RESUMEN
Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury.
Asunto(s)
Antibacterianos/farmacología , Ciego/inmunología , Ciego/microbiología , Diabetes Mellitus Tipo 1/inmunología , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/fisiología , Animales , Enfermedades Autoinmunes , Bacterias/clasificación , Bacterias/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Código de Histonas , Intestinos/inmunología , Masculino , Redes y Vías Metabólicas , Metagenoma , Ratones , Ratones Endogámicos NOD , MicroARNsRESUMEN
The analysis of single-cell genomics data presents several statistical challenges, and extensive efforts have been made to produce methods for the analysis of this data that impute missing values, address sampling issues and quantify and correct for noise. In spite of such efforts, no consensus on best practices has been established and all current approaches vary substantially based on the available data and empirical tests. The k-Nearest Neighbor Graph (kNN-G) is often used to infer the identities of, and relationships between, cells and is the basis of many widely used dimensionality-reduction and projection methods. The kNN-G has also been the basis for imputation methods using, e.g., neighbor averaging and graph diffusion. However, due to the lack of an agreed-upon optimal objective function for choosing hyperparameters, these methods tend to oversmooth data, thereby resulting in a loss of information with regard to cell identity and the specific gene-to-gene patterns underlying regulatory mechanisms. In this paper, we investigate the tuning of kNN- and diffusion-based denoising methods with a novel non-stochastic method for optimally preserving biologically relevant informative variance in single-cell data. The framework, Denoising Expression data with a Weighted Affinity Kernel and Self-Supervision (DEWÄKSS), uses a self-supervised technique to tune its parameters. We demonstrate that denoising with optimal parameters selected by our objective function (i) is robust to preprocessing methods using data from established benchmarks, (ii) disentangles cellular identity and maintains robust clusters over dimension-reduction methods, (iii) maintains variance along several expression dimensions, unlike previous heuristic-based methods that tend to oversmooth data variance, and (iv) rarely involves diffusion but rather uses a fixed weighted kNN graph for denoising. Together, these findings provide a new understanding of kNN- and diffusion-based denoising methods. Code and example data for DEWÄKSS is available at https://gitlab.com/Xparx/dewakss/-/tree/Tjarnberg2020branch.
Asunto(s)
Algoritmos , Genómica/métodos , Análisis de la Célula Individual/métodos , Aprendizaje Automático Supervisado , Animales , Línea Celular , Bases de Datos Genéticas , Humanos , Ratones , RNA-Seq , Saccharomyces cerevisiaeRESUMEN
IL-4 activates macrophages to adopt distinct phenotypes associated with clearance of helminth infections and tissue repair, but the phenotype depends on the cellular lineage of these macrophages. The molecular basis of chromatin remodeling in response to IL-4 stimulation in tissue-resident and monocyte-derived macrophages is not understood. In this study, we find that IL-4 activation of different lineages of peritoneal macrophages in mice is accompanied by lineage-specific chromatin remodeling in regions enriched with binding motifs of the pioneer transcription factor PU.1. PU.1 motif is similarly associated with both tissue-resident and monocyte-derived IL-4-induced accessible regions but has different lineage-specific DNA shape features and predicted cofactors. Mutation studies based on natural genetic variation between C57BL/6 and BALB/c mouse strains indicate that accessibility of these IL-4-induced regions can be regulated through differences in DNA shape without direct disruption of PU.1 motifs. We propose a model whereby DNA shape features of stimulation-dependent genomic elements contribute to differences in the accessible chromatin landscape of alternatively activated macrophages on different genetic backgrounds that may contribute to phenotypic variations in immune responses.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , ADN/genética , Macrófagos Peritoneales/fisiología , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Animales , Sitios de Unión/genética , Inmunidad/genética , Interleucina-4/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocitos/fisiología , Mutación/genética , Unión Proteica/genéticaRESUMEN
The p53 gene contains homozygous mutations in ~50-60% of human cancers. About 90% of these mutations encode missense mutant proteins that span ~190 different codons localized in the DNA-binding domain of the gene and protein. These mutations produce a protein with a reduced capacity to bind to a specific DNA sequence that regulates the p53 transcriptional pathway. Eight of these mutations are localized in codons that account for ~28% of the total p53 mutations and these alleles appear to be selected for preferentially in human cancers of many tissue types. This article explores the question 'Why are there hotspot mutations in the p53 gene in human cancers?' Four possible reasons for this are considered; (1) the hotspot mutant alleles produce a protein that has a highly altered structure, (2) environmental mutagens produce allele-specific changes in the p53 gene, (3) these mutations arise at selected sites in the gene due to a specific DNA sequence, such as a methylated cytosine residue in a CpG dinucleotide, which has a higher mutation rate changing C to T nucleotides, (4) along with the observed change in mutant p53 proteins, which produce a loss of function (DNA binding and transcription), some mutant proteins have an allele-specific gain of function that promotes cancer. Evidence is presented that demonstrates the first three possibilities all contribute some property to this list of hotspot mutations. The fourth possibility remains to be tested.
Asunto(s)
Genes p53 , Mutación Missense , Proteína p53 Supresora de Tumor/genética , Mutación con Ganancia de Función , Frecuencia de los Genes , Humanos , Proteína p53 Supresora de Tumor/químicaRESUMEN
Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.
Asunto(s)
Isocitrato Deshidrogenasa/genética , Factores de Transcripción SOXB1/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína Nuclear Ligada al Cromosoma X/genética , Animales , Apoptosis , Astrocitoma/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Factor de Unión a CCCTC/metabolismo , Diferenciación Celular , Células Cultivadas , Metilación de ADN , Epigénesis Genética , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ratones , Ratones SCID , Clasificación del Tumor , Invasividad Neoplásica , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Interferencia de ARN , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Proteína Nuclear Ligada al Cromosoma X/antagonistas & inhibidores , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
BACKGROUND: Inflammatory bowel diseases (IBD) are believed to be driven by dysregulated interactions between the host and the gut microbiota. Our goal is to characterize and infer relationships between mucosal T cells, the host tissue environment, and microbial communities in patients with IBD who will serve as basis for mechanistic studies on human IBD. METHODS: We characterized mucosal CD4 T cells using flow cytometry, along with matching mucosal global gene expression and microbial communities data from 35 pinch biopsy samples from patients with IBD. We analyzed these data sets using an integrated framework to identify predictors of inflammatory states and then reproduced some of the putative relationships formed among these predictors by analyzing data from the pediatric RISK cohort. RESULTS: We identified 26 predictors from our combined data set that were effective in distinguishing between regions of the intestine undergoing active inflammation and regions that were normal. Network analysis on these 26 predictors revealed SAA1 as the most connected node linking the abundance of the genus Bacteroides with the production of IL17 and IL22 by CD4 T cells. These SAA1-linked microbial and transcriptome interactions were further reproduced with data from the pediatric IBD RISK cohort. CONCLUSIONS: This study identifies expression of SAA1 as an important link between mucosal T cells, microbial communities, and their tissue environment in patients with IBD. A combination of T cell effector function data, gene expression and microbial profiling can distinguish between intestinal inflammatory states in IBD regardless of disease types.
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Linfocitos T CD4-Positivos/inmunología , Microbioma Gastrointestinal/inmunología , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Proteína Amiloide A Sérica/fisiología , Adulto , Biopsia , Estudios de Casos y Controles , Niño , Colon/inmunología , Colon/microbiología , Colon/patología , Expresión Génica , Humanos , Inmunidad Celular , Enfermedades Inflamatorias del Intestino/patología , Interleucina-17/biosíntesis , Interleucinas/biosíntesis , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Células Th17/inmunología , Interleucina-22RESUMEN
Increasing incidence of inflammatory bowel diseases, such as Crohn's disease, in developed nations is associated with changes to the microbial environment, such as decreased prevalence of helminth colonization and alterations to the gut microbiota. We find that helminth infection protects mice deficient in the Crohn's disease susceptibility gene Nod2 from intestinal abnormalities by inhibiting colonization by an inflammatory Bacteroides species. Resistance to Bacteroides colonization was dependent on type 2 immunity, which promoted the establishment of a protective microbiota enriched in Clostridiales. Additionally, we show that individuals from helminth-endemic regions harbor a similar protective microbiota and that deworming treatment reduced levels of Clostridiales and increased Bacteroidales. These results support a model of the hygiene hypothesis in which certain individuals are genetically susceptible to the consequences of a changing microbial environment.
Asunto(s)
Infecciones por Bacteroides/inmunología , Bacteroides/inmunología , Enfermedad de Crohn/genética , Microbioma Gastrointestinal/inmunología , Intestinos/inmunología , Proteína Adaptadora de Señalización NOD2/genética , Tricuriasis/inmunología , Trichuris/inmunología , Animales , Clostridiales/inmunología , Infecciones por Clostridium/inmunología , Enfermedad de Crohn/inmunología , Predisposición Genética a la Enfermedad , Hipótesis de la Higiene , Intestinos/microbiología , Intestinos/parasitología , Ratones , Ratones MutantesRESUMEN
BACKGROUND: Obesity, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD) are serious health concerns, especially in Western populations. Antibiotic exposure and high-fat diet (HFD) are important and modifiable factors that may contribute to these diseases. METHODS: To investigate the relationship of antibiotic exposure with microbiome perturbations in a murine model of growth promotion, C57BL/6 mice received lifelong sub-therapeutic antibiotic treatment (STAT), or not (control), and were fed HFD starting at 13 weeks. To characterize microbiota changes caused by STAT, the V4 region of the 16S rRNA gene was examined from collected fecal samples and analyzed. RESULTS: In this model, which included HFD, STAT mice developed increased weight and fat mass compared to controls. Although results in males and females were not identical, insulin resistance and NAFLD were more severe in the STAT mice. Fecal microbiota from STAT mice were distinct from controls. Compared with controls, STAT exposure led to early conserved diet-independent microbiota changes indicative of an immature microbial community. Key taxa were identified as STAT-specific and several were found to be predictive of disease. Inferred network models showed topological shifts concurrent with growth promotion and suggest the presence of keystone species. CONCLUSIONS: These studies form the basis for new models of type 2 diabetes and NAFLD that involve microbiome perturbation.
Asunto(s)
Adiposidad , Antibacterianos/farmacología , Dieta Alta en Grasa/efectos adversos , Microbioma Gastrointestinal/efectos de los fármacos , Resistencia a la Insulina , Hepatopatías/etiología , Hepatopatías/metabolismo , Adiposidad/efectos de los fármacos , Animales , Biodiversidad , Composición Corporal , Peso Corporal , Citocinas/sangre , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Hormonas/sangre , Mediadores de Inflamación/sangre , Insulina/metabolismo , Metabolismo de los Lípidos , Hepatopatías/microbiología , Metagenoma , Metagenómica , Ratones , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/microbiología , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Factores de TiempoRESUMEN
16S ribosomal RNA (rRNA) gene and other environmental sequencing techniques provide snapshots of microbial communities, revealing phylogeny and the abundances of microbial populations across diverse ecosystems. While changes in microbial community structure are demonstrably associated with certain environmental conditions (from metabolic and immunological health in mammals to ecological stability in soils and oceans), identification of underlying mechanisms requires new statistical tools, as these datasets present several technical challenges. First, the abundances of microbial operational taxonomic units (OTUs) from amplicon-based datasets are compositional. Counts are normalized to the total number of counts in the sample. Thus, microbial abundances are not independent, and traditional statistical metrics (e.g., correlation) for the detection of OTU-OTU relationships can lead to spurious results. Secondly, microbial sequencing-based studies typically measure hundreds of OTUs on only tens to hundreds of samples; thus, inference of OTU-OTU association networks is severely under-powered, and additional information (or assumptions) are required for accurate inference. Here, we present SPIEC-EASI (SParse InversE Covariance Estimation for Ecological Association Inference), a statistical method for the inference of microbial ecological networks from amplicon sequencing datasets that addresses both of these issues. SPIEC-EASI combines data transformations developed for compositional data analysis with a graphical model inference framework that assumes the underlying ecological association network is sparse. To reconstruct the network, SPIEC-EASI relies on algorithms for sparse neighborhood and inverse covariance selection. To provide a synthetic benchmark in the absence of an experimentally validated gold-standard network, SPIEC-EASI is accompanied by a set of computational tools to generate OTU count data from a set of diverse underlying network topologies. SPIEC-EASI outperforms state-of-the-art methods to recover edges and network properties on synthetic data under a variety of scenarios. SPIEC-EASI also reproducibly predicts previously unknown microbial associations using data from the American Gut project.