RESUMEN
Patients afflicted with Stimulator of interferon gene (STING) gain-of-function mutations frequently present with debilitating interstitial lung disease (ILD) that is recapitulated in mice expressing the STINGV154M mutation (VM). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in initiating ILD. To identify STING-expressing non-hematopoietic cell types required for the development of ILD, we use a conditional knockin (CKI) model and direct expression of the VM allele to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted VM expression results in enhanced recruitment of immune cells to the lung associated with elevated chemokine expression and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of STING-associated vasculopathy with onset in infancy (SAVI) patients or patients afflicted with other ILD-related disorders.
Asunto(s)
Células Endoteliales , Mutación con Ganancia de Función , Pulmón , Proteínas de la Membrana , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Ratones , Pulmón/patología , Pulmón/metabolismo , Linfocitos/metabolismo , Enfermedades Pulmonares Intersticiales/patología , Enfermedades Pulmonares Intersticiales/genética , Enfermedades Pulmonares Intersticiales/metabolismo , Ratones Endogámicos C57BL , HumanosRESUMEN
A detailed understanding of the innate immune mechanisms involved in restricting SARS-CoV-2 infection and how the virus disrupts these processes could reveal new strategies to boost antiviral mechanisms and develop therapeutics for COVID-19. Here, we identify cellular nucleic acid-binding protein (CNBP) as a key host factor controlling SARS-CoV-2 infection. In response to RNA-sensing pathways, CNBP is phosphorylated and translocates from the cytosol to the nucleus where it binds to the interferon-ß enhancer to initiate transcription. Because SARS-CoV-2 evades immune detection by the host's RNA-sensing pathways, CNBP is largely retained in the cytosol where it restricts SARS-CoV-2 directly, leading to a battle between the host and SARS-CoV-2 that extends beyond antiviral immune signaling pathways. We further demonstrated that CNBP binds SARS-CoV-2 viral RNA directly and competes with the viral nucleocapsid protein to prevent viral RNA and nucleocapsid protein from forming liquid-liquid phase separation (LLPS) condensates critical for viral replication. Consequently, cells and animals lacking CNBP have higher viral loads, and CNBP-deficient mice succumb rapidly to infection. Altogether, these findings identify CNBP as a key antiviral factor for SARS-CoV-2, functioning both as a regulator of antiviral IFN gene expression and a cell-intrinsic restriction factor that disrupts LLPS to limit viral replication and spread. In addition, our studies also highlight viral condensates as important targets and strategies for the development of drugs to combat COVID-19.
Asunto(s)
COVID-19 , Interferones , Animales , Ratones , Proteínas de la Nucleocápside , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/fisiología , Factores de Transcripción , Replicación ViralRESUMEN
The cGMP-AMP Synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway plays a critical role in sensing dsDNA localized to the cytosol, resulting in the activation of a robust inflammatory response. While cGAS-STING signaling is essential for antiviral immunity, aberrant STING activation is observed in amyotrophic lateral sclerosis (ALS), lupus, and autoinflammatory diseases such as Aicardi-Goutières syndrome (AGS) and STING associated vasculopathy with onset in infancy (SAVI). Significant efforts have therefore focused on the development of STING inhibitors. In a concurrent submission, we reported that BB-Cl-amidine inhibits STING-dependent signaling in the nanomolar range, both in vitro and in vivo. Considering this discovery, we sought to generate analogs with higher potency and proteome-wide selectivity. Herein, we report the development of LB244, which displays nanomolar potency and inhibits STING signaling with markedly enhanced proteome-wide selectivity. Moreover, LB244 mirrored the efficacy of BB-Cl-amidine in vivo. In summary, our data identify novel chemical entities that inhibit STING signaling and provide a scaffold for the development of therapeutics for treating STING-dependent inflammatory diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Autoinmunes del Sistema Nervioso , Humanos , Proteoma , Antivirales , GMP Cíclico , NucleotidiltransferasasRESUMEN
Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys148. Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.
Asunto(s)
Interferón Tipo I , Proteínas de la Membrana , Ratones , Animales , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Interferón Tipo I/metabolismo , FN-kappa B/metabolismo , ADNRESUMEN
This study reveals a previously uncharacterized mechanism to restrict intestinal inflammation via a regulatory RNA transcribed from a noncoding genomic locus. We identified a novel transcript of the lncRNA HOXA11os specifically expressed in the distal colon that is reduced to undetectable levels in colitis. HOXA11os is localized to mitochondria under basal conditions and interacts with a core subunit of complex 1 of the electron transport chain (ETC) to maintain its activity. Deficiency of HOXA11os in colonic myeloid cells results in complex I deficiency, dysfunctional oxidative phosphorylation (OXPHOS), and the production of mitochondrial reactive oxygen species (mtROS). As a result, HOXA11os-deficient mice develop spontaneous intestinal inflammation and are hypersusceptible to colitis. Collectively, these studies identify a new regulatory axis whereby a lncRNA maintains intestinal homeostasis and restricts inflammation in the colon through the regulation of complex I activity.
Asunto(s)
Colitis , ARN Largo no Codificante , Animales , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Colitis/genética , Colitis/metabolismo , Inflamación/metabolismo , Mitocondrias/genética , Homeostasis , Mucosa Intestinal/metabolismoRESUMEN
PYHIN proteins AIM2 and IFI204 sense pathogen DNA, while other PYHINs have been shown to regulate host gene expression through as-yet unclear mechanisms. We characterize mouse PYHIN IFI207, which we find is not involved in DNA sensing but rather is required for cytokine promoter induction in macrophages. IFI207 co-localizes with both active RNA polymerase II (RNA Pol II) and IRF7 in the nucleus and enhances IRF7-dependent gene promoter induction. Generation of Ifi207-/- mice shows no role for IFI207 in autoimmunity. Rather, IFI207 is required for the establishment of a Klebsiella pneumoniae lung infection and for Klebsiella macrophage phagocytosis. These insights into IFI207 function illustrate that PYHINs can have distinct roles in innate immunity independent of DNA sensing and highlight the need to better characterize the whole mouse locus, one gene at a time.
Asunto(s)
Citocinas , Klebsiella pneumoniae , Ratones , Animales , Klebsiella pneumoniae/genética , Proteínas Nucleares/metabolismo , Inmunidad Innata , ADNRESUMEN
γδ T cells reside at mucosal and epithelial barriers, and they often accumulate at sites of inflammation, both infectious and autoimmune, as well as in certain tumors. However, progress in understanding their function is considerably hampered by a lack of full understanding of the ligands recognized by TCR-γδ and how expression of these ligands is regulated. We recently developed a soluble human TCR-γδ (Vγ9Vδ1) tetramer from a synovial γδ T cell clone of a Lyme arthritis patient and observed that it stains monocytes activated by Borrelia burgdorferi. Those findings are extended in the current study to further examine the physiological regulation of ligand expression on monocytes. The TCR-γδ ligand is induced by a variety of TLR agonists and requires NF-κB activation. Of particular interest is that ligand expression also requires caspase activation of the inflammasome and is dependent on active metabolism, mitochondrial reactive oxygen species, and activation of gasdermin-D. Consistent with these observations, the TCR-γδ ligand is expressed by a subset of metabolically active CD14+CD16+ monocytes and colocalizes intracellularly with mitochondria. The findings suggest a model in which synovial γδ T cell ligand is a self-antigen whose surface expression is increased by inflammatory conditions and mitochondrial stress.
Asunto(s)
Gasderminas , Receptores de Antígenos de Linfocitos T gamma-delta , Humanos , Ligandos , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades antiviral immunity through the expression of viral proteins that block detection, signaling, interferon (IFN) induction, and IFN-stimulated gene (ISG) expression1, 2. Weak induction of type I IFNs is associated with a hyperinflammatory response in patients that develop severe COVID-193, 4, 5. Here we uncover a role for cellular nucleic acid-binding protein (CNBP) in restricting SARS-CoV-2. Typically, CNBP resides in the cytosol and, in response to RNA sensing pathways, undergoes phosphorylation, nuclear translocation, and IFNß enhancer DNA binding to turn on IFNß gene transcription. In SARS-CoV-2-infected cells CNBP coordinates IFNß gene transcription. In addition, CNBP binds SARS-CoV-2 viral RNA directly. CNBP competes with the nucleocapsid (N) protein and prevents viral RNA and nucleocapsid protein from undergoing liquid-liquid phase separation (LLPS) forming condensates critical for viral replication. Consequently, cells and animals lacking CNBP have higher viral loads and CNBP-deficient mice succumb rapidly to infection. Altogether, these findings identify CNBP as a key antiviral factor for SARS-CoV-2, functioning both as a regulator of antiviral IFN gene expression and a cell intrinsic restriction factor that disrupts LLPS to limit viral replication and spread.
RESUMEN
Type I interferons (IFNs) are innate immune cytokines required to establish cellular host defense. Precise control of IFN gene expression is crucial to maintaining immune homeostasis. Here, we demonstrated that cellular nucleic acid-binding protein (CNBP) was required for the production of type I IFNs in response to RNA virus infection. CNBP deficiency markedly impaired IFN production in macrophages and dendritic cells that were infected with a panel of RNA viruses or stimulated with synthetic double-stranded RNA. Furthermore, CNBP-deficient mice were more susceptible to influenza virus infection than were wild-type mice. Mechanistically, CNBP was phosphorylated and translocated to the nucleus, where it directly binds to the promoter of IFNb in response to RNA virus infection. Furthermore, CNBP controlled the recruitment of IFN regulatory factor (IRF) 3 and IRF7 to IFN promoters for the maximal induction of IFNb gene expression. These studies reveal a previously unrecognized role for CNBP as a transcriptional regulator of type I IFN genes engaged downstream of RNA virus-mediated innate immune signaling, which provides an additional layer of control for IRF3- and IRF7-dependent type I IFN gene expression and the antiviral innate immune response.
Asunto(s)
Inmunidad , Interferón Tipo I/metabolismo , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/virología , Virus ARN/inmunología , Proteínas de Unión al ARN/metabolismo , Células A549 , Animales , Células HEK293 , Humanos , Inmunidad/efectos de los fármacos , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Ratones Endogámicos C57BL , Poli I-C/farmacología , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Virus ARN/efectos de los fármacos , ARN Viral/metabolismo , Transducción de Señal/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Alterations in the cGAS-STING DNA-sensing pathway affect intestinal homeostasis. We sought to delineate the functional role of STING in intestinal inflammation. Increased STING expression was a feature of intestinal inflammation in mice with colitis and in humans afflicted with inflammatory bowel disease. Mice bearing an allele rendering STING constitutively active exhibited spontaneous colitis and dysbiosis, as well as progressive chronic intestinal inflammation and fibrosis. Bone marrow chimera experiments revealed STING accumulation in intestinal macrophages and monocytes as the initial driver of inflammation. Depletion of Gram-negative bacteria prevented STING accumulation in these cells and alleviated intestinal inflammation. STING accumulation occurred at the protein rather than transcript level, suggesting post-translational stabilization. We found that STING was ubiquitinated in myeloid cells, and this K63-linked ubiquitination could be elicited by bacterial products, including cyclic di-GMP. Our findings suggest a positive feedback loop wherein dysbiosis foments the accumulation of STING in intestinal myeloid cells, driving intestinal inflammation.
Asunto(s)
Colitis/inmunología , Disbiosis/inmunología , Inmunidad Innata/inmunología , Proteínas de la Membrana/inmunología , Células Mieloides/inmunología , Ubiquitinación/inmunología , Animales , Estudios de Casos y Controles , Femenino , Humanos , Inflamación/inmunología , Intestinos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunologíaRESUMEN
Detection of DNA is an important determinant of host-defense but also a driver of autoinflammatory and autoimmune diseases. Failure to degrade self-DNA in DNAseII or III(TREX1)-deficient mice results in activation of the cGAS-STING pathway. Deficiency of cGAS or STING in these models ameliorates disease manifestations. However, the contribution of the cGAS-STING pathway, relative to endosomal TLRs, in systemic lupus erythematosus (SLE) is controversial. In fact, STING deficiency failed to rescue, and actually exacerbated, disease manifestations in Fas-deficient SLE-prone mice. We have now extended these observations to a chronic model of SLE induced by the i.p. injection of TMPD (pristane). We found that both cGAS- and STING-deficiency not only failed to rescue mice from TMPD-induced SLE, but resulted in increased autoantibody production and higher proteinuria levels compared to cGAS STING sufficient mice. Further, we generated cGASKOFaslpr mice on a pure MRL/Faslpr background using Crispr/Cas9 and found slightly exacerbated, and not attenuated, disease. We hypothesized that the cGAS-STING pathway constrains TLR activation, and thereby limits autoimmune manifestations in these two models. Consistent with this premise, mice lacking cGAS and Unc93B1 or STING and Unc93B1 developed minimal systemic autoimmunity as compared to cGAS or STING single knock out animals. Nevertheless, TMPD-driven lupus in B6 mice was abrogated upon AAV-delivery of DNAse I, implicating a DNA trigger. Overall, this study demonstrated that the cGAS-STING pathway does not promote systemic autoimmunity in murine models of SLE. These data have important implications for cGAS-STING-directed therapies being developed for the treatment of systemic autoimmunity.
Asunto(s)
Autoinmunidad , Susceptibilidad a Enfermedades , Lupus Eritematoso Sistémico/etiología , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Animales , Autoanticuerpos/inmunología , Autoinmunidad/genética , Biomarcadores , Modelos Animales de Enfermedad , Expresión Génica , Inmunofenotipificación , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Nucleotidiltransferasas/genéticaRESUMEN
Long non-coding RNAs are important regulators of biological processes including immune responses. The immunoregulatory functions of lncRNAs have been revealed primarily in murine models with limited understanding of lncRNAs in human immune responses. Here, we identify lncRNA LUCAT1 which is upregulated in human myeloid cells stimulated with lipopolysaccharide and other innate immune stimuli. Targeted deletion of LUCAT1 in myeloid cells increases expression of type I interferon stimulated genes in response to LPS. By contrast, increased LUCAT1 expression results in a reduction of the inducible ISG response. In activated cells, LUCAT1 is enriched in the nucleus where it associates with chromatin. Further, LUCAT1 limits transcription of interferon stimulated genes by interacting with STAT1 in the nucleus. Together, our study highlights the role of the lncRNA LUCAT1 as a post-induction feedback regulator which functions to restrain the immune response in human cells.
Asunto(s)
Regulación de la Expresión Génica , Interferones/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Fenómenos Biológicos , Cromatina/metabolismo , Citocinas/metabolismo , Retroalimentación , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad Innata/efectos de los fármacos , Lipopolisacáridos/efectos adversos , Ratones , Células Mieloides/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Proteínas , Factor de Transcripción STAT1/metabolismo , Células THP-1RESUMEN
Activated macrophages undergo a metabolic switch to aerobic glycolysis, accumulating Krebs' cycle intermediates that alter transcription of immune response genes. We extended these observations by defining fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization, and capacity to induce cell death. In mice, the administration of DMF protects against lipopolysaccharide shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics that include DMF, for the treatment of multiple sclerosis.
Asunto(s)
Cisteína/análogos & derivados , Dimetilfumarato/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Fiebre Mediterránea Familiar/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Proteínas de Unión a Fosfato/metabolismo , Piroptosis/efectos de los fármacos , Animales , Caspasas/metabolismo , Ciclo del Ácido Cítrico/efectos de los fármacos , Cisteína/metabolismo , Dimetilfumarato/uso terapéutico , Femenino , Células HEK293 , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Lipopolisacáridos/inmunología , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas de Unión a Fosfato/genética , Procesamiento Proteico-Postraduccional , Piroptosis/inmunologíaRESUMEN
Functional peptides encoded by short open reading frames are emerging as important mediators of fundamental biological processes. In this study, we identified a micropeptide produced from a putative long noncoding RNA (lncRNAs) that is important in controlling innate immunity. By studying lncRNAs in mice macrophages, we identified lncRNA 1810058I24Rik, which was downregulated in both human and murine myeloid cells exposed to LPS as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed that this transcript was localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 aa micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene mitochondrial micropeptide-47 (Mm47). Crispr-Cas9-mediated deletion of Mm47, as well as small interfering RNA studies in mice primary macrophages, showed that the transcriptional response downstream of TLR4 was intact in cells lacking Mm47. In contrast, Mm47-deficient or knockdown cells were compromised for Nlrp3 inflammasome responses. Activation of Nlrc4 or Aim2 inflammasomes were intact in cells lacking Mm47. This study therefore identifies, to our knowledge, a novel mitochondrial micropeptide Mm47 that is required for the activation of the Nlrp3 inflammasome. This work further highlights the functional activity of short open reading frame-encoded peptides and underscores their importance in innate immunity.
Asunto(s)
Citosol/metabolismo , Inflamasomas/metabolismo , Macrófagos/fisiología , Mitocondrias/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fragmentos de Péptidos/metabolismo , ARN Largo no Codificante/genética , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Humanos , Inmunidad Innata/genética , Lipopolisacáridos/inmunología , Ratones , Mitocondrias/genética , Fragmentos de Péptidos/genética , ARN Interferente Pequeño/genéticaRESUMEN
The mitochondrial antiviral signaling protein (MAVS) orchestrates host antiviral innate immune response to RNA virus infection. However, how MAVS signaling is controlled to eradicate virus while preventing self-destructive inflammation remains obscure. Here, we show that protein geranylgeranylation, a posttranslational lipid modification of proteins, limits MAVS-mediated immune signaling by targeting Rho family small guanosine triphosphatase Rac1 into the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) at the mitochondria-ER junction. Protein geranylgeranylation and subsequent palmitoylation promote Rac1 translocation into MAMs upon viral infection. MAM-localized Rac1 limits MAVS' interaction with E3 ligase Trim31 and hence inhibits MAVS ubiquitination, aggregation, and activation. Rac1 also facilitates the recruitment of caspase-8 and cFLIPL to the MAVS signalosome and the subsequent cleavage of Ripk1 that terminates MAVS signaling. Consistently, mice with myeloid deficiency of protein geranylgeranylation showed improved survival upon influenza A virus infection. Our work revealed a critical role of protein geranylgeranylation in regulating antiviral innate immune response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inmunidad Innata/fisiología , Neuropéptidos/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Prenilación de Proteína/inmunología , Proteína de Unión al GTP rac1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Animales , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Masculino , Ratones Noqueados , Neuropéptidos/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/mortalidad , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína RCA2 de Unión a GTPRESUMEN
Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).
Asunto(s)
Cromatina/química , Cromatina/genética , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , ARN Largo no Codificante/genética , ARN/química , Telomerasa/química , Animales , Células Cultivadas , Cromosomas , Células Madre Embrionarias/citología , Genoma , Ratones , Regiones Promotoras Genéticas , ARN/genética , Telomerasa/genéticaRESUMEN
An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Recently, cellular nucleic acid-binding protein (CNBP) was identified as a regulator of nuclear factor-kappaB (NF-κB)-dependent proinflammatory cytokine gene expression. Here, we generated mice lacking CNBP and found that CNBP regulates a very restricted gene signature that includes IL-12ß. CNBP resides in the cytosol of macrophages and translocates to the nucleus in response to diverse microbial pathogens and pathogen-derived products. Cnbp-deficient macrophages induced canonical NF-κB/Rel signaling normally but were impaired in their ability to control the activation of c-Rel, a key driver of IL-12ß gene transcription. The nuclear translocation and DNA-binding activity of c-Rel required CNBP. Lastly, Cnbp-deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL-12ß, as well as a reduced T helper type 1 (Th1) cell IFN-γ response essential to controlling parasite replication. Collectively, these findings identify CNBP as important regulator of c-Rel-dependent IL-12ß gene transcription and Th1 immunity.
Asunto(s)
Inmunidad Celular , Subunidad p40 de la Interleucina-12/inmunología , Proteínas de Unión al ARN/inmunología , Células TH1/inmunología , Transcripción Genética/inmunología , Animales , Interferón gamma/genética , Interferón gamma/inmunología , Subunidad p40 de la Interleucina-12/genética , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/inmunología , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Proto-Oncogénicas c-rel/inmunología , Proteínas de Unión al ARN/genética , Células TH1/citologíaRESUMEN
An inducible gene expression program is a hallmark of the host inflammatory response. Recently, long intergenic non-coding RNAs (lincRNAs) have been shown to regulate the magnitude, duration, and resolution of these responses. Among these is lincRNA-Cox2, a dynamically regulated gene that broadly controls immune gene expression. To evaluate the in vivo functions of this lincRNA, we characterized multiple models of lincRNA-Cox2-deficient mice. LincRNA-Cox2-deficient macrophages and murine tissues had altered expression of inflammatory genes. Transcriptomic studies from various tissues revealed that deletion of the lincRNA-Cox2 locus also strongly impaired the basal and inducible expression of the neighboring gene prostaglandin-endoperoxide synthase (Ptgs2), encoding cyclooxygenase-2, a key enzyme in the prostaglandin biosynthesis pathway. By utilizing different genetic manipulations in vitro and in vivo, we found that lincRNA-Cox2 functions through an enhancer RNA mechanism to regulate Ptgs2. More importantly, lincRNA-Cox2 also functions in trans, independently of Ptgs2, to regulate critical innate immune genes in vivo.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Inmunidad/genética , Modelos Genéticos , ARN Largo no Codificante/metabolismo , Animales , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Regulación de la Expresión Génica , Células HEK293 , Humanos , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , ARN/metabolismo , Empalme del ARN/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/metabolismo , Transcripción GenéticaRESUMEN
Toll-like receptors TLR7 and TLR9 are both implicated in the activation of autoreactive B cells and other cell types associated with systemic lupus erythematosus (SLE) pathogenesis. However, Tlr9-/- autoimmune-prone strains paradoxically develop more severe disease. We have now leveraged the negative regulatory role of TLR9 to develop an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II+ cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. These mice are distinguished by a high frequency of OVA-specific Tbet+, IFN-γ+, and FasL-expressing Th1 cells as well as autoantibody-producing B cells. Unexpectedly, contrary to what occurs in most models of SLE, they also developed skin lesions that are very similar to those of human cutaneous lupus erythematosus (CLE) as far as clinical appearance, histological changes, and gene expression. FasL was a key effector mechanism in the skin, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, our model provides a relevant system for exploring the pathophysiology of CLE as well as the negative regulatory role of TLR9.
Asunto(s)
Proteína Ligando Fas/metabolismo , Lupus Eritematoso Cutáneo/inmunología , Glicoproteínas de Membrana/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/deficiencia , Animales , Autoanticuerpos/biosíntesis , Linfocitos B/inmunología , Linfocitos B/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Interferón Tipo I/metabolismo , Lupus Eritematoso Cutáneo/metabolismo , Lupus Eritematoso Cutáneo/patología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ovalbúmina/inmunología , Piel/inmunología , Piel/patología , Células TH1/inmunología , Células TH1/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismoRESUMEN
Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1ß that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.