RESUMO
Interleukin-17 (IL-17)-producing helper T (TH17) cells are heterogenous and consist of nonpathogenic TH17 (npTH17) cells that contribute to tissue homeostasis and pathogenic TH17 (pTH17) cells that mediate tissue inflammation. Here, we characterize regulatory pathways underlying TH17 heterogeneity and discover substantial differences in the chromatin landscape of npTH17 and pTH17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTH17 cells share accessible chromatin configurations with regulatory T cells, whereas pTH17 cells exhibit features of both npTH17 cells and type 1 helper T (TH1) cells. Integrating single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq), we infer self-reinforcing and mutually exclusive regulatory networks controlling different cell states and predicted transcription factors regulating TH17 cell pathogenicity. We validate that BACH2 promotes immunomodulatory npTH17 programs and restrains proinflammatory TH1-like programs in TH17 cells in vitro and in vivo. Furthermore, human genetics implicate BACH2 in multiple sclerosis. Overall, our work identifies regulators of TH17 heterogeneity as potential targets to mitigate autoimmunity.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Cromatina , Células Th17 , Células Th17/imunologia , Células Th17/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Animais , Cromatina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/genética , Camundongos Knockout , Células Th1/imunologia , Humanos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Inflamação/imunologia , Inflamação/genética , Análise de Célula Única , Esclerose Múltipla/imunologia , Esclerose Múltipla/genética , FemininoRESUMO
While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.
Assuntos
Autoimunidade , Intestinos/imunologia , Células-Tronco/metabolismo , Células Th17/imunologia , Animais , Movimento Celular , Células Clonais , Encefalomielite Autoimune Experimental/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Homeostase , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , RNA/metabolismo , RNA-Seq , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CXCR6/metabolismo , Receptores de Interleucina/metabolismo , Reprodutibilidade dos Testes , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Análise de Célula Única , Baço/metabolismoRESUMO
CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.
Assuntos
Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/parasitologia , Colo/microbiologia , Colo/parasitologia , Microbioma Gastrointestinal , Heligmosomatoidea/patogenicidade , Enteropatias Parasitárias/parasitologia , Animais , Bactérias/imunologia , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Colo/imunologia , Colo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Heligmosomatoidea/imunologia , Interações Hospedeiro-Patógeno , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Enteropatias Parasitárias/genética , Enteropatias Parasitárias/imunologia , Enteropatias Parasitárias/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nematospiroides dubius/imunologia , Nematospiroides dubius/patogenicidade , Nippostrongylus/imunologia , Nippostrongylus/patogenicidade , Fenótipo , Salmonella enterica/imunologia , Salmonella enterica/patogenicidade , Análise de Célula Única , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , TranscriptomaRESUMO
Neuroimmune interactions have emerged as critical modulators of allergic inflammation, and type 2 innate lymphoid cells (ILC2s) are an important cell type for mediating these interactions. Here, we show that ILC2s expressed both the neuropeptide calcitonin gene-related peptide (CGRP) and its receptor. CGRP potently inhibited alarmin-driven type 2 cytokine production and proliferation by lung ILC2s both in vitro and in vivo. CGRP induced marked changes in ILC2 expression programs in vivo and in vitro, attenuating alarmin-driven proliferative and effector responses. A distinct subset of ILCs scored highly for a CGRP-specific gene signature after in vivo alarmin stimulation, suggesting CGRP regulated this response. Finally, we observed increased ILC2 proliferation and type 2 cytokine production as well as exaggerated responses to alarmins in mice lacking the CGRP receptor. Together, these data indicate that endogenous CGRP is a critical negative regulator of ILC2 responses in vivo.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Linfócitos/fisiologia , Neuropeptídeos/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Alarminas/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Proliferação de Células , Células Cultivadas , Retroalimentação Fisiológica , Imunidade Inata , Interleucina-33/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroimunomodulação , Neuropeptídeos/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/genética , Transdução de Sinais , Células Th2/imunologiaRESUMO
Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.
Assuntos
Ciclo Celular , Linhagem da Célula , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Antioxidantes/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/patologia , Código de Barras de DNA Taxonômico , Ácidos Graxos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Lentivirus/genética , Recidiva Local de Neoplasia/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Oncogênicas/antagonistas & inibidores , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Gene regulation is a complex and tightly controlled process that defines cell identity, health and disease, and response to pharmacologic and environmental signals. Recently developed DNA-targeting platforms, including zinc finger proteins, transcription activator-like effectors (TALEs) and the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, have enabled the recruitment of transcriptional modulators and epigenome-modifying factors to any genomic site, leading to new insights into the function of epigenetic marks in gene expression. Additionally, custom transcriptional and epigenetic regulation is facilitating refined control over cell function and decision making. The unique properties of the CRISPR-Cas9 system have created new opportunities for high-throughput genetic screens and multiplexing targets to manipulate complex gene expression patterns. This Review summarizes recent technological developments in this area and their application to biomedical challenges. We also discuss remaining limitations and necessary future directions for this field.
Assuntos
Epigênese Genética/fisiologia , Epigenômica , Transcrição Gênica/fisiologia , Animais , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Engenharia Genética/métodosRESUMO
Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Epigênese Genética , Epigenômica/métodos , Elementos Reguladores de Transcrição/genética , Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Globinas/genética , Células HEK293 , Humanos , Células K562 , Lentivirus/genética , RNA Guia de Cinetoplastídeos/genética , Proteínas Repressoras/genética , Proteínas Virais/genéticaRESUMO
Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia de Proteínas/métodos , Edição de RNA , Fatores de Transcrição/genética , Ativação Transcricional , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Ribonucleases/genética , Pequeno RNA não TraduzidoRESUMO
Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.
Assuntos
Distrofina/genética , Éxons , Terapia Genética/métodos , Edição de RNA , RNA Mensageiro/genética , Dedos de Zinco/genética , Animais , Sequência de Bases , Distrofina/biossíntese , Distrofina/química , Eletroporação , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Mioblastos/metabolismo , Mioblastos/patologia , Fases de Leitura Aberta , Plasmídeos/química , Plasmídeos/genética , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Deleção de SequênciaRESUMO
Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.
Assuntos
Distrofina/biossíntese , Distrofina/genética , Endonucleases/metabolismo , Marcação de Genes , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofina/metabolismo , Endonucleases/genética , Exoma , Genoma Humano , Células HEK293 , Humanos , Distrofia Muscular de Duchenne/metabolismo , Fases de LeituraRESUMO
Here we introduce a mostly natural sequencing-by-synthesis (mnSBS) method for single-cell RNA sequencing (scRNA-seq), adapted to the Ultima genomics platform, and systematically benchmark it against current scRNA-seq technology. mnSBS uses mostly natural, unmodified nucleotides and only a low fraction of fluorescently labeled nucleotides, which allows for high polymerase processivity and lower costs. We demonstrate successful application in four scRNA-seq case studies of different technical and biological types, including 5' and 3' scRNA-seq, human peripheral blood mononuclear cells from a single individual and in multiplex, as well as Perturb-Seq. Benchmarking shows that results from mnSBS-based scRNA-seq are very similar to those using Illumina sequencing, with minor differences in results related to the position of reads relative to annotated gene boundaries, owing to single-end reads of Ultima being closer to gene ends than reads from Illumina. The method is thus compatible with state-of-the-art scRNA-seq libraries independent of the sequencing technology. We expect mnSBS to be of particular utility for cost-effective large-scale scRNA-seq projects.
Assuntos
Perfilação da Expressão Gênica , Leucócitos Mononucleares , Humanos , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Análise da Expressão Gênica de Célula Única , Análise de Célula Única/métodos , NucleotídeosRESUMO
E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the balance of DC1, DC2, migratory DC and macrophage states and a gradient of DC maturation. Family members grouped into co-functional modules that were enriched for physical interactions and impacted specific programs through substrate transcription factors. E3s and their adaptors co-regulated the same processes, but partnered with different substrate recognition adaptors to impact distinct aspects of the DC life cycle. Genetic interactions were more prevalent within than between modules, and a deep learning model, comßVAE, predicts the outcome of new combinations by leveraging modularity. The E3 regulatory network was associated with heritable variation and aberrant gene expression in immune cells in human inflammatory diseases. Our study provides a general approach to dissect gene function.
RESUMO
Genome sequencing studies have identified millions of somatic variants in cancer, but it remains challenging to predict the phenotypic impact of most. Experimental approaches to distinguish impactful variants often use phenotypic assays that report on predefined gene-specific functional effects in bulk cell populations. Here, we develop an approach to functionally assess variant impact in single cells by pooled Perturb-seq. We measured the impact of 200 TP53 and KRAS variants on RNA profiles in over 300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison with orthogonal assays. We discovered that KRAS variants did not merely fit into discrete functional categories, but spanned a continuum of gain-of-function phenotypes, and that their functional impact could not have been predicted solely by their frequency in patient cohorts. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, with potential applications in multiple disease settings.
Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Mapeamento Cromossômico , Humanos , Neoplasias Pulmonares/genética , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Recent technological advances have enabled massively parallel chromatin profiling with scATAC-seq (single-cell assay for transposase accessible chromatin by sequencing). Here we present ATAC with select antigen profiling by sequencing (ASAP-seq), a tool to simultaneously profile accessible chromatin and protein levels. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA for clonal tracking, capturing three distinct modalities in single cells. ASAP-seq uses a bridging approach that repurposes antibody:oligonucleotide conjugates designed for existing technologies that pair protein measurements with single-cell RNA sequencing. Together with DOGMA-seq, an adaptation of CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) for measuring gene activity across the central dogma of gene regulation, we demonstrate the utility of systematic multi-omic profiling by revealing coordinated and distinct changes in chromatin, RNA and surface proteins during native hematopoietic differentiation and peripheral blood mononuclear cell stimulation and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.
Assuntos
RNA-Seq/métodos , Análise de Célula Única/métodos , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Cromatina/metabolismo , DNA Mitocondrial/genética , Epigenômica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hematopoese , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.
Assuntos
Antígenos CD58/imunologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Melanoma/patologia , Análise de Célula Única/métodos , Evasão Tumoral , Antígenos CD58/genética , Antígenos CD58/metabolismo , Sistemas CRISPR-Cas , Técnicas de Cocultura , Biologia Computacional/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Epitopos/genética , Técnicas de Inativação de Genes , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/patologia , Melanoma/tratamento farmacológico , Melanoma/imunologia , Análise de Sequência de RNA , Evasão Tumoral/genéticaRESUMO
Interleukin-27 (IL-27) is an immunoregulatory cytokine that suppresses inflammation through multiple mechanisms, including induction of IL-10, but the transcriptional network mediating its diverse functions remains unclear. Combining temporal RNA profiling with computational algorithms, we predict 79 transcription factors induced by IL-27 in T cells. We validate 11 known and discover 5 positive (Cebpb, Fosl2, Tbx21, Hlx, and Atf3) and 2 negative (Irf9 and Irf8) Il10 regulators, generating an experimentally refined regulatory network for Il10. We report two central regulators, Prdm1 and Maf, that cooperatively drive the expression of signature genes induced by IL-27 in type 1 regulatory T cells, mediate IL-10 expression in all T helper cells, and determine the regulatory phenotype of colonic Foxp3+ regulatory T cells. Prdm1/Maf double-knockout mice develop spontaneous colitis, phenocopying ll10-deficient mice. Our work provides insights into IL-27-driven transcriptional networks and identifies two shared Il10 regulators that orchestrate immunoregulatory programs across T helper cell subsets.
Assuntos
Redes Reguladoras de Genes/genética , Interleucina-10/metabolismo , Interleucina-27/metabolismo , Células Th1/metabolismo , Animais , Humanos , Camundongos , Camundongos KnockoutRESUMO
CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.
Assuntos
Proteínas de Bactérias/metabolismo , Endonucleases/metabolismo , Inativação Gênica , Pró-Proteína Convertase 9/genética , RNA Guia de Cinetoplastídeos/genética , Staphylococcus aureus/enzimologia , Animais , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Colesterol/sangue , Endonucleases/genética , Terapia Genética , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/sangue , RNA Guia de Cinetoplastídeos/metabolismo , Staphylococcus aureus/genética , Transcrição GênicaRESUMO
Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-α and IL-1ß. These inflammatory cytokines promote apoptosis and senescence of cells in diseased tissue and extracellular matrix breakdown. Stem cell-based therapies are being considered for the treatment of musculoskeletal diseases, but the presence of these inflammatory cytokines will have similar deleterious action on therapeutic cells delivered to these environments. Methods that prevent inflammatory-induced apoptosis and proinflammatory signaling, in cell and pathway-specific manners are needed. In this study we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing to alter cell response to inflammatory environments by repressing inflammatory cytokine cell receptors, specifically TNFR1 and IL1R1. We targeted CRISPR/Cas9-based repressors to TNFR1 and IL1R1 gene regulatory elements in human adipose-derived stem cells (hADSCs) and investigated the functional outcomes of repression of these genes. Efficient signaling regulation was demonstrated in engineered hADSCs, as activity of the downstream transcription factor NF-κB was significantly reduced or maintained at baseline levels in the presence of TNF-α or IL-1ß. Pellet culture of undifferentiated hADSCs demonstrated improved survival in engineered hADSCs treated with TNF-α or IL-1ß, while having little effect on their immunomodulatory properties. Furthermore, engineered hADSCs demonstrated improved chondrogenic differentiation capacity in the presence of TNF-α or IL-1ß, as shown by superior production of glycosaminglycans in this inflammatory environment. Overall this work demonstrates a novel method for modulating cell response to inflammatory signaling that has applications in engineering cells delivered to inflammatory environments, and as a direct gene therapy to protect endogenous cells exposed to chronic inflammation, as observed in a broad spectrum of degenerative musculoskeletal pathology.