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1.
Exp Mol Med ; 52(9): 1452-1465, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32929226

RESUMO

Intratumor heterogeneity is a common characteristic across diverse cancer types and presents challenges to current standards of treatment. Advancements in high-throughput sequencing and imaging technologies provide opportunities to identify and characterize these aspects of heterogeneity. Notably, transcriptomic profiling at a single-cell resolution enables quantitative measurements of the molecular activity that underlies the phenotypic diversity of cells within a tumor. Such high-dimensional data require computational analysis to extract relevant biological insights about the cell types and states that drive cancer development, pathogenesis, and clinical outcomes. In this review, we highlight emerging themes in the computational analysis of single-cell transcriptomics data and their applications to cancer research. We focus on downstream analytical challenges relevant to cancer research, including how to computationally perform unified analysis across many patients and disease states, distinguish neoplastic from nonneoplastic cells, infer communication with the tumor microenvironment, and delineate tumoral and microenvironmental evolution with trajectory and RNA velocity analysis. We include discussions of challenges and opportunities for future computational methodological advancements necessary to realize the translational potential of single-cell transcriptomic profiling in cancer.

2.
Sci Adv ; 6(26): eaba4353, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32637608

RESUMO

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.

3.
Nature ; 582(7811): 234-239, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499652

RESUMO

On average, Peruvian individuals are among the shortest in the world1. Here we show that Native American ancestry is associated with reduced height in an ethnically diverse group of Peruvian individuals, and identify a population-specific, missense variant in the FBN1 gene (E1297G) that is significantly associated with lower height. Each copy of the minor allele (frequency of 4.7%) reduces height by 2.2 cm (4.4 cm in homozygous individuals). To our knowledge, this is the largest effect size known for a common height-associated variant. FBN1 encodes the extracellular matrix protein fibrillin 1, which is a major structural component of microfibrils. We observed less densely packed fibrillin-1-rich microfibrils with irregular edges in the skin of individuals who were homozygous for G1297 compared with individuals who were homozygous for E1297. Moreover, we show that the E1297G locus is under positive selection in non-African populations, and that the E1297 variant shows subtle evidence of positive selection specifically within the Peruvian population. This variant is also significantly more frequent in coastal Peruvian populations than in populations from the Andes or the Amazon, which suggests that short stature might be the result of adaptation to factors that are associated with the coastal environment in Peru.


Assuntos
Estatura/genética , Fibrilina-1/genética , Mutação de Sentido Incorreto , Seleção Genética , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Hereditariedade , Humanos , Índios Sul-Americanos/genética , Masculino , Microfibrilas/química , Microfibrilas/genética , Peru
4.
Sci Transl Med ; 12(545)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461333

RESUMO

It is challenging to quickly diagnose slowly progressing diseases. To prioritize multiple related diagnoses, we developed G-PROB (Genetic Probability tool) to calculate the probability of different diseases for a patient using genetic risk scores. We tested G-PROB for inflammatory arthritis-causing diseases (rheumatoid arthritis, systemic lupus erythematosus, spondyloarthropathy, psoriatic arthritis, and gout). After validating on simulated data, we tested G-PROB in three cohorts: 1211 patients identified by International Classification of Diseases (ICD) codes within the eMERGE database, 245 patients identified through ICD codes and medical record review within the Partners Biobank, and 243 patients first presenting with unexplained inflammatory arthritis and with final diagnoses by record review within the Partners Biobank. Calibration of G-probabilities with disease status was high, with regression coefficients from 0.90 to 1.08 (1.00 is ideal). G-probabilities discriminated true diagnoses across the three cohorts with pooled areas under the curve (95% CI) of 0.69 (0.67 to 0.71), 0.81 (0.76 to 0.84), and 0.84 (0.81 to 0.86), respectively. For all patients, at least one disease could be ruled out, and in 45% of patients, a likely diagnosis was identified with a 64% positive predictive value. In 35% of cases, the clinician's initial diagnosis was incorrect. Initial clinical diagnosis explained 39% of the variance in final disease, which improved to 51% (P < 0.0001) after adding G-probabilities. Converting genotype information before a clinical visit into an interpretable probability value for five different inflammatory arthritides could potentially be used to improve the diagnostic efficiency of rheumatic diseases in clinical practice.

5.
Proc Natl Acad Sci U S A ; 117(10): 5532-5541, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32079724

RESUMO

The role of stromal fibroblasts in chronic inflammation is unfolding. In rheumatoid arthritis, leukocyte-derived cytokines TNF and IL-17A work together, activating fibroblasts to become a dominant source of the hallmark cytokine IL-6. However, IL-17A alone has minimal effect on fibroblasts. To identify key mediators of the synergistic response to TNF and IL-17A in human synovial fibroblasts, we performed time series, dose-response, and gene-silencing transcriptomics experiments. Here we show that in combination with TNF, IL-17A selectively induces a specific set of genes mediated by factors including cut-like homeobox 1 (CUX1) and IκBζ (NFKBIZ). In the promoters of CXCL1, CXCL2, and CXCL3, we found a putative CUX1-NF-κB binding motif not found elsewhere in the genome. CUX1 and NF-κB p65 mediate transcription of these genes independent of LIFR, STAT3, STAT4, and ELF3. Transcription of NFKBIZ, encoding the atypical IκB factor IκBζ, is IL-17A dose-dependent, and IκBζ only mediates the transcriptional response to TNF and IL-17A, but not to TNF alone. In fibroblasts, IL-17A response depends on CUX1 and IκBζ to engage the NF-κB complex to produce chemoattractants for neutrophil and monocyte recruitment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Inflamação/metabolismo , Interleucina-17/fisiologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Artrite Reumatoide/genética , Células Cultivadas , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Quimiocinas CXC/genética , Fatores Quimiotáticos/genética , Fibroblastos/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Inflamação/genética , Interleucina-17/farmacologia , Interleucina-6/genética , Metaloproteinase 3 da Matriz/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Líquido Sinovial , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/genética , Transcriptoma/efeitos da radiação , Fator de Necrose Tumoral alfa/farmacologia
6.
Nat Methods ; 16(12): 1289-1296, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740819

RESUMO

The emerging diversity of single-cell RNA-seq datasets allows for the full transcriptional characterization of cell types across a wide variety of biological and clinical conditions. However, it is challenging to analyze them together, particularly when datasets are assayed with different technologies, because biological and technical differences are interspersed. We present Harmony (https://github.com/immunogenomics/harmony), an algorithm that projects cells into a shared embedding in which cells group by cell type rather than dataset-specific conditions. Harmony simultaneously accounts for multiple experimental and biological factors. In six analyses, we demonstrate the superior performance of Harmony to previously published algorithms while requiring fewer computational resources. Harmony enables the integration of ~106 cells on a personal computer. We apply Harmony to peripheral blood mononuclear cells from datasets with large experimental differences, five studies of pancreatic islet cells, mouse embryogenesis datasets and the integration of scRNA-seq with spatial transcriptomics data.


Assuntos
Análise de Célula Única/métodos , Algoritmos , Animais , Sequência de Bases , Conjuntos de Dados como Assunto , Células HEK293 , Humanos , Células Jurkat , Camundongos
9.
Nat Immunol ; 20(7): 902-914, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209404

RESUMO

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.


Assuntos
Rim/imunologia , Nefrite Lúpica/imunologia , Biomarcadores , Biópsia , Análise por Conglomerados , Biologia Computacional/métodos , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Interferons/metabolismo , Rim/metabolismo , Rim/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Anotação de Sequência Molecular , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Célula Única , Transcriptoma
10.
Nat Immunol ; 20(7): 928-942, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31061532

RESUMO

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Perfilação da Expressão Gênica , Membrana Sinovial/metabolismo , Transcriptoma , Artrite Reumatoide/patologia , Autoimunidade/genética , Biomarcadores , Biologia Computacional/métodos , Estudos Transversais , Citocinas/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Membrana Sinovial/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fluxo de Trabalho
11.
Nat Immunol ; 20(7): 915-927, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31110316

RESUMO

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.


Assuntos
Perfilação da Expressão Gênica , Interferon Tipo I/metabolismo , Queratinócitos/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Transcriptoma , Biópsia , Linhagem da Célula/genética , Biologia Computacional/métodos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Perfilação da Expressão Gênica/métodos , Humanos , Nefrite Lúpica/patologia , Ligação Proteica , Transdução de Sinais , Análise de Célula Única , Pele/imunologia , Pele/metabolismo , Pele/patologia
12.
Nat Commun ; 10(1): 687, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737409

RESUMO

How innate T cells (ITC), including invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, maintain a poised effector state has been unclear. Here we address this question using low-input and single-cell RNA-seq of human lymphocyte populations. Unbiased transcriptomic analyses uncover a continuous 'innateness gradient', with adaptive T cells at one end, followed by MAIT, iNKT, γδ T and natural killer cells at the other end. Single-cell RNA-seq reveals four broad states of innateness, and heterogeneity within canonical innate and adaptive populations. Transcriptional and functional data show that innateness is characterized by pre-formed mRNA encoding effector functions, but impaired proliferation marked by decreased baseline expression of ribosomal genes. Together, our data shed new light on the poised state of ITC, in which innateness is defined by a transcriptionally-orchestrated trade-off between rapid cell growth and rapid effector function.


Assuntos
Proliferação de Células/fisiologia , Linfócitos/metabolismo , Feminino , Ontologia Genética , Humanos , Imunidade Inata/fisiologia , Imunofenotipagem , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/fisiologia , Masculino , Células T Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/metabolismo
13.
Sci Transl Med ; 10(463)2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333237

RESUMO

High-dimensional single-cell analyses have improved the ability to resolve complex mixtures of cells from human disease samples; however, identifying disease-associated cell types or cell states in patient samples remains challenging because of technical and interindividual variation. Here, we present mixed-effects modeling of associations of single cells (MASC), a reverse single-cell association strategy for testing whether case-control status influences the membership of single cells in any of multiple cellular subsets while accounting for technical confounders and biological variation. Applying MASC to mass cytometry analyses of CD4+ T cells from the blood of rheumatoid arthritis (RA) patients and controls revealed a significantly expanded population of CD4+ T cells, identified as CD27- HLA-DR+ effector memory cells, in RA patients (odds ratio, 1.7; P = 1.1 × 10-3). The frequency of CD27- HLA-DR+ cells was similarly elevated in blood samples from a second RA patient cohort, and CD27- HLA-DR+ cell frequency decreased in RA patients who responded to immunosuppressive therapy. Mass cytometry and flow cytometry analyses indicated that CD27- HLA-DR+ cells were associated with RA (meta-analysis P = 2.3 × 10-4). Compared to peripheral blood, synovial fluid and synovial tissue samples from RA patients contained about fivefold higher frequencies of CD27- HLA-DR+ cells, which comprised ~10% of synovial CD4+ T cells. CD27- HLA-DR+ cells expressed a distinctive effector memory transcriptomic program with T helper 1 (TH1)- and cytotoxicity-associated features and produced abundant interferon-γ (IFN-γ) and granzyme A protein upon stimulation. We propose that MASC is a broadly applicable method to identify disease-associated cell populations in high-dimensional single-cell data.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linfócitos T CD4-Positivos/imunologia , Subpopulações de Linfócitos T/imunologia , Idoso , Proliferação de Células , Citotoxicidade Imunológica , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Células Th1/imunologia , Transcriptoma/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
14.
Genome Biol ; 19(1): 168, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30340504

RESUMO

BACKGROUND: Cytokines are critical to human disease and are attractive therapeutic targets given their widespread influence on gene regulation and transcription. Defining the downstream regulatory mechanisms influenced by cytokines is central to defining drug and disease mechanisms. One promising strategy is to use interactions between expression quantitative trait loci (eQTLs) and cytokine levels to define target genes and mechanisms. RESULTS: In a clinical trial for anti-IL-6 in patients with systemic lupus erythematosus, we measure interferon (IFN) status, anti-IL-6 drug exposure, and whole blood genome-wide gene expression at three time points. We show that repeat transcriptomic measurements increases the number of cis eQTLs identified compared to using a single time point. We observe a statistically significant enrichment of in vivo eQTL interactions with IFN status and anti-IL-6 drug exposure and find many novel interactions that have not been previously described. Finally, we find transcription factor binding motifs interrupted by eQTL interaction SNPs, which point to key regulatory mediators of these environmental stimuli and therefore potential therapeutic targets for autoimmune diseases. In particular, genes with IFN interactions are enriched for ISRE binding site motifs, while those with anti-IL-6 interactions are enriched for IRF4 motifs. CONCLUSIONS: This study highlights the potential to exploit clinical trial data to discover in vivo eQTL interactions with therapeutically relevant environmental variables.


Assuntos
Citocinas/genética , Regulação da Expressão Gênica , Lúpus Eritematoso Sistêmico/genética , Locos de Características Quantitativas/genética , Humanos
15.
Arthritis Res Ther ; 20(1): 139, 2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29996944

RESUMO

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 µg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.


Assuntos
Artrite Reumatoide/patologia , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Membrana Sinovial/patologia , Criopreservação , Humanos
16.
Nat Genet ; 50(4): 621-629, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29632380

RESUMO

We introduce an approach to identify disease-relevant tissues and cell types by analyzing gene expression data together with genome-wide association study (GWAS) summary statistics. Our approach uses stratified linkage disequilibrium (LD) score regression to test whether disease heritability is enriched in regions surrounding genes with the highest specific expression in a given tissue. We applied our approach to gene expression data from several sources together with GWAS summary statistics for 48 diseases and traits (average N = 169,331) and found significant tissue-specific enrichments (false discovery rate (FDR) < 5%) for 34 traits. In our analysis of multiple tissues, we detected a broad range of enrichments that recapitulated known biology. In our brain-specific analysis, significant enrichments included an enrichment of inhibitory over excitatory neurons for bipolar disorder, and excitatory over inhibitory neurons for schizophrenia and body mass index. Our results demonstrate that our polygenic approach is a powerful way to leverage gene expression data for interpreting GWAS signals.


Assuntos
Expressão Gênica , Predisposição Genética para Doença , Transtorno Bipolar/genética , Índice de Massa Corporal , Encéfalo/metabolismo , Cromatina/genética , Epigênese Genética , Perfilação da Expressão Gênica/estatística & dados numéricos , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Humanos , Doenças do Sistema Imunitário/genética , Desequilíbrio de Ligação , Modelos Genéticos , Herança Multifatorial , Neurônios/metabolismo , Esquizofrenia/genética , Distribuição Tecidual/genética
17.
Nat Commun ; 9(1): 789, 2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29476097

RESUMO

Fibroblasts regulate tissue homeostasis, coordinate inflammatory responses, and mediate tissue damage. In rheumatoid arthritis (RA), synovial fibroblasts maintain chronic inflammation which leads to joint destruction. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to pathology. Here, we show functional and transcriptional differences between fibroblast subsets from human synovial tissues using bulk transcriptomics of targeted subpopulations and single-cell transcriptomics. We identify seven fibroblast subsets with distinct surface protein phenotypes, and collapse them into three subsets by integrating transcriptomic data. One fibroblast subset, characterized by the expression of proteins podoplanin, THY1 membrane glycoprotein and cadherin-11, but lacking CD34, is threefold expanded in patients with RA relative to patients with osteoarthritis. These fibroblasts localize to the perivascular zone in inflamed synovium, secrete proinflammatory cytokines, are proliferative, and have an in vitro phenotype characteristic of invasive cells. Our strategy may be used as a template to identify pathogenic stromal cellular subsets in other complex diseases.


Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Artrite Reumatoide/genética , Caderinas/genética , Caderinas/metabolismo , Células Cultivadas , Humanos , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Transcriptoma
18.
Curr Opin Rheumatol ; 30(1): 65-71, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28984647

RESUMO

PURPOSE OF REVIEW: Stroma is a broad term referring to the connective tissue matrix in which other cells reside. It is composed of diverse cell types with functions such as extracellular matrix maintenance, blood and lymph vessel development, and effector cell recruitment. The tissue microenvironment is determined by the molecular characteristics and relative abundances of different stromal cells such as fibroblasts, endothelial cells, pericytes, and mesenchymal precursor cells. Stromal cell heterogeneity is explained by embryonic developmental lineage, stages of differentiation to other cell types, and activation states. Interaction between immune and stromal cell types is critical to wound healing, cancer, and a wide range of inflammatory diseases. Here, we review recent studies of inflammatory diseases that use functional genomics and single-cell technologies to identify and characterize stromal cell types associated with pathogenesis. RECENT FINDINGS: High dimensional strategies using mRNA sequencing, mass cytometry, and fluorescence activated cell-sorting with fresh primary tissue samples are producing detailed views of what is happening in diseased tissue in rheumatoid arthritis, inflammatory bowel disease, and cancer. Fibroblasts positive for CD90 (Thy-1) are enriched in the synovium of rheumatoid arthritis patients. Single-cell RNA-seq studies will lead to more discoveries about the stroma in the near future. SUMMARY: Stromal cells form the microenvironment of inflamed and diseased tissues. Functional genomics is producing an increasingly detailed view of subsets of stromal cells with pathogenic functions in rheumatic diseases and cancer. Future genomics studies will discover disease mechanisms by perturbing molecular pathways with chemokines and therapies known to affect patient outcomes. Functional genomics studies with large sample sizes of patient tissues will identify patient subsets with different disease phenotypes or treatment responses.


Assuntos
Artrite Reumatoide/genética , Doenças Inflamatórias Intestinais/genética , RNA Mensageiro/metabolismo , Células Estromais/citologia , Artrite Reumatoide/imunologia , Diferenciação Celular , Quimiocinas , Células Endoteliais/citologia , Células Endoteliais/imunologia , Fibroblastos/citologia , Fibroblastos/imunologia , Citometria de Fluxo , Genômica , Humanos , Doenças Inflamatórias Intestinais/imunologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Pericitos/citologia , Pericitos/imunologia , Doenças Reumáticas/genética , Doenças Reumáticas/imunologia , Análise de Sequência de RNA , Análise de Célula Única , Células Estromais/imunologia , Membrana Sinovial/citologia , Antígenos Thy-1/imunologia
19.
Nat Genet ; 49(4): 504-510, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28191890

RESUMO

Recent research has uncovered an important role for de novo variation in neurodevelopmental disorders. Using aggregated data from 9,246 families with autism spectrum disorder, intellectual disability, or developmental delay, we found that ∼1/3 of de novo variants are independently present as standing variation in the Exome Aggregation Consortium's cohort of 60,706 adults, and these de novo variants do not contribute to neurodevelopmental risk. We further used a loss-of-function (LoF)-intolerance metric, pLI, to identify a subset of LoF-intolerant genes containing the observed signal of associated de novo protein-truncating variants (PTVs) in neurodevelopmental disorders. LoF-intolerant genes also carry a modest excess of inherited PTVs, although the strongest de novo-affected genes contribute little to this excess, thus suggesting that the excess of inherited risk resides in lower-penetrant genes. These findings illustrate the importance of population-based reference cohorts for the interpretation of candidate pathogenic variants, even for analyses of complex diseases and de novo variation.


Assuntos
Variação Genética/genética , Transtornos do Neurodesenvolvimento/genética , Transtorno do Espectro Autista/genética , Exoma/genética , Predisposição Genética para Doença/genética , Humanos , Deficiência Intelectual/genética , Fenótipo
20.
Nature ; 542(7639): 110-114, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28150777

RESUMO

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linfócitos B/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Artrite Reumatoide/sangue , Linfócitos B/patologia , Diferenciação Celular , Movimento Celular , Quimiocina CXCL13/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Interleucinas/metabolismo , Fatores Ativadores de Macrófagos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptores CXCR5/deficiência , Receptores CXCR5/metabolismo , Receptores de Quimiocinas/metabolismo , Proteínas Repressoras/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Líquido Sinovial/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo
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