RESUMO
Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells.
Assuntos
Proteínas de Ciclo Celular , Cromatina , Redes Reguladoras de Genes , Fatores de Transcrição , Transcrição Gênica , Cromatina/metabolismo , Cromatina/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Regulação da Expressão Gênica , Complexo Mediador/metabolismo , Complexo Mediador/genética , Análise de Célula Única , Transcriptoma , Coesinas , Proteínas que Contêm BromodomínioRESUMO
Janus kinase (JAK) inhibitors improve antitumor responses.
Assuntos
Inibidores de Janus Quinases , Janus Quinases , Neoplasias , Humanos , Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Janus Quinases/metabolismo , Janus Quinases/antagonistas & inibidores , Inibidores de Janus Quinases/uso terapêutico , Animais , CamundongosRESUMO
Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.
Assuntos
Imunidade Inata , Linfopoese , Animais , Camundongos , Colite , Ligantes , Transdução de SinaisRESUMO
Infection-induced T cell responses must be properly tempered and terminated to prevent immuno-pathology. Using transgenic mice, we demonstrate that T cell intrinsic STAT1 signaling is required to curb inflammation during acute infection with Toxoplasma gondii. Specifically, we report that mice lacking STAT1 selectively in T cells expel parasites but ultimately succumb to lethal immuno-pathology characterized by aberrant Th1-type responses with reduced IL-10 and increased IL-13 production. We also find that, unlike STAT1, STAT3 is not required for induction of IL-10 or suppression of IL-13 during acute toxoplasmosis. Each of these findings was confirmed in vitro and ChIP-seq data mining showed that STAT1 and STAT3 co-localize at the Il10 locus, as well as loci encoding other transcription factors that regulate IL-10 production, most notably Maf and Irf4. These data advance basic understanding of how infection-induced T cell responses are managed to prevent immuno-pathology and provide specific insights on the anti-inflammatory properties of STAT1, highlighting its role in shaping the character of Th1-type responses.
RESUMO
Changes in metabolism of macrophages are required to sustain macrophage activation in response to different stimuli. We showed that the cytokine TGF-ß (transforming growth factor-ß) regulates glycolysis in macrophages independently of inflammatory cytokine production and affects survival in mouse models of sepsis. During macrophage activation, TGF-ß increased the expression and activity of the glycolytic enzyme PFKL (phosphofructokinase-1 liver type) and promoted glycolysis but suppressed the production of proinflammatory cytokines. The increase in glycolysis was mediated by an mTOR-c-MYC-dependent pathway, whereas the inhibition of cytokine production was due to activation of the transcriptional coactivator SMAD3 and suppression of the activity of the proinflammatory transcription factors AP-1, NF-κB, and STAT1. In mice with LPS-induced endotoxemia and experimentally induced sepsis, the TGF-ß-induced enhancement in macrophage glycolysis led to decreased survival, which was associated with increased blood coagulation. Analysis of septic patient cohorts revealed that the expression of PFKL, TGFBRI (which encodes a TGF-ß receptor), and F13A1 (which encodes a coagulation factor) in myeloid cells positively correlated with COVID-19 disease. Thus, these results suggest that TGF-ß is a critical regulator of macrophage metabolism and could be a therapeutic target in patients with sepsis.
Assuntos
COVID-19 , Sepse , Camundongos , Animais , Fator de Crescimento Transformador beta/metabolismo , Lipopolissacarídeos/toxicidade , COVID-19/metabolismo , Macrófagos/metabolismo , Sepse/metabolismo , Inflamação/metabolismo , Citocinas/metabolismo , GlicóliseRESUMO
Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.
Assuntos
Imunidade Inata , Fator 1 de Transcrição de Linfócitos T , Animais , Camundongos , Linfócitos , Tecido Linfoide/metabolismo , Fator 1 de Transcrição de Linfócitos T/metabolismoRESUMO
CD4+ T helper 17 (TH17) cells protect barrier tissues but also trigger autoimmunity. The mechanisms behind these opposing processes remain unclear. Here, we found that the transcription factor EGR2 controlled the transcriptional program of pathogenic TH17 cells in the central nervous system (CNS) but not that of protective TH17 cells at barrier sites. EGR2 was significantly elevated in myelin-reactive CD4+ T cells from patients with multiple sclerosis and mice with autoimmune neuroinflammation. The EGR2 transcriptional program was intricately woven within the TH17 cell transcriptional regulatory network and showed high interconnectivity with core TH17 cell-specific transcription factors. Mechanistically, EGR2 enhanced TH17 cell differentiation and myeloid cell recruitment to the CNS by upregulating pathogenesis-associated genes and myelomonocytic chemokines. T cell-specific deletion of Egr2 attenuated neuroinflammation without compromising the host's ability to control infections. Our study shows that EGR2 regulates tissue-specific and disease-specific functions in pathogenic TH17 cells in the CNS.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Camundongos , Diferenciação Celular , Sistema Nervoso Central , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias , Células Th1 , Células Th17 , Fatores de Transcrição , Virulência , HumanosRESUMO
BACKGROUND: Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. METHODS: We evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy. RESULTS: Genome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. CONCLUSIONS: Gain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.).
Assuntos
Doenças Autoimunes , Fármacos Dermatológicos , Janus Quinases , Escleroderma Sistêmico , Janus Quinases/antagonistas & inibidores , Nitrilas , Pirazóis/uso terapêutico , Pirazóis/farmacologia , Pirimidinas , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/genética , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/genética , Mutação de Sentido Incorreto , Mutação com Ganho de Função , Fármacos Dermatológicos/uso terapêutico , Anti-Inflamatórios/uso terapêuticoRESUMO
Protein kinases play a major role in cellular activation processes, including signal transduction by diverse immunoreceptors. Given their roles in cell growth and death and in the production of inflammatory mediators, targeting kinases has proven to be an effective treatment strategy, initially as anticancer therapies, but shortly thereafter in immune-mediated diseases. Herein, we provide an overview of the status of small molecule inhibitors specifically generated to target protein kinases relevant to immune cell function, with an emphasis on those approved for the treatment of immune-mediated diseases. The development of inhibitors of Janus kinases that target cytokine receptor signalling has been a particularly active area, with Janus kinase inhibitors being approved for the treatment of multiple autoimmune and allergic diseases as well as COVID-19. In addition, TEC family kinase inhibitors (including Bruton's tyrosine kinase inhibitors) targeting antigen receptor signalling have been approved for haematological malignancies and graft versus host disease. This experience provides multiple important lessons regarding the importance (or not) of selectivity and the limits to which genetic information informs efficacy and safety. Many new agents are being generated, along with new approaches for targeting kinases.
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Doenças do Sistema Imunitário , Proteínas Quinases , Humanos , Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Doenças do Sistema Imunitário/tratamento farmacológicoRESUMO
The proprotein convertase subtilisin/kexins (PCSKs) regulate biological actions by cleaving immature substrate proteins. The archetype PCSK, FURIN, promotes the pathogenicity of viruses by proteolytically processing viral proteins. FURIN has also important regulatory functions in both innate and adaptive immune responses but its role in the CD8+ CTLs remains enigmatic. We used a T-cell-specific FURIN deletion in vivo to demonstrate that FURIN promotes host response against the CTL-dependent lymphocytic choriomeningitis virus by virtue of restricting viral burden and augmenting interferon gamma (IFNG) production. We also characterized Furin KO CD8+ T cells ex vivo, including after their activation with FURIN regulating cytokines IL12 or TGFB1. Furin KO CD8+ T cells show an inherently activated phenotype characterized by the upregulation of effector genes and increased frequencies of CD44+ , TNF+ , and IFNG+ cells. In the activated CTLs, FURIN regulates the productions of IL2, TNF, and GZMB and the genes associated with the TGFBR-signaling pathway. FURIN also controls the expression of Eomes, Foxo1, and Bcl6 and the levels of ITGAE and CD62L, which implies a role in the development of CTL memory. Collectively, our data suggest that the T-cell expressed FURIN is important for host responses in viral infections, CTL homeostasis/activation, and memory development.
Assuntos
Coriomeningite Linfocítica , Linfócitos T Citotóxicos , Camundongos , Animais , Linfócitos T CD8-Positivos , Furina/genética , Camundongos Endogâmicos C57BL , Vírus da Coriomeningite Linfocítica , Memória ImunológicaRESUMO
The U.S. Food and Drug Administration approval of dupilumab for moderate-to-severe atopic dermatitis shifted the paradigm from use of broad, systemic immunosuppressants to a safer, targeted treatment and led to the emergence of newer interleukin (IL)-4/IL-13 directed biologics and small molecule therapies, namely Janus kinase (JAK) inhibitors (JAKi). Tralokinumab and emerging (not yet approved) lebrikizumab, which both target IL-13, are alternative biologics to dupilumab. The emerging anti-IL-31 receptor nemolizumab is likely to be used second-line to other biologics, primarily for pruritus. Three JAKi are currently in use for treating atopic dermatitis, 2 of which, abrocitinib and upadacitinib, are U.S. Food and Drug Administration-approved. This review provides an in-depth, practical discussion on use of these biologics and JAKi that are approved or have completed phase 3 clinical trials in pediatric patients and adults, comparing the groups of medications based on available efficacy and safety data.
Assuntos
Produtos Biológicos , Dermatite Atópica , Adulto , Humanos , Criança , Dermatite Atópica/tratamento farmacológico , Interleucina-13 , Imunossupressores/uso terapêutico , Prurido/tratamento farmacológico , Produtos Biológicos/uso terapêutico , Resultado do TratamentoRESUMO
T cells are critical orchestrators of the adaptive immune response that optimally eliminate a specific pathogen. Aberrant T-cell development and function are implicated in a broad range of human disease including immunodeficiencies, autoimmune diseases, and allergic diseases. Accordingly, therapies targeting T cells and their effector cytokines have markedly improved the care of patients with immune dysregulatory diseases. Newer discoveries concerning T-cell-mediated antitumor immunity and T-cell exhaustion have further prompted development of highly effective and novel treatment modalities for malignancies, including checkpoint inhibitors and antigen-reactive T cells. Recent discoveries are also uncovering the depth and variability of T-cell phenotypes: while T cells have long been described using a subset-based classification system, next-generation sequencing technologies suggest an astounding degree of complexity and heterogeneity at the single-cell level.
Assuntos
Neoplasias , Linfócitos T , Humanos , Imunidade Adaptativa , Citocinas , Imunidade Celular , Linfócitos T CD8-PositivosRESUMO
Activated lymphocytes adapt their metabolism to meet the energetic and biosynthetic demands imposed by rapid growth and proliferation. Common gamma chain (cγ) family cytokines are central to these processes, but the role of downstream signal transducer and activator of transcription 5 (STAT5) signaling, which is engaged by all cγ members, is poorly understood. Using genome-, transcriptome-, and metabolome-wide analyses, we demonstrate that STAT5 is a master regulator of energy and amino acid metabolism in CD4+ T helper cells. Mechanistically, STAT5 localizes to an array of enhancers and promoters for genes encoding essential enzymes and transporters, where it facilitates p300 recruitment and epigenetic remodeling. We also find that STAT5 licenses the activity of two other key metabolic regulators, the mTOR signaling pathway and the MYC transcription factor. Building on the latter, we present evidence for transcriptome-wide cooperation between STAT5 and MYC in both normal and transformed T cells. Together, our data provide a molecular framework for transcriptional programing of T cell metabolism downstream of cγ cytokines and highlight the JAK-STAT pathway in mediating cellular growth and proliferation.
Assuntos
Janus Quinases , Fator de Transcrição STAT5 , Fator de Transcrição STAT5/genética , Transdução de Sinais , Fatores de Transcrição STAT , Linfócitos T Auxiliares-Indutores , CitocinasRESUMO
The discovery of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway arose from investigations of how cells respond to interferons (IFNs), revealing a paradigm in cell signaling conserved from slime molds to mammals. These discoveries revealed mechanisms underlying rapid gene expression mediated by a wide variety of extracellular polypeptides including cytokines, interleukins, and related factors. This knowledge has provided numerous insights into human disease, from immune deficiencies to cancer, and was rapidly translated to new drugs for autoimmune, allergic, and infectious diseases, including COVID-19. Despite these advances, major challenges and opportunities remain.
Assuntos
COVID-19 , Janus Quinases , Animais , Citocinas/metabolismo , Humanos , Interferons/metabolismo , Janus Quinases/metabolismo , Mamíferos/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de SinaisRESUMO
The four Janus kinase (JAK) proteins and seven signal transducer and activator of transcription (STAT) transcription factors mediate intracellular signal transduction downstream of cytokine receptors, which are implicated in the pathology of autoimmune, allergic and inflammatory diseases. Development of targeted small-molecule therapies such as JAK inhibitors, which have varied selective inhibitory profiles, has enabled a paradigm shift in the treatment of diverse disorders. JAK inhibitors suppress intracellular signalling mediated by multiple cytokines involved in the pathological processes of rheumatoid arthritis and many other immune and inflammatory diseases, and therefore have the capacity to target multiple aspects of those diseases. In addition to rheumatoid arthritis, JAK inhibition has potential for treatment of autoimmune diseases including systemic lupus erythematosus, spondyloarthritis, inflammatory bowel disease and alopecia areata, in which stimulation of innate immunity activates adaptive immunity, leading to generation of autoreactive T cells and activation and differentiation of B cells. JAK inhibitors are also effective in the treatment of allergic disorders, such as atopic dermatitis, and can even be used for the COVID-19-related cytokine storm. Mechanism-based treatments targeting JAK-STAT pathways have the potential to provide positive outcomes by minimizing the use of glucocorticoids and/or non-specific immunosuppressants in the treatment of systemic immune-mediated inflammatory diseases.
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COVID-19 , Reumatologia , Humanos , Janus Quinases , SARS-CoV-2 , Fatores de Transcrição STAT/metabolismoRESUMO
Not simply an attribute of the adaptive immune system, immunological memory can be viewed on multiple levels. Accordingly, the molecular basis of memory comprises multiple mechanisms. The advent of new sequencing technologies has greatly enhanced the understanding of gene regulation and lymphocyte specification, and improved measurement of chromatin states affords new insights into the epigenomic and transcriptomic programs that underlie memory. Beyond canonical genes, the involvement of long noncoding RNAs (lncRNAs) is becoming increasingly apparent, and it appears that there are more than two to three times as many lncRNAs as protein-coding genes. lncRNAs can directly interact with DNA, RNA, and proteins, and a single lncRNA can contain multiple modular domains and thus interact with different classes of molecules. Yet, most lncRNAs have not been tested for function, and even fewer knockout mice have been generated. It is therefore timely to consider new potential mechanisms that may contribute to immune memory.
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RNA Longo não Codificante , Animais , Cromatina , Epigenômica , Regulação da Expressão Gênica , Linfócitos , Camundongos , RNA Longo não Codificante/metabolismoRESUMO
Since its discovery, the Janus kinase-signal transduction and activation of transcription (JAK-STAT) pathway has become recognized as a central mediator of widespread and varied human physiological processes. The field of JAK-STAT biology, particularly its clinical relevance, continues to be shaped by 2 important advances. First, the increased use of genomic sequencing has led to the discovery of novel clinical syndromes caused by mutations in JAK and STAT genes. This has provided insights regarding the consequences of aberrant JAK-STAT signaling for immunity, lymphoproliferation, and malignancy. In addition, since the approval of ruxolitinib and tofacitinib, the therapeutic use of JAK inhibitors (jakinibs) has expanded to include a large spectrum of diseases. Efficacy and safety data from over a decade of clinical studies have provided additional mechanistic insights while improving the care of patients with inflammatory and neoplastic conditions. This review discusses major advances in the field, focusing on updates in genetic diseases and in studies of clinical jakinibs in human disease.
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Doenças Genéticas Inatas/tratamento farmacológico , Inibidores de Janus Quinases/uso terapêutico , Janus Quinases/imunologia , Fatores de Transcrição STAT/imunologia , Animais , Citocinas/imunologia , Doenças Genéticas Inatas/imunologia , Humanos , Janus Quinases/genética , Mutação , Fatores de Transcrição STAT/genética , Transdução de SinaisRESUMO
Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8+ T cell effector differentiation, providing insight into phenotypes of patients with APDS.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Memória Imunológica , Doenças da Imunodeficiência Primária/enzimologia , Transcrição Gênica , Viroses/enzimologia , Adolescente , Adulto , Animais , Apoptose , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Estudos de Casos e Controles , Criança , Cromatina/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/imunologia , Modelos Animais de Doenças , Ativação Enzimática , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/imunologia , Transdução de Sinais , Viroses/genética , Viroses/imunologiaRESUMO
MicroRNAs (miRNAs, miRs) regulate cell fate decisions by post-transcriptionally tuning networks of mRNA targets. We used miRNA-directed pathway discovery to reveal a regulatory circuit that influences Ig class switch recombination (CSR). We developed a system to deplete mature, activated B cells of miRNAs, and performed a rescue screen that identified the miR-221/222 family as a positive regulator of CSR. Endogenous miR-221/222 regulated B cell CSR to IgE and IgG1 in vitro, and miR-221/222-deficient mice exhibited defective IgE production in allergic airway challenge and polyclonal B cell activation models in vivo. We combined comparative Ago2-HITS-CLIP and gene expression analyses to identify mRNAs bound and regulated by miR-221/222 in primary B cells. Interrogation of these putative direct targets uncovered functionally relevant downstream genes. Genetic depletion or pharmacological inhibition of Foxp1 and Arid1a confirmed their roles as key modulators of CSR to IgE and IgG1.