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Deleterious somatic mutations in DNA methyltransferase 3 alpha (DNMT3A) and TET mehtylcytosine dioxygenase 2 (TET2) are associated with clonal expansion of hematopoietic cells and higher risk of cardiovascular disease (CVD). Here, we investigated roles of DNMT3A and TET2 in normal human monocyte-derived macrophages (MDM), in MDM isolated from individuals with DNMT3A or TET2 mutations, and in macrophages isolated from human atherosclerotic plaques. We found that loss of function of DNMT3A or TET2 resulted in a type I interferon response due to impaired mitochondrial DNA integrity and activation of cGAS signaling. DNMT3A and TET2 normally maintained mitochondrial DNA integrity by regulating the expression of transcription factor A mitochondria (TFAM) dependent on their interactions with RBPJ and ZNF143 at regulatory regions of the TFAM gene. These findings suggest that targeting the cGAS-type I IFN pathway may have therapeutic value in reducing risk of CVD in patients with DNMT3A or TET2 mutations.
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Enfermedades Cardiovasculares , ADN Metiltransferasa 3A/metabolismo , Proteínas de Unión al ADN/metabolismo , Dioxigenasas/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Humanos , Interferones/metabolismo , Macrófagos/metabolismo , Mitocondrias/genética , Mutación/genética , Nucleotidiltransferasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismoRESUMEN
Although the fetal immune system is considered tolerogenic, preterm infants can suffer from severe intestinal inflammation, including necrotizing enterocolitis (NEC). Here, we demonstrate that human fetal intestines predominantly contain tumor necrosis factor-α (TNF-α)+CD4+CD69+ T effector memory (Tem) cells. Single-cell RNA sequencing of fetal intestinal CD4+ T cells showed a T helper 1 phenotype and expression of genes mediating epithelial growth and cell cycling. Organoid co-cultures revealed a dose-dependent, TNF-α-mediated effect of fetal intestinal CD4+ T cells on intestinal stem cell (ISC) development, in which low T cell numbers supported epithelial development, whereas high numbers abrogated ISC proliferation. CD4+ Tem cell frequencies were higher in inflamed intestines from preterm infants with NEC than in healthy infant intestines and showed enhanced TNF signaling. These findings reveal a distinct population of TNF-α-producing CD4+ T cells that promote mucosal development in fetal intestines but can also mediate inflammation upon preterm birth.
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Linfocitos T CD4-Positivos/inmunología , Feto/inmunología , Memoria Inmunológica/inmunología , Intestinos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Células Epiteliales/citología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Feto/metabolismo , Humanos , Recién Nacido , Mucosa Intestinal/embriología , Mucosa Intestinal/crecimiento & desarrollo , Mucosa Intestinal/inmunología , Intestinos/embriología , Intestinos/crecimiento & desarrollo , Ratones Endogámicos C57BL , Embarazo , Células Madre/citología , Células Madre/inmunología , Células Madre/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in ß cells. Here, a mouse genetics approach shows that removal of TCF4 from ß cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.
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Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Hígado/metabolismo , Redes y Vías Metabólicas , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Animales , Animales Recién Nacidos , Dieta Alta en Grasa , Ayuno/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , Proteína 2 Similar al Factor de Transcripción 7/genética , Activación TranscripcionalRESUMEN
Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132-requiring upregulation of neddylation to restore proteasomal function and proteasomal stress-led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature-delayed closure of the ischiopubic rami-correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.
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Discapacidad Intelectual , Linfopenia , Humanos , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Transducción de Señal/genética , Discapacidad Intelectual/genética , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfopenia/genéticaRESUMEN
Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.
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Receptores ErbB/inmunología , Interleucina-13/inmunología , Interleucina-33/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Células Th2/inmunología , Anfirregulina/inmunología , Anfirregulina/metabolismo , Animales , Línea Celular , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Expresión Génica/genética , Expresión Génica/inmunología , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nematospiroides dubius/inmunología , Nematospiroides dubius/fisiología , Nocardia/inmunología , Nocardia/fisiología , Nocardiosis/inmunología , Nocardiosis/metabolismo , Nocardiosis/microbiología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/metabolismo , Infecciones por Strongylida/parasitología , Células Th2/metabolismoRESUMEN
BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16â 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.
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Aterosclerosis , Redes Reguladoras de Genes , Análisis de la Célula Individual , Humanos , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Ratones , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Masculino , Placa Aterosclerótica , Progresión de la Enfermedad , Femenino , Macrófagos/metabolismo , Macrófagos/patología , Ratones Noqueados , Receptores de LDL/genética , Receptores de LDL/metabolismo , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologíaRESUMEN
BACKGROUND: Cell phenotype switching is increasingly being recognized in atherosclerosis. However, our understanding of the exact stimuli for such cellular transformations and their significance for human atherosclerosis is still evolving. Intraplaque hemorrhage is thought to be a major contributor to plaque progression in part by stimulating the influx of CD163+ macrophages. Here, we explored the hypothesis that CD163+ macrophages cause plaque progression through the induction of proapoptotic endothelial-to-mesenchymal transition (EndMT) within the fibrous cap. METHODS: Human coronary artery sections from CVPath's autopsy registry were selected for pathological analysis. Athero-prone ApoE-/- and ApoE-/-/CD163-/- mice were used for in vivo studies. Human peripheral blood mononuclear cell-induced macrophages and human aortic endothelial cells were used for in vitro experiments. RESULTS: In 107 lesions with acute coronary plaque rupture, 55% had pathological evidence of intraplaque hemorrhage in nonculprit vessels/lesions. Thinner fibrous cap, greater CD163+ macrophage accumulation, and a larger number of CD31/FSP-1 (fibroblast specific protein-1) double-positive cells and TUNEL (terminal deoxynucleotidyl transferase-dUTP nick end labeling) positive cells in the fibrous cap were observed in nonculprit intraplaque hemorrhage lesions, as well as in culprit rupture sections versus nonculprit fibroatheroma sections. Human aortic endothelial cells cultured with supernatants from hemoglobin/haptoglobin-exposed macrophages showed that increased mesenchymal marker proteins (transgelin and FSP-1) while endothelial markers (VE-cadherin and CD31) were reduced, suggesting EndMT induction. Activation of NF-κB (nuclear factor kappa ß) signaling by proinflammatory cytokines released from CD163+ macrophages directly regulated the expression of Snail, a critical transcription factor during EndMT induction. Western blot analysis for cleaved caspase-3 and microarray analysis of human aortic endothelial cells indicated that apoptosis was stimulated during CD163+ macrophage-induced EndMT. Additionally, CD163 deletion in athero-prone mice suggested that CD163 is required for EndMT and plaque progression. Using single-cell RNA sequencing from human carotid endarterectomy lesions, a population of EndMT was detected, which demonstrated significant upregulation of apoptosis-related genes. CONCLUSIONS: CD163+ macrophages provoke EndMT, which may promote plaque progression through fibrous cap thinning.
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Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Macrófagos , Placa Aterosclerótica , Receptores de Superficie Celular , Humanos , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Animales , Antígenos CD/metabolismo , Antígenos CD/genética , Macrófagos/metabolismo , Macrófagos/patología , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Ratones , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Masculino , Ratones Noqueados para ApoE , Ratones Endogámicos C57BL , Apoptosis , Femenino , Transición Epitelial-Mesenquimal , Vasos Coronarios/patología , Vasos Coronarios/metabolismoRESUMEN
In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.
RESUMEN
BACKGROUND: Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events. METHODS: Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells. RESULTS: Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P=0.0034) and adjusted multivariate model (hazard ratio, 1.56; P=0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P=0.018) and of plaque hemorrhage (hazard ratio, 1.7; P=0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFß-triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells. CONCLUSIONS: Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications.
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Metilación de ADN , Células Endoteliales , Epigénesis Genética , Placa Aterosclerótica , Humanos , Masculino , Femenino , Anciano , Pronóstico , Persona de Mediana Edad , Células Endoteliales/patología , Células Endoteliales/metabolismo , Factores de Edad , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/cirugía , Células Cultivadas , Factores de Riesgo , Medición de RiesgoRESUMEN
BACKGROUND: Studies in humans and mice using the expression of an X-linked gene or lineage tracing, respectively, have suggested that clones of smooth muscle cells (SMCs) exist in human atherosclerotic lesions but are limited by either spatial resolution or translatability of the model. METHODS: Phenotypic clonality can be detected by X-chromosome inactivation patterns. We investigated whether clones of SMCs exist in unstable human atheroma using RNA in situ hybridization (BaseScope) to identify a naturally occurring 24-nucleotide deletion in the 3'UTR of the X-linked BGN (biglycan) gene, a proteoglycan highly expressed by SMCs. BGN-specific BaseScope probes were designed to target the wild-type or deletion mRNA. Three different coronary artery plaque types (erosion, rupture, and adaptive intimal thickening) were selected from heterozygous females for the deletion BGN. Hybridization of target RNA-specific probes was used to visualize the spatial distribution of mutants. A clonality index was calculated from the percentage of each probe in each region of interest. Spatial transcriptomics were used to identify differentially expressed transcripts within clonal and nonclonal regions. RESULTS: Less than one-half of regions of interest in the intimal plaque were considered clonal with the mean percent regions of interest with clonality higher in the intimal plaque than in the media. This was consistent for all plaque types. The relationship of the dominant clone in the intimal plaque and media showed significant concordance. In comparison with the nonclonal lesions, the regions with SMC clonality had lower expression of genes encoding cell growth suppressors such as CD74, SERF-2 (small EDRK-rich factor 2), CTSB (cathepsin B), and HLA-DPA1 (major histocompatibility complex, class II, DP alpha 1), among others. CONCLUSIONS: Our novel approach to examine clonality suggests atherosclerosis is primarily a disease of polyclonally and to a lesser extent clonally expanded SMCs and may have implications for the development of antiatherosclerotic therapies.
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Aterosclerosis , Placa Aterosclerótica , Femenino , Humanos , Ratones , Animales , Músculo Liso Vascular/metabolismo , Aterosclerosis/patología , Placa Aterosclerótica/patología , Células Clonales/patología , Proliferación Celular , Miocitos del Músculo Liso/metabolismo , ARNRESUMEN
BACKGROUND: Women presenting with coronary artery disease more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. METHODS: Gene regulatory networks were created using RNAseq gene expression data from human carotid atherosclerotic plaques. The networks were prioritized based on sex bias, relevance for smooth muscle biology, and coronary artery disease genetic enrichment. Network expression was linked to histologically determined plaque phenotypes. In addition, their expression in plaque cell types was studied at single-cell resolution using single-cell RNAseq. Finally, their relevance for disease progression was studied in female and male Apoe-/- mice fed a Western diet for 18 and 30 weeks. RESULTS: Here, we identify multiple sex-stratified gene regulatory networks from human carotid atherosclerotic plaques. Prioritization of the female networks identified 2 main SMC gene regulatory networks in late-stage atherosclerosis. Single-cell RNA sequencing mapped these female networks to 2 SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like network was mostly expressed in plaques that were vulnerable in women. Finally, the mice ortholog of key driver gene MFGE8 (milk fat globule EGF and factor V/VIII domain containing) showed retained expression in advanced plaques from female mice but was downregulated in male mice during atherosclerosis progression. CONCLUSIONS: Female atherosclerosis is characterized by gene regulatory networks that are active in fibrous vulnerable plaques rich in myofibroblast-like SMCs.
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Aterosclerosis , Enfermedad de la Arteria Coronaria , Placa Aterosclerótica , Femenino , Masculino , Humanos , Ratones , Animales , Placa Aterosclerótica/patología , Redes Reguladoras de Genes , Miofibroblastos/metabolismo , Enfermedad de la Arteria Coronaria/patología , Aterosclerosis/patología , Miocitos del Músculo Liso/metabolismoRESUMEN
OBJECTIVES: How the local inflammatory environment regulates epigenetic changes in the context of inflammatory arthritis remains unclear. Here we assessed the transcriptional and active enhancer profile of monocytes derived from the inflamed joints of JIA patients, a model well-suited for studying inflammatory arthritis. METHODS: RNA sequencing and H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) were used to analyse the transcriptional and epigenetic profile, respectively, of JIA synovial fluid-derived monocytes. RESULTS: Synovial-derived monocytes display an activated phenotype, which is regulated on the epigenetic level. IFN signalling-associated genes are increased and epigenetically altered in synovial monocytes, indicating a driving role for IFN in establishing the local inflammatory phenotype. Treatment of synovial monocytes with the Janus-associated kinase (JAK) inhibitor ruxolitinib, which inhibits IFN signalling, transformed the activated enhancer landscape and reduced disease-associated gene expression, thereby inhibiting the inflammatory phenotype. CONCLUSION: This study provides novel insights into epigenetic regulation of inflammatory arthritis patient-derived monocytes and highlights the therapeutic potential of epigenetic modulation for the treatment of inflammatory rheumatic diseases.
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Artritis , Monocitos , Humanos , Monocitos/metabolismo , Epigénesis Genética , Artritis/metabolismo , Líquido Sinovial/metabolismo , FenotipoRESUMEN
Side-chain rotamer prediction is one of the most critical late stages in protein 3D structure building. Highly advanced and specialized algorithms (e.g., FASPR, RASP, SCWRL4, and SCWRL4v) optimize this process by use of rotamer libraries, combinatorial searches, and scoring functions. We seek to identify the sources of key rotamer errors as a basis for correcting and improving the accuracy of protein modeling going forward. In order to evaluate the aforementioned programs, we process 2496 high-quality single-chained all-atom filtered 30% homology protein 3D structures and use discretized rotamer analysis to compare original with calculated structures. Among 513,024 filtered residue records, increased amino acid residue-dependent rotamer errorsâassociated in particular with polar and charged amino acid residues (ARG, LYS, and GLN)âclearly correlate with increased amino acid residue solvent accessibility and an increased residue tendency toward the adoption of non-canonical off rotamers which modeling programs struggle to predict accurately. Understanding the impact of solvent accessibility now appears key to improved side-chain prediction accuracies.
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Aminoácidos , Proteínas , Solventes , Proteínas/química , Aminoácidos/química , Algoritmos , Conformación ProteicaRESUMEN
BACKGROUND: Although sex differences in coronary artery disease are widely accepted with women developing more stable atherosclerosis than men, the underlying pathobiology of such differences remains largely unknown. In coronary artery disease, recent integrative systems biological studies have inferred gene regulatory networks (GRNs). Within these GRNs, key driver genes have shown great promise but have thus far been unidentified in women. METHODS: We generated sex-specific GRNs of the atherosclerotic arterial wall in 160 women and age-matched men in the STARNET study (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task). We integrated the female GRNs with single-cell RNA-sequencing data of the human atherosclerotic plaque and single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic smooth muscle cell (SMC) lineage-tracing mice. RESULTS: By comparing sex-specific GRNs, we observed clear sex differences in network activity within the atherosclerotic tissues. Genes more active in women were associated with mesenchymal cells and endothelial cells, whereas genes more active in men were associated with the immune system. We determined that key drivers of GRNs active in female coronary artery disease were predominantly found in (SMCs by single-cell sequencing of the human atherosclerotic plaques, and higher expressed in female plaque SMCs, as well. To study the functions of these female SMC key drivers in atherosclerosis, we examined single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic SMC lineage-tracing mice. The female key drivers were found to be expressed by phenotypically modulated SMCs and affected by Klf4, suggesting that sex differences in atherosclerosis involve phenotypic switching of plaque SMCs. CONCLUSIONS: Our systems approach provides novel insights into molecular mechanisms that underlie sex differences in atherosclerosis. To discover sex-specific therapeutic targets for atherosclerosis, an increased emphasis on sex-stratified approaches in the analysis of multi-omics data sets is warranted.
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Aterosclerosis/genética , Redes Reguladoras de Genes/genética , Miocitos del Músculo Liso/metabolismo , Animales , Aterosclerosis/fisiopatología , Diferenciación Celular , Femenino , Humanos , Ratones , FenotipoRESUMEN
Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.
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Artritis/inmunología , Colitis/inmunología , Factores de Transcripción Forkhead/metabolismo , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Proliferación Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Células HEK293 , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Líquido Sinovial/citología , Linfocitos T Reguladores/metabolismo , Vía de Señalización Wnt , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
RATIONALE: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. OBJECTIVE: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. METHODS AND RESULTS: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4+ and CD8+ T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. CONCLUSIONS: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.
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Enfermedades de las Arterias Carótidas/genética , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Linfocitos/metabolismo , Células Mieloides/metabolismo , Miocitos del Músculo Liso/metabolismo , Placa Aterosclerótica , Análisis de la Célula Individual , Transcriptoma , Anciano , Anciano de 80 o más Años , Animales , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Transdiferenciación Celular , Secuenciación de Inmunoprecipitación de Cromatina , Bases de Datos Genéticas , Células Endoteliales/patología , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Linfocitos/patología , Masculino , Ratones , Persona de Mediana Edad , Células Mieloides/patología , Miocitos del Músculo Liso/patología , Fenotipo , RNA-SeqRESUMEN
The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.
Asunto(s)
Células Madre Adultas/metabolismo , Envejecimiento/genética , Acumulación de Mutaciones , Tasa de Mutación , Especificidad de Órganos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Preescolar , Colon/metabolismo , Análisis Mutacional de ADN , Femenino , Genes Relacionados con las Neoplasias/genética , Humanos , Incidencia , Intestino Delgado/metabolismo , Hígado/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Células Madre Multipotentes/metabolismo , Neoplasias/epidemiología , Neoplasias/genética , Organoides/metabolismo , Mutación Puntual/genética , Adulto JovenRESUMEN
AIMS: Our objective was to better understand the genetic bases of dilated cardiomyopathy (DCM), a leading cause of systolic heart failure. METHODS AND RESULTS: We conducted the largest genome-wide association study performed so far in DCM, with 2719 cases and 4440 controls in the discovery population. We identified and replicated two new DCM-associated loci on chromosome 3p25.1 [lead single-nucleotide polymorphism (SNP) rs62232870, P = 8.7 × 10-11 and 7.7 × 10-4 in the discovery and replication steps, respectively] and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 × 10-8 and 1.4 × 10-3 in the discovery and replication steps, respectively), while confirming two previously identified DCM loci on chromosomes 10 and 1, BAG3 and HSPB7. A genetic risk score constructed from the number of risk alleles at these four DCM loci revealed a 3-fold increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). In silico annotation and functional 4C-sequencing analyses on iPSC-derived cardiomyocytes identify SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, in silico and data mining annotations, and to a lesser extent functional analysis, strongly suggest SMARCB1 as the candidate culprit gene. CONCLUSION: This study provides a better understanding of the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure.
Asunto(s)
Cardiomiopatía Dilatada , Insuficiencia Cardíaca Sistólica , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Reguladoras de la Apoptosis , Cardiomiopatía Dilatada/genética , Cromosomas , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Insuficiencia Cardíaca Sistólica/genética , Humanos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. METHODS: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. RESULTS: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. CONCLUSIONS: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.
Asunto(s)
Aterosclerosis/genética , Aterosclerosis/patología , Factores de Transcripción de Tipo Kruppel/genética , Miocitos del Músculo Liso/patología , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/patología , Animales , Femenino , Humanos , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Noqueados , Fenotipo , Análisis de Secuencia de ARN/métodosRESUMEN
Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.