonly pathological hallmark of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), a devastating disorder characterized by a selective motor neurons degeneration. Whether such activation might represent a causal event rather than a secondary epiphenomenon remains elusive. Here, we show that CNS-delivery of IL-4-via a lentiviral-mediated gene therapy strategy-skews microglia to proliferate, inducing these cells to adopt the phenotype of slowly proliferating cells. Transcriptome analysis revealed that IL-4-treated microglia express a broad number of genes normally encoded by embryonic microglia. Since embryonic microglia sustain CNS development, we then hypothesized that turning adult microglia to acquire such phenotype via IL-4 might be an efficient in vivo strategy to sustain motor neuron survival in ALS. IL-4 gene therapy in SOD1G93A mice resulted in a general amelioration of clinical outcomes during the early slowly progressive phase of the disease. However, such approach did not revert neurodegenerative processes occurring in the late and fast progressing phase of the disease.
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Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/terapia , Trasplante de Médula Ósea , Terapia Genética/métodos , Interleucina-4/genética , Microglía/metabolismo , Proteínas del Tejido Nervioso/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis/genética , Interleucina-4/administración & dosificación , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patología , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Cultivo Primario de Células , Transducción de Señal , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa-1/deficiencia , Superóxido Dismutasa-1/genética , Transcriptoma , Trasplante HomólogoRESUMEN
Oxidative stress plays a fundamental role in many conditions. Specifically, redox imbalance inhibits endothelial cell (EC) growth, inducing cell death and senescence. We used global transcriptome profiling to investigate the involvement of noncoding-RNAs in these phenotypes. By RNA-sequencing, transcriptome changes were analyzed in human ECs exposed to H2O2, highlighting a pivotal role of p53-signaling. Bioinformatic analysis and validation in p53-silenced ECs, identified several p53-targets among both mRNAs and long noncoding-RNAs (lncRNAs), including MALAT1 and NEAT1. Among microRNAs (miRNAs), miR-192-5p was the most induced by H2O2 treatment, in a p53-dependent manner. Down-modulated mRNA-targets of miR-192-5p were involved in cell cycle, DNA repair and stress response. Accordingly, miR-192-5p overexpression significantly decreased EC proliferation, inducing cell death. A central role of the p53-pathway was also confirmed by the analysis of differential exon usage: Upon H2O2 treatment, the expression of p53-dependent 5'-isoforms of MDM2 and PVT1 increased selectively. The transcriptomic alterations identified in H2O2-treated ECs were also observed in other physiological and pathological conditions where redox control plays a fundamental role, such as ECs undergoing replicative senescence, skeletal muscles of critical limb-ischemia patients and the peripheral-blood mononuclear cells of long-living individuals. Collectively, these findings indicate a prominent role of noncoding-RNAs in oxidative stress response.
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Regulación de la Expresión Génica/fisiología , Estrés Oxidativo/fisiología , ARN no Traducido/biosíntesis , Proteína p53 Supresora de Tumor/metabolismo , Anciano , Anciano de 80 o más Años , Línea Celular , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Oxidantes/farmacología , Oxidación-Reducción , TranscriptomaRESUMEN
In multiple sclerosis, the pathological interaction between autoreactive Th cells and mononuclear phagocytes in the CNS drives initiation and maintenance of chronic neuroinflammation. Here, we found that intrathecal transplantation of neural stem/precursor cells (NPCs) in mice with experimental autoimmune encephalomyelitis (EAE) impairs the accumulation of inflammatory monocyte-derived cells (MCs) in the CNS, leading to improved clinical outcome. Secretion of IL-23, IL-1, and TNF-α, the cytokines required for terminal differentiation of Th cells, decreased in the CNS of NPC-treated mice, consequently inhibiting the induction of GM-CSF-producing pathogenic Th cells. In vivo and in vitro transcriptome analyses showed that NPC-secreted factors inhibit MC differentiation and activation, favoring the switch toward an antiinflammatory phenotype. Tgfb2-/- NPCs transplanted into EAE mice were ineffective in impairing MC accumulation within the CNS and failed to drive clinical improvement. Moreover, intrathecal delivery of TGF-ß2 during the effector phase of EAE ameliorated disease severity. Taken together, these observations identify TGF-ß2 as the crucial mediator of NPC immunomodulation. This study provides evidence that intrathecally transplanted NPCs interfere with the CNS-restricted inflammation of EAE by reprogramming infiltrating MCs into antiinflammatory myeloid cells via secretion of TGF-ß2.
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Monocitos/fisiología , Esclerosis Múltiple/metabolismo , Células-Madre Neurales/trasplante , Factor de Crecimiento Transformador beta2/fisiología , Animales , Encéfalo/inmunología , Encéfalo/patología , Diferenciación Celular , Células Cultivadas , Citocinas/biosíntesis , Citocinas/metabolismo , Células Dendríticas/fisiología , Femenino , Inmunomodulación , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Microglía/inmunología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Células-Madre Neurales/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , TranscriptomaRESUMEN
Systems biology provides opportunities to fully understand the genes and pathways in disease pathogenesis. We used literature knowledge and unbiased multiple data meta-analysis paradigms to analyze microarray datasets across different mouse strains and acute allergic asthma models. Our combined gene-driven and pathway-driven strategies generated a stringent signature list totaling 933 genes with 41% (440) asthma-annotated genes and 59% (493) ignorome genes, not previously associated with asthma. Within the list, we identified inflammation, circadian rhythm, lung-specific insult response, stem cell proliferation domains, hubs, peripheral genes, and super-connectors that link the biological domains (Il6, Il1ß, Cd4, Cd44, Stat1, Traf6, Rela, Cadm1, Nr3c1, Prkcd, Vwf, Erbb2). In conclusion, this novel bioinformatics approach will be a powerful strategy for clinical and across species data analysis that allows for the validation of experimental models and might lead to the discovery of novel mechanistic insights in asthma.