RESUMEN
Microglia are innate immune cells in the brain. Transcription factor IRF8 (interferon regulatory factor 8) is highly expressed in microglia. However, its role in postnatal microglia development is unknown. We demonstrate that IRF8 binds stepwise to enhancer regions of postnatal microglia along with Sall1 and PU.1, reaching a maximum after day 14. IRF8 binding correlated with a stepwise increase in chromatin accessibility, which preceded the initiation of microglia-specific transcriptome. Constitutive and postnatal Irf8 deletion led to a loss of microglia identity and gain of disease-associated microglia (DAM)-like genes. Combined analysis of single-cell (sc)RNA sequencing and single-cell transposase-accessible chromatin with sequencing (scATAC-seq) revealed a correlation between chromatin accessibility and transcriptome at a single-cell level. IRF8 was also required for microglia-specific DNA methylation patterns. Last, in the 5xFAD model, constitutive and postnatal Irf8 deletion reduced the interaction of microglia with amyloidß plaques and the size of plaques, lessening neuronal loss. Together, IRF8 sets the epigenetic landscape, which is required for postnatal microglia gene expression.
Asunto(s)
Epigénesis Genética , Factores Reguladores del Interferón , Ratones Noqueados , Microglía , Transcriptoma , Microglía/metabolismo , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Animales , Ratones , Metilación de ADN , Ratones Endogámicos C57BL , Cromatina/metabolismo , Cromatina/genética , Encéfalo/metabolismo , Análisis de la Célula Individual , Proteínas Proto-Oncogénicas , TransactivadoresRESUMEN
Memory B cells (MBCs) differentiate into plasma cells (PCs) or germinal centers (GCs) upon antigen recall. How this decision is programmed is not understood. We found that the relative strength between two antagonistic transcription factors, B lymphocyte-induced maturation protein 1 (BLIMP1) and BTB domain and CNC homolog 2 (BACH2), progressively increases in favor of BLIMP1 in antigen-responding B cells through the course of primary responses. MBC subsets that preferentially produce secondary GCs expressed comparatively higher BACH2 but lower BLIMP1 than those predisposed for PC development. Skewing the BLIMP1-BACH2 balance in otherwise fate-predisposed MBC subsets could switch their fate preferences. Underlying the changing BLIMP1-over-BACH2 balance, we observed progressively increased accessibilities at chromatin loci that are specifically opened in PCs, particularly those that contain interferon-sensitive response elements (ISREs) and are controlled by interferon regulatory factor 4 (IRF4). IRF4 is upregulated by B cell receptor, CD40 or innate receptor signaling and it induces graded levels of PC-specifying epigenetic imprints according to the strength of stimulation. By analyzing history-stamped GC B cells, we found progressively increased chromatin accessibilities at PC-specific, IRF4-controlled gene loci over time. Therefore, the cumulative stimulation history of B cells is epigenetically recorded in an IRF4-dependent manner, determines the relative strength between BLIMP1 and BACH2 in individual MBCs and dictates their probabilities to develop into GCs or PCs upon restimulation.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Diferenciación Celular , Epigénesis Genética , Centro Germinal , Memoria Inmunológica , Factores Reguladores del Interferón , Células B de Memoria , Células Plasmáticas , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Animales , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Ratones , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Células B de Memoria/inmunología , Células B de Memoria/metabolismo , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Centro Germinal/inmunología , Centro Germinal/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal , Activación de Linfocitos/genéticaRESUMEN
Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.
Asunto(s)
Linfocitos B , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Factor de Transcripción Ikaros , Linfopoyesis , Factor de Transcripción PAX5 , Transactivadores , Animales , Factor de Transcripción Ikaros/metabolismo , Factor de Transcripción Ikaros/genética , Factor de Transcripción PAX5/metabolismo , Factor de Transcripción PAX5/genética , Ratones , Transactivadores/metabolismo , Transactivadores/genética , Linfocitos B/metabolismo , Linfocitos B/inmunología , Linfopoyesis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteolisis , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Diferenciación Celular , Proteína 1 Similar al Factor de Transcripción 7/metabolismo , Proteína 1 Similar al Factor de Transcripción 7/genética , Factor de Transcripción 3/metabolismo , Factor de Transcripción 3/genética , Recombinación V(D)J , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Inmunoglobulina de Cadenas Ligeras Subrogadas/metabolismo , Inmunoglobulina de Cadenas Ligeras Subrogadas/genética , Transcripción GenéticaRESUMEN
The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.
Asunto(s)
Células Dendríticas , Receptores Tipo II del Factor de Necrosis Tumoral , Humanos , Diferenciación Celular , Linaje de la Célula , Factores Reguladores del Interferón/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Timo/metabolismo , Factores de Necrosis Tumoral/metabolismoRESUMEN
The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1; Fos-Jun) cooperate to promote the effector functions of T cells, but NFAT in the absence of AP-1 imposes a negative feedback program of T cell hyporesponsiveness (exhaustion). Here, we show that basic leucine zipper ATF-like transcription factor (BATF) and interferon regulatory factor 4 (IRF4) cooperate to counter T cell exhaustion in mouse tumor models. Overexpression of BATF in CD8+ T cells expressing a chimeric antigen receptor (CAR) promoted the survival and expansion of tumor-infiltrating CAR T cells, increased the production of effector cytokines, decreased the expression of inhibitory receptors and the exhaustion-associated transcription factor TOX and supported the generation of long-lived memory T cells that controlled tumor recurrence. These responses were dependent on BATF-IRF interaction, since cells expressing a BATF variant unable to interact with IRF4 did not survive in tumors and did not effectively delay tumor growth. BATF may improve the antitumor responses of CAR T cells by skewing their phenotypes and transcriptional profiles away from exhaustion and towards increased effector function.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD8-positivos/inmunología , Factores Reguladores del Interferón/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias/inmunología , Receptores Quiméricos de Antígenos/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción NFATC/metabolismo , Recurrencia Local de Neoplasia/inmunología , Factor de Transcripción AP-1/metabolismoRESUMEN
Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We showed active chromatin remodeling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Therefore, our study represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Inflamación/inmunología , Neutrófilos/inmunología , Activación Transcripcional/genética , Animales , Células CHO , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Cricetulus , Femenino , Factores Reguladores del Interferón/metabolismo , Factor 6 Similar a Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Factores de Transcripción del Factor Regulador X/metabolismo , Factor de Transcripción ReIB/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.
Asunto(s)
Linaje de la Célula , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Células Dendríticas/metabolismo , Elementos de Facilitación Genéticos , Factores Reguladores del Interferón/metabolismo , Monocitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células de la Médula Ósea , Células Cultivadas , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/genética , Células Dendríticas/inmunología , Epigénesis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Monocitos/inmunología , Células Progenitoras Mieloides/inmunología , Fenotipo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Bacterias/patogenicidad , Infecciones Bacterianas/microbiología , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD4-Positivos/parasitología , Colon/microbiología , Colon/parasitología , Microbioma Gastrointestinal , Heligmosomatoidea/patogenicidad , Parasitosis Intestinales/parasitología , Animales , Bacterias/inmunología , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Citrobacter rodentium/inmunología , Citrobacter rodentium/patogenicidad , Colon/inmunología , Colon/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Heligmosomatoidea/inmunología , Interacciones Huésped-Patógeno , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Parasitosis Intestinales/genética , Parasitosis Intestinales/inmunología , Parasitosis Intestinales/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Nematospiroides dubius/inmunología , Nematospiroides dubius/patogenicidad , Nippostrongylus/inmunología , Nippostrongylus/patogenicidad , Fenotipo , Salmonella enterica/inmunología , Salmonella enterica/patogenicidad , Análisis de la Célula Individual , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , TranscriptomaRESUMEN
The activities, ontogeny, and mechanisms of lineage expansion of eosinophils are less well resolved than those of other immune cells, despite the use of biological therapies targeting the eosinophilia-promoting cytokine interleukin (IL)-5 or its receptor, IL-5Rα. We combined single-cell proteomics and transcriptomics and generated transgenic IL-5Rα reporter mice to revisit eosinophilopoiesis. We reconciled human and murine eosinophilopoiesis and provided extensive cell-surface immunophenotyping and transcriptomes at different stages along the continuum of eosinophil maturation. We used these resources to show that IL-5 promoted eosinophil-lineage expansion via transit amplification, while its deletion or neutralization did not compromise eosinophil maturation. Informed from our resources, we also showed that interferon response factor-8, considered an essential promoter of myelopoiesis, was not intrinsically required for eosinophilopoiesis. This work hence provides resources, methods, and insights for understanding eosinophil ontogeny, the effects of current precision therapeutics, and the regulation of eosinophil development and numbers in health and disease.
Asunto(s)
Linaje de la Célula , Eosinófilos , Interleucina-5 , Ratones Transgénicos , Proteómica , Análisis de la Célula Individual , Transcriptoma , Eosinófilos/inmunología , Eosinófilos/metabolismo , Animales , Interleucina-5/metabolismo , Interleucina-5/genética , Humanos , Ratones , Proteómica/métodos , Análisis de la Célula Individual/métodos , Diferenciación Celular/inmunología , Ratones Endogámicos C57BL , Perfilación de la Expresión Génica/métodos , Subunidad alfa del Receptor de Interleucina-5/metabolismo , Subunidad alfa del Receptor de Interleucina-5/genética , Mielopoyesis/genética , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Ratones NoqueadosRESUMEN
Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor T cell responses. Jag2-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered T cell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Factores Reguladores del Interferón , Proteína Jagged-2 , Neoplasias Pulmonares , Ratones Noqueados , Macrófagos Asociados a Tumores , Animales , Humanos , Ratones , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-2/metabolismo , Proteína Jagged-2/genética , Proteína Jagged-2/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Receptor Notch1/metabolismo , Receptor Notch1/genética , Receptores Notch/metabolismo , Transducción de Señal , Escape del Tumor/inmunología , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismoRESUMEN
Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Inflamasomas/metabolismo , Factores Reguladores del Interferón/metabolismo , Proteína Inhibidora de la Apoptosis Neuronal/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al Calcio/genética , Células Cultivadas , Citocinas/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Flagelina/metabolismo , Factores Reguladores del Interferón/antagonistas & inhibidores , Factores Reguladores del Interferón/genética , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína Inhibidora de la Apoptosis Neuronal/genética , Regiones Promotoras Genéticas , Unión Proteica , Pseudomonas aeruginosa/patogenicidad , Piroptosis , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Salmonella typhimurium/patogenicidad , Transcripción GenéticaRESUMEN
Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT.
Asunto(s)
Neoplasias de la Mama/patología , Exosomas/patología , ARN no Traducido/metabolismo , Células del Estroma/patología , Microambiente Tumoral , Neoplasias de la Mama/metabolismo , Proteína 58 DEAD Box/metabolismo , Exosomas/metabolismo , Humanos , Factores Reguladores del Interferón/metabolismo , Células MCF-7 , Metástasis de la Neoplasia , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Receptores Inmunológicos , Receptores de Reconocimiento de Patrones/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Células del Estroma/metabolismo , Virosis/metabolismoRESUMEN
The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.
Asunto(s)
Micronúcleos con Defecto Cromosómico , Nucleotidiltransferasas , Transducción de Señal , Humanos , Células HeLa , Radiación , Replicación del ADN , Daño del ADN , Factor 3 Regulador del Interferón/metabolismo , Transcripción Genética , Factores Reguladores del Interferón/metabolismo , Técnicas de Inactivación de Genes , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Cinética , Transfección , Nucleosomas/metabolismo , Morfolinas , Purinas , Hidroxiurea , Línea Celular Tumoral , Virus Vaccinia/fisiología , Vaccinia/inmunología , Vaccinia/metabolismoRESUMEN
Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.
Asunto(s)
Células Dendríticas/citología , Elementos de Facilitación Genéticos/genética , Factores Reguladores del Interferón/metabolismo , Macrófagos/citología , Monocitos/citología , Animales , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Linaje de la Célula , Células Dendríticas/inmunología , Regulación de la Expresión Génica , Factores Reguladores del Interferón/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Células Madre/citología , Células Tumorales CultivadasRESUMEN
Classical type 1 dendritic cells (cDC1s) are required for antiviral and antitumor immunity, which necessitates an understanding of their development. Development of the cDC1 progenitor requires an E-protein-dependent enhancer located 41 kilobases downstream of the transcription start site of the transcription factor Irf8 (+41-kb Irf8 enhancer), but its maturation instead requires the Batf3-dependent +32-kb Irf8 enhancer. To understand this switch, we performed single-cell RNA sequencing of the common dendritic cell progenitor (CDP) and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2 and Zeb2. Genetic epistasis among these factors revealed that Nfil3 expression is required for the transition from Zeb2hi and Id2lo CDPs to Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E-protein activity to exclude plasmacytoid dendritic cell potential and explains the switch in Irf8 enhancer usage during cDC1 development.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Células Dendríticas/citología , Elementos de Facilitación Genéticos/genética , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Factores Reguladores del Interferón/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Represoras/metabolismo , Células Madre/citologíaRESUMEN
Immune checkpoint blockade therapy has shifted the paradigm for cancer treatment. However, the majority of patients lack effective responses due to insufficient T cell infiltration in tumors. Here we show that expression of mitochondrial uncoupling protein 2 (UCP2) in tumor cells determines the immunostimulatory feature of the tumor microenvironment (TME) and is positively associated with prolonged survival. UCP2 reprograms the immune state of the TME by altering its cytokine milieu in an interferon regulatory factor 5-dependent manner. Consequently, UCP2 boosts the conventional type 1 dendritic cell- and CD8+ T cell-dependent anti-tumor immune cycle and normalizes the tumor vasculature. Finally we show, using either a genetic or pharmacological approach, that induction of UCP2 sensitizes melanomas to programmed cell death protein-1 blockade treatment and elicits effective anti-tumor responses. Together, this study demonstrates that targeting the UCP2 pathway is a potent strategy for alleviating the immunosuppressive TME and overcoming the primary resistance of programmed cell death protein-1 blockade.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Melanoma Experimental/inmunología , Neoplasias Cutáneas/inmunología , Microambiente Tumoral/inmunología , Proteína Desacopladora 2/inmunología , Animales , Antineoplásicos Inmunológicos/farmacología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Resistencia a Antineoplásicos/inmunología , Femenino , Humanos , Inmunoterapia/métodos , Factores Reguladores del Interferón/inmunología , Factores Reguladores del Interferón/metabolismo , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/mortalidad , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/mortalidad , Análisis de Supervivencia , Resultado del Tratamiento , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismoRESUMEN
Conventional dendritic cells (cDCs) consist of two major functionally and phenotypically distinct subsets, cDC1 and cDC2, whose development is dependent on distinct sets of transcription factors. Interferon regulatory factor 8 (IRF8) is required at multiple stages of cDC1 development, but its role in committed cDC1 remains unclear. Here, we used Xcr1-cre to delete Irf8 in committed cDC1 and demonstrate that Irf8 is required for maintaining the identity of cDC1. In the absence of Irf8, committed cDC1 acquired the transcriptional, functional, and chromatin accessibility properties of cDC2. This conversion was independent of Irf4 and was associated with the decreased accessibility of putative IRF8, Batf3, and composite AP-1-IRF (AICE)-binding elements, together with increased accessibility of cDC2-associated transcription-factor-binding elements. Thus, IRF8 expression by committed cDC1 is required for preventing their conversion into cDC2-like cells.
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Células Dendríticas , Factores Reguladores del Interferón , Células Dendríticas/metabolismo , Epigénesis Genética , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismoRESUMEN
Tumors are populated by antigen-presenting cells (APCs) including macrophage subsets with distinct origins and functions. Here, we examined how cancer impacts mononuclear phagocytic APCs in a murine model of breast cancer. Tumors induced the expansion of monocyte-derived tumor-associated macrophages (TAMs) and the activation of type 1 dendritic cells (DC1s), both of which expressed and required the transcription factor interferon regulatory factor-8 (IRF8). Although DC1s mediated cytotoxic T lymphocyte (CTL) priming in tumor-draining lymph nodes, TAMs promoted CTL exhaustion in the tumor, and IRF8 was required for TAMs' ability to present cancer cell antigens. TAM-specific IRF8 deletion prevented exhaustion of cancer-cell-reactive CTLs and suppressed tumor growth. Tumors from patients with immune-infiltrated renal cell carcinoma had abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. Furthermore, the TAM-IRF8 signature co-segregated with CTL exhaustion signatures across multiple cancer types. Thus, CTL exhaustion is promoted by TAMs via IRF8.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Animales , Ratones , Macrófagos Asociados a Tumores , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Linfocitos T Citotóxicos , Células DendríticasRESUMEN
Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified an Irf8 superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kb Irf8 enhancer is required for pre-cDC1 specification, while the +32-kb Irf8 enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggesting cis dependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kb Irf8 enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located in cis Thus, the +41-kb Irf8 enhancer controls the subsequent activation of the +32-kb Irf8 enhancer in a manner that is independent of associated lncRNA transcription.