RESUMEN
Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.
Asunto(s)
Células Madre Hematopoyéticas/citología , Transducción de Señal , Tretinoina/farmacología , Vitamina A/administración & dosificación , Animales , Vías Biosintéticas , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Supervivencia Celular , Dieta , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Poli I-C/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análisis de la Célula Individual , Estrés Fisiológico , Vitamina A/farmacología , Vitaminas/administración & dosificación , Vitaminas/farmacologíaRESUMEN
During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.
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Macrófagos , Vía de Pentosa Fosfato , Ribosamonofosfatos , eIF-2 Quinasa , Animales , Ribosamonofosfatos/metabolismo , Ratones , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Isomerasas Aldosa-Cetosa/metabolismo , Isomerasas Aldosa-Cetosa/genética , ARN/metabolismo , ARN/genética , Poli I-C/farmacología , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos/inmunología , Replicación Viral , FosforilaciónRESUMEN
Inflammation induced during infection can both promote and suppress immunity. This contradiction suggests that inflammatory cytokines affect the immune system in a context-dependent manner. Here we show that nonspecific bystander inflammation conditions naive CD4(+) T cells for enhanced peripheral Foxp3 induction and reduced effector differentiation. This results in inhibition of immune responses in vivo via a Foxp3-dependent effect on antigen-specific naive CD4(+) T cell precursors. Such conditioning may have evolved to allow immunity to infection while limiting subsequent autoimmunity caused by release of self-antigens in the wake of infection. Furthermore, this phenomenon suggests a mechanistic explanation for the idea that early tuning of the immune system by infection affects the long-term quality of immune regulation.
Asunto(s)
Asma/inmunología , Autoinmunidad/inmunología , Efecto Espectador/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Diabetes Mellitus/inmunología , Factores de Transcripción Forkhead/inmunología , Inflamación , Autotolerancia/inmunología , Animales , Autoantígenos , Efecto Espectador/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Línea Celular Tumoral , Citocinas/efectos de los fármacos , Citocinas/farmacología , Metilación de ADN , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Perfilación de la Expresión Génica , Inductores de Interferón/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Tolerancia Periférica/inmunología , Poli I-C/farmacología , Regiones Promotoras Genéticas , Linfopoyetina del Estroma TímicoRESUMEN
In utero infection and maternal inflammation can adversely impact fetal brain development. Maternal systemic illness, even in the absence of direct fetal brain infection, is associated with an increased risk of neuropsychiatric disorders in affected offspring. The cell types mediating the fetal brain response to maternal inflammation are largely unknown, hindering the development of novel treatment strategies. Here, we show that microglia, the resident phagocytes of the brain, highly express receptors for relevant pathogens and cytokines throughout embryonic development. Using a rodent maternal immune activation (MIA) model in which polyinosinic:polycytidylic acid is injected into pregnant mice, we demonstrate long-lasting transcriptional changes in fetal microglia that persist into postnatal life. We find that MIA induces widespread gene expression changes in neuronal and non-neuronal cells; importantly, these responses are abolished by selective genetic deletion of microglia, indicating that microglia are required for the transcriptional response of other cortical cell types to MIA. These findings demonstrate that microglia play a crucial durable role in the fetal response to maternal inflammation, and should be explored as potential therapeutic cell targets.
Asunto(s)
Encéfalo , Inflamación , Microglía , Poli I-C , Animales , Microglía/metabolismo , Microglía/inmunología , Femenino , Embarazo , Ratones , Encéfalo/patología , Encéfalo/inmunología , Encéfalo/metabolismo , Inflamación/patología , Inflamación/genética , Poli I-C/farmacología , Feto , Ratones Endogámicos C57BL , Regulación del Desarrollo de la Expresión Génica , Neuronas/metabolismoRESUMEN
Apoptosis, a major form of programmed cell death, is an essential component of host defense against invading intracellular pathogens. Viruses encode inhibitors of apoptosis to evade host responses during infection, and to support their own replication and survival. Therefore, hosts and their viruses are entangled in a constant evolutionary arms race to control apoptosis. Until now, apoptosis in the context of the antiviral immune system has been almost exclusively studied in vertebrates. This limited phyletic sampling makes it impossible to determine whether a similar mechanism existed in the last common ancestor of animals. Here, we established assays to probe apoptosis in the sea anemone Nematostella vectensis, a model species of Cnidaria, a phylum that diverged approximately 600 million years ago from the rest of animals. We show that polyinosinic:polycytidylic acid (poly I:C), a synthetic long double-stranded RNA mimicking viral RNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), is sufficient to induce apoptosis in N. vectensis. Furthermore, at the transcriptomic level, apoptosis related genes are significantly enriched upon poly(I:C) exposure in N. vectensis as well as bilaterian invertebrates. Our phylogenetic analysis of caspase family genes in N. vectensis reveals conservation of all four caspase genes involved in apoptosis in mammals and revealed a cnidarian-specific caspase gene which was strongly upregulated. Altogether, our findings suggest that apoptosis in response to a viral challenge is a functionally conserved mechanism that can be traced back to the last common ancestor of Bilateria and Cnidaria.
Asunto(s)
Apoptosis , ARN Bicatenario , Anémonas de Mar , Animales , Anémonas de Mar/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , Filogenia , Poli I-C/farmacología , Cnidarios/genética , Evolución BiológicaRESUMEN
RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-ß (IFN-ß), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360-510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway.
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Proteína 58 DEAD Box , ARN Helicasas DEAD-box , Inmunidad Innata , Receptores Inmunológicos , Humanos , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/inmunología , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/inmunología , Receptores Inmunológicos/metabolismo , Poli I-C/inmunología , Poli I-C/farmacología , ARN Helicasas/metabolismo , ARN Helicasas/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Células HEK293RESUMEN
Tripartite-motif protein-56 (TRIM56) positively regulates the induction of type I interferon response via the TLR3 pathway by enhancing IRF3 activation and depends on its C-terminal residues 621-750 for interacting with the adaptor TRIF. However, the precise underlying mechanism and detailed TRIM56 determinants remain unclear. Herein, we show ectopic expression of murine TRIM56 also enhances TLR3-dependent interferon-ß promoter activation, suggesting functional conservation. We found that endogenous TRIM56 and TRIF formed a complex early (0.5-2 h) after poly-I:C stimulation and that TRIM56 overexpression also promoted activation of NF-κB by poly-I:C but not that by TNF-α or IL-1ß, consistent with a specific effect on TRIF prior to the bifurcation of NF-κB and IRF3. Using transient transfection and Tet-regulated cell lines expressing various TRIM56 mutants, we demonstrated the Coiled-coil domain and a segment spanning residues â¼434-610, but not the B-box or residues 355-433, were required for TRIM56 augmentation of TLR3 signaling. Moreover, alanine substitution at each putative phosphorylation site, Ser471, Ser475, and Ser710, abrogated TRIM56 function. Concordantly, mutants bearing Ser471Ala, Ser475Ala, or Ser710Ala, or lacking the Coiled-coil domain, all lost the capacity to enhance poly-I:C-induced establishment of an antiviral state. Furthermore, the Ser710Ala mutation disrupted the TRIM56-TRIF association. Using phospho-specific antibodies, we detected biphasic phosphorylation of TRIM56 at Ser471 and Ser475 following TLR3 stimulation, with the early phase occurring at â¼0.5 to 1 h, prior to IRF3 phosphorylation. Together, these data reveal novel molecular details critical for the TRIM56 augmentation of TLR3-dependent antiviral response and highlight important roles for TRIM56 scaffolding and phosphorylation.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Inmunidad Innata , Receptor Toll-Like 3 , Proteínas de Motivos Tripartitos , Animales , Humanos , Ratones , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Células HEK293 , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , FN-kappa B/metabolismo , Fosforilación , Poli I-C/farmacología , Dominios Proteicos , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Dendritic cell (DC) activation by viral RNA sensors such as TLR3 and MDA-5 is critical for initiating antiviral immunity. Optimal DC activation is promoted by type I interferon (IFN) signaling which is believed to occur in either autocrine or paracrine fashion. Here, we show that neither autocrine nor paracrine type I IFN signaling can fully account for DC activation by poly(I:C) in vitro and in vivo. By controlling the density of type I IFN-producing cells in vivo, we establish that instead a quorum of type I IFN-producing cells is required for optimal DC activation and that this process proceeds at the level of an entire lymph node. This collective behavior, governed by type I IFN diffusion, is favored by the requirement for prolonged cytokine exposure to achieve DC activation. Furthermore, collective DC activation was found essential for the development of innate and adaptive immunity in lymph nodes. Our results establish how collective rather than cell-autonomous processes can govern the initiation of immune responses.
Asunto(s)
Células Dendríticas/fisiología , Interferón Tipo I/metabolismo , Ganglios Linfáticos/citología , Percepción de Quorum/fisiología , Animales , Linfocitos T CD8-positivos/fisiología , Recuento de Células , Células Dendríticas/efectos de los fármacos , Inmunidad Innata/inmunología , Inflamación/patología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Interferón Tipo I/farmacología , Ganglios Linfáticos/inmunología , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Poli I-C/farmacologíaRESUMEN
The transcription factor GATA-3 is expressed and required for differentiation and function throughout the T lymphocyte lineage. Despite evidence it may also be expressed in multipotent hematopoietic stem cells (HSCs), any role for GATA-3 in these cells has remained unclear. Here we found GATA-3 was in the cytoplasm in quiescent long-term stem cells from steady-state bone marrow but relocated to the nucleus when HSCs cycled. Relocation depended on signaling via the mitogen-activated protein kinase p38 and was associated with a diminished capacity for long-term reconstitution after transfer into irradiated mice. Deletion of Gata3 enhanced the repopulating capacity and augmented the self-renewal of long-term HSCs in cell-autonomous fashion without affecting the cell cycle. Our observations position GATA-3 as a regulator of the balance between self-renewal and differentiation in HSCs that acts downstream of the p38 signaling pathway.
Asunto(s)
Factor de Transcripción GATA3/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Animales , Factor de Transcripción GATA3/genética , Eliminación de Gen , Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Ligandos , Ratones , Ratones Noqueados , Poli I-C/farmacología , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The production of type I interferon is essential for viral clearance but is kept under tight control to avoid unnecessary tissue damage from hyperinflammatory responses. Here we found that OASL1 inhibited translation of IRF7, the master transcription factor for type I interferon, and thus negatively regulated the robust production of type I interferon during viral infection. OASL1 inhibited the translation of IRF7 mRNA by binding to the 5' untranslated region (UTR) of IRF7 and possibly by inhibiting scanning of the 43S preinitiation complex along the message. Oasl1-/- mice were resistant to viral infection because of the greater abundance of type I interferon, which suggests that OASL1 could be a potential therapeutic target for boosting the production of type I interferon during viral infection.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Regulación de la Expresión Génica , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Biosíntesis de Proteínas , 2',5'-Oligoadenilato Sintetasa/genética , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Línea Celular , Virus de la Encefalomiocarditis/inmunología , Homocigoto , Humanos , Inductores de Interferón/administración & dosificación , Inductores de Interferón/farmacología , Interferón Tipo I/biosíntesis , Interferón Tipo I/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Poli I-C/administración & dosificación , Poli I-C/farmacología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/metabolismo , Virosis/genética , Virosis/inmunologíaRESUMEN
Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.
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Antígenos CD/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Cadenas alfa de Integrinas/metabolismo , Melanoma Experimental/inmunología , Poli I-C/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/farmacología , Animales , Presentación de Antígeno/inmunología , Línea Celular Tumoral , Células Dendríticas/citología , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Pregnant women are exposed to various microbes, some of which can harm the mother and/or fetus and can lead to life-long morbidity and even death. The syncytiotrophoblast (STB) covers the placental villi and comes into direct contact with pathogens contained in the maternal blood and plays a key role in placental host defense. However, the precise mechanisms whereby the STB recognizes and responds to pathogenic microbes remain unclear. In this study, we comprehensively analyzed the expression of functional pattern recognition receptors, which are responsible for tissue defense against pathogens, in a primary STB model differentiated from highly purified human term cytotrophoblasts (CTBs). Screening for mRNA expression and multiplex cytokine/chemokine production demonstrated that differentiated CTBs (dCTBs) predominantly expressed dsRNA receptors, including TLR3, MDA5, and RIG-I. We confirmed that term human placentas also expressed TLR3. Transcriptome analysis revealed common and unique responses of dCTBs to a synthetic dsRNA (polyinosinic-polycytidylic acid) compared with human peripheral mononuclear cells. Moreover, polyinosinic-polycytidylic acid induced the release of type I and type III IFNs (IFN-ß, IFN-λ1, IFN-λ2, IFN-λ3), as well as mRNA expression of IFN-stimulated genes (IFIT1, MX1, and OAS1). dCTBs underwent apoptosis via the mitochondrial pathway in response to dsRNA stimulation. These results suggest that dsRNA receptors expressed on the STB are key players in antiviral defense in the placenta. Elucidation of the underpinnings of these defense processes can help us better understand the pathophysiology of viral infections during pregnancy.
Asunto(s)
Placenta , Trofoblastos , Humanos , Femenino , Embarazo , Placenta/metabolismo , Poli I-C/farmacología , Receptor Toll-Like 3/metabolismo , Receptores de Reconocimiento de Patrones/genética , ARN Bicatenario , ARN MensajeroRESUMEN
Circular RNAs (circRNAs) generated via back-splicing are enhanced by flanking complementary sequences. Expression levels of circRNAs vary under different conditions, suggesting participation of protein factors in their biogenesis. Using genome-wide siRNA screening that targets all human unique genes and an efficient circRNA expression reporter, we identify double-stranded RNA-binding domain containing immune factors NF90/NF110 as key regulators in circRNA biogenesis. NF90/NF110 promote circRNA production in the nucleus by associating with intronic RNA pairs juxtaposing the circRNA-forming exon(s); they also interact with mature circRNAs in the cytoplasm. Upon viral infection, circRNA expression is decreased, in part owing to the nuclear export of NF90/NF110 to the cytoplasm. Meanwhile, NF90/NF110 released from circRNP complexes bind to viral mRNAs as part of their functions in antiviral immune response. Our results therefore implicate a coordinated regulation of circRNA biogenesis and function by NF90/NF110 in viral infection.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Unión Proteica , ARN Mensajero/metabolismo , ARN Viral/metabolismo , ARN/biosíntesis , Virosis/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/inmunología , Poli I-C/farmacología , ARN/química , ARN/genética , Interferencia de ARN , Procesamiento Postranscripcional del ARN , Empalme del ARN , Estabilidad del ARN , ARN Circular , ARN Mensajero/genética , ARN Viral/genética , Transfección , Virosis/genética , Virosis/inmunologíaRESUMEN
Various infections trigger a storm of proinflammatory cytokines in which IL-6 acts as a major contributor and leads to diffuse alveolar damage in patients. However, the metabolic regulatory mechanisms of IL-6 in lung injury remain unclear. Polyriboinosinic-polyribocytidylic acid [poly(I:C)] activates pattern recognition receptors involved in viral sensing and is widely used in alternative animal models of RNA virus-infected lung injury. In this study, intratracheal instillation of poly(I:C) with or without an IL-6-neutralizing antibody model was combined with metabonomics, transcriptomics, and so forth to explore the underlying molecular mechanisms of IL-6-exacerbated lung injury. We found that poly(I:C) increased the IL-6 concentration, and the upregulated IL-6 further induced lung ferroptosis, especially in alveolar epithelial type II cells. Meanwhile, lung regeneration was impaired. Mechanistically, metabolomic analysis showed that poly(I:C) significantly decreased glycolytic metabolites and increased bile acid intermediate metabolites that inhibited the bile acid nuclear receptor farnesoid X receptor (FXR), which could be reversed by IL-6-neutralizing antibody. In the ferroptosis microenvironment, IL-6 receptor monoclonal antibody tocilizumab increased FXR expression and subsequently increased the Yes-associated protein (YAP) concentration by enhancing PKM2 in A549 cells. FXR agonist GW4064 and liquiritin, a potential natural herbal ingredient as an FXR regulator, significantly attenuated lung tissue inflammation and ferroptosis while promoting pulmonary regeneration. Together, the findings of the present study provide the evidence that IL-6 promotes ferroptosis and impairs regeneration of alveolar epithelial type II cells during poly(I:C)-induced murine lung injury by regulating the FXR-PKM2-YAP axis. Targeting FXR represents a promising therapeutic strategy for IL-6-associated inflammatory lung injury.
Asunto(s)
Ferroptosis , Interleucina-6 , Pulmón , Poli I-C , Receptores Citoplasmáticos y Nucleares , Ferroptosis/efectos de los fármacos , Animales , Poli I-C/farmacología , Interleucina-6/metabolismo , Ratones , Receptores Citoplasmáticos y Nucleares/metabolismo , Pulmón/patología , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Ratones Endogámicos C57BL , Masculino , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Lesión Pulmonar/tratamiento farmacológico , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Pigs serve as a crucial source of protein in the human diet and play a fundamental role in ensuring food security. However, infectious diseases caused by bacteria or viruses are a major threat to effective global pig farming, jeopardizing human health. Peripheral blood mononuclear cells (PBMCs) are a mixture of immune cells that play crucial roles in immunity and disease resistance in pigs. Previous studies on the gene expression regulation patterns of PBMCs have concentrated on a single immune stimulus or immune cell subpopulation, which has limited our comprehensive understanding of the mechanisms of the pig immune response. RESULTS: Here, we integrated and re-analyzed RNA-seq data published online for porcine PBMC stimulated by lipopolysaccharide (LPS), polyinosinic acid (PolyI:C), and various unknown microorganisms (EM). The results revealed that gene expression and its functional characterization are highly specific to the pathogen, identifying 603, 254, and 882 pathogen-specific genes and 38 shared genes, respectively. Notably, LPS and PolyI:C stimulation directly triggered inflammatory and immune-response pathways, while exposure to mixed microbes (EM) enhanced metabolic processes. These pathogen-specific genes were enriched in immune trait-associated quantitative trait loci (QTL) and eGenes in porcine immune tissues and were implicated in specific cell types. Furthermore, we discussed the roles of eQTLs rs3473322705 and rs1109431654 in regulating pathogen- and cell-specific genes CD300A and CD93, using cellular experiments. Additionally, by integrating genome-wide association studies datasets from 33 complex traits and diseases in humans, we found that pathogen-specific genes were significantly enriched for immune traits and metabolic diseases. CONCLUSIONS: We systematically analyzed the gene expression profiles of the three stimulations and demonstrated pathogen-specific and cell-specific gene regulation across different stimulations in porcine PBMCs. These findings enhance our understanding of shared and distinct regulatory mechanisms of genetic variants in pig immune traits.
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Leucocitos Mononucleares , Lipopolisacáridos , Poli I-C , Sitios de Carácter Cuantitativo , Animales , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/inmunología , Porcinos , Poli I-C/farmacología , Lipopolisacáridos/farmacología , Perfilación de la Expresión Génica , Transcriptoma , Regulación de la Expresión GénicaRESUMEN
The RNA-binding protein PKR serves as a crucial antiviral innate immune factor that globally suppresses translation by sensing viral double-stranded RNA (dsRNA) and by phosphorylating the translation initiation factor eIF2α. Recent findings have unveiled that single-stranded RNAs (ssRNAs), including in vitro transcribed (IVT) mRNA, can also bind to and activate PKR. However, the precise mechanism underlying PKR activation by ssRNAs, remains incompletely understood. Here, we developed a NanoLuc Binary Technology (NanoBiT)-based in vitro PKR dimerization assay to assess the impact of ssRNAs on PKR dimerization. Our findings demonstrate that, akin to double-stranded polyinosinic:polycytidylic acid (polyIC), an encephalomyocarditis virus (EMCV) RNA, as well as NanoLuc luciferase (Nluc) mRNA, can induce PKR dimerization. Conversely, homopolymeric RNA lacking secondary structure fails to promote PKR dimerization, underscoring the significance of secondary structure in this process. Furthermore, adenovirus VA RNA 1, another ssRNA, impedes PKR dimerization by competing with Nluc mRNA. Additionally, we observed structured ssRNAs capable of forming G-quadruplexes induce PKR dimerization. Collectively, our results indicate that ssRNAs have the ability to either induce or inhibit PKR dimerization, thus representing potential targets for the development of antiviral and anti-inflammatory agents.
Asunto(s)
Virus de la Encefalomiocarditis , Multimerización de Proteína , ARN Bicatenario , ARN Viral , eIF-2 Quinasa , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/química , Humanos , ARN Viral/metabolismo , ARN Viral/genética , ARN Viral/química , Virus de la Encefalomiocarditis/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/química , Poli I-C/farmacología , Conformación de Ácido NucleicoRESUMEN
CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.
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Antígeno CD47 , Leucemia Mieloide Aguda , Antígeno CD47/metabolismo , Antígeno CD47/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Humanos , Animales , Ratones , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Sulfonamidas/farmacología , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Antígenos de Diferenciación/metabolismo , Fagocitosis/efectos de los fármacos , Poli I-C/farmacologíaRESUMEN
INTRODUCTION: A wide range of positive, negative, and cognitive symptoms compose the clinical presentation of schizophrenia. Schizophrenia is a multifactorial disorder in which genetic and environmental risk factors interact for a full emergence of the disorder. Infectious challenges during pregnancy are a well-known environmental risk factor for schizophrenia. Also, genetic variants affecting the function of fractalkine signaling between neurons and microglia were linked to schizophrenia. Translational animal models recapitulating these complex gene-environment associations have a great potential to untangle schizophrenia neurobiology and propose new therapeutic strategies. METHODS: Given that genetic variants affecting the function of fractalkine signaling between neurons and microglia were linked to schizophrenia, we compared the outcomes of a well-characterized model of maternal immune activation induced using the viral mimetic polyinosinic:polycytidylic acid (Poly I:C) in wild-type versus fractalkine receptor knockout mice. Possible behavioral and immune alterations were assessed in male and female offspring during adulthood. Considering the role of the hippocampus in schizophrenia, microglial analyses and bulk RNA sequencing were performed within this region to assess the neuroimmune dynamics at play. Males and females were examined separately. RESULTS: Offspring exposed to the dual challenge paradigm exhibited symptoms relevant to schizophrenia and unpredictably to mood disorders. Males displayed social and cognitive deficits related to schizophrenia, while females mainly presented anxiety-like behaviors related to mood disorders. Hippocampal microglia in females exposed to the dual challenge were hypertrophic, indicative of an increased surveillance, whereas those in males showed on the other end of the spectrum blunted morphologies with a reduced phagocytosis. Hippocampal bulk-RNA sequencing further revealed a downregulation in females of genes related to GABAergic transmission, which represents one of the main proposed causes of mood disorders. CONCLUSIONS: Building on previous results, we identified in the current study distinctive behavioral phenotypes in female mice exposed to a dual genetic and environmental challenge, thus proposing a new model of neurodevelopmentally-associated mood and affective symptoms. This paves the way to future sex-specific investigations into the susceptibility to developmental challenges using animal models based on genetic and immune vulnerability as presented here.
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Microglía , Efectos Tardíos de la Exposición Prenatal , Embarazo , Humanos , Ratones , Femenino , Masculino , Animales , Quimiocina CX3CL1 , Poli I-C/farmacología , Conducta Animal/fisiología , Perfilación de la Expresión Génica , Hipocampo , Modelos Animales de EnfermedadRESUMEN
Maternal immune activation (MIA) during pregnancy increases the risk for the unborn foetus to develop neurodevelopmental conditions such as autism spectrum disorder and schizophrenia later in life. MIA mouse models recapitulate behavioural and biological phenotypes relevant to both conditions, and are valuable models to test novel treatment approaches. Selenium (Se) has potent anti-inflammatory properties suggesting it may be an effective prophylactic treatment against MIA. The aim of this study was to determine if Se supplementation during pregnancy can prevent adverse effects of MIA on offspring brain and behaviour in a mouse model. Selenium was administered via drinking water (1.5 ppm) to pregnant dams from gestational day (GD) 9 to birth, and MIA was induced at GD17 using polyinosinic:polycytidylic acid (poly-I:C, 20 mg/kg via intraperitoneal injection). Foetal placenta and brain cytokine levels were assessed using a Luminex assay and brain elemental nutrients assessed using inductively coupled plasma- mass spectrometry. Adult offspring were behaviourally assessed using a reinforcement learning paradigm, the three-chamber sociability test and the open field test. MIA elevated placental IL-1ß and IL-17, and Se supplementation successfully prevented this elevation. MIA caused an increase in foetal brain calcium, which was prevented by Se supplement. MIA caused in offspring a female-specific reduction in sociability, which was recovered by Se, and a male-specific reduction in social memory, which was not recovered by Se. Exposure to poly-I:C or selenium, but not both, reduced performance in the reinforcement learning task. Computational modelling indicated that this was predominantly due to increased exploratory behaviour, rather than reduced rate of learning the location of the food reward. This study demonstrates that while Se may be beneficial in ameliorating sociability deficits caused by MIA, it may have negative effects in other behavioural domains. Caution in the use of Se supplementation during pregnancy is therefore warranted.
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Trastorno del Espectro Autista , Efectos Tardíos de la Exposición Prenatal , Selenio , Ratones , Animales , Femenino , Embarazo , Masculino , Humanos , Conducta Animal/fisiología , Selenio/farmacología , Placenta , Modelos Animales de Enfermedad , Poli I-C/farmacología , Suplementos DietéticosRESUMEN
Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.