RESUMEN
Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.
Asunto(s)
Homeostasis , Quinasas Janus , Macrófagos , Ratones Noqueados , Factores de Transcripción STAT , Transducción de Señal , Animales , Ratones , Macrófagos/inmunología , Macrófagos/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Ratones Endogámicos C57BL , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , TYK2 Quinasa/metabolismo , TYK2 Quinasa/genética , Regulación de la Expresión GénicaRESUMEN
SARS-CoV-2 surveillance by wastewater-based epidemiology is poised to provide a complementary approach to sequencing individual cases. However, robust quantification of variants and de novo detection of emerging variants remains challenging for existing strategies. We deep sequenced 3,413 wastewater samples representing 94 municipal catchments, covering >59% of the population of Austria, from December 2020 to February 2022. Our system of variant quantification in sewage pipeline designed for robustness (termed VaQuERo) enabled us to deduce the spatiotemporal abundance of predefined variants from complex wastewater samples. These results were validated against epidemiological records of >311,000 individual cases. Furthermore, we describe elevated viral genetic diversity during the Delta variant period, provide a framework to predict emerging variants and measure the reproductive advantage of variants of concern by calculating variant-specific reproduction numbers from wastewater. Together, this study demonstrates the power of national-scale WBE to support public health and promises particular value for countries without extensive individual monitoring.
Asunto(s)
COVID-19 , Monitoreo Epidemiológico Basado en Aguas Residuales , Humanos , Aguas Residuales , SARS-CoV-2/genética , COVID-19/epidemiología , ARN ViralRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gave rise to an international public health emergency in 3 months after its emergence in Wuhan, China. Typically for an RNA virus, random mutations occur constantly leading to new lineages, incidental with a higher transmissibility. The highly infective alpha lineage, firstly discovered in the UK, led to elevated mortality and morbidity rates as a consequence of Covid-19, worldwide. Wastewater surveillance proved to be a powerful tool for early detection and subsequent monitoring of the dynamics of SARS-CoV-2 and its variants in a defined catchment. Using a combination of sequencing and RT-qPCR approaches, we investigated the total SARS-CoV-2 concentration and the emergence of the alpha lineage in wastewater samples in Vienna, Austria linking it to clinical data. Based on a non-linear regression model and occurrence of signature mutations, we conclude that the alpha variant was present in Vienna sewage samples already in December 2020, even one month before the first clinical case was officially confirmed and reported by the health authorities. This provides evidence that a well-designed wastewater monitoring approach can provide a fast snapshot and may detect the circulating lineages in wastewater weeks before they are detectable in the clinical samples. Furthermore, declining 14 days prevalence data with simultaneously increasing SARS-CoV-2 total concentration in wastewater indicate a different shedding behavior for the alpha variant. Overall, our results support wastewater surveillance to be a suitable approach to spot early circulating SARS-CoV-2 lineages based on whole genome sequencing and signature mutations analysis.
Asunto(s)
COVID-19 , Monitoreo Epidemiológico Basado en Aguas Residuales , COVID-19/epidemiología , Humanos , SARS-CoV-2/genética , Aguas ResidualesRESUMEN
OBJECTIVE: The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. METHODS: The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. RESULTS: We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. CONCLUSIONS: Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation.
Asunto(s)
Proteína Forkhead Box O1/metabolismo , Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Adulto , Animales , Desdiferenciación Celular , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
OBJECTIVES: We explore the importance of SARS-CoV-2 sentinel surveillance testing in primary care during a regional COVID-19 outbreak in Austria. DESIGN: Prospective cohort study. SETTING: A single sentinel practice serving 22 829 people in the ski-resort of Schladming-Dachstein. PARTICIPANTS: All 73 patients presenting with mild-to-moderate flu-like symptoms between 24 February and 03 April, 2020. INTERVENTION: Nasopharyngeal sampling to detect SARS-CoV-2 using real-time reverse transcriptase-quantitative PCR (RT-qPCR). OUTCOME MEASURES: We compared RT-qPCR at presentation with confirmed antibody status. We split the outbreak in two parts, by halving the period from the first to the last case, to characterise three cohorts of patients with confirmed infection: early acute (RT-qPCR reactive) in the first half; and late acute (reactive) and late convalescent (non-reactive) in the second half. For each cohort, we report the number of cases detected, the accuracy of RT-qPCR, the duration and variety of symptoms, and the number of viral clades present. RESULTS: Twenty-two patients were diagnosed with COVID-19 (eight early acute, seven late acute and seven late convalescent), 44 patients tested SARS-CoV-2 negative and 7 were excluded. The sensitivity of RT-qPCR was 100% among all acute cases, dropping to 68.1% when including convalescent. Test specificity was 100%. Mean duration of symptoms for each group were 2 days (range 1-4) among early acute, 4.4 days (1-7) among late acute and 8 days (2-12) among late convalescent. Confirmed infection was associated with loss of taste. Acute infection was associated with loss of taste, nausea/vomiting, breathlessness, sore throat and myalgia; but not anosmia, fever or cough. Transmission clusters of three viral clades (G, GR and L) were identified. CONCLUSIONS: RT-qPCR testing in primary care can rapidly and accurately detect SARS-CoV-2 among people with flu-like illness in a heterogeneous viral outbreak. Targeted testing in primary care can support national sentinel surveillance of COVID-19.
Asunto(s)
COVID-19 , SARS-CoV-2 , Austria , Estudios de Cohortes , Humanos , Atención Primaria de Salud , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
The BTB zinc finger transcription factor MAZR (also known as PATZ1) controls, partially in synergy with the transcription factor Runx3, the development of CD8 lineage T cells. Here we explored the role of MAZR as well as combined activities of MAZR/Runx3 during cytotoxic T lymphocyte (CTL) and memory CD8+ T cell differentiation. In contrast to the essential role of Runx3 for CTL effector function, the deletion of MAZR had a mild effect on the generation of CTLs in vitro. However, a transcriptome analysis demonstrated that the combined deletion of MAZR and Runx3 resulted in much more widespread downregulation of CTL signature genes compared to single Runx3 deletion, indicating that MAZR partially compensates for loss of Runx3 in CTLs. Moreover, in line with the findings made in vitro, the analysis of CTL responses to LCMV infection revealed that MAZR and Runx3 cooperatively regulate the expression of CD8α, Granzyme B and perforin in vivo. Interestingly, while memory T cell differentiation is severely impaired in Runx3-deficient mice, the deletion of MAZR leads to an enlargement of the long-lived memory subset and also partially restored the differentiation defect caused by loss of Runx3. This indicates distinct functions of MAZR and Runx3 in the generation of memory T cell subsets, which is in contrast to their cooperative roles in CTLs. Together, our study demonstrates complex interplay between MAZR and Runx3 during CTL and memory T cell differentiation, and provides further insight into the molecular mechanisms underlying the establishment of CTL and memory T cell pools.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/inmunología , Memoria Inmunológica/inmunología , Proteínas de Neoplasias/inmunología , Proteínas Represoras/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno/inmunología , Activación de Linfocitos/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/virologíaRESUMEN
CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , COVID-19 , Epítopos de Linfocito T , Antígenos HLA-A/inmunología , Inmunidad Celular , Mutación , SARS-CoV-2 , Linfocitos T CD8-positivos/patología , COVID-19/genética , COVID-19/inmunología , COVID-19/patología , Proliferación Celular , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Interferón gamma/inmunología , Péptidos/genética , Péptidos/inmunología , SARS-CoV-2/genética , SARS-CoV-2/inmunologíaAsunto(s)
Proteína Potenciadora del Homólogo Zeste 2/genética , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Leucemia Mielomonocítica Crónica/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/genética , Proteínas ras/genética , Carcinogénesis/genética , Línea Celular Tumoral , Células HL-60 , Humanos , Células Mieloides/efectos de los fármacosRESUMEN
Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. However, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluripotency. Four intergenic elements form a super-enhancer, and most of the elements contribute to Fgf5 induction at distinct time points. A fifth, poised enhancer located in the first intron contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. Despite low individual enhancer activity, together these elements strongly induce Fgf5 expression in a super-additive fashion that involves strong accumulation of RNA polymerase II at the intronic enhancer. Finally, we observe a strong anti-correlation between RNA polymerase II levels at enhancers and their distance to the closest promoter, and we identify candidate elements with properties similar to the intronic enhancer.
Asunto(s)
Elementos de Facilitación Genéticos , Factor 5 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Animales , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Exones , Factor 5 de Crecimiento de Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/genética , Histonas/metabolismo , Intrones , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , ARN Polimerasa II/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Análisis de la Célula Individual , Transcripción Genética , Proteína Fluorescente RojaRESUMEN
Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.
Asunto(s)
Diabetes Gestacional/inmunología , Muerte Fetal/etiología , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Linfocitos T Reguladores/inmunología , Timo/inmunología , Adipocitos/patología , Animales , Proliferación Celular , Diabetes Gestacional/etiología , Diabetes Gestacional/metabolismo , Diabetes Gestacional/patología , Células Epiteliales/inmunología , Femenino , Feto/inmunología , Feto/metabolismo , Feto/patología , Glucosa/metabolismo , Intolerancia a la Glucosa/genética , Humanos , Grasa Intraabdominal/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Placenta/inmunología , Placenta/patología , Embarazo , Receptor Activador del Factor Nuclear kappa-B/deficiencia , Receptor Activador del Factor Nuclear kappa-B/genética , Linfocitos T Reguladores/citología , Timo/citología , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
Superspreading events shaped the coronavirus disease 2019 (COVID-19) pandemic, and their rapid identification and containment are essential for disease control. Here, we provide a national-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading during the first wave of infections in Austria, a country that played a major role in initial virus transmissions in Europe. Capitalizing on Austria's well-developed epidemiological surveillance system, we identified major SARS-CoV-2 clusters during the first wave of infections and performed deep whole-genome sequencing of more than 500 virus samples. Phylogenetic-epidemiological analysis enabled the reconstruction of superspreading events and charts a map of tourism-related viral spread originating from Austria in spring 2020. Moreover, we exploited epidemiologically well-defined clusters to quantify SARS-CoV-2 mutational dynamics, including the observation of low-frequency mutations that progressed to fixation within the infection chain. Time-resolved virus sequencing unveiled viral mutation dynamics within individuals with COVID-19, and epidemiologically validated infector-infectee pairs enabled us to determine an average transmission bottleneck size of 103 SARS-CoV-2 particles. In conclusion, this study illustrates the power of combining epidemiological analysis with deep viral genome sequencing to unravel the spread of SARS-CoV-2 and to gain fundamental insights into mutational dynamics and transmission properties.
Asunto(s)
COVID-19/epidemiología , COVID-19/transmisión , Mutación/genética , SARS-CoV-2/genética , Austria/epidemiología , Secuencia de Bases , COVID-19/genética , COVID-19/virología , Interacciones Huésped-Patógeno/genética , Humanos , Tasa de Mutación , FilogeniaRESUMEN
This is a report on the first identified cases of coronavirus disease 2019 (COVID-19) in Austria. The first documented case was a person who stayed in Kühtai, Tyrol, from 24 to 26 January 2020, and had been infected by a Chinese instructor in Starnberg (Germany) between 20 and 22 January. This counts as a German case since her diagnosis was eventually made in Munich (Germany) on 28 January. On 25 February, two cases imported from Italy were diagnosed in Innsbruck but again no secondary cases were identified in Austria. The first three infections of Austrian inhabitants were detected on 27 February in Vienna. The two resulting clusters finally included 6 (source of initial infection unknown) and 61 cases. Most likely, Italy was the source of the latter cluster. On 12 March the first fatal case of COVID-19 in Austria was reported, a 69-year-old Viennese who died in a Vienna hospital after returning from a cruise ship tour in Italy. On 6 March three autochthonously acquired cases were reported in the Tyrol, all related to the ski resort Ischgl. Of the first 14 Islandic COVID-19 cases infected in Ischgl, 11 had already returned to Iceland on 29 February. We consider that the incriminated barkeeper, who tested PCR positive on 7 March, was neither the primary case nor a superspreader. In our opinion, undetected transmission of SARS-CoV2 had been ongoing in Ischgl prior to the first laboratory confirmed cases. Our data also underline that the introduction of SARS-CoV2 into Austria was not one single event.
Asunto(s)
Betacoronavirus , Infecciones por Coronavirus , Pandemias , Neumonía Viral , Adulto , Austria , COVID-19 , Femenino , Humanos , Masculino , Persona de Mediana Edad , SARS-CoV-2 , Adulto JovenRESUMEN
In mammalian genomes, a subset of genes is regulated by genomic imprinting, resulting in silencing of one parental allele. Imprinting is essential for cerebral cortex development, but prevalence and functional impact in individual cells is unclear. Here, we determined allelic expression in cortical cell types and established a quantitative platform to interrogate imprinting in single cells. We created cells with uniparental chromosome disomy (UPD) containing two copies of either the maternal or the paternal chromosome; hence, imprinted genes will be 2-fold overexpressed or not expressed. By genetic labeling of UPD, we determined cellular phenotypes and transcriptional responses to deregulated imprinted gene expression at unprecedented single-cell resolution. We discovered an unexpected degree of cell-type specificity and a novel function of imprinting in the regulation of cortical astrocyte survival. More generally, our results suggest functional relevance of imprinted gene expression in glial astrocyte lineage and thus for generating cortical cell-type diversity.
Asunto(s)
Corteza Cerebral/metabolismo , Impresión Genómica , Transcriptoma , Disomía Uniparental , Animales , Astrocitos/clasificación , Astrocitos/metabolismo , Corteza Cerebral/citología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , RNA-Seq , Análisis de la Célula IndividualRESUMEN
The Bruton tyrosine kinase (BTK) inhibitor ibrutinib provides effective treatment for patients with chronic lymphocytic leukemia (CLL), despite extensive heterogeneity in this disease. To define the underlining regulatory dynamics, we analyze high-resolution time courses of ibrutinib treatment in patients with CLL, combining immune-phenotyping, single-cell transcriptome profiling, and chromatin mapping. We identify a consistent regulatory program starting with a sharp decrease of NF-κB binding in CLL cells, which is followed by reduced activity of lineage-defining transcription factors, erosion of CLL cell identity, and acquisition of a quiescence-like gene signature. We observe patient-to-patient variation in the speed of execution of this program, which we exploit to predict patient-specific dynamics in the response to ibrutinib based on the pre-treatment patient samples. In aggregate, our study describes time-dependent cellular, molecular, and regulatory effects for therapeutic inhibition of B cell receptor signaling in CLL, and it establishes a broadly applicable method for epigenome/transcriptome-based treatment monitoring.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/efectos de los fármacos , Cromatina/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pirazoles/antagonistas & inhibidores , Pirazoles/metabolismo , Pirazoles/uso terapéutico , Pirimidinas/antagonistas & inhibidores , Pirimidinas/metabolismo , Pirimidinas/uso terapéutico , Adenina/análogos & derivados , Epigenoma , Epigenómica , Perfilación de la Expresión Génica , Heterogeneidad Genética/efectos de los fármacos , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Aprendizaje Automático , Piperidinas , Receptores de Antígenos de Linfocitos B/efectos de los fármacos , Análisis de Secuencia de ARN , Factores de Transcripción , TranscriptomaRESUMEN
Tumor associated inflammation predicts response to immune checkpoint blockade in human melanoma. Current theories on regulation of inflammation center on anti-tumor T cell responses. Here we show that tumor associated B cells are vital to melanoma associated inflammation. Human B cells express pro- and anti-inflammatory factors and differentiate into plasmablast-like cells when exposed to autologous melanoma secretomes in vitro. This plasmablast-like phenotype can be reconciled in human melanomas where plasmablast-like cells also express T cell-recruiting chemokines CCL3, CCL4, CCL5. Depletion of B cells in melanoma patients by anti-CD20 immunotherapy decreases tumor associated inflammation and CD8+ T cell numbers. Plasmablast-like cells also increase PD-1+ T cell activation through anti-PD-1 blockade in vitro and their frequency in pretherapy melanomas predicts response and survival to immune checkpoint blockade. Tumor associated B cells therefore orchestrate and sustain melanoma inflammation and may represent a predictor for survival and response to immune checkpoint blockade therapy.
Asunto(s)
Inflamación/metabolismo , Melanoma/metabolismo , Anticuerpos Monoclonales/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Humanos , Inmunoterapia , Técnicas In Vitro , Inflamación/inmunología , Melanoma/inmunología , Melanoma/terapia , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
Aberrations in genes coding for subunits of the BRG1/BRM associated factor (BAF) chromatin remodeling complexes are highly abundant in human cancers. Currently, it is not understood how these mostly loss-of-function mutations contribute to cancer development and how they can be targeted therapeutically. The cancer-type-specific occurrence patterns of certain subunit mutations suggest subunit-specific effects on BAF complex function, possibly by the formation of aberrant residual complexes. Here, we systematically characterize the effects of individual subunit loss on complex composition, chromatin accessibility and gene expression in a panel of knockout cell lines deficient for 22 BAF subunits. We observe strong, specific and sometimes discordant alterations dependent on the targeted subunit and show that these explain intracomplex codependencies, including the synthetic lethal interactions SMARCA4-ARID2, SMARCA4-ACTB and SMARCC1-SMARCC2. These data provide insights into the role of different BAF subcomplexes in genome-wide chromatin organization and suggest approaches to therapeutically target BAF-mutant cancers.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Mutación , Neoplasias/patología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Factores de Transcripción/genética , TranscriptomaRESUMEN
RATIONALE: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.
Asunto(s)
Células Endoteliales/metabolismo , Vesículas Extracelulares/metabolismo , Interferón Tipo I/biosíntesis , Mitocondrias/metabolismo , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Adulto , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/inmunología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Lipopolisacáridos/toxicidad , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/inmunología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Adulto JovenRESUMEN
The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.
Asunto(s)
Ácido Fólico/metabolismo , Regulación de la Expresión Génica , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/genética , Técnicas de Inactivación de Genes , Humanos , Mutación con Pérdida de Función , Unión Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Transporte de Proteínas , Transducción de Señal , Transcripción GenéticaRESUMEN
Multiple sclerosis (MS) is a human neurodegenerative disease characterized by the invasion of autoreactive T cells from the periphery into the CNS. Application of pan-histone deacetylase inhibitors (HDACi) ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model for MS, suggesting that HDACi might be a potential therapeutic strategy for MS. However, the function of individual HDAC members in the pathogenesis of EAE is not known. In this study we report that mice with a T cell-specific deletion of HDAC1 (using the Cd4-Cre deleter strain; HDAC1-cKO) were completely resistant to EAE despite the ability of HDAC1cKO CD4+ T cells to differentiate into Th17 cells. RNA sequencing revealed STAT1 as a prominent upstream regulator of differentially expressed genes in activated HDAC1-cKO CD4+ T cells and this was accompanied by a strong increase in phosphorylated STAT1 (pSTAT1). This suggests that HDAC1 controls STAT1 activity in activated CD4+ T cells. Increased pSTAT1 levels correlated with a reduced expression of the chemokine receptors Ccr4 and Ccr6, which are important for the migration of T cells into the CNS. Finally, EAE susceptibility was restored in WT:HDAC1-cKO mixed BM chimeric mice, indicating a cell-autonomous defect. Our data demonstrate a novel pathophysiological role for HDAC1 in EAE and provide evidence that selective inhibition of HDAC1 might be a promising strategy for the treatment of MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Histona Desacetilasa 1/metabolismo , Esclerosis Múltiple/metabolismo , Factor de Transcripción STAT1/metabolismo , Células Th17/fisiología , Animales , Movimiento Celular , Células Cultivadas , Quimera , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Histona Desacetilasa 1/genética , Humanos , Ratones , Ratones Noqueados , Esclerosis Múltiple/inmunología , Receptores CCR4/metabolismo , Receptores CCR6/metabolismo , Factor de Transcripción STAT1/genéticaRESUMEN
Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail-like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.