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How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB.
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Cromatina/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Interleucina-1alfa/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Sitios de Unión , Células Cultivadas , Quimiocinas/metabolismo , Cromatina/química , Cromatina/genética , Células HeLa , Humanos , Quinasas Quinasa Quinasa PAM/genética , FN-kappa B/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL-4 and IL-13 induce AD-like features in keratinocytes in vitro. However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure is superior to model AD compared to simple 2D cell culture with cytokines. For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single-cell RNA sequencing data (scRNA-seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL-4 and IL-13 induced the closest in vivo-like AD phenotype which was observed in the scRNA-seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM-associated genes such as POSTN. While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo. Taken together, our comprehensive study shows that the simple model using IL-4 or IL-13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro, which may aid the discovery of novel treatment options.
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Dermatitis Atópica , Fibroblastos , Interleucina-13 , Interleucina-4 , Queratinocitos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Células Th2 , Humanos , Fibroblastos/metabolismo , Interleucina-4/farmacología , Interleucina-4/metabolismo , Interleucina-13/metabolismo , Interleucina-13/farmacología , Citocinas/metabolismo , Técnicas de Cocultivo , RNA-Seq , Células Cultivadas , Piel/patologíaRESUMEN
BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting in decreased quality of life. Histamine and specifically the H4 receptor play a key role in the inflammatory process in AD and serve as targets for novel therapeutic approaches. OBJECTIVE: In the present study we aimed to elucidate the immunopathological mechanisms with which the H4 receptor impacts TH2 cells and contributes to AD pathophysiology. METHODS: Total CD4+ T cells obtained from healthy or AD individuals and in vitro differentiated TH2 cells were cultured under different conditions and the mRNA expression or protein production of target molecules were determined using quantitative real-time PCR and ELISA. RESULTS: H4 receptor mRNA expression was upregulated concentration dependent upon IL-4 stimulation in in vitro differentiated TH2 cells progressively during the differentiation. Transcriptomic analysis of in vitro differentiated TH2 versus TH1 cells revealed that the H4 receptor among other genes represents one of the highly upregulated genes in TH2 cells. Most importantly, increased amounts of IL-5 and IL-13 mRNA expression were detected in in vitro differentiated TH2 cells as well as protein secretion in the presence of histamine or of the H4 receptor-selective-agonist when compared to the untreated control. CONCLUSION: We show for the first time an H4 receptor dependent upregulation of the pro-inflammatory cytokines IL-5 and IL-13 in human TH2 cells by histamine. This suggests that the blockade of the H4 receptor may lead to downregulation of these cytokines and amelioration of AD symptoms as reported in first clinical studies.
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Dermatitis Atópica , Interleucina-13 , Interleucina-5 , Receptores Histamínicos H4 , Células Th2 , Humanos , Células Th2/inmunología , Células Th2/metabolismo , Dermatitis Atópica/inmunología , Dermatitis Atópica/metabolismo , Interleucina-13/metabolismo , Interleucina-5/metabolismo , Receptores Histamínicos H4/metabolismo , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Células CultivadasRESUMEN
Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease. Up to 40% of cases are associated with heterozygous mutations in myosin binding protein C (cMyBP-C, MYBPC3). Most of these mutations lead to premature termination codons (PTC) and patients show reduction of functional cMyBP-C. This so-called haploinsufficiency most likely contributes to disease development. We analyzed mechanisms underlying haploinsufficiency using cardiac tissue from HCM-patients with truncation mutations in MYBPC3 (MYBPC3trunc). We compared transcriptional activity, mRNA and protein expression to donor controls. To differentiate between HCM-specific and general hypertrophy-induced mechanisms we used patients with left ventricular hypertrophy due to aortic stenosis (AS) as an additional control. We show that cMyBP-C haploinsufficiency starts at the mRNA level, despite hypertrophy-induced increased transcriptional activity. Gene set enrichment analysis (GSEA) of RNA-sequencing data revealed an increased expression of NMD-components. Among them, Up-frameshift protein UPF3B, a regulator of NMD was upregulated in MYBPC3trunc patients and not in AS-patients. Strikingly, we show that in sarcomeres UPF3B but not UPF1 and UPF2 are localized to the Z-discs, the presumed location of sarcomeric protein translation. Our data suggest that cMyBP-C haploinsufficiency in HCM-patients is established by UPF3B-dependent NMD during the initial translation round at the Z-disc.
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Cardiomiopatía Hipertrófica , Miocitos Cardíacos , Humanos , Cardiomiopatía Hipertrófica/metabolismo , Haploinsuficiencia , Hipertrofia/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
The kidney is a complex organ, which consists of multiple components with highly diverse cell types. A detailed understanding of these cell types in health and disease is crucial for the future development of preventive and curative treatment strategies. In recent years, single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) technology has opened up completely new possibilities in investigating the variety of renal cell populations in physiological and pathological states. Here, we systematically assessed differences between scRNAseq and snRNAseq approaches in transcriptome analysis of murine kidneys after ischemia-reperfusion injury. We included tissues from control kidneys and from kidneys harvested 1 wk after mild (17-min clamping time) and severe (27-min clamping time) transient unilateral ischemia. Our findings revealed important methodological differences in the discovery of inflammatory cells, tubular cells, and other specialized cell types. Although the scRNAseq approach was advantageous for investigating immune cells, the snRNAseq approach allowed superior insights into healthy and damaged tubular cells. Apart from differences in the quantitative discovery rate, we found important qualitative discrepancies in the captured transcriptomes with crucial consequences for the interpretation of cell states and molecular functions. Together, we provide an overview of method-dependent differences between scRNAseq and snRNAseq results from identical postischemic kidney tissues. Our results highlight the importance of choosing the right approach for specific research questions.NEW & NOTEWORTHY Single-cell and single-nucleus RNA sequencing technologies provide powerful new tools to examine complex tissues such as the kidney. This research reference paper provides practical information on the differences between the two technologies when examining murine kidneys after ischemia-reperfusion injury. The results will serve those who are debating which protocols to use in their given study.
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Daño por Reperfusión , Transcriptoma , Animales , Isquemia/metabolismo , Riñón/metabolismo , Ratones , Daño por Reperfusión/patologíaRESUMEN
OBJECTIVES: Giant cell arteritis (GCA) is the most common primary vasculitis, preferentially affecting the aorta and its large-calibre branches. An imbalance between proinflammatory CD4+ T helper cell subsets and regulatory T cells (Tregs) is thought to be involved in the pathogenesis of GCA and Treg dysfunction has been associated with active disease. Our work aims to explore the aetiology of Treg dysfunction and the way it is affected by remission-inducing immunomodulatory regimens. METHODS: A total of 41 GCA patients were classified into active disease (n=14) and disease in remission (n=27). GCA patients' and healthy blood donors' (HD) Tregs were sorted and subjected to transcriptome and phenotypic analysis. RESULTS: Transcriptome analysis revealed 27 genes, which were differentially regulated between GCA-derived and HD-derived Tregs. Among those, we identified transcription factors, glycolytic enzymes and IL-2 signalling mediators. We confirmed the downregulation of forkhead box P3 (FOXP3) and interferon regulatory factor 4 (IRF4) at protein level and identified the ineffective induction of glycoprotein A repetitions predominant (GARP) and CD25 as well as the reduced T cell receptor (TCR)-induced calcium influx as correlates of Treg dysfunction in GCA. Inhibition of glycolysis in HD-derived Tregs recapitulated most identified dysfunctions of GCA Tregs, suggesting the central pathogenic role of the downregulation of the glycolytic enzymes. Separate analysis of the subgroup of tocilizumab-treated patients identified the recovery of the TCR-induced calcium influx and the Treg suppressive function to associate with disease remission. CONCLUSIONS: Our findings suggest that low glycolysis and calcium signalling account for Treg dysfunction and inflammation in GCA.
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Factores de Transcripción Forkhead/genética , Arteritis de Células Gigantes/tratamiento farmacológico , Arteritis de Células Gigantes/genética , Factores Reguladores del Interferón/genética , Linfocitos T Reguladores/fisiología , Anciano , Anticuerpos Monoclonales Humanizados/uso terapéutico , Calcio/metabolismo , Señalización del Calcio/genética , Estudios de Casos y Controles , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Arteritis de Células Gigantes/inmunología , Glucólisis/genética , Humanos , Agentes Inmunomoduladores/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2/genética , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , FenotipoRESUMEN
BACKGROUND AND AIMS: Programmed death 1 (PD-1) checkpoint inhibition has shown promising results in patients with hepatocellular carcinoma, inducing objective responses in approximately 20% of treated patients. The roles of other coinhibitory molecules and their individual contributions to T-cell dysfunction in liver cancer, however, remain largely elusive. APPROACH AND RESULTS: We performed a comprehensive mRNA profiling of cluster of differentiation 8 (CD8) T cells in a murine model of autochthonous liver cancer by comparing the transcriptome of naive, functional effector, and exhausted, tumor-specific CD8 T cells. Subsequently, we functionally validated the role of identified genes in T-cell exhaustion. Our results reveal a unique transcriptome signature of exhausted T cells and demonstrate that up-regulation of the inhibitory immune receptor T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitor motif domains (TIGIT) represents a hallmark in the process of T-cell exhaustion in liver cancer. Compared to PD-1, expression of TIGIT more reliably identified exhausted CD8 T cells at different stages of their differentiation. In combination with PD-1 inhibition, targeting of TIGIT with antagonistic antibodies resulted in synergistic inhibition of liver cancer growth in immunocompetent mice. Finally, we demonstrate expression of TIGIT on tumor-infiltrating CD8 T cells in tissue samples of patients with hepatocellular carcinoma and intrahepatic cholangiocarcinoma and identify two subsets of patients based on differential expression of TIGIT on tumor-specific T cells. CONCLUSIONS: Our transcriptome analysis provides a valuable resource for the identification of key pathways involved in T-cell exhaustion in patients with liver cancer and identifies TIGIT as a potential target in checkpoint combination therapies.
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Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Colangiocarcinoma/genética , Colangiocarcinoma/inmunología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Receptores Inmunológicos/genética , Transcriptoma , Anciano , Animales , Neoplasias de los Conductos Biliares/patología , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Colangiocarcinoma/patología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores Inmunológicos/antagonistas & inhibidores , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacosRESUMEN
BACKGROUND: In hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis. METHODS: Global miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined. RESULTS: HDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3'UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs. CONCLUSIONS: This study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis.
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Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials.
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Terapia Genética , Vectores Genéticos , Animales , Daño del ADN , Expresión Génica , Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Vectores Genéticos/genética , Células Madre Hematopoyéticas , Humanos , Aprendizaje Automático , Ratones , Mutagénesis InsercionalRESUMEN
Given the intimate link between inflammation and dysregulated cell proliferation in cancer, we investigated cytokine-triggered gene expression in different cell cycle stages. Transcriptome analysis revealed that G1 release through cyclin-dependent kinase 6 (CDK6) and CDK4 primes and cooperates with the cytokine-driven gene response. CDK6 physically and functionally interacts with the NF-κB subunit p65 in the nucleus and is found at promoters of many transcriptionally active NF-κB target genes. CDK6 recruitment to distinct chromatin regions of inflammatory genes was essential for proper loading of p65 to its cognate binding sites and for the function of p65 coactivators, such as TRIP6. Furthermore, cytokine-inducible nuclear translocation and chromatin association of CDK6 depends on the kinase activity of TAK1 and p38. These results have widespread biological implications, as aberrant CDK6 expression or activation that is frequently observed in human tumors modulates NF-κB to shape the cytokine and chemokine repertoires in chronic inflammation and cancer.
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Cromatina/metabolismo , Quinasa 6 Dependiente de la Ciclina/fisiología , FN-kappa B/genética , Ciclo Celular/genética , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/fisiología , Quinasa 6 Dependiente de la Ciclina/análisis , Quinasa 6 Dependiente de la Ciclina/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Interleucina-1/metabolismo , Interleucina-1/fisiología , Interleucina-8/genética , Interleucina-8/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/fisiología , Regiones Promotoras Genéticas , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Preeclampsia is associated with an increased cardiovascular morbidity of mother and offspring, thus contributing to a substantial burden in women and children's health. It has been proven that endothelial progenitor cell (EPC) numbers and functional characteristics are impaired in cardiovascular disease and preeclampsia, although causative factors for the latter have remained elusive. MicroRNA (miRNA) modifications are a potential mechanism through which exposure to an altered environment translates into the development of chronic disease. In this study, we examined whether development of preeclampsia corresponds to alterations of miRNAs in maternal- and cord-blood-derived EPC. To test this end, we analyzed maternal and neonatal miRNAs via RNA sequencing from endothelial cells of preeclamptic and healthy controls in different cell culture passages. We were able to demonstrate differentially represented miRNAs in all groups. Hsa-miR-1270 showed significantly different levels in cord blood EPC from preeclampsia versus control and was negatively correlated with mRNA levels of its predicted targets ANGPTL7 and TFRC. Transfection with an hsa-miR-1270 inhibitor decreased the tube formation capacity and chemotactic motility but did not change proliferation in vitro. Target predictions and gene set enrichment analyses identified alternative splicing as a significantly enriched pathway for hsa-miR-1270. The top miRNAs in three other groups were predicted to target transcriptional and developmental pathways. Here, we showed for the first time significantly different levels of miRNAs and differently represented mRNA levels of predicted target genes in EPC derived from preeclampsia. Understanding the effects of preeclampsia on the epigenetic mechanisms of EPC will be crucial and may provide initial insights for further evaluation of the benefits of therapies targeting this cell population.
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Células Progenitoras Endoteliales/metabolismo , MicroARNs/genética , Preeclampsia/genética , Adulto , Proteína 7 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/genética , Antígenos CD/genética , Estudios de Casos y Controles , Proliferación Celular , Células Cultivadas , Quimiotaxis , Femenino , Sangre Fetal/citología , Sangre Fetal/metabolismo , Perfilación de la Expresión Génica , Humanos , Recién Nacido , Masculino , MicroARNs/sangre , MicroARNs/metabolismo , Neovascularización Patológica/genética , Preeclampsia/sangre , Preeclampsia/metabolismo , Embarazo , ARN Mensajero/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Transferrina/genética , Adulto JovenRESUMEN
Morbidity and mortality rates in acute lung injury (ALI) increase with age. As alveolar epithelial type II cells (AE2) are crucial for lung function and repair, we hypothesized that aging promotes senescence in AE2 and contributes to the severity and impaired regeneration in ALI. ALI was induced with 2.5 µg lipopolysaccharide/g body weight in young (3 mo) and old (18 mo) mice that were euthanized 24 h, 72 h, and 10 days later. Lung function, pulmonary surfactant activity, stereology, cell senescence, and single-cell RNA sequencing analyses were performed to investigate AE2 function in aging and ALI. In old mice, surfactant activity was severely impaired. A 60% mortality rate and lung function decline were observed in old, but not in young, mice with ALI. AE2 of young mice adapted to injury by increasing intracellular surfactant volume and proliferation rate. In old mice, however, this adaptive response was compromised, and AE2 of old mice showed signs of cell senescence, increased inflammatory signaling, and impaired surfactant metabolism in ALI. These findings provide evidence that ALI promotes a limited proliferation rate, increased inflammatory response, and surfactant dysfunction in old, but not in young, mice, supporting an impaired regenerative capacity and reduced survival rate in ALI with advancing age.
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Lesión Pulmonar Aguda/metabolismo , Envejecimiento , Células Epiteliales Alveolares/metabolismo , Surfactantes Pulmonares/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Células Epiteliales Alveolares/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismoRESUMEN
RATIONALE: Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing. OBJECTIVES: We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency. METHODS: Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed. MEASUREMENTS AND MAIN RESULTS: On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice. CONCLUSIONS: We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.
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Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Macrófagos Alveolares/metabolismo , Proteinosis Alveolar Pulmonar/metabolismo , Proteinosis Alveolar Pulmonar/terapia , Animales , Humanos , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1+ vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto+ vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap - including 1700012B09Rik, 1700026L06Rik and Fam183b - we provide extended experimental evidence for a ciliary function.
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Cilios/metabolismo , Feto/metabolismo , Factores de Transcripción Forkhead/metabolismo , Organizadores Embrionarios/metabolismo , Organogénesis , Mucosa Respiratoria/embriología , Animales , Diferenciación Celular/genética , Línea Celular , Regulación hacia Abajo/genética , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Estudios de Asociación Genética , Genoma , Proteínas Fluorescentes Verdes/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Ratones , Especificidad de Órganos/genética , Organogénesis/genética , Reproducibilidad de los Resultados , Mucosa Respiratoria/citología , Fracciones Subcelulares/metabolismo , Transcriptoma/genéticaRESUMEN
Interleukin (IL)-36α - one of the novel members of the IL-1 family of cytokines - is a potent regulator of dendritic and T cells and plays an important role in inflammatory processes like experimental skin inflammation in mice and in mouse models for human psoriasis. Here, we demonstrate that C/EBPß, a transcription factor required for the selective expression of inflammatory genes, is a key activator of the Il36A gene in murine macrophages. RNAi-mediated suppression of C/EBPß expression in macrophages (C/EBPß(low) cells) significantly impaired Il36A gene induction following challenge with LPS. Despite the presence of five predicted C/EBP binding sites, luciferase reporter assays demonstrated that C/EBPß confers responsiveness to LPS primarily through a half-CREâ¢C/EBP element in the proximal Il36A promoter. Electrophoretic mobility shift assays showed that C/EBPß but not CREB proteins interact with this critical half-CREâ¢C/EBP element. In addition, overexpression of C/EBPß in C/EBPß(low) cells enhanced the expression of Il36A whereas CREB-1 had no effect. Finally, chromatin immunoprecipitation confirmed that C/EBPß but neither CREB-1, ATF-2 nor ATF4 is directly recruited to the proximal promoter region of the Il36A gene. Together, these findings demonstrate an essential role of C/EBPß in the regulation of the Il36A gene via the proximal half-CREâ¢C/EBP element in response to inflammatory stimuli.
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Proteína beta Potenciadora de Unión a CCAAT/fisiología , Inflamación/genética , Interleucina-1/genética , Macrófagos/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , Codón Iniciador/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Psoriasis/genética , Elementos Reguladores de la TranscripciónRESUMEN
The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser(536), p38, and c-jun was inhibited, and basal inhibitory p65-Ser(468) phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1-dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1-associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.
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Monocitos/inmunología , FN-kappa B/inmunología , Proteína Fosfatasa 1/inmunología , Transducción de Señal/inmunología , Factor de Transcripción AP-1/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos/inmunología , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/inmunología , Glucógeno Sintasa Quinasa 3/metabolismo , Células HeLa , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Quinasa I-kappa B/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Factor de Transcripción ReIA/metabolismo , Transcriptoma/efectos de los fármacos , Transcriptoma/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. It is known that untranslated regions of the RNA can play a role in these processes. Here we show that THOC5, a member of the TREX (transcription/export) complex, plays a role in expression of only a subset of constitutively active genes, however transcriptome analysis reveals that more than 90% of IEG were not induced by serum in THOC5 depleted cells. Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts, were not released from chromatin in THOC5 depleted cells. Genes in another group, including Myc and Smad7 transcripts, were released with shortening of 3'UTR by alternative cleavage, and were spliced but export was impaired in THOC5 depleted cells. By interactome analysis using THOC5 as bait, we show that upon stimulation with serum THOC5 forms a complex with polyadenylation-specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3'UTR of THOC5 target genes. These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3'end-processing.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Genes Inmediatos-Precoces , Proteínas Nucleares/metabolismo , Procesamiento de Término de ARN 3' , Regiones no Traducidas 3' , Animales , Línea Celular , Ratones , Células 3T3 NIH , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Empalme del ARN , Proteína smad7/genética , Proteína smad7/metabolismo , Transcripción GenéticaRESUMEN
Kaposi's sarcoma (KS) is a mesenchymal tumour, which is caused by Kaposi's sarcoma herpesvirus (KSHV) and develops under inflammatory conditions. KSHV-infected endothelial spindle cells, the neoplastic cells in KS, show increased invasiveness, attributed to the elevated expression of metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). The majority of these spindle cells harbour latent KSHV genomes, while a minority undergoes lytic reactivation with subsequent production of new virions and viral or cellular chemo- and cytokines, which may promote tumour invasion and dissemination. In order to better understand KSHV pathogenesis, we investigated cellular mechanisms underlying the lytic reactivation of KSHV. Using a combination of small molecule library screening and siRNA silencing we found a STE20 kinase family member, MAP4K4, to be involved in KSHV reactivation from latency and to contribute to the invasive phenotype of KSHV-infected endothelial cells by regulating COX-2, MMP-7, and MMP-13 expression. This kinase is also highly expressed in KS spindle cells in vivo. These findings suggest that MAP4K4, a known mediator of inflammation, is involved in KS aetiology by regulating KSHV lytic reactivation, expression of MMPs and COX-2, and, thereby modulating invasiveness of KSHV-infected endothelial cells.
Asunto(s)
Células Endoteliales/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Herpesvirus Humano 8/fisiología , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas de Neoplasias/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Sarcoma de Kaposi/metabolismo , Activación Viral/fisiología , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Células Endoteliales/patología , Células Endoteliales/virología , Femenino , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Inflamación/virología , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/biosíntesis , Metaloproteinasa 7 de la Matriz/genética , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patologíaRESUMEN
BACKGROUND: Natural killer (NK) cells have been detected in the lesional skin of patients with inflammatory skin diseases, where high levels of histamine are also present. Therefore, we investigated the effect of histamine, in particular via the histamine H4 receptor (H4R), on gene expression levels in human NK cells. METHODS: Comprehensive microarray-based mRNA expression profiling was performed to assess the gene expression levels in human NK cells in response to H4R stimulation in an unbiased approach. The expression of selected cytokines and chemokines was quantified by real-time PCR and enzyme-linked immunosorbent assay. RESULTS: The microarray analysis identified only few genes which were differentially regulated upon H4R stimulation. In follow-up studies, a significant upregulation of CCL3 and CCL4 at the mRNA level and in addition for CCL3 also at the protein level via the H4R was observed. CONCLUSION: The elevated expression levels of chemokines in response to H4R stimulation might foster the inflammation in allergic skin diseases and characterize the H4R as a promising therapeutic target.
Asunto(s)
Citocinas/biosíntesis , Dermatitis Atópica/inmunología , Células Asesinas Naturales/inmunología , ARN Mensajero/biosíntesis , Receptores Acoplados a Proteínas G/inmunología , Receptores Histamínicos/inmunología , Linfocitos T CD4-Positivos/inmunología , Quimiocina CCL3/biosíntesis , Quimiocina CCL4/biosíntesis , Humanos , Inflamación/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Receptores Histamínicos H4RESUMEN
Histone deacetylase (HDAC) 3, as a cofactor in co-repressor complexes containing silencing mediator for retinoid or thyroid-hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR), has been shown to repress gene transcription in a variety of contexts. Here, we reveal a novel role for HDAC3 as a positive regulator of IL-1-induced gene expression. Various experimental approaches involving RNAi-mediated knockdown, conditional gene deletion or small molecule inhibitors indicate a positive role of HDAC3 for transcription of the majority of IL-1-induced human or murine genes. This effect was independent from the gene regulatory effects mediated by the broad-spectrum HDAC inhibitor trichostatin A (TSA) and thus suggests IL-1-specific functions for HDAC3. The stimulatory function of HDAC3 for inflammatory gene expression involves a mechanism that uses binding to NF-κB p65 and its deacetylation at various lysines. NF-κB p65-deficient cells stably reconstituted to express acetylation mimicking forms of p65 (p65 K/Q) had largely lost their potential to stimulate IL-1-triggered gene expression, implying that the co-activating property of HDAC3 involves the removal of inhibitory NF-κB p65 acetylations at K122, 123, 314 and 315. These data describe a novel function for HDAC3 as a co-activator in inflammatory signaling pathways and help to explain the anti-inflammatory effects frequently observed for HDAC inhibitors in (pre)clinical use.