RESUMEN
Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.
Asunto(s)
Inflamación/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Mitocondrias/enzimología , Succinato Deshidrogenasa/metabolismo , Ácido Succínico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Ciclo del Ácido Cítrico , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/genética , Interleucina-10/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/metabolismo , Malonatos/farmacología , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Análisis de Secuencia de ARN , Succinato Deshidrogenasa/genética , TranscriptomaRESUMEN
Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.
Asunto(s)
COVID-19 , Proteasas 3C de Coronavirus , Células Epiteliales , Inflamasomas , Proteínas NLR , SARS-CoV-2 , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Caspasa 3/metabolismo , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/metabolismo , Células Epiteliales/metabolismo , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Pulmón/metabolismo , Pulmón/virología , Proteínas NLR/genética , Proteínas NLR/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptosis , SARS-CoV-2/enzimología , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidadRESUMEN
Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.
Asunto(s)
Apoptosis/inmunología , Macrófagos/inmunología , Necroptosis/inmunología , Piroptosis/inmunología , Infecciones por Salmonella/inmunología , Salmonella/inmunología , Animales , Caspasa 1/deficiencia , Caspasa 1/genética , Caspasa 12/deficiencia , Caspasa 12/genética , Caspasa 8/genética , Caspasas Iniciadoras/deficiencia , Caspasas Iniciadoras/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
Inflammatory bowel disease is a chronic, relapsing condition with two subtypes, Crohn's disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWASs) in UC implicate a FCGR2A variant that alters the binding affinity of the antibody receptor it encodes, FcγRIIA, for immunoglobulin G (IgG). Here, we aimed to understand the mechanisms whereby changes in FcγRIIA affinity would affect inflammation in an IgA-dominated organ. We found a profound induction of anti-commensal IgG and a concomitant increase in activating FcγR signaling in the colonic mucosa of UC patients. Commensal-IgG immune complexes engaged gut-resident FcγR-expressing macrophages, inducing NLRP3- and reactive-oxygen-species-dependent production of interleukin-1ß (IL-1ß) and neutrophil-recruiting chemokines. These responses were modulated by the FCGR2A genotype. In vivo manipulation of macrophage FcγR signal strength in a mouse model of UC determined the magnitude of intestinal inflammation and IL-1ß-dependent type 17 immunity. The identification of an important contribution of IgG-FcγR-dependent inflammation to UC has therapeutic implications.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Colitis Ulcerosa/inmunología , Microbioma Gastrointestinal/inmunología , Inmunoglobulina G/inmunología , Interleucina-1beta/inmunología , Células Th17/inmunología , Animales , Colitis/inducido químicamente , Colitis/inmunología , Colitis/microbiología , Colitis/patología , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Sulfato de Dextran/toxicidad , Regulación de la Expresión Génica , Genotipo , Humanos , Inflamación , Interleucina-8/biosíntesis , Interleucina-8/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Macrófagos/inmunología , Ratones , Fagocitos/inmunología , ARN Mensajero/biosíntesis , Especies Reactivas de Oxígeno , Receptores de IgG/biosíntesis , Receptores de IgG/genética , Receptores de IgG/inmunologíaRESUMEN
Mechano-immunity, the intersection between cellular or tissue mechanics and immune cell function, is emerging as an important factor in many inflammatory diseases. Mechano-sensing defines how cells detect mechanical changes in their environment. Mechano-response defines how cells adapt to such changes, e.g. form synapses, signal or migrate. Inflammasomes are intracellular immune sensors that detect changes in tissue and cell homoeostasis during infection or injury. We and others recently found that mechano-sensing of tissue topology (swollen tissue), topography (presence and distribution of foreign solid implant) or biomechanics (stiffness), alters inflammasome activity. Once activated, inflammasomes induce the secretion of inflammatory cytokines, but also change cellular mechanical properties, which influence how cells move, change their shape, and interact with other cells. When overactive, inflammasomes lead to chronic inflammation. This clearly places inflammasomes as important players in mechano-immunity. Here, we discuss a model whereby inflammasomes integrate pathogen- and tissue-injury signals, with changes in tissue mechanics, to shape the downstream inflammatory responses and allow cell and tissue mechano-adaptation. We will review the emerging evidence that supports this model.
Asunto(s)
Citocinas , Inflamasomas , Humanos , InflamaciónRESUMEN
SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.
Asunto(s)
Cuerpos Extraños , Inflamasomas , Humanos , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Prótesis e ImplantesRESUMEN
The alarm cytokine interleukin-1ß (IL-1ß) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1ß production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1ß; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1ß from its pro-form. However, despite the important role of IL-1ß in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1ß processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1ß processing, providing a previously unrecognized control of IL-1ß in tissue-resident macrophages.
Asunto(s)
Epoprostenol/inmunología , Interleucina-10/inmunología , Interleucina-1beta/inmunología , Macrófagos Peritoneales/inmunología , Animales , Epoprostenol/genética , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-10/genética , Interleucina-1beta/genética , Macrófagos Peritoneales/patología , Ratones , Ratones TransgénicosRESUMEN
The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male breast. In this study, we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo-anatomy highlights the resemblance of the male gland to that of neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is similarly low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distribution of leukocytes within the ducts and stroma is similar to the mammary gland of female sheep and females of other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster near the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology.
Asunto(s)
Glándulas Mamarias Animales , Animales , Masculino , Glándulas Mamarias Animales/anatomía & histología , Glándulas Mamarias Animales/metabolismo , Ovinos , Modelos Animales , Femenino , Proliferación Celular/fisiología , Células Epiteliales/metabolismoRESUMEN
Inflammation driven by the NLRP3 inflammasome in macrophages is an important contributor to chronic metabolic diseases that affect growing numbers of individuals. Many of these diseases involve the pathologic accumulation of endogenous lipids or their oxidation products, which can activate NLRP3. Other endogenous lipids, however, can inhibit the activation of NLRP3. The intracellular mechanisms by which these lipids modulate NLRP3 activity are now being identified. This review discusses emerging evidence suggesting that organelle stress, particularly involving mitochondria, lysosomes, and the endoplasmic reticulum, may be key in lipid-induced modification of NLRP3 inflammasome activity.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Estrés del Retículo Endoplásmico , Humanos , Lípidos , MitocondriasRESUMEN
Infection of human macrophages with Salmonella enterica serovar Typhimurium (S. Typhimurium) leads to inflammasome activation. Inflammasomes are multiprotein complexes facilitating caspase-1 activation and subsequent gasdermin D-mediated cell death and IL-1ß and IL-18 cytokine release. The NAIP/NLRC4 inflammasome is activated by multiple bacterial protein ligands, including flagellin from the flagellum and the needle protein PrgI from the S. Typhimurium type III secretion system. In this study, we show that transfected ultrapure flagellin from S Typhimurium induced cell death and cytokine secretion in THP-1 cells and primary human monocyte-derived macrophages. In THP-1 cells, NAIP/NLRC4 and NLRP3 played redundant roles in inflammasome activation during infection with S. Typhimurium. Knockout of NAIP or NLRC4 in THP-1 cells revealed that flagellin, but not PrgI, now activated the NLRP3 inflammasome through a reactive oxygen species- and/or cathepsin-dependent mechanism that was independent of caspase-4/5 activity. In conclusion, our data suggest that NLRP3 can be activated by flagellin to act as a "safety net" to maintain inflammasome activation under conditions of suboptimal NAIP/NLRC4 activation, as observed in THP-1 cells, possibly explaining the redundant role of NLRP3 and NAIP/NLRC4 during S. Typhimurium infection.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/metabolismo , Inflamasomas/metabolismo , Macrófagos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína Inhibidora de la Apoptosis Neuronal/metabolismo , Salmonella typhi/fisiología , Fiebre Tifoidea/inmunología , Apoptosis , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas de Unión al Calcio/genética , Caspasas Iniciadoras/metabolismo , Flagelina , Humanos , Proteína Inhibidora de la Apoptosis Neuronal/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Células THP-1 , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.
Asunto(s)
Genes p53 , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Cinética , Mutación , Desplegamiento Proteico , Agregado de ProteínasRESUMEN
The mammalian innate immune system deals with invading pathogens and stress by activating pattern-recognition receptors (PRRs) in the host. Initially proposed to be triggered by the discrimination of defined molecular signatures from pathogens rather than from self, it is now clear that PRRs can also be activated by endogenous ligands, bacterial metabolites and, following pathogen-induced alterations of cellular processes, changes in the F-actin cytoskeleton. These processes are collectively referred to as effector-triggered immunity (ETI). Here, we summarize the molecular and conceptual advances in our understanding of cell autonomous innate immune responses against bacterial pathogens, and discuss how classical activation of PRRs and ETI interplay to drive inflammatory responses.
Asunto(s)
Bacterias/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Animales , Humanos , Inflamación/patologíaRESUMEN
Leucine-rich repeat kinase 2 (LRRK2) encodes a complex protein that includes kinase and GTPase domains. Genome-wide association studies have identified dominant LRRK2 alleles that predispose their carriers to late-onset idiotypic Parkinson's disease (PD) and also to autoimmune disorders such as Crohn's disease. Considerable evidence indicates that PD initiation and progression involve activation of innate immune functions in microglia, which are brain-resident macrophages. Here we asked whether LRRK2 modifies inflammatory signaling and how this modification might contribute to PD and Crohn's disease. We used RNA-Seq-based high-resolution transcriptomics to compare gene expression in activated primary macrophages derived from WT and Lrrk2 knockout mice. Remarkably, expression of a single gene, Rap guanine nucleotide exchange factor 3 (Rapgef3), was strongly up-regulated in the absence of LRRK2 and down-regulated in its presence. We observed similar regulation of Rapgef3 expression in cells treated with a highly specific inhibitor of LRRK2 protein kinase activity. Rapgef3 encodes an exchange protein, activated by cAMP 1 (EPAC-1), a guanine nucleotide exchange factor that activates the small GTPase Rap-1. Rap-1 mediates cell adhesion, polarization, and directional motility, and our results indicate that LRRK2 modulates chemotaxis of microglia and macrophages. Dominant PD-associated LRRK2 alleles may suppress EPAC-1 activity, further restricting motility and preventing efficient migration of microglia to sites of neuronal damage. Functional analysis in vivo in a subclinical infection model also indicated that Lrrk2 subtly modifies the inflammatory response. These results indicate that LRRK2 modulates the expression of genes involved in murine immune cell chemotaxis.
Asunto(s)
Adhesión Celular , Polaridad Celular , Quimiotaxis , Regulación de la Expresión Génica , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Activación de Macrófagos , Macrófagos/enzimología , Animales , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Ratones Noqueados , Microglía/enzimología , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
The detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, the delivery of single molecules remains to be a challenge. Currently, there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position and then image the cellular response. Here, we have combined light-sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local-delivery selective-plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and ionic feedback current at the nanopipette tip to control the position from which the molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2 µm thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited the aggregates of amyloid-ß, which causes proteotoxicity relevant to Alzheimer's disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in the three-dimensional (3D) mode enables the live detection of MyDD88 accumulation and the formation of myddosome signaling complexes, as a result of the aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells.
Asunto(s)
Nanotecnología , Imagen Individual de Molécula , Membrana Celular , Microscopía de Fuerza AtómicaRESUMEN
Inflammasomes enable cells to respond to pathogens or biological damage, but the specific signals being used to convey these messages are not always clear. A new paper identifies two potential microbiota-derived metabolites, the bile acid analogues BAA485 and BAA473, as the first small molecules to activate the pyrin inflammasome. These results suggest that microbiota may be able to modulate this inflammatory process which, in turn, may contribute to the maintenance of intestinal homeostasis.
Asunto(s)
Ácidos y Sales Biliares/inmunología , Microbioma Gastrointestinal/inmunología , Inflamasomas/inmunología , Intestinos/inmunología , Pirina/inmunología , Animales , Humanos , Inflamación/inmunologíaRESUMEN
In the published article, the Fig. 2 was published incorrectly. The correct Fig. 2 is given below.
RESUMEN
Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1ß release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases.
Asunto(s)
Cardiolipinas/farmacología , Macrófagos/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Animales , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Unión Competitiva , Cardiolipinas/química , Cardiolipinas/metabolismo , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL10/metabolismo , Células HEK293 , Humanos , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Monocitos/citología , Monocitos/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Unión Proteica , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The IUPHAR/BPS Guide to PHARMACOLOGY (GtoPdb, www.guidetopharmacology.org) and its precursor IUPHAR-DB, have captured expert-curated interactions between targets and ligands from selected papers in pharmacology and drug discovery since 2003. This resource continues to be developed in conjunction with the International Union of Basic and Clinical Pharmacology (IUPHAR) and the British Pharmacological Society (BPS). As previously described, our unique model of content selection and quality control is based on 96 target-class subcommittees comprising 512 scientists collaborating with in-house curators. This update describes content expansion, new features and interoperability improvements introduced in the 10 releases since August 2015. Our relationship matrix now describes â¼9000 ligands, â¼15 000 binding constants, â¼6000 papers and â¼1700 human proteins. As an important addition, we also introduce our newly funded project for the Guide to IMMUNOPHARMACOLOGY (GtoImmuPdb, www.guidetoimmunopharmacology.org). This has been 'forked' from the well-established GtoPdb data model and expanded into new types of data related to the immune system and inflammatory processes. This includes new ligands, targets, pathways, cell types and diseases for which we are recruiting new IUPHAR expert committees. Designed as an immunopharmacological gateway, it also has an emphasis on potential therapeutic interventions.
Asunto(s)
Bases de Datos Farmacéuticas , Fenómenos del Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Animales , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Ligandos , Farmacología , Proteínas/efectos de los fármacosRESUMEN
Analysis of oxylipins by liquid chromatography mass spectrometry (LC/MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC/MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC/DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross-section values in nitrogen (DTCCSN2), and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DTCCSN2 and m/ z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift-time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium-infected murine bone-marrow-derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Eicosanoides/análisis , Ácidos Grasos/análisis , Oxilipinas/análisis , Espectrometría de Masas en Tándem/métodos , Animales , Células Cultivadas , Humanos , Espectrometría de Movilidad Iónica/métodos , Ratones Endogámicos C57BLRESUMEN
The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1ß processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1ß processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.