RESUMEN
The discovery of long noncoding RNAs (lncRNA) has provided a new perspective on gene regulation in diverse biological contexts. lncRNAs are remarkably versatile molecules that interact with RNA, DNA, or proteins to promote or restrain the expression of protein-coding genes. Activation of immune cells is associated with dynamic changes in expression of genes, the products of which combat infectious microorganisms, initiate repair, and resolve inflammatory responses in cells and tissues. Recent evidence indicates that lncRNAs play important roles in directing the development of diverse immune cells and controlling the dynamic transcriptional programs that are a hallmark of immune cell activation. The importance of these molecules is underscored by their newly recognized roles in inflammatory diseases. In this review, we discuss the contribution of lncRNAs in the development and activation of immune cells and their roles in immune-related diseases. We also discuss challenges faced in identifying biological functions for this large and complex class of genes.
Asunto(s)
Enfermedades del Sistema Inmune/genética , Inmunidad/genética , ARN Largo no Codificante/inmunología , Animales , Regulación de la Expresión Génica , HumanosRESUMEN
Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.
Asunto(s)
Regulación de la Expresión Génica , Inflamación/genética , Macrófagos/inmunología , ARN Largo no Codificante/metabolismo , Animales , Cromátides/metabolismo , Eliminación de Gen , Humanos , Listeria monocytogenes/fisiología , Listeriosis/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , ARN Largo no Codificante/genética , Infecciones por Respirovirus/inmunología , Virus Sendai/fisiología , Receptores Toll-Like/metabolismo , TranscriptomaRESUMEN
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
Alzheimer's disease (AD) is characterized by the progressive destruction and dysfunction of central neurons. AD patients commonly have unprovoked seizures compared with age-matched controls. Amyloid peptide-related inflammation is thought to be an important aspect of AD pathogenesis. We previously reported that NLRP3 inflammasome KO mice, when bred into APPswe/PS1ΔE9 (APP/PS1) mice, are completely protected from amyloid-induced AD-like disease, presumably because they cannot produce mature IL1ß or IL18. To test the role of IL18, we bred IL18KO mice with APP/PS1 mice. Surprisingly, IL18KO/APP/PS1 mice developed a lethal seizure disorder that was completely reversed by the anticonvulsant levetiracetam. IL18-deficient AD mice showed a lower threshold in chemically induced seizures and a selective increase in gene expression related to increased neuronal activity. IL18-deficient AD mice exhibited increased excitatory synaptic proteins, spine density, and basal excitatory synaptic transmission that contributed to seizure activity. This study identifies a role for IL18 in suppressing aberrant neuronal transmission in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Convulsiones/metabolismo , Transmisión Sináptica , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Amiloide/genética , Animales , Inflamasomas/genética , Interleucina-18/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Levetiracetam , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piracetam/análogos & derivados , Piracetam/farmacología , Convulsiones/tratamiento farmacológico , Convulsiones/genética , Convulsiones/patologíaRESUMEN
Tracheal cytotoxin (TCT), a monomer of DAP-type peptidoglycan from Bordetella pertussis, causes cytopathology in the respiratory epithelia of mammals and robustly triggers the Drosophila Imd pathway. PGRP-LE, a cytosolic innate immune sensor in Drosophila, directly recognizes TCT and triggers the Imd pathway, yet the mechanisms by which TCT accesses the cytosol are poorly understood. In this study, we report that CG8046, a Drosophila SLC46 family transporter, is a novel transporter facilitating cytosolic recognition of TCT, and plays a crucial role in protecting flies against systemic Escherichia coli infection. In addition, mammalian SLC46A2s promote TCT-triggered NOD1 activation in human epithelial cell lines, indicating that SLC46As is a conserved group of peptidoglycan transporter contributing to cytosolic immune recognition.
Asunto(s)
Citosol/inmunología , Proteínas de Drosophila/metabolismo , Inmunidad Innata , Peptidoglicano/inmunología , Simportadores/metabolismo , Factores de Virulencia de Bordetella/inmunología , Animales , Línea Celular , Pared Celular/inmunología , Pared Celular/metabolismo , Citosol/metabolismo , Drosophila/inmunología , Drosophila/microbiología , Escherichia coli/fisiología , Células HEK293 , Humanos , Peptidoglicano/química , Peptidoglicano/metabolismo , Transducción de Señal , Factores de Virulencia de Bordetella/química , Factores de Virulencia de Bordetella/metabolismoRESUMEN
Bacterial sepsis involves a complex interaction between the host immune response and bacterial LPS. LPS binds Toll-like receptor (TLR) 4, which leads to the release of proinflammatory cytokines that are essential for a potent innate immune response against pathogens. The innate immune system is tightly regulated, as excessive inflammation can lead to organ failure and death. MicroRNAs have recently emerged as important regulators of the innate immune system. Here we determined the function of miR-718, which is conserved across mammals and overlaps with the 5' UTR of the interleukin 1 receptor-associated kinase (IRAK1) gene. As IRAK1 is a key component of innate immune signaling pathways that are downstream of most TLRs, we hypothesized that miR-718 helps regulate the innate immune response. Activation of TLR4, but not TLR3, induced the expression of miR-718 in macrophages. miR-718 expression was also induced in the spleens of mice upon LPS injection. miR-718 modulates PI3K/Akt signaling by directly down-regulating phosphatase and tensin homolog (PTEN), thereby promoting phosphorylation of Akt, which leads to a decrease in proinflammatory cytokine production. Phosphorylated Akt induces let-7e expression, which, in turn, down-regulates TLR4 and further diminishes TLR4-mediated proinflammatory signals. Decreased miR-718 expression is associated with bacterial burden during Neisseria gonorrhoeae infection and alters the infection dynamics of N. gonorrhoeae in vitro Furthermore, miR-718 regulates the induction of LPS tolerance in macrophages. We propose a role for miR-718 in controlling TLR4 signaling and inflammatory cytokine signaling through a negative feedback regulation loop involving down-regulation of TLR4, IRAK1, and NF-κB.
Asunto(s)
Regiones no Traducidas 5' , Citocinas/metabolismo , Macrófagos/metabolismo , MicroARNs/metabolismo , Fosfohidrolasa PTEN/metabolismo , Transducción de Señal , Animales , Citocinas/genética , Gonorrea/genética , Gonorrea/metabolismo , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Noqueados , MicroARNs/genética , Neisseria gonorrhoeae/metabolismo , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Approximately 75% of the human genome is transcribed and many of these spliced transcripts contain primate-specific Alu elements, the most abundant mobile element in the human genome. The majority of exonized Alu elements are located in long noncoding RNAs (lncRNAs) and the untranslated regions of mRNA, with some performing molecular functions. To further assess the potential for Alu elements to be repurposed as functional RNA domains, we investigated the distribution and evolution of Alu elements in spliced transcripts. Our analysis revealed that Alu elements are underrepresented in mRNAs and lncRNAs, suggesting that most exonized Alu elements arising in the population are rare or deleterious to RNA function. When mRNAs and lncRNAs retain exonized Alu elements, they have a clear preference for Alu dimers, left monomers, and right monomers. mRNAs often acquire Alu elements when their genes are duplicated within Alu-rich regions. In lncRNAs, reverse-oriented Alu elements are significantly enriched and are not restricted to the 3' and 5' ends. Both lncRNAs and mRNAs primarily contain the Alu J and S subfamilies that were amplified relatively early in primate evolution. Alu J subfamilies are typically overrepresented in lncRNAs, whereas the Alu S dimer is overrepresented in mRNAs. The sequences of Alu dimers tend to be constrained in both lncRNAs and mRNAs, whereas the left and right monomers are constrained within particular Alu subfamilies and classes of RNA. Collectively, these findings suggest that Alu-containing RNAs are capable of forming stable structures and that some of these Alu domains might have novel biological functions.
Asunto(s)
Regiones no Traducidas 3' , Regiones no Traducidas 5' , Elementos Alu , Genoma Humano , ARN Largo no Codificante/química , Biología Computacional/métodos , Evolución Molecular , Exones , Humanos , ARN Largo no Codificante/genéticaRESUMEN
Natural antisense transcripts (NATs) are a class of long noncoding RNAs (lncRNAs) that are complementary to other protein-coding genes. Although thousands of NATs are encoded by mammalian genomes, their functions in innate immunity are unknown. In this study, we identified and characterized a novel NAT, AS-IL1α, which is partially complementary to IL-1α. Similar to IL-1α, AS-IL1α is expressed at low levels in resting macrophages and is induced following infection with Listeria monocytogenes or stimulation with TLR ligands (Pam3CSK4, LPS, polyinosinic-polycytidylic acid). Inducible expression of IL-1α mRNA and protein were significantly reduced in macrophages expressing shRNA that target AS-IL1α. AS-IL1α is located in the nucleus and did not alter the stability of IL-1α mRNA. Instead, AS-IL1α was required for the recruitment of RNA polymerase II to the IL-1α promoter. In summary, our studies identify AS-IL1α as an important regulator of IL-1α transcription during the innate immune response.
Asunto(s)
Regulación de la Expresión Génica , Mediadores de Inflamación , Interleucina-1alfa/genética , ARN sin Sentido/genética , ARN no Traducido/genética , Transcripción Genética , Animales , Línea Celular , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Sitios Genéticos , Ligandos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Interferencia de ARN , Receptores Toll-Like/metabolismoRESUMEN
The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fisher's Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures.
Asunto(s)
Farmacorresistencia Viral/genética , Genética de Población , Subtipo H1N1 del Virus de la Influenza A/genética , Selección Genética , Teorema de Bayes , Flujo Genético , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/genética , Gripe Humana/virología , Mutación , Oseltamivir/farmacologíaRESUMEN
Influenza A virus (IAV) has a segmented genome that allows for the exchange of genome segments between different strains. This reassortment accelerates evolution by breaking linkage, helping IAV cross species barriers to potentially create highly virulent strains. Challenges associated with monitoring the process of reassortment in molecular detail have limited our understanding of its evolutionary implications. We applied a novel deep sequencing approach with quantitative analysis to assess the in vitro temporal evolution of genomic reassortment in IAV. The combination of H1N1 and H3N2 strains reproducibly generated a new H1N2 strain with the hemagglutinin and nucleoprotein segments originating from H1N1 and the remaining six segments from H3N2. By deep sequencing the entire viral genome, we monitored the evolution of reassortment, quantifying the relative abundance of all IAV genome segments from the two parent strains over time and measuring the selection coefficients of the reassorting segments. Additionally, we observed several mutations coemerging with reassortment that were not found during passaging of pure parental IAV strains. Our results demonstrate how reassortment of the segmented genome can accelerate viral evolution in IAV, potentially enabled by the emergence of a small number of individual mutations.
Asunto(s)
Alphainfluenzavirus/genética , Genoma Viral , Virus Reordenados/genética , Selección Genética , Animales , Biología Computacional , Perros , Evolución Molecular , Frecuencia de los Genes , Genotipo , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Límite de Detección , Células de Riñón Canino Madin Darby , Nucleoproteínas/genética , Análisis de Secuencia de ARNRESUMEN
The transcriptional repressor B lymphocyte-induced maturation protein 1 (BLIMP1) is a master regulator of B and T cell differentiation. To examine the role of BLIMP1 in innate immunity, we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal infection induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO macrophages identified the murine chemokine (C-C motif) ligand 8, CCL8, as a direct target of Blimp1-mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8, and consequently Blimp1 CKO mice had higher levels of circulating CCL8, resulting in increased neutrophils in the peripheral blood, promoting a more aggressive antibacterial response. Mice lacking the Ccl8 gene were more susceptible to L. monocytogenes infection than were wild-type mice. Although CCL8 failed to recruit neutrophils directly, it was chemotactic for γ/δ T cells, and CCL8-responsive γ/δ T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on γ/δ T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host defenses by suppressing expression of the chemokine CCL8.
Asunto(s)
Quimiocina CCL8/inmunología , Regulación de la Expresión Génica/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Macrófagos/inmunología , Factores de Transcripción/inmunología , Animales , Quimiocina CCL8/genética , Regulación de la Expresión Génica/genética , Listeriosis/genética , Macrófagos/patología , Ratones , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/patología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Factores de Transcripción/genética , Transcripción Genética/genética , Transcripción Genética/inmunologíaRESUMEN
BACKGROUND & AIMS: The alimentary tract contains a diffuse endocrine system comprising enteroendocrine cells that secrete peptides or biogenic amines to regulate digestion, insulin secretion, food intake, and energy homeostasis. Lineage analysis in the stomach revealed that a significant fraction of endocrine cells in the gastric corpus did not arise from Neurogenin3 (Neurog3)-expressing cells, unlike enteroendocrine cells elsewhere in the digestive tract. We aimed to isolate enriched serotonin-secreting and enterochromaffin-like (ECL) cells from the stomach and to clarify their cellular origin. METHODS: We used Neurogenic differentiation 1 (NeuroD1) and Neurog3 lineage analysis and examined the differentiation of serotonin-producing and ECL cells in stomach tissues of NeuroD1-cre;ROSA(tdTom), tryptophan hydroxylase 1 (Tph1)-cyan fluorescent protein (CFP), c-Kit(wsh/wsh), and Neurog3Cre;ROSA(tdTom) mice by immunohistochemistry. We used fluorescence-activated cell sorting to isolate each cell type for gene expression analysis. We also performed RNA sequencing analysis of ECL cells. RESULTS: Neither serotonin-secreting nor ECL cells of the corpus arose from cells expressing NeuroD1. Serotonin-secreting cells expressed a number of mast cell genes but not genes associated with endocrine differentiation; they did not develop in c-Kit(wsh/wsh) mice and were labeled with transplanted bone marrow cells. RNA sequencing analysis of ECL cells revealed high expression levels of many genes common to endocrine cells, including transcription factors, hormones, ion channels, and solute transporters but not markers of bone marrow cells. CONCLUSIONS: Serotonin-expressing cells of the gastric corpus of mice appear to be bone marrow-derived mucosal mast cells. Gene expression analysis of ECL cells indicated that they are endocrine cells of epithelial origin that do not express the same transcription factors as their intestinal enteroendocrine cell counterparts.
Asunto(s)
Linaje de la Célula , Células Enterocromafines/patología , Células Enteroendocrinas/patología , Serotonina/metabolismo , Estómago/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Diferenciación Celular , Células Enterocromafines/metabolismo , Células Enteroendocrinas/metabolismo , Mucosa Gástrica/metabolismo , Mastocitos/metabolismo , Mastocitos/patología , Ratones , Ratones Transgénicos , Modelos Animales , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Evolución Molecular , Genoma Viral , Subtipo H1N1 del Virus de la Influenza A/genética , Oseltamivir/farmacología , Animales , Línea Celular , Perros , Ensayos Analíticos de Alto Rendimiento , Técnicas In Vitro , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Concentración 50 Inhibidora , Mutación , Ensayo de Placa ViralRESUMEN
Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces systemic lupus erythematosus-like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL can also trigger the production of proinflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (ΔCS) mouse line. ΔCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity.
Asunto(s)
Proteína Ligando Fas/metabolismo , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Animales , Apoptosis/inmunología , Células 3T3 BALB , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Proteína Ligando Fas/inmunología , Citometría de Flujo , Inmunosupresores/toxicidad , Enfermedades Renales/patología , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Terpenos/toxicidad , TranscriptomaRESUMEN
The innate immune system senses nucleic acids by germline-encoded pattern recognition receptors. RNA is sensed by Toll-like receptor members TLR3, TLR7 and TLR8, or by the RNA helicases RIG-I (also known as DDX58) and MDA-5 (IFIH1). Little is known about sensors for cytoplasmic DNA that trigger antiviral and/or inflammatory responses. The best characterized of these responses involves activation of the TANK-binding kinase (TBK1)-interferon regulatory factor 3 (IRF3) signalling axis to trigger transcriptional induction of type I interferon genes. A second, less well-defined pathway leads to the activation of an 'inflammasome' that, via caspase-1, controls the catalytic cleavage of the pro-forms of the cytokines IL1beta and IL18 (refs 6, 7). Using mouse and human cells, here we identify the PYHIN (pyrin and HIN domain-containing protein) family member absent in melanoma 2 (AIM2) as a receptor for cytosolic DNA, which regulates caspase-1. The HIN200 domain of AIM2 binds to DNA, whereas the pyrin domain (but not that of the other PYHIN family members) associates with the adaptor molecule ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) to activate both NF-kappaB and caspase-1. Knockdown of Aim2 abrogates caspase-1 activation in response to cytoplasmic double-stranded DNA and the double-stranded DNA vaccinia virus. Collectively, these observations identify AIM2 as a new receptor for cytoplasmic DNA, which forms an inflammasome with the ligand and ASC to activate caspase-1.
Asunto(s)
Caspasa 1/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citosol/metabolismo , ADN/metabolismo , Inflamación/metabolismo , Proteínas Nucleares/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Muerte Celular , Línea Celular , Proteínas del Citoesqueleto/genética , ADN/inmunología , Proteínas de Unión al ADN , Activación Enzimática , Humanos , Inflamación/enzimología , Inflamación/patología , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/genética , Poli dA-dT/inmunología , Unión Proteica , Virus Vaccinia/inmunologíaRESUMEN
Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors â¼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.
Asunto(s)
Anopheles/genética , Genoma de los Insectos , Insectos Vectores/genética , Animales , Anopheles/clasificación , Brasil , Cromosomas de Insectos/genética , Elementos Transponibles de ADN , Evolución Molecular , Femenino , Variación Genética , Interacciones Huésped-Parásitos , Proteínas de Insectos/genética , Insectos Vectores/clasificación , Resistencia a los Insecticidas , Insecticidas/farmacología , Malaria/parasitología , Masculino , Anotación de Secuencia Molecular , Filogenia , Sintenía , TranscriptomaRESUMEN
Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
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ARN Helicasas DEAD-box/inmunología , Interferón beta/inmunología , Membrana Mucosa/virología , Fiebre del Valle del Rift/enzimología , Fiebre del Valle del Rift/inmunología , Virus de la Fiebre del Valle del Rift/fisiología , Animales , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Células Dendríticas/inmunología , Células Dendríticas/virología , Femenino , Humanos , Interferón beta/genética , Macrófagos/inmunología , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Membrana Mucosa/inmunología , Fiebre del Valle del Rift/prevención & control , Fiebre del Valle del Rift/virología , Transducción de Señal , Receptores Toll-Like/genética , Receptores Toll-Like/inmunologíaRESUMEN
Plasmodium falciparum (P. falciparum) induces trained innate immune responses in vitro, where initial stimulation of adherent PBMCs with P. falciparum-infected RBCs (iRBCs) results in hyperresponsiveness to subsequent ligation of TLR2. This response correlates with the presence of T and B lymphocytes in adherent PBMCs, suggesting that innate immune training is partially due to adaptive immunity. We found that T cell-depleted PBMCs and purified monocytes alone did not elicit hyperproduction of IL-6 and TNF-α under training conditions. Analysis of P. falciparum-trained PBMCs showed that DCs did not develop under control conditions, and IL-6 and TNF-α were primarily produced by monocytes and DCs. Transwell experiments isolating purified monocytes from either PBMCs or purified CD4+ T cells, but allowing diffusion of secreted proteins, enabled monocytes trained with iRBCs to hyperproduce IL-6 and TNF-α after TLR restimulation. Purified monocytes stimulated with IFN-γ hyperproduced IL-6 and TNF-α, whereas blockade of IFN-γ in P. falciparum-trained PBMCs inhibited trained responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from patients with malaria showed persistently open chromatin at genes that appeared to be trained in vitro. Together, these findings indicate that the trained immune response of monocytes to P. falciparum is not completely cell intrinsic but depends on soluble signals from lymphocytes.
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Linfocitos , Malaria Falciparum , Monocitos , Cromatina , Humanos , Interleucina-6/genética , Linfocitos/inmunología , Malaria Falciparum/inmunología , Monocitos/inmunología , Plasmodium falciparum , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PFRED a software application for the design, analysis, and visualization of antisense oligonucleotides and siRNA is described. The software provides an intuitive user-interface for scientists to design a library of siRNA or antisense oligonucleotides that target a specific gene of interest. Moreover, the tool facilitates the incorporation of various design criteria that have been shown to be important for stability and potency. PFRED has been made available as an open-source project so the code can be easily modified to address the future needs of the oligonucleotide research community. A compiled version is available for downloading at https://github.com/pfred/pfred-gui/releases/tag/v1.0 as a java Jar file. The source code and the links for downloading the precompiled version can be found at https://github.com/pfred.
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Biología Computacional/métodos , Cartilla de ADN/genética , Oligonucleótidos Antisentido/genética , Algoritmos , Biblioteca de Genes , Genómica , Oligonucleótidos/genética , ARN Interferente Pequeño/genética , Programas Informáticos , Interfaz Usuario-ComputadorRESUMEN
Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.