RESUMO
Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.
Assuntos
Imunidade nas Mucosas/imunologia , Interleucina-18/imunologia , Mucosa Intestinal/imunologia , Animais , Citocinas/imunologia , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/metabolismo , Células Epiteliais/imunologia , Feminino , Células Caliciformes/imunologia , Interleucina-18/biossíntese , Mucosa Intestinal/metabolismo , Intestino Delgado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/imunologia , Ratos , Ratos Sprague-Dawley , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologiaRESUMO
Adaptive immunity provides life-long protection by generating central and effector memory T cells and the most recently described tissue resident memory T (TRM) cells. However, the cellular origin of CD4 TRM cells and their contribution to host defense remain elusive. Using IL-17A tracking-fate mouse models, we found that a significant fraction of lung CD4 TRM cells derive from IL-17A-producing effector (TH17) cells following immunization with heat-killed Klebsiella pneumonia (Kp). These exTH17 TRM cells are maintained in the lung by IL-7, produced by lymphatic endothelial cells. During a memory response, neither antibodies, γδ T cells, nor circulatory T cells are sufficient for the rapid host defense required to eliminate Kp. Conversely, using parabiosis and depletion studies, we demonstrated that exTH17 TRM cells play an important role in bacterial clearance. Thus, we delineate the origin and function of airway CD4 TRM cells during bacterial infection, offering novel strategies for targeted vaccine design.
Assuntos
Infecções por Klebsiella/imunologia , Células Th17/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Toxina Diftérica/farmacologia , Modelos Animais de Doenças , Feminino , Memória Imunológica , Interleucina-17/genética , Interleucina-17/metabolismo , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/patogenicidade , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/citologia , Células Th17/metabolismoRESUMO
The immune system safeguards organ integrity by employing a balancing act of inflammatory and immunosuppressive mechanisms designed to neutralize foreign invaders and resolve injury. Maintaining or restoring a state of immune homeostasis is particularly challenging at barrier sites where constant exposure to immunogenic environmental agents may induce destructive inflammation. Recent studies underscore the role of epithelial and mesenchymal barrier cells in regulating immune cell function and local homeostatic and inflammatory responses. Here, we highlight immunoregulatory circuits engaging epithelial and mesenchymal cells in the intestine, airways, and skin and discuss how immune communications with hematopoietic cells and the microbiota orchestrate local immune homeostasis and inflammation.
Assuntos
Epitélio/imunologia , Homeostase , Inflamação/imunologia , Mesoderma/imunologia , Animais , Células Epiteliais/imunologia , Humanos , Infecções/imunologia , Intestinos/citologia , Intestinos/imunologia , Intestinos/fisiologia , Mesoderma/citologia , Sistema Respiratório/imunologiaRESUMO
The intestinal mucosal barrier controlling the resident microbiome is dependent on a protective mucus layer generated by goblet cells, impairment of which is a hallmark of the inflammatory bowel disease, ulcerative colitis. Here, we show that IL-18 is critical in driving the pathologic breakdown of barrier integrity in a model of colitis. Deletion of Il18 or its receptor Il18r1 in intestinal epithelial cells (Δ/EC) conferred protection from colitis and mucosal damage in mice. In contrast, deletion of the IL-18 negative regulator Il18bp resulted in severe colitis associated with loss of mature goblet cells. Colitis and goblet cell loss were rescued in Il18bp(-/-);Il18r(Δ/EC) mice, demonstrating that colitis severity is controlled at the level of IL-18 signaling in intestinal epithelial cells. IL-18 inhibited goblet cell maturation by regulating the transcriptional program instructing goblet cell development. These results inform on the mechanism of goblet cell dysfunction that underlies the pathology of ulcerative colitis.
Assuntos
Colite Ulcerativa/patologia , Colite Ulcerativa/fisiopatologia , Interleucina-18/imunologia , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/metabolismo , Sulfato de Dextrana , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Feminino , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Subunidade alfa de Receptor de Interleucina-18/genética , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Mucosa Intestinal/fisiopatologia , Masculino , Camundongos , Transdução de SinaisRESUMO
The mechanism by which cells undergo death determines whether dying cells trigger inflammatory responses or remain immunologically silent. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. Here, we identify a mechanism by which mitochondria and downstream proapoptotic caspases regulate the activation of antiviral immunity. In the absence of active caspases, mitochondrial outer membrane permeabilization by Bax and Bak results in the expression of type I interferons (IFNs). This induction is mediated by mitochondrial DNA-dependent activation of the cGAS/STING pathway and results in the establishment of a potent state of viral resistance. Our results show that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response and to inactivate this response in a caspase-dependent manner. This mechanism provides a dual control, which determines whether mitochondria initiate an immunologically silent or a proinflammatory type of cell death.
Assuntos
Apoptose , Caspases/metabolismo , Interferon Tipo I/metabolismo , Transdução de Sinais , Animais , DNA Mitocondrial/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Interferon Tipo I/imunologia , Camundongos , Camundongos Knockout , Viroses/imunologiaRESUMO
Tissue-resident innate lymphoid cells (ILCs) help sustain barrier function and respond to local signals. ILCs are traditionally classified as ILC1, ILC2 or ILC3 on the basis of their expression of specific transcription factors and cytokines1. In the skin, disease-specific production of ILC3-associated cytokines interleukin (IL)-17 and IL-22 in response to IL-23 signalling contributes to dermal inflammation in psoriasis. However, it is not known whether this response is initiated by pre-committed ILCs or by cell-state transitions. Here we show that the induction of psoriasis in mice by IL-23 or imiquimod reconfigures a spectrum of skin ILCs, which converge on a pathogenic ILC3-like state. Tissue-resident ILCs were necessary and sufficient, in the absence of circulatory ILCs, to drive pathology. Single-cell RNA-sequencing (scRNA-seq) profiles of skin ILCs along a time course of psoriatic inflammation formed a dense transcriptional continuum-even at steady state-reflecting fluid ILC states, including a naive or quiescent-like state and an ILC2 effector state. Upon disease induction, the continuum shifted rapidly to span a mixed, ILC3-like subset also expressing cytokines characteristic of ILC2s, which we inferred as arising through multiple trajectories. We confirmed the transition potential of quiescent-like and ILC2 states using in vitro experiments, single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and in vivo fate mapping. Our results highlight the range and flexibility of skin ILC responses, suggesting that immune activities primed in healthy tissues dynamically adapt to provocations and, left unchecked, drive pathological remodelling.
Assuntos
Imunidade Inata/imunologia , Linfócitos/imunologia , Linfócitos/patologia , Psoríase/imunologia , Psoríase/patologia , Pele/imunologia , Pele/patologia , Animais , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Modelos Animais de Doenças , Feminino , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-23/imunologia , Análise de Classes Latentes , Linfócitos/classificação , Masculino , Camundongos , Psoríase/genética , RNA Citoplasmático Pequeno/genética , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Cytokines were the first modern immunotherapies to produce durable responses in patients with advanced cancer, but they have only modest efficacy and limited tolerability1,2. In an effort to identify alternative cytokine pathways for immunotherapy, we found that components of the interleukin-18 (IL-18) pathway are upregulated on tumour-infiltrating lymphocytes, suggesting that IL-18 therapy could enhance anti-tumour immunity. However, recombinant IL-18 previously did not demonstrate efficacy in clinical trials3. Here we show that IL-18BP, a high-affinity IL-18 decoy receptor, is frequently upregulated in diverse human and mouse tumours and limits the anti-tumour activity of IL-18 in mice. Using directed evolution, we engineered a 'decoy-resistant' IL-18 (DR-18) that maintains signalling potential but is impervious to inhibition by IL-18BP. Unlike wild-type IL-18, DR-18 exerted potent anti-tumour effects in mouse tumour models by promoting the development of poly-functional effector CD8+ T cells, decreasing the prevalence of exhausted CD8+ T cells that express the transcriptional regulator of exhaustion TOX, and expanding the pool of stem-like TCF1+ precursor CD8+ T cells. DR-18 also enhanced the activity and maturation of natural killer cells to effectively treat anti-PD-1 resistant tumours that have lost surface expression of major histocompatibility complex class I molecules. These results highlight the potential of the IL-18 pathway for immunotherapeutic intervention and implicate IL-18BP as a major therapeutic barrier.
Assuntos
Imunoterapia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-18/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Estimativa de Kaplan-Meier , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Receptores de Interleucina-18/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Mutations that result in loss of function of Nod2, an intracellular receptor for bacterial peptidoglycan, are associated with Crohn's disease. Here we found that the E3 ubiquitin ligase Pellino3 was an important mediator in the Nod2 signaling pathway. Pellino3-deficient mice had less induction of cytokines after engagement of Nod2 and had exacerbated disease in various experimental models of colitis. Furthermore, expression of Pellino3 was lower in the colons of patients with Crohn's disease. Pellino3 directly bound to the kinase RIP2 and catalyzed its ubiquitination. Loss of Pellino3 led to attenuation of Nod2-induced ubiquitination of RIP2 and less activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs). Our findings identify RIP2 as a substrate for Pellino3 and Pellino3 as an important mediator in the Nod2 pathway and regulator of intestinal inflammation.
Assuntos
Colite/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Citrobacter rodentium/imunologia , Colite/genética , Colite/imunologia , Colite/microbiologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Adulto JovemRESUMO
Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.
Assuntos
Pressão Hidrostática , Imunidade Inata , Canais Iônicos/metabolismo , Mecanotransdução Celular/imunologia , Animais , Endotelina-1/metabolismo , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/microbiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Transdução de SinaisRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Activated CD4 T cells proliferate rapidly and remodel epigenetically before exiting the cell cycle and engaging acquired effector functions. Metabolic reprogramming from the naive state is required throughout these phases of activation1. In CD4 T cells, T-cell-receptor ligation-along with co-stimulatory and cytokine signals-induces a glycolytic anabolic program that is required for biomass generation, rapid proliferation and effector function2. CD4 T cell differentiation (proliferation and epigenetic remodelling) and function are orchestrated coordinately by signal transduction and transcriptional remodelling. However, it remains unclear whether these processes are regulated independently of one another by cellular biochemical composition. Here we demonstrate that distinct modes of mitochondrial metabolism support differentiation and effector functions of mouse T helper 1 (TH1) cells by biochemically uncoupling these two processes. We find that the tricarboxylic acid cycle is required for the terminal effector function of TH1 cells through succinate dehydrogenase (complex II), but that the activity of succinate dehydrogenase suppresses TH1 cell proliferation and histone acetylation. By contrast, we show that complex I of the electron transport chain, the malate-aspartate shuttle and mitochondrial citrate export are required to maintain synthesis of aspartate, which is necessary for the proliferation of T helper cells. Furthermore, we find that mitochondrial citrate export and the malate-aspartate shuttle promote histone acetylation, and specifically regulate the expression of genes involved in T cell activation. Combining genetic, pharmacological and metabolomics approaches, we demonstrate that the differentiation and terminal effector functions of T helper cells are biochemically uncoupled. These findings support a model in which the malate-aspartate shuttle, mitochondrial citrate export and complex I supply the substrates needed for proliferation and epigenetic remodelling early during T cell activation, whereas complex II consumes the substrates of these pathways, which antagonizes differentiation and enforces terminal effector function. Our data suggest that transcriptional programming acts together with a parallel biochemical network to enforce cell state.
Assuntos
Diferenciação Celular , Mitocôndrias/metabolismo , Células Th1/citologia , Células Th1/imunologia , Acetilação , Animais , Ácido Aspártico/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Transporte de Elétrons , Feminino , Histonas/metabolismo , Humanos , Ativação Linfocitária/genética , Malatos/metabolismo , Masculino , Camundongos , Succinato Desidrogenase/metabolismo , Células Th1/metabolismo , Transcrição GênicaRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system in vivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony-stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted in existing MISTRG mice. By targeting the G-CSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.
Assuntos
Neutrófilos , Receptores de Fator Estimulador de Colônias de Granulócitos , Humanos , Camundongos , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos/genética , CitocinasRESUMO
γδ T cells are an abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells' origin in the thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. N6-methyladenosine (m6A) represents one of the most common posttranscriptional modifications of messenger RNA (mRNA) in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here, we show that depletion of the m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal Salmonella typhimurium infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components Jagged1 and Notch2. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Linfócitos Intraepiteliais , RNA Mensageiro , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Linfócitos Intraepiteliais/enzimologia , Linfócitos Intraepiteliais/imunologia , Proteína Jagged-1/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais/genéticaRESUMO
Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression.
Assuntos
Infecções por Cardiovirus/imunologia , Regulação da Expressão Gênica , Interferon Tipo I/imunologia , Receptor 3 Toll-Like/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/mortalidade , Infecções por Cardiovirus/virologia , Vírus da Encefalomiocardite/imunologia , Homeostase , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/genética , Camundongos , Camundongos Knockout , Transdução de Sinais , Taxa de Sobrevida , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/imunologia , Receptor 3 Toll-Like/genética , Ubiquitina/genética , Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential1,2. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, we show that ribosomes associate with a large number of RNAs that were previously annotated as 'non-protein coding'. Although the idea that such non-canonical ORFs can encode functional proteins is controversial3,4, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF 'hidden' within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.
Assuntos
Imunidade nas Mucosas/genética , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , RNA Longo não Codificante/genética , Animais , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Colite/genética , Colite/imunologia , Colite/metabolismo , Imunidade nas Mucosas/efeitos dos fármacos , Interleucina-12/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , RNA Longo não Codificante/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Salmonella typhimurium/imunologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Comprehensive and accurate comparisons of transcriptomic distributions of cells from samples taken from two different biological states, such as healthy versus diseased individuals, are an emerging challenge in single-cell RNA sequencing (scRNA-seq) analysis. Current methods for detecting differentially abundant (DA) subpopulations between samples rely heavily on initial clustering of all cells in both samples. Often, this clustering step is inadequate since the DA subpopulations may not align with a clear cluster structure, and important differences between the two biological states can be missed. Here, we introduce DA-seq, a targeted approach for identifying DA subpopulations not restricted to clusters. DA-seq is a multiscale method that quantifies a local DA measure for each cell, which is computed from its k nearest neighboring cells across a range of k values. Based on this measure, DA-seq delineates contiguous significant DA subpopulations in the transcriptomic space. We apply DA-seq to several scRNA-seq datasets and highlight its improved ability to detect differences between distinct phenotypes in severe versus mildly ill COVID-19 patients, melanomas subjected to immune checkpoint therapy comparing responders to nonresponders, embryonic development at two time points, and young versus aging brain tissue. DA-seq enabled us to detect differences between these phenotypes. Importantly, we find that DA-seq not only recovers the DA cell types as discovered in the original studies but also reveals additional DA subpopulations that were not described before. Analysis of these subpopulations yields biological insights that would otherwise be undetected using conventional computational approaches.
Assuntos
Envelhecimento/genética , COVID-19/genética , Linhagem da Célula/genética , Melanoma/genética , RNA Citoplasmático Pequeno/genética , Neoplasias Cutâneas/genética , Envelhecimento/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Encéfalo/citologia , Encéfalo/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Linhagem da Célula/imunologia , Citocinas/genética , Citocinas/imunologia , Conjuntos de Dados como Assunto , Células Dendríticas/imunologia , Células Dendríticas/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Melanoma/imunologia , Melanoma/patologia , Monócitos/imunologia , Monócitos/virologia , Fenótipo , RNA Citoplasmático Pequeno/imunologia , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença , Análise de Célula Única/métodos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T/imunologia , Linfócitos T/virologia , TranscriptomaRESUMO
Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of the Daedalus locus, which is proximal to Ikaros, led to a severe reduction in early lymphoid progenitors, exerting control over the earliest fate decisions during lymphoid lineage commitment. Daedalus locus deletion led to alterations in Ikaros isoform expression and a significant reduction in Ikaros protein. The Daedalus locus may function through direct DNA interaction as Hi-C analysis demonstrated an interaction between the two loci. Finally, we identify an Ikaros-regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage-specific manner. Daedalus appears to act as a gatekeeper of Ikaros's broad lineage-specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. These findings represent a key illustration of how a transcription factor with broad lineage expression must work in concert with noncoding elements to orchestrate hematopoietic lineage commitment.
Assuntos
Hematopoese/genética , Fator de Transcrição Ikaros/genética , Linfopoese/genética , RNA não Traduzido/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , CamundongosRESUMO
Suppressor of cytokine signaling (SOCS) proteins are key regulators of CD4(+) T cell differentiation, and in particular, we have recently shown that SOCS2 inhibits the development of Th2 cells and allergic immune responses. Interestingly, transcriptome analyses have identified SOCS2 as being preferentially expressed in both natural regulatory T cells (Tregs) and inducible Tregs (iTregs); however, the role of SOCS2 in Foxp3(+) Treg function or development has not been fully elucidated. In this study, we show that despite having no effect on natural Treg development or function, SOCS2 is highly expressed in iTregs and required for the stable expression of Foxp3 in iTregs in vitro and in vivo. Indeed, SOCS2-deficient CD4(+) T cells upregulated Foxp3 following in vitro TGF-ß stimulation, but failed to maintain stable expression of Foxp3. Moreover, in vivo generation of iTregs following OVA feeding was impaired in the absence of SOCS2 and could be rescued in the presence of IL-4 neutralizing Ab. Following IL-4 stimulation, SOCS2-deficient Foxp3(+) iTregs secreted elevated IFN-γ and IL-13 levels and displayed enhanced STAT6 phosphorylation. Therefore, we propose that SOCS2 regulates iTreg stability by downregulating IL-4 signaling. Moreover, SOCS2 is essential to maintain the anti-inflammatory phenotype of iTregs by preventing the secretion of proinflammatory cytokines. Collectively, these results suggest that SOCS2 may prevent IL-4-induced Foxp3(+) iTreg instability. Foxp3(+) iTregs are key regulators of immune responses at mucosal surfaces; therefore, this dual role of SOCS2 in both Th2 and Foxp3(+) iTregs reinforces SOCS2 as a potential therapeutic target for Th2-biased diseases.