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BACKGROUND: Myeloid cells are critical determinants of the sustained inflammation in Crohn's Disease (CD). Targeting such cells may be an effective therapeutic approach for refractory CD patients. Bromodomain and extra-terminal domain protein inhibitors (iBET) are potent anti-inflammatory agents; however, they also possess wide-ranging toxicities. In the current study, we make use of a BET inhibitor containing an esterase sensitive motif (ESM-iBET), which is cleaved by carboxylesterase-1 (CES1), a highly expressed esterase in mononuclear myeloid cells. METHODS: We profiled CES1 protein expression in the intestinal biopsies, peripheral blood, and CD fistula tract (fCD) cells of CD patients using mass cytometry. The anti-inflammatory effect of ESM-iBET or its control (iBET) were evaluated in healthy donor CD14+ monocytes and fCD cells, using cytometric beads assay or RNA-sequencing. RESULTS: CES1 was specifically expressed in monocyte, macrophage, and dendritic cell populations in the intestinal tissue, peripheral blood, and fCD cells of CD patients. ESM-iBET inhibited IL1ß, IL6, and TNFα secretion from healthy donor CD14+ monocytes and fCD immune cells, with 10- to 26-fold more potency over iBET in isolated CD14+ monocytes. Transcriptomic analysis revealed that ESM-iBET inhibited multiple inflammatory pathways, including TNF, JAK-STAT, NF-kB, NOD2, and AKT signaling, with superior potency over iBET. CONCLUSIONS: We demonstrate specific CES1 expression in mononuclear myeloid cell subsets in peripheral blood and inflamed tissues of CD patients. We report that low dose ESM-iBET accumulates in CES1-expressing cells and exerts robust anti-inflammatory effects, which could be beneficial in refractory CD patients.
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Antiinflamatorios , Enfermedad de Crohn , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Hidrolasas de Éster Carboxílico , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/metabolismo , Humanos , Mediadores de Inflamación , Interleucina-6 , Células Mieloides/metabolismo , FN-kappa B , Proteínas Proto-Oncogénicas c-akt , ARN , Factor de Necrosis Tumoral alfaRESUMEN
On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.
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SARS-CoV-2RESUMEN
AIMS: GSK3358699 is a mononuclear myeloid-targeted bromodomain and extra-terminal domain (BET) family inhibitor which demonstrates immunomodulatory effects in vitro. This phase 1, randomized, first-in-human study evaluated the safety, pharmacokinetics, and pharmacodynamics of GSK3358699 in healthy male participants (NCT03426995). METHODS: Part A (N = 23) included three dose-escalating periods of 1-40 mg of GSK3358699 or placebo in two cohorts in a single ascending-dose crossover design. Part C (N = 25) was planned as an initial dose of 10 mg of GSK3358699 or placebo daily for 14 days followed by selected doses in four sequential cohorts. RESULTS: In part A, exposure to GSK3358699 and its metabolite GSK3206944 generally increased with increasing doses. The median initial half-life ranged from 0.7 to 1.1 (GSK3358699) and 2.1 to 2.9 (GSK3206944) hours after a single dose of 1-40 mg. GSK3206944 concentrations in monocytes were quantifiable at 1-hour post-dose following 10 mg of GSK3358699 and 1 and 4 hours post-dose following 20-40 mg. Mean predicted percentage inhibition of ex vivo lipopolysaccharide-induced monocyte chemoattractant protein (MCP)-1 reached 75% with 40 mg of GSK3358699. GSK3358699 did not inhibit interleukin (IL)-6 and tumour necrosis factor (TNF). The most common adverse event (AE) was headache. Four AEs of nonsustained ventricular tachycardia were observed across parts A and C. One serious AE of atrial fibrillation (part C) required hospitalization. CONCLUSIONS: Single doses of GSK3358699 are generally well tolerated with significant metabolite concentrations detected in target cells. A complete assessment of pharmacodynamics was limited by assay variability. A causal relationship could not be excluded for cardiac-related AEs, resulting in an inability to identify a suitable repeat-dose regimen and study termination.
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Relación Dosis-Respuesta a Droga , Área Bajo la Curva , Estudios Cruzados , Método Doble Ciego , Voluntarios Sanos , Humanos , MasculinoRESUMEN
BACKGROUND AND AIMS: Histone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD]. METHODS: CES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn's disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter. RESULTS: CES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis. CONCLUSIONS: We demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.
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Hidrolasas de Éster Carboxílico , Colitis , Enfermedad de Crohn , Inhibidores de Histona Desacetilasas , Enfermedades Inflamatorias del Intestino , Animales , Hidrolasas de Éster Carboxílico/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Lipopolisacáridos , Ratones , Monocitos , Células MieloidesRESUMEN
As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-γ is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-γ to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-γ alone was sufficient to generate macrophages (IFN-γ MÏ) that were phagocytic and responsive to polarization. We demonstrate that IFN-γ MÏ are potent activators of T lymphocytes that produce IL-17 and IFN-γ. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-γ MÏ and demonstrate that these markers are enriched in the skin of patients with inflamed psoriasis. Collectively, we show that IFN-γ can drive human monocyte to macrophage differentiation, leading to bona fide macrophages with inflammatory characteristics.
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Diferenciación Celular/fisiología , Inflamación/metabolismo , Interferón gamma/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Psoriasis/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Humanos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Fenotipo , Proteómica/métodos , Piel/metabolismoRESUMEN
Macrophages are key immune cells in the activation and regulation of immune responses. These cells are present in all tissues under homeostatic conditions and in many disease settings. Macrophages can exhibit a wide range of phenotypes depending on local and systemic cues that drive the differentiation and activation process. Macrophage heterogeneity is also defined by their ontogeny. Tissue macrophages can either derive from circulating blood monocytes or are seeded as tissue-resident macrophages during embryonic development. In humans, the study of in vivo-generated macrophages is often difficult with laborious and cell-changing isolation procedures. Therefore, translatable, reproducible, and robust in vitro models for human macrophages in health and disease are necessary. Most of the methods for studying monocyte-derived macrophages are based on the use of limited factors to differentiate the monocytes into macrophages. Current knowledge shows that the in vivo situation is more complex, and a wide range of molecules in the tissue microenvironment promote and impact on monocyte to macrophage differentiation as well as activation. In this review, macrophage heterogeneity is discussed and the human in vitro models that can be applied for research, especially for monocyte-derived macrophages. We also focus on new molecules (IL-34, platelet factor 4, etc.) used to generate macrophages expressing different phenotypes.
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Macrófagos/metabolismo , Diferenciación Celular , Humanos , Macrófagos/citología , Modelos Biológicos , Monocitos/citología , FenotipoRESUMEN
The signalling receptor for LPS, CD14, is a key marker of, and facilitator for, pro-inflammatory macrophage function. Pro-inflammatory macrophage differentiation remains a process facilitating a broad array of disease pathologies, and has recently emerged as a potential target against cytokine storm in COVID19. Here, we perform a whole-genome CRISPR screen to identify essential nodes regulating CD14 expression in myeloid cells, using the differentiation of THP-1 cells as a starting point. This strategy uncovers many known pathways required for CD14 expression and regulating macrophage differentiation while additionally providing a list of novel targets either promoting or limiting this process. To speed translation of these results, we have then taken the approach of independently validating hits from the screen using well-curated small molecules. In this manner, we identify pharmacologically tractable hits that can either increase CD14 expression on non-differentiated monocytes or prevent CD14 upregulation during macrophage differentiation. An inhibitor for one of these targets, MAP2K3, translates through to studies on primary human monocytes, where it prevents upregulation of CD14 following M-CSF induced differentiation, and pro-inflammatory cytokine production in response to LPS. Therefore, this screening cascade has rapidly identified pharmacologically tractable nodes regulating a critical disease-relevant process.
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Diferenciación Celular/efectos de los fármacos , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Biomarcadores , Células Cultivadas , Citocinas/metabolismo , Humanos , Inmunofenotipificación , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/efectos adversos , Macrófagos/efectos de los fármacos , Células THP-1RESUMEN
T helper (Th) cells are CD4+ effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis.
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Benzazepinas/farmacología , Histona Demetilasas/metabolismo , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Pirimidinas/farmacología , Células Th17/metabolismo , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Benzazepinas/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Código de Histonas/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Humanos , Interleucina-17/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Cultivo Primario de Células , Pirimidinas/uso terapéutico , RNA-Seq , Espondilitis Anquilosante/tratamiento farmacológico , Espondilitis Anquilosante/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Factores de Transcripción/metabolismoRESUMEN
Monocytes and macrophages are key drivers in the pathogenesis of inflammatory diseases. Epigenetic targets have been shown to control the transcriptional profile and phenotype of these cells. Since histone deacetylase protein inhibitors demonstrate profound anti-inflammatory activity, we wanted to test whether HDAC inhibition within monocytes and macrophages could be applied to suppress inflammation in vivo. ESM technology conjugates an esterase-sensitive motif (ESM) onto small molecules to allow targeting of cells that express carboxylesterase 1 (CES1), such as mononuclear myeloid cells. This study utilized an ESM-HDAC inhibitor to target monocytes and macrophages in mice in both an acute response model and an atherosclerosis model. We demonstrate that the molecule blocks the maturation of peritoneal macrophages and inhibits pro-inflammatory cytokine production in both models but to a lesser extent in the atherosclerosis model. Despite regulating the inflammatory response, ESM-HDAC528 did not significantly affect plaque size or phenotype, although histological classification of the plaques demonstrated a significant shift to a less severe phenotype. We hereby show that HDAC inhibition in myeloid cells impairs the maturation and activation of peritoneal macrophages but shows limited efficacy in a model of atherosclerosis.
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Non-alcoholic fatty liver disease (NAFLD) is a leading form of chronic liver disease with large unmet need. Non-alcoholic steatohepatitis (NASH), a progressive variant of NAFLD, can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. To identify potential new therapeutics for NASH, we used a computational approach based on Connectivity Map (CMAP) analysis, which pointed us to bromodomain and extra-terminal motif (BET) inhibitors for treating NASH. To experimentally validate this hypothesis, we tested a small-molecule inhibitor of the BET family of proteins, GSK1210151A (I-BET151), in the STAM mouse NASH model at two different dosing timepoints (onset of NASH and progression to fibrosis). I-BET151 decreased the non-alcoholic fatty liver disease activity score (NAS), a clinical endpoint for assessing the severity of NASH, as well as progression of liver fibrosis and interferon-γ expression. Transcriptional characterization of these mice through RNA-sequencing was consistent with predictions from the CMAP analysis of a human NASH signature and pointed to alterations in molecular mechanisms related to interferon signaling and cholesterol biosynthesis, as well as reversal of gene expression patterns linked to fibrotic markers. Altogether, these results suggest that inhibition of BET proteins may present a novel therapeutic opportunity in the treatment of NASH and liver fibrosis.
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Biología Computacional/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/administración & dosificación , Cirrosis Hepática/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Animales , Colesterol/biosíntesis , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Interferón gamma/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/inmunología , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/inmunología , Análisis de Secuencia de ARN , Índice de Severidad de la EnfermedadRESUMEN
Epigenetic modifiers are an emerging class of anti-tumor drugs, potent in multiple cancer contexts. Their effect on spontaneously developing autoimmune diseases has been little explored. We report that a short treatment with I-BET151, a small-molecule inhibitor of a family of bromodomain-containing transcriptional regulators, irreversibly suppressed development of type-1 diabetes in NOD mice. The inhibitor could prevent or clear insulitis, but had minimal influence on the transcriptomes of infiltrating and circulating T cells. Rather, it induced pancreatic macrophages to adopt an anti-inflammatory phenotype, impacting the NF-κB pathway in particular. I-BET151 also elicited regeneration of islet ß-cells, inducing proliferation and expression of genes encoding transcription factors key to ß-cell differentiation/function. The effect on ß cells did not require T cell infiltration of the islets. Thus, treatment with I-BET151 achieves a 'combination therapy' currently advocated by many diabetes investigators, operating by a novel mechanism that coincidentally dampens islet inflammation and enhances ß-cell regeneration.
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Diabetes Mellitus Tipo 1/genética , Epigénesis Genética , Células Secretoras de Insulina/patología , Macrófagos/patología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Epigénesis Genética/efectos de los fármacos , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Inflamación/patología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos NOD , Monocitos/citología , Monocitos/efectos de los fármacos , FN-kappa B/metabolismo , Fenotipo , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Delayed neuronal loss and brain atrophy after cerebral ischemia contribute to stroke and dementia pathology, but the mechanisms are poorly understood. Phagocytic removal of neurons is generally assumed to be beneficial and to occur only after neuronal death. However, we report herein that inhibition of phagocytosis can prevent delayed loss and death of functional neurons after transient brain ischemia. Two phagocytic proteins, Mer receptor tyrosine kinase (MerTK) and Milk fat globule EGF-like factor 8 (MFG-E8), were transiently up-regulated by macrophages/microglia after focal brain ischemia in vivo. Strikingly, deficiency in either protein completely prevented long-term functional motor deficits after cerebral ischemia and strongly reduced brain atrophy as a result of inhibiting phagocytosis of neurons. Correspondingly, in vitro glutamate-stressed neurons reversibly exposed the "eat-me" signal phosphatidylserine, leading to their phagocytosis by microglia; this neuronal loss was prevented in the absence of microglia and reduced if microglia were genetically deficient in MerTK or MFG-E8, both of which mediate phosphatidylserine-recognition. Thus, phagocytosis of viable neurons contributes to brain pathology and, surprisingly, blocking this process is strongly beneficial. Therefore, inhibition of specific phagocytic pathways may present therapeutic targets for preventing delayed neuronal loss after transient cerebral ischemia.
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Antígenos de Superficie/metabolismo , Isquemia Encefálica/metabolismo , Proteínas de la Leche/metabolismo , Neuronas/metabolismo , Fagocitosis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Antígenos de Superficie/genética , Atrofia , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Infarto Encefálico/genética , Infarto Encefálico/metabolismo , Isquemia Encefálica/genética , Muerte Celular , Células Cultivadas , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Proteínas de la Leche/genética , Neuronas/patología , Proteínas Proto-Oncogénicas/genética , Ratas , Ratas Mutantes , Proteínas Tirosina Quinasas Receptoras/genética , Factores de Tiempo , Regulación hacia Arriba , Tirosina Quinasa c-MerRESUMEN
In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.
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Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Zinc/química , Línea Celular Tumoral , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores de Histona Desacetilasas/síntesis química , Histona Desacetilasas/genética , Humanos , Modelos Moleculares , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Oxadiazoles/química , Relación Estructura-Actividad , Zinc/metabolismoRESUMEN
The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.
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Inhibidores Enzimáticos/farmacología , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Secuencia de Aminoácidos , Animales , Biocatálisis/efectos de los fármacos , Dominio Catalítico , Células Cultivadas , Inhibidores Enzimáticos/metabolismo , Evolución Molecular , Histonas/química , Histonas/metabolismo , Humanos , Concentración 50 Inhibidora , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/clasificación , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Macrófagos/enzimología , Macrófagos/metabolismo , Metilación/efectos de los fármacos , Ratones , Modelos Moleculares , Especificidad por Sustrato , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
It has been postulated that the G protein-coupled receptor, GPR55, is a third cannabinoid receptor. Given that the ligands at the CB(1) and CB(2) receptors are effective analgesic and anti-inflammatory agents, the role of GPR55 in hyperalgesia associated with inflammatory and neuropathic pain has been investigated. As there are no well-validated GPR55 tool compounds, a GPR55 knockout (GPR55(-/-)) mouse line was generated and fully backcrossed onto the C57BL/6 strain. General phenotypic analysis of GPR55(-/-) mice revealed no obvious primary differences, compared with wild-type (GPR55(+/+)) littermates. GPR55(-/-) mice were then tested in the models of adjuvant-induced inflammation and partial nerve ligation. Following intraplantar administration of Freund's complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in GPR55(-/-) mice up to 14 days post-injection. Cytokine profiling experiments showed that at 14 days post-FCA injection there were increased levels of IL-4, IL-10, IFN gamma and GM-CSF in paws from the FCA-injected GPR55(-/-) mice when compared with the FCA-injected GPR55(+/+) mice. This suggests that GPR55 signalling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55(-/-) mice. In the model of neuropathic hypersensitivity, GPR55(-/-) mice also failed to develop mechanical hyperalgesia up to 28 days post-ligation. These data clearly suggest that the manipulation of GPR55 may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.
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Hiperalgesia/metabolismo , Mediadores de Inflamación/fisiología , Neuralgia/metabolismo , Receptores de Cannabinoides/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Hiperalgesia/genética , Inflamación/genética , Inflamación/metabolismo , Inflamación/fisiopatología , Mediadores de Inflamación/metabolismo , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Neuralgia/genética , Dimensión del Dolor/métodos , Estimulación Física/métodos , Receptores de Cannabinoides/deficiencia , Receptores de Cannabinoides/genéticaRESUMEN
BACKGROUND: Alzheimer's disease is characterized by the accumulation of neuritic plaques, containing activated microglia and beta-amyloid peptides (Abeta). Fibrillar Abeta can activate microglia, resulting in production of toxic and inflammatory mediators like hydrogen peroxide, nitric oxide, and cytokines. We have recently found that microglial proliferation is regulated by hydrogen peroxide derived from NADPH oxidase. Thus, in this study, we investigated whether Abeta can stimulate microglial proliferation and cytokine production via activation of NADPH oxidase to produce hydrogen peroxide. METHODS: Primary mixed glial cultures were prepared from the cerebral cortices of 7-day-old Wistar rats. At confluency, microglial cells were isolated by tapping, replated, and treated either with or without Abeta. Hydrogen peroxide production by cells was measured with Amplex Red and peroxidase. Microglial proliferation was assessed under a microscope 0, 24 and 48 hours after plating. TNF-alpha and IL-1beta levels in the culture medium were assessed by ELISA. RESULTS: We found that 1 muM fibrillar (but not soluble) Abeta1-40 peptide induced microglial proliferation and caused release of hydrogen peroxide, TNF-alpha and IL-1beta from microglial cells. Proliferation was prevented by the NADPH oxidase inhibitor apocynin (10 microM), by the hydrogen peroxide-degrading enzyme catalase (60 U/ml), and by its mimetics EUK-8 and EUK-134 (20 microM); as well as by an antibody against TNF-alpha and by a soluble TNF receptor inhibitor. Production of TNF-alpha and IL-1beta, measured after 24 hours of Abeta treatment, was also prevented by apocynin, catalase and EUKs, but the early release (measured after 1 hour of Abeta treatment) of TNF-alpha was insensitive to apocynin or catalase. CONCLUSION: These results indicate that Abeta1-40-induced microglial proliferation is mediated both by microglial release of TNF-alpha and production of hydrogen peroxide from NADPH oxidase. This suggests that TNF-alpha and NADPH oxidase, and its products, are potential targets to prevent Abeta-induced inflammatory neurodegeneration.
RESUMEN
Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1beta or TNF-alpha. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1beta or TNF-alpha, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1beta and TNF-alpha rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1beta or TNF-alpha is mediated by hydrogen peroxide from NADPH oxidase.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Microglía/citología , Microglía/metabolismo , NADPH Oxidasas/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/farmacología , Interleucina-1/metabolismo , Interleucina-1/farmacología , Microglía/efectos de los fármacos , Microglía/inmunología , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Inflammation-activated glia are seen in many CNS pathologies and may kill neurons through the release of cytotoxic mediators, such as nitric oxide from inducible NO synthase (iNOS), and possibly superoxide from NADPH oxidase (NOX). We set out to determine the relative role of these species in inducing neuronal death, and to test the dual-key hypothesis that the production of both species simultaneously is required for significant neuronal death. METHODS: Primary co-cultures of cerebellar granule neurons and glia from rats were used to investigate the effect of NO (from iNOS, following lipopolysaccharide (LPS) and/or cytokine addition) or superoxide/hydrogen peroxide (from NOX, following phorbol 12-myristate 13-acetate (PMA), ATP analogue (BzATP), interleukin-1beta (IL-1beta) or arachidonic acid (AA) addition) on neuronal survival. RESULTS: Induction of glial iNOS caused little neuronal death. Similarly, activation of NOX alone resulted in little or no neuronal death. However, if NOX was activated (by PMA or BzATP) in the presence of iNOS (induced by LPS and interferon-gamma) then substantial delayed neuronal death occurred over 48 hours, which was prevented by inhibitors of iNOS (1400W), NOX (apocynin) or a peroxynitrite decomposer (FeTPPS). Neurons and glia were also found to stain positive for nitrotyrosine (a putative marker of peroxynitrite) only when both iNOS and NOX were simultaneously active. If NOX was activated by weak stimulators (IL-1beta, AA or the fibrillogenic prion peptide PrP106-126) in the presence of iNOS, it caused microglial proliferation and delayed neurodegeneration, which was prevented by iNOS or NOX inhibitors, a peroxynitrite decomposer or a NMDA-receptor antagonist (MK-801). CONCLUSION: These results suggest a dual-key mechanism, whereby glial iNOS or microglial NOX activation alone is relatively benign, but if activated simultaneously are synergistic in killing neurons, through generating peroxynitrite. This mechanism may mediate inflammatory neurodegeneration in response to cytokines, bacteria, ATP, arachidonate and pathological prions, in which case neurons may be protected by iNOS or NOX inhibitors, or scavengers of NO, superoxide or peroxynitrite.
RESUMEN
Inflammatory-activated glia are seen in numerous central nervous system (CNS) pathologies and can kill nearby neurons through the release of cytotoxic mediators. Glia, when activated, can express the inducible isoform of nitric oxide synthase (iNOS) producing high levels of nitric oxide (NO), which can kill neurons in certain conditions. We show, however, that inflammatory activation of glia in a mature culture of cerebellar granule neurons and glia causes little or no neuronal death under normal (21%) oxygen conditions. Similarly, hypoxia (2% oxygen) or low levels of an NO donor (100 microM DETA/NO) caused little or no neuronal death in nonactivated cultures. If inflammatory activation of glia or addition of NO donor was combined with hypoxia, however, extensive neuronal death occurred. Death in both cases was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker MK-801, implying that death was mediated by the glutamate receptor. Low levels of NO were found to increase the apparent K(M) of cellular oxygen consumption for oxygen, probably due to NO-induced inhibition of mitochondrial respiration, in competition with oxygen, at cytochrome oxidase. Necrotic death, induced by hypoxia plus DETA/NO, was increased further by deoxyglucose, an inhibitor of glycolysis, suggesting that necrosis was mediated by energy depletion. Hypoxia was found to be a potent stimulator of microglia proliferation, but this proliferation was not significant in inflammatory-activated cultures. These results suggest that low levels of NO can induce neuronal death under hypoxic conditions, mediated by glutamate after NO inhibition of respiration in competition with oxygen. Brain inflammation can thus sensitize to hypoxia-induced death, which may be important in pathologies such as stroke, neurodegeneration, and brain aging.