RESUMEN
Inflammageing leads to uncontrolled leukocyte trafficking in response to inflammatory insults. Here, we used a zymosan-induced peritonitis mouse model on inflammation to investigate the role of the PEPITEM pathway on leukocyte migration in ageing. We then analysed whether PEPITEM could modulate leukocyte migration in older adults. We observed a loss of functionality in the PEPITEM pathway, which normally controls leukocyte trafficking in response to inflammation, in older adults and aged mice and show that this can be rescued by supplementation with PEPITEM. Thus, leading to the exciting possibility that PEPITEM supplementation may represent a potential pre-habilitation geroprotective agent to rejuvenate immune functions.
RESUMEN
The existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity, regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength. Simultaneously, PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin, which sequesters RANKL, thereby limiting osteoclast activity and bone resorption. In disease models, PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus, PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover.
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Resorción Ósea , Osteoblastos , Osteogénesis , Animales , Humanos , Osteoblastos/metabolismo , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Ratones , Resorción Ósea/patología , Resorción Ósea/metabolismo , Anabolizantes/farmacología , Anabolizantes/uso terapéutico , Remodelación Ósea/efectos de los fármacos , Osteoporosis/patología , Osteoporosis/metabolismo , Osteoporosis/tratamiento farmacológico , Ligando RANK/metabolismo , Osteoclastos/metabolismo , Osteoclastos/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Osteoprotegerina/metabolismo , Femenino , Transducción de Señal/efectos de los fármacos , Péptidos/farmacología , Masculino , Ratones Endogámicos C57BL , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patologíaRESUMEN
Leukocyte trafficking shows strong diurnal rhythmicity and is tightly regulated by circadian rhythms. As we age, leukocyte trafficking becomes dysregulated, contributing to the increased systemic, low-grade, chronic inflammation observed in older adults. Ageing is also associated with diminished circadian outputs and a dysregulation of the circadian rhythm. Despite this, there is little evidence to show the direct impact of age-associated dampening of circadian rhythms on the dysregulation of leukocyte trafficking. Here, we review the core mammalian circadian clock machinery and discuss the changes that occur in this biological system in ageing. In particular, we focus on the changes that occur to leukocyte trafficking rhythmicity with increasing age and consider how this impacts inflammation and the development of immune-mediated inflammatory disorders (IMIDs). We aim to encourage future ageing biology research to include a circadian approach in order to fully elucidate whether age-related circadian changes occur as a by-product of healthy ageing, or if they play a significant role in the development of IMIDs.
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Envejecimiento/inmunología , Quimiotaxis de Leucocito/inmunología , Ritmo Circadiano/inmunología , Inflamación/inmunología , Animales , HumanosRESUMEN
INTRODUCTION: The Endosialin/CD248/TEM1 protein is expressed in adipose tissue and its expression increases with obesity. Recently, genetic deletion of CD248 has been shown to protect mice against atherosclerosis on a high fat diet. OBJECTIVES: We investigated the effect of high fat diet feeding on visceral fat pads and circulating lipid profiles in CD248 knockout mice compared to controls. METHODS: From 10 weeks old, CD248-/- and +/+ mice were fed either chow (normal) diet or a high fat diet for 13 weeks. After 13 weeks the metabolic profiles and relative quantities of circulating lipid species were assessed using ultra high performance liquid chromatography-quadrupole time-of flight mass spectrometry (UHPLC-MS) with high resolution accurate mass (HRAM) capability. RESULTS: We demonstrate a specific reduction in the size of the perirenal fat pad in CD248-/- mice compared to CD248+/+, despite similar food intake. More strikingly, we identify significant, diet-dependent differences in the serum metabolic phenotypes of CD248 null compared to age and sex-matched wildtype control mice. Generalised protection from HFD-induced lipid accumulation was observed in CD248 null mice compared to wildtype, with particular reduction noted in the lysophosphatidylcholines, phosphatidylcholines, cholesterol and carnitine. CONCLUSIONS: Overall these results show a clear and protective metabolic consequence of CD248 deletion in mice, implicating CD248 in lipid metabolism or trafficking and opening new avenues for further investigation using anti-CD248 targeting agents.
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Antígenos CD/genética , Antígenos CD/metabolismo , Cromatografía Liquida , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Espectrometría de Masas en Tándem , Tejido Adiposo/metabolismo , Animales , Antígenos de Neoplasias , Carnitina/metabolismo , Colesterol , Cromatografía Líquida de Alta Presión , Dieta Alta en Grasa , Femenino , Grasa Intraabdominal/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Obesidad/metabolismo , Fosfatidilcolinas/metabolismo , TranscriptomaRESUMEN
The COVID-19 pandemic is an unprecedented healthcare emergency causing mortality and illness across the world. Although primarily affecting the lungs, the SARS-CoV-2 virus also affects the cardiovascular system. In addition to cardiac effects, e.g. myocarditis, arrhythmias, and myocardial damage, the vasculature is affected in COVID-19, both directly by the SARS-CoV-2 virus, and indirectly as a result of a systemic inflammatory cytokine storm. This includes the role of the vascular endothelium in the recruitment of inflammatory leucocytes where they contribute to tissue damage and cytokine release, which are key drivers of acute respiratory distress syndrome (ARDS), in disseminated intravascular coagulation, and cardiovascular complications in COVID-19. There is also evidence linking endothelial cells (ECs) to SARS-CoV-2 infection including: (i) the expression and function of its receptor angiotensin-converting enzyme 2 (ACE2) in the vasculature; (ii) the prevalence of a Kawasaki disease-like syndrome (vasculitis) in COVID-19; and (iii) evidence of EC infection with SARS-CoV-2 in patients with fatal COVID-19. Here, the Working Group on Atherosclerosis and Vascular Biology together with the Council of Basic Cardiovascular Science of the European Society of Cardiology provide a Position Statement on the importance of the endothelium in the underlying pathophysiology behind the clinical presentation in COVID-19 and identify key questions for future research to address. We propose that endothelial biomarkers and tests of function (e.g. flow-mediated dilatation) should be evaluated for their usefulness in the risk stratification of COVID-19 patients. A better understanding of the effects of SARS-CoV-2 on endothelial biology in both the micro- and macrovasculature is required, and endothelial function testing should be considered in the follow-up of convalescent COVID-19 patients for early detection of long-term cardiovascular complications.
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COVID-19/virología , Enfermedades Cardiovasculares/virología , Endotelio Vascular/virología , SARS-CoV-2/patogenicidad , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/fisiopatología , Fármacos Cardiovasculares/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Citocinas/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Interacciones Huésped-Patógeno , Humanos , Mediadores de Inflamación/metabolismo , Pronóstico , Medición de Riesgo , Factores de Riesgo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Internalización del Virus , Tratamiento Farmacológico de COVID-19RESUMEN
Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-ß1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-ß1-stimulated cremaster muscle, while in the ApoE-/- model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic.
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Plaquetas , Vesículas Extracelulares , Animales , Células Endoteliales , Humanos , Inflamación , Ratones , Monocitos , Complejo GPIb-IX de Glicoproteína PlaquetariaRESUMEN
Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.
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Agregación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Polisacáridos/farmacología , Receptores de Superficie Celular/sangre , Animales , Biopolímeros/farmacología , Calcio/sangre , Humanos , Ratones , Ratones Noqueados , Quinasa Syk/sangreRESUMEN
Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.
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Calcio/metabolismo , Daño por Reperfusión Miocárdica/genética , Proteína ORAI1/genética , Tetraspaninas/genética , Trombosis de la Vena/genética , Factor de von Willebrand/genética , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Pollos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Histamina/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Transporte Iónico/efectos de los fármacos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteína ORAI1/metabolismo , Transducción de Señal , Tetraspaninas/metabolismo , Trombina/farmacología , Trombosis de la Vena/metabolismo , Trombosis de la Vena/patología , Factor de von Willebrand/metabolismoRESUMEN
The Src family kinases (SFK) are a group of signalling molecules with important regulatory functions in inflammation and haemostasis. Leucocytes and platelets express multiple isoforms of the SFKs. Previous studies used broad-spectrum pharmacological inhibitors, or murine models deficient in multiple SFK isoforms, to demonstrate the functional consequences of deficiencies in SFK signalling. Here, we hypothesized that individual SFK operate in a non-redundant fashion in the thrombo-inflammatory recruitment of monocyte during atherosclerosis. Using in vitro adhesion assays and single SFK knockout mice crossed with the ApoE-/- model of atherosclerosis, we find that SFK signalling regulates platelet-dependent recruitment of monocytes. However, loss of a single SFK, Fgr or Lyn, reduced platelet-mediated monocyte recruitment in vitro. This translated into a significant reduction in the burden of atherosclerotic disease in Fgr-/- /ApoE-/- or Lyn-/- /ApoE-/- animals. SFK signalling is not redundant in thrombo-inflammatory vascular disease and individual SFK may represent targets for therapeutic intervention.
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Apolipoproteínas E/genética , Enfermedad de la Arteria Coronaria/genética , Monocitos/metabolismo , Proteínas Proto-Oncogénicas/genética , Familia-src Quinasas/genética , Animales , Aorta/metabolismo , Aorta/patología , Apolipoproteínas E/deficiencia , Adhesión Celular , Enfermedad de la Arteria Coronaria/etiología , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Dieta Alta en Grasa/efectos adversos , Femenino , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/patología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas/deficiencia , Transducción de Señal , Familia-src Quinasas/deficienciaRESUMEN
OBJECTIVE/BACKGROUND: In a pilot study, a relationship between abdominal aortic aneurysm (AAA) diameter and serum interleukin (IL)-1α levels was reported, and that endothelial cell (EC) activation in vitro in response to serum from patients with AAA was blocked by anti-IL-1α antibodies. The aim of the present study was to further investigate the relationship between serum IL-1α and asymptomatic infrarenal AAA size, morphology, and growth rates. METHODS: Serum IL-1α was measured using enzyme linked immunosorbent assay in 101 patients with asymptomatic, infrarenal AAA and related to aneurysm size, morphology, and growth rates. RESULTS: IL-1α was measured in 101 patients. There was no statistically significant difference in mean age between men and women. IL-1α was detectable in 62.4% of patients; median IL-1α titre was 3.26 pg/mL. There was no statistically significant relationship between IL-1α and maximum AAA antero-posterior diameter as measured by ultrasound (p = .649), AAA morphology (aortic length [p = .394], sac [p = .369], and thrombus volume [p = .629]) as measured on computed tomography, absolute increase in AAA diameter (p = .214), or AAA growth rate (p = .230). CONCLUSION: IL-1α is detectable in the majority of patients with infrarenal AAA, but the cause and clinical significance of this novel observation remains unknown.
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Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/sangre , Aneurisma de la Aorta Abdominal/patología , Interleucina-1alfa/sangre , Anciano , Anciano de 80 o más Años , Aorta Abdominal/diagnóstico por imagen , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Aortografía/métodos , Enfermedades Asintomáticas , Biomarcadores/sangre , Angiografía por Tomografía Computarizada , Dilatación Patológica , Progresión de la Enfermedad , Femenino , Humanos , Masculino , UltrasonografíaRESUMEN
The recruitment of blood leukocytes across the endothelium to sites of tissue infection is central to inflammation, but also promotes chronic inflammatory diseases. A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane molecular scissor that is implicated in leukocyte transmigration by proteolytically cleaving its endothelial substrates. These include VE-cadherin, a homotypic adhesion molecule that regulates endothelial barrier function, and transmembrane chemokines CX3CL1 and CXCL16, which have receptors on leukocytes. However, a definitive role for endothelial ADAM10 in transmigration of freshly isolated primary leukocytes under flow has not been demonstrated, and the relative importance of distinct ADAM10 substrates is unknown. Emerging evidence suggests that ADAM10 can be regarded as six different molecular scissors with different substrate specificities, depending on which of six TspanC8 tetraspanins it is associated with, but TspanC8s remain unstudied in leukocyte transmigration. In the current study, ADAM10 knockdown on primary HUVECs was found to impair transmigration of freshly isolated human peripheral blood T lymphocytes, but not neutrophils or B lymphocytes, in an in vitro flow assay. This impairment was due to delayed transmigration rather than a complete block, and was overcome in the presence of neutrophils. Transmigration of purified lymphocytes was dependent on ADAM10 regulation of VE-cadherin, but not CX3CL1 and CXCL16. Tspan5 and Tspan17, the two most closely related TspanC8s by sequence, were the only TspanC8s that regulated VE-cadherin expression and were required for lymphocyte transmigration. Therefore endothelial Tspan5- and Tspan17-ADAM10 complexes may regulate inflammation by maintaining normal VE-cadherin expression and promoting T lymphocyte transmigration.
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Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Antígenos CD/genética , Cadherinas/genética , Proteínas de la Membrana/metabolismo , Linfocitos T/fisiología , Tetraspaninas/metabolismo , Migración Transendotelial y Transepitelial , Proteína ADAM10/deficiencia , Proteína ADAM10/genética , Proteína ADAM10/inmunología , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/inmunología , Antígenos CD/metabolismo , Linfocitos B/inmunología , Linfocitos B/fisiología , Cadherinas/metabolismo , Células Cultivadas , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/inmunología , Quimiocina CXCL16 , Quimiocinas CXC/genética , Quimiocinas CXC/inmunología , Células Endoteliales/inmunología , Células Endoteliales/fisiología , Humanos , Inflamación/inmunología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Neutrófilos/inmunología , Neutrófilos/fisiología , Receptores Depuradores/genética , Receptores Depuradores/inmunología , Linfocitos T/inmunología , Tetraspaninas/genética , Tetraspaninas/inmunologíaRESUMEN
Two major monocyte subsets, CD14+CD16- (classical) and CD14+/dimCD16+ (nonclassical/intermediate), have been described. Each has different functions ascribed in its interactions with vascular endothelial cells (EC), including migration and promoting inflammation. Although monocyte subpopulations have been studied in isolated systems, their influence on EC and on the course of inflammation has been ignored. In this study, using unstimulated or cytokine-activated EC, we observed significant differences in the recruitment, migration, and reverse migration of human monocyte subsets. Associated with this, and based on their patterns of cytokine secretion, there was a difference in their capacity to activate EC and support the secondary recruitment of flowing neutrophils. High levels of TNF were detected in cocultures with nonclassical/intermediate monocytes, the blockade of which significantly reduced neutrophil recruitment. In contrast, classical monocytes secreted high levels of IL-6, the blockade of which resulted in increased neutrophil recruitment. When cocultures contained both monocyte subsets, or when conditioned supernatant from classical monocytes cocultures (IL-6hi) was added to nonclassical/intermediate monocyte cocultures (TNFhi), the activating effects of TNF were dramatically reduced, implying that when present, the anti-inflammatory activities of IL-6 were dominant over the proinflammatory activities of TNF. These changes in neutrophil recruitment could be explained by regulation of E-selectin on the cocultured EC. This study suggests that recruited human monocyte subsets trigger a regulatory pathway of cytokine-mediated signaling at the EC interface, and we propose that this is a mechanism for limiting the phlogistic activity of newly recruited monocytes.
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Quimiotaxis de Leucocito/inmunología , Células Endoteliales/inmunología , Inflamación/inmunología , Monocitos/inmunología , Transducción de Señal/inmunología , Separación Celular , Citometría de Flujo , Humanos , Interleucina-6/inmunología , Reacción en Cadena de la Polimerasa , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Fish oil supplementation is of great medical and public interest with epidemiological evidence of health benefits in humans, in particular by conferring protection against heart diseases. Its anti-inflammatory properties have also been reported. Initial results from short-lived mouse strains showed that fish oil can increase lifespan, affecting pathways like inflammation and oxidation thought to be involved in the regulation of aging. Could fish oil and its omega-3 fatty acids act as geroprotectors? Probably not. A new study by Strong et al. challenges the role for fish oil supplementation in aging. Using a large cohort of genetically heterogeneous mice in three sites, part of the Interventions Testing Program of the NIA, Strong et al. show that fish oil supplementation at either low or high dosages has no effect on the lifespan of male or female mice. Although it is still possible that fish oil supplementation has health benefits for specific age-related diseases, it does not appear to slow aging or have longevity benefits.
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Aceites de Pescado , Longevidad , Animales , Antioxidantes , Suplementos Dietéticos , Femenino , Inhibidores de Glicósido Hidrolasas , Humanos , Masculino , Ratones , Factor 2 Relacionado con NF-E2RESUMEN
The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin - the latter with and without rapamycin, and two drugs previously examined: 17-α-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-α-estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male-specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α-glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.
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Antioxidantes/farmacología , Estradiol/farmacología , Inhibidores de Glicósido Hidrolasas/farmacología , Longevidad/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , alfa-Glucosidasas/metabolismo , Acarbosa/farmacología , Animales , Medicamentos Herbarios Chinos/farmacología , Aceites de Pescado/farmacología , Fuerza de la Mano , Masculino , Masoprocol/farmacología , Metformina/farmacología , Ratones , Prueba de Desempeño de Rotación con Aceleración Constante , Sirolimus/farmacología , Análisis de Supervivencia , Ácido Ursodesoxicólico/farmacologíaRESUMEN
A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15 was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Plaquetas/metabolismo , Membrana Celular/metabolismo , Endotelio Vascular/metabolismo , Proteínas de la Membrana/metabolismo , Tetraspaninas/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Plaquetas/citología , Línea Celular , Membrana Celular/enzimología , Células Cultivadas , Endotelio Vascular/citología , Activación Enzimática , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Propiedades de Superficie , Tetraspanina 29/química , Tetraspanina 29/genética , Tetraspanina 29/metabolismo , Tetraspaninas/química , Tetraspaninas/genéticaRESUMEN
Thrombosis is a common, life-threatening consequence of systemic infection; however, the underlying mechanisms that drive the formation of infection-associated thrombi are poorly understood. Here, using a mouse model of systemic Salmonella Typhimurium infection, we determined that inflammation in tissues triggers thrombosis within vessels via ligation of C-type lectin-like receptor-2 (CLEC-2) on platelets by podoplanin exposed to the vasculature following breaching of the vessel wall. During infection, mice developed thrombi that persisted for weeks within the liver. Bacteria triggered but did not maintain this process, as thrombosis peaked at times when bacteremia was absent and bacteria in tissues were reduced by more than 90% from their peak levels. Thrombus development was triggered by an innate, TLR4-dependent inflammatory cascade that was independent of classical glycoprotein VI-mediated (GPVI-mediated) platelet activation. After infection, IFN-γ release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-γ, or depletion of monocytic-lineage cells or CLEC-2 on platelets markedly inhibited the process. Together, our data indicate that infection-driven thrombosis follows local inflammation and upregulation of podoplanin and platelet activation. The identification of this pathway offers potential therapeutic opportunities to control the devastating consequences of infection-driven thrombosis without increasing the risk of bleeding.
Asunto(s)
Plaquetas/metabolismo , Lectinas Tipo C/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Trombosis/metabolismo , Animales , Plaquetas/patología , Interferón gamma/genética , Interferón gamma/metabolismo , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Infecciones por Salmonella/complicaciones , Infecciones por Salmonella/genética , Infecciones por Salmonella/patología , Trombosis/etiología , Trombosis/genética , Trombosis/patología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3 zeta delta (14.3.3.ζδ) protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin-induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type 1 diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to that of healthy age-matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Control of patient T cell trafficking is re-established by treatment with exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic ischemia-reperfusion injury, Salmonella infection, uveitis and Sjögren's syndrome, PEPITEM reduced T cell recruitment into inflamed tissues.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos B/citología , Regulación de la Expresión Génica , Homeostasis , Inflamación/inmunología , Linfocitos T/citología , Proteínas 14-3-3/metabolismo , Adiponectina/metabolismo , Adulto , Factores de Edad , Anciano , Envejecimiento , Animales , Artritis Reumatoide/sangre , Cadherinas/metabolismo , Adhesión Celular , Movimiento Celular , Diabetes Mellitus Tipo 1/sangre , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lisofosfolípidos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Péptidos/química , Receptores de Adiponectina/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Adulto JovenRESUMEN
Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro "vascular" models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment. Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNFα) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy. In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNFα-stimulated endothelium. Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.
Asunto(s)
Comunicación Celular/fisiología , Leucocitos/citología , Células del Estroma/citología , Humanos , Inflamación/sangre , Inflamación/patología , Células Madre Mesenquimatosas/citologíaRESUMEN
Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy.