RESUMEN
Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.
Asunto(s)
Adenosina , Astrocitos , Encéfalo , Metabolismo Energético , Neuronas , Transducción de Señal , Animales , Femenino , Masculino , Ratones , Ratas , Adenosina/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Encéfalo/citología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glucosa/metabolismo , Hipocampo/metabolismo , Hipocampo/citología , Ácido Láctico/metabolismo , Ratones Endogámicos C57BL , Plasticidad Neuronal , Neuronas/metabolismo , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/efectos de los fármacos , Receptor de Adenosina A2B/genética , Receptor de Adenosina A2B/metabolismo , Reconocimiento en Psicología/fisiología , Sueño/genética , Sueño/fisiología , Sinapsis/metabolismoRESUMEN
Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline.
Asunto(s)
Vías Auditivas/fisiopatología , Disfunción Cognitiva/metabolismo , Eritrocitos/metabolismo , Hipoxia/metabolismo , Receptor de Adenosina A2B/metabolismo , 2,3-Difosfoglicerato/metabolismo , Envejecimiento/patología , Animales , Bisfosfoglicerato Mutasa/genética , Bisfosfoglicerato Mutasa/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Cóclea/fisiopatología , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/genética , Disfunción Cognitiva/fisiopatología , Activación Enzimática , Eliminación de Gen , Glucólisis , Hipoxia/complicaciones , Hipoxia/genética , Hipoxia/fisiopatología , Inflamación/complicaciones , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Microglía/metabolismo , Microglía/patología , Receptor de Adenosina A2B/deficienciaRESUMEN
The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.
Asunto(s)
Envejecimiento/metabolismo , Obesidad/metabolismo , Receptor de Adenosina A2B/metabolismo , Adolescente , Adulto , Animales , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Receptor de Adenosina A2B/deficiencia , Transducción de Señal , Adulto JovenRESUMEN
Elsa N. Bou Ghanem works in the field of innate immune senescence, inflammation, and host defense. In this mSphere of Influence article, she reflects on how "Adenosine A2B receptor deficiency promotes host defenses against Gram-negative bacterial pneumonia" by Barletta et al. (K. E. Barletta, R. E. Cagnina, M. D. Burdick, J. Linden, and B. Mehrad, Am J Respir Crit Care Med 186:1044-1050, 2012, https://doi.org/10.1164/rccm.201204-0622OC) impacted her own work examining the role of the extracellular adenosine pathway in neutrophil responses and host defense against pneumococcal pneumonia.
Asunto(s)
Interacciones Huésped-Patógeno , Neutrófilos/inmunología , Neumonía Bacteriana/inmunología , Neumonía Neumocócica/inmunología , Receptor de Adenosina A2B/inmunología , Animales , Pulmón/microbiología , Ratones , Receptor de Adenosina A2B/deficiencia , Streptococcus pneumoniae/inmunologíaRESUMEN
Although the cardioprotective effect of adenosine is undisputed, the role of the adenosine A2b receptor (A2bR) in ischemic cardiac remodeling is not defined. In this study we aimed to unravel the role A2bR plays in modulating the immune response and the healing mechanisms after myocardial infarction. Genetic and pharmacological (PSB603) inactivation of A2bR as well as activation of A2bR with BAY60-6583 does not alter cardiac remodeling of the infarcted (50-min left anterior descending artery occlusion/reperfusion) murine heart. Flow cytometry of immune cell subsets identified a significant increase in B cells, NK cells, CD8 and CD4 T cells, as well as FoxP3-expressing regulatory T cells in the injured heart in A2bR-deficient mice. Analysis of T-cell function revealed that expression and secretion of interleukin (IL)-2, interferon (IFN)γ, and tumor necrosis factor (TNF)α by T cells is under A2bR control. In addition, we found substantial cellular heterogeneity in the response of immune cells and cardiomyocytes to A2bR deficiency: while in the absence of A2bR, expression of IL-6 was greatly reduced in cardiomyocytes and immune cells except T cells, and expression of IL-1ß was strongly reduced in cardiomyocytes, granulocytes, and B cells as determined by quantitative PCR. Our findings indicate that A2bR signaling in the ischemic heart triggers substantial changes in cardiac immune cell composition of the lymphoid lineage and induces a profound cell type-specific downregulation of IL-6 and IL-1ß. This suggests the presence of a targetable adenosine-A2bR-IL-6-axis triggered by adenosine formed by the ischemic heart. NEW & NOTEWORTHY Genetic deletion and pharmacological inactivation/activation of A2bR does not alter cardiac remodeling after MI but is associated by compensatory upregulation of various pro- and anti-inflammatory immune cell subsets (B cells, NK cells, CD8 and CD4 T cells, regulatory T cells). In the inflamed heart, A2bR modulates the expression of IL-2, IFNγ, TNFα in T cells and of IL-6 in cardiomyocytes, monocytes, granulocytes and B cells. This suggests an important adenosine-IL-6 axis, which is controlled by A2bR via local adenosine.
Asunto(s)
Interleucina-6/metabolismo , Linfocitos/metabolismo , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Receptor de Adenosina A2B/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Modelos Animales de Enfermedad , Femenino , Interleucina-1beta/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Linfocitos/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/inmunología , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/patología , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/genética , Transducción de Señal , Remodelación VentricularRESUMEN
Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.
Asunto(s)
Adenosina/sangre , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/enzimología , Eritrocitos Anormales/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/sangre , Receptor de Adenosina A2B/sangre , Adenosina Desaminasa/sangre , Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Adenosina-5'-(N-etilcarboxamida)/farmacología , Agammaglobulinemia/sangre , Agammaglobulinemia/enzimología , Agammaglobulinemia/genética , Anemia de Células Falciformes/genética , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/sangre , Eritrocitos/efectos de los fármacos , Eritrocitos/enzimología , Eritrocitos/metabolismo , Eritrocitos Anormales/efectos de los fármacos , Eritrocitos Anormales/enzimología , Hemoglobina Falciforme/genética , Hemoglobina Falciforme/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/genética , Inmunodeficiencia Combinada Grave/sangre , Inmunodeficiencia Combinada Grave/enzimología , Inmunodeficiencia Combinada Grave/genética , Transducción de SeñalRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a lethal, fibroproliferative disease. Pulmonary hypertension (PH) can develop secondary to IPF and increase mortality. Alternatively, activated macrophages (AAMs) contribute to the pathogenesis of both IPF and PH. Here we hypothesized that adenosine signaling through the ADORA2B on AAMs impacts the progression of these disorders and that conditional deletion of ADORA2B on myeloid cells would have a beneficial effect in a model of these diseases. Conditional knockout mice lacking ADORA2B on myeloid cells (Adora2B(f/f)-LysM(Cre)) were exposed to the fibrotic agent bleomycin (BLM; 0.035 U/g body weight, i.p.). At 14, 17, 21, 25, or 33 d after exposure, SpO2, bronchoalveolar lavage fluid (BALF), and histologic analyses were performed. On day 33, lung function and cardiovascular analyses were determined. Markers for AAM and mediators of fibrosis and PH were assessed. Adora2B(f/f)-LysM(Cre) mice presented with attenuated fibrosis, improved lung function, and no evidence of PH compared with control mice exposed to BLM. These findings were accompanied by reduced expression of CD206 and arginase-1, markers for AAMs. A 10-fold reduction in IL-6 and a 5-fold decrease in hyaluronan, both linked to lung fibrosis and PH, were also observed. These data suggest that activation of the ADORA2B on macrophages plays an active role in the pathogenesis of lung fibrosis and PH.
Asunto(s)
Hipertensión Pulmonar/etiología , Fibrosis Pulmonar Idiopática/etiología , Receptor de Adenosina A2B/deficiencia , Animales , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Humanos , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/fisiopatología , Activación de Macrófagos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/fisiología , Receptor de Adenosina A2B/genética , Receptor de Adenosina A2B/fisiologíaRESUMEN
The type 2 immune response evoked by intestinal nematode parasites contributes to worm expulsion and tolerance to associated tissue damage. We investigated whether this host response is affected by blocking signaling by the putative endogenous danger signal adenosine, which can be released during inflammation and host cell damage. Specific blockade of the A2B adenosine receptor (A2BAR) inhibited worm elimination and the development of innate and adaptive components of the type 2 primary and memory response. Infected mice lacking A2BAR exhibited decreased M2 macrophage and eosinophil recruitment and reduced IL-4 and IL-13 cytokine production. Additionally, shortly after infection, upregulation of the alarmin IL-33, which drives type 2 immunity, and activation of innate lymphoid type 2 (ILC2) cells was inhibited, while exogenous IL-33 restored ILC2 cell activation and type 2 cytokine expression. Thus, adenosine acts as a danger-associated molecular pattern (DAMP) that initiates helminth-induced type 2 immune responses through A2BAR.
Asunto(s)
Adenosina/metabolismo , Nematodos/inmunología , Nematospiroides dubius/inmunología , Receptor de Adenosina A2B/metabolismo , Infecciones por Strongylida/inmunología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Adenosina A2B/deficienciaRESUMEN
Adenosine is a key endogenous signaling molecule that regulates immune responses. A(2B) adenosine receptor (AR) is a relatively low-affinity receptor for adenosine, and the activation of A(2B)AR is believed to require pathological level of adenosine that is associated with ischemia, inflammation, trauma, or other types of stress. The role of A(2B)AR in the pathogenesis of multiple sclerosis (MS) is still unclear. In this study, we discovered that A(2B)AR was upregulated both in the peripheral blood leukocytes of MS patients and the peripheral lymphoid tissues of experimental autoimmune encephalomyelitis (EAE) mice. A(2B)AR-specific antagonists, CVT-6883 and MRS-1754, alleviated the clinical symptoms of EAE and protected the CNS from immune damage. A(2B)AR-knockout mice also developed less severe EAE. Further study indicated that blocking or deleting A(2B)AR inhibited Th17 cell differentiation by blocking IL-6 production from APCs such as dendritic cells. In dendritic cells, A(2B)AR was also upregulated during the development of EAE. CVT-6883 and genetic deletion of A(2B)AR significantly reduced adenosine-mediated IL-6 production. The phospholipase Cß-protein kinase C and p38 MAPK pathways were found to be involved in the A(2B)AR-mediated IL-6 production. Our findings not only revealed the pathological role of A(2B)AR in EAE, but also suggested that this receptor might be a new therapeutic target for the development of anti-MS drugs.
Asunto(s)
Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-6/antagonistas & inhibidores , Receptor de Adenosina A2B/metabolismo , Células Th17/inmunología , Adulto , Animales , Células Cultivadas , Técnicas de Cocultivo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/prevención & control , Femenino , Inhibidores de Crecimiento/antagonistas & inhibidores , Inhibidores de Crecimiento/fisiología , Humanos , Interleucina-6/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptor de Adenosina A2B/deficiencia , Células Th17/metabolismo , Células Th17/patología , Regulación hacia Arriba/inmunologíaRESUMEN
BACKGROUND: Liver ischemia-reperfusion injury (IRI) is a known risk factor for the postoperative outcome of patients undergoing liver surgery/transplantation. Attempts to protect from organ damage require multidisciplinary strategies and are of emerging interest in view of patients with higher age and American Society of Anesthesiology status. Ischemic preconditioning has been successfully applied to prevent from IRI during liver resection/transplantation. Because even short periods of ischemia during preconditioning inevitably lead to hypoxia and formation of anti-inflammatory/cytoprotective acting adenosine, we reasoned that short nonischemic hypoxia also protects against hepatic IRI. METHODS: Mice underwent hypoxic preconditioning (HPC) by breathing 10% oxygen for 10 min followed by 10 min of 21% oxygen before left liver lobe ischemia (45 min) and reperfusion (4 hr). The interactions of hypoxiaâadenosineâadenosine receptors were tested by pharmacologic antagonism at adenosine receptor (AR) sites in wild-type mice and in mice with genetic deletions at the A1, A2A, A2B, and A3 ARs. Hepatocellular damage, inflammation, and metabolic effects were quantified by enzyme activities, cytokines, liver myeloperoxidase, blood adenosine, and tissue AMP, respectively. RESULTS: Hepatoprotection by HPC was significant in wild-type and A1, A2A, and A3 AR knockout mice as quantified by lower alanine aminotransferase serum activities, cytokine levels, histologic damage scores, tissue myeloperoxidase concentrations, and preserved AMP concentrations. Protection by HPC was blunted in mice pretreated with the A2B AR antagonist MRS1754 or in A2B AR knockout mice. CONCLUSIONS: Because liver protective effects of HPC are negated when the A2B receptor is nonfunctional, the hypoxiaâadenosineâA2B receptor pathway plays a critical role in the prevention of warm IRI in vivo. Hypoxic activation of this pathway warrants use of selective A2B AR agonists or even intermittent hypoxia (e.g., in deceased organ donors) to protect from liver IRI.
Asunto(s)
Hipoxia/fisiopatología , Precondicionamiento Isquémico , Hígado/irrigación sanguínea , Receptor de Adenosina A2B/fisiología , Daño por Reperfusión/prevención & control , Isquemia Tibia , Acetamidas/farmacología , Adenosina/fisiología , Animales , Hepatocitos/patología , Hepatocitos/fisiología , Hígado/patología , Hígado/fisiopatología , Trasplante de Hígado/patología , Trasplante de Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Purinas/farmacología , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/efectos de los fármacos , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Transducción de Señal/fisiologíaRESUMEN
Renal ischemia is among the leading causes of acute kidney injury (AKI). Previous studies have shown that extracellular adenosine is a prominent tissue-protective cue elicited during ischemia, including signaling events through the adenosine receptor 2b (Adora2b). To investigate the functional role of Adora2b signaling in cytokine-mediated inflammatory pathways, we screened wild-type and Adora2b-deficient mice undergoing renal ischemia for expression of a range of inflammatory cytokines. These studies demonstrated a selective and robust increase of TNF-α levels in Adora2b-deficient mice following renal ischemia and reperfusion. Based on these findings, we next sought to understand the contribution of TNF-α on ischemic AKI through a combination of loss- and gain-of-function studies. Loss of TNF-α, through either Ab blockade or study of Tnf-α-deficient animals, resulted in significantly attenuated tissue injury and improved kidney function following renal ischemia. Conversely, transgenic mice with overexpression of TNF-α had significantly pronounced susceptibility to AKI. Furthermore, neutrophil depletion or reconstitution of Adora2b(-/-) mice with Tnf-α-deficient neutrophils rescued their phenotype. In total, these data demonstrate a critical role of adenosine signaling in constraining neutrophil-dependent production of TNF-α and implicate therapies targeting TNF-α in the treatment of ischemic AKI.
Asunto(s)
Lesión Renal Aguda/inmunología , Lesión Renal Aguda/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptor de Adenosina A2B/fisiología , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Lesión Renal Aguda/genética , Animales , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Isquemia/genética , Isquemia/inmunología , Isquemia/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/patología , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/genética , Reperfusión/métodos , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/deficienciaRESUMEN
RATIONALE: Activation of the adenosine A(2B) receptor (A(2B)R) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. OBJECTIVES: To test the hypothesis that absence of adenosine A(2B) receptor signaling promotes host defense against bacterial pneumonia. METHODS: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A(2B)R. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. MEASUREMENTS AND MAIN RESULTS: A(2B)R(-/-) mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow-derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A(2B)R(-/-) and wild-type mice, but A(2B)R(-/-) neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A(2B)R(-/-) mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. CONCLUSIONS: These data suggest that the absence of A(2B)R signaling enhances antimicrobial activity in gram-negative bacterial pneumonia.
Asunto(s)
Infecciones por Klebsiella/inmunología , Klebsiella pneumoniae , Neutrófilos/inmunología , Neumonía Bacteriana/inmunología , Receptor de Adenosina A2B/deficiencia , Animales , Células de la Médula Ósea/metabolismo , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/metabolismo , Neumonía Bacteriana/metabolismo , Receptor de Adenosina A2B/inmunología , Transducción de SeñalRESUMEN
Asthma is a chronic condition with high morbidity and healthcare costs, and cockroach allergens are an established cause of urban pediatric asthma. A better understanding of cell types involved in promoting lung inflammation could provide new targets for the treatment of chronic pulmonary disease. Because of its role in regulating myeloid cell-dependent inflammatory processes, we examined A(2B) R expression by myeloid cells in a cockroach allergen model of murine asthma-like pulmonary inflammation. Both systemic and myeloid tissue-specific A(2B) R deletion significantly decreased pulmonary inflammatory cell recruitment, airway mucin production, and proinflammatory cytokine secretion after final allergen challenge in sensitized mice. A(2B) R deficiency resulted in a dramatic reduction on Th2-type airways responses with decreased pulmonary eosinophilia without augmenting neutrophilia, and decreased lung IL-4, IL-5, and IL-13 production. Chemokine analysis demonstrated that eotaxin 1 and 2 secretion in response to repeated allergen challenge is myeloid cell A(2B) R dependent. In contrast, there were no differences in the levels of the CXC chemokines keratinocyte-derived chemokine and MIP-2 in the myeloid cell A(2B) R-deficient mice, strengthening A(2B) R involvement in the development of Th2-type airways inflammation. Proinflammatory TNF-α, IFN-γ, and IL-17 secretion were also reduced in systemic and myeloid tissue-specific A(2B) R deletion mouse lines. Our results demonstrate Th2-type predominance for A(2B) R expression by myeloid cells as a mechanism of development of asthma-like pulmonary inflammation.
Asunto(s)
Alérgenos/toxicidad , Mediadores de Inflamación/administración & dosificación , Células Mieloides/inmunología , Células Mieloides/patología , Receptor de Adenosina A2B/administración & dosificación , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/patología , Animales , Blattellidae/inmunología , Enfermedad Crónica , Femenino , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/fisiología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Mieloides/metabolismo , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/fisiología , Hipersensibilidad Respiratoria/metabolismoRESUMEN
The nuclear factor kappa B (NFκB) pathway controls a variety of processes, including inflammation, and thus, the regulation of NFκB has been a continued focus of study. Here, we report a newly identified regulation of this pathway, involving direct binding of the transcription factor NFκB1 (the p105 subunit of NFκB) to the C-terminus of the A(2B) adenosine receptor (A(2B)AR), independent of ligand activation. Intriguingly, binding of A(2B)AR to specific sites on p105 prevents polyubiquitylation and degradation of p105 protein. Ectopic expression of the A(2B)AR increases p105 levels and inhibits NFκB activation, whereas p105 protein levels are reduced in cells from A(2B)AR-knockout mice. In accordance with the known regulation of expression of anti- and pro-inflammatory cytokines by p105, A(2B)AR-null mice generate less interleukin (IL)-10, and more IL-12 and tumor necrosis factor (TNF-α). Taken together, our results show that the A(2B)AR inhibits NFκB activation by physically interacting with p105, thereby blocking its polyubiquitylation and degradation. Our findings unveil a surprising function for the A(2B)AR, and provide a novel mechanistic insight into the control of the NFκB pathway and inflammation.
Asunto(s)
Inflamación/metabolismo , Inflamación/patología , Subunidad p50 de NF-kappa B/metabolismo , Receptor de Adenosina A2B/metabolismo , Animales , Citocinas/biosíntesis , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Poliubiquitina/metabolismo , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteolisis , Receptor de Adenosina A2B/química , Receptor de Adenosina A2B/deficiencia , Técnicas del Sistema de Dos Híbridos , UbiquitinaciónRESUMEN
Adenosine plays a role in physiological and pathological conditions, and A(2) adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A(2B)AR and its relationship to the A(2A)AR in coronary flow (CF) changes using A(2B) single-knockout (KO) and A(2A/2B) double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A(2A)KO and A(2B)KO and A(2A/2B)DKO mice. BAY 60-6583 (a selective A(2B) agonist) had no effect on CF in A(2B)KO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min(-1)·g(-1)). 5'-N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A(2B)KO mice (maximum of 34.6 ± 4.7 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Also, CGS-21680 (a selective A(2A) agonist) increased CF in A(2B)KO mice (maximum of 29 ± 1.9 ml·min(-1)·g(-1)) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min(-1)·g(-1)). SCH-58261 (an A(2A)-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A(2B)KO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min(-1)·g(-1)) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min(-1)·g(-1)). NECA did not induce any increase in CF in A(2A/2B)DKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min(-1)·g(-1)). Furthermore, the mitochondrial ATP-sensitive K(+) (K(ATP)) channel blocker 5-hydroxydecanoate had no effect on the NECA-induced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min(-1)·g(-1)), A(2A)KO (12.5 ± 2.3 ml·min(-1)·g(-1)), and A(2B)KO (16.2 ± 0.8 ml·min(-1)·g(-1)) mice was significantly blunted by the K(ATP) channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min(-1)·g(-1), respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min(-1)·g(-1)) and BAY 60-6583-induced (16.4 ± 1.60 ml·min(-1)·g(-1)) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min(-1)·g(-1), respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A(2A)ARs in A(2B)KO mice and demonstrates that both A(2A)ARs and A(2B)ARs induce CF changes through K(ATP) channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders.
Asunto(s)
Circulación Coronaria , Vasos Coronarios/metabolismo , Canales KATP/metabolismo , Receptor de Adenosina A2A/deficiencia , Receptor de Adenosina A2B/deficiencia , Vasodilatación , Animales , Arteriolas/metabolismo , Velocidad del Flujo Sanguíneo , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca , Canales KATP/antagonistas & inhibidores , Mesenterio/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Perfusión , Bloqueadores de los Canales de Potasio/farmacología , Agonistas del Receptor Purinérgico P1/farmacología , Antagonistas de Receptores Purinérgicos P1/farmacología , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2B/genética , Vasodilatación/efectos de los fármacos , Función Ventricular Izquierda , Presión VentricularRESUMEN
All four adenosine receptor subtypes have been shown to play a role in cardioprotection, and there is evidence that all four subtypes may be expressed in cardiomyocytes. There is also increasing evidence that optimal adenosine cardioprotection requires the activation of more than one receptor subtype. The purpose of this study was to determine whether adenosine A(2A) and/or A(2B) receptors modulate adenosine A(1) receptor-mediated cardioprotection. Isolated perfused hearts of wild-type (WT), A(2A) knockout (KO), and A(2B)KO mice, perfused at constant pressure and constant heart rate, underwent 30 min of global ischemia and 60 min of reperfusion. The adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA; 200 nM) was administrated 10 min before ischemia and for the first 10 min of reperfusion. Treatment with CHA significantly improved postischemic left ventricular developed pressure (74 ± 4% vs. 44 ± 4% of preischemic left ventricular developed pressure at 60 min of reperfusion) and reduced infarct size (30 ± 2% with CHA vs. 52 ± 5% in control) in WT hearts, effects that were blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). Treatments with the A(2A) receptor agonist CGS-21680 (200 nM) and the A(2B) agonist BAY 60-6583 (200 nM) did not exert any beneficial effects. Deletion of adenosine A(2A) or A(2B) receptor subtypes did not alter ischemia-reperfusion injury, but CHA failed to exert a cardioprotective effect in hearts of mice from either KO group. These findings indicate that both adenosine A(2A) and A(2B) receptors are required for adenosine A(1) receptor-mediated cardioprotection, implicating a role for interactions among receptor subtypes.
Asunto(s)
Agonistas del Receptor de Adenosina A1/farmacología , Adenosina/análogos & derivados , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Receptor de Adenosina A1/efectos de los fármacos , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Adenosina/farmacología , Antagonistas del Receptor de Adenosina A1/farmacología , Agonistas del Receptor de Adenosina A2/farmacología , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Perfusión , Receptor Cross-Talk , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/deficiencia , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/genética , Factores de Tiempo , Función Ventricular Izquierda/efectos de los fármacos , Presión Ventricular/efectos de los fármacosRESUMEN
Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5'-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A(2B) receptor (A(2B)R) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A(2B)R signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A(2B)R in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A(2B)R activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders.
Asunto(s)
Óxido Nítrico Sintasa de Tipo III/fisiología , Erección Peniana/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptor de Adenosina A2B/fisiología , 5'-Nucleotidasa/metabolismo , Adenosina/fisiología , Adenosina Trifosfato/fisiología , Animales , Línea Celular , Estimulación Eléctrica , Células Endoteliales/fisiología , Activación Enzimática , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Pene/inervación , Pene/fisiología , Receptor de Adenosina A2B/deficiencia , Receptor de Adenosina A2B/genética , Transducción de Señal/fisiología , Estrés MecánicoRESUMEN
OBJECTIVE: Macrophage- and vascular-derived matrix metalloproteinase (MMP)-9 plays an important role in neointima formation after vascular injury. The A2b adenosine receptor (A2bAR) elevates cyclic adenosine monophosphate and suppresses tumor necrosis factor-α (TNF-α) levels at baseline and after vascular injury. Considering the influences of TNF-α on MMP-9 expression and activity, here we examined the effect of the A2bAR on the expression of MMP-9 and its potential dependency on TNF-α. MATERIALS AND METHODS: We applied protein activity and mRNA analyses of MMP-9 in macrophages derived from A2bAR knockout (KO) and TNF-α receptor KO mice. We employed guidewire-induced femoral artery injuries on A2bAR KO and control mice and analyzed by immunohistochemistry MMP-9 expression in the neointima area. RESULTS: MMP-9 activity is somewhat less in resident A2bAR KO macrophages compared with wild-type cells. However, MMP-9 is increased in activated macrophages from A2bAR KO when TNF-α is further elevated, or in wild-type cells after TNF-α treatment. In accordance, A2bAR activation downregulates MMP-9 expression in wild-type macrophages, which is ablated in TNF-α receptor KO cells. A greater vascular lesion after femoral artery injury in A2bAR KO mice is associated with elevated TNF-α levels and augmented MMP-9, compared to control mice. CONCLUSIONS: Ablation of the A2bAR in activated macrophages increases MMP-9. A2bAR activation reduces MMP-9 expression, which depends on TNF-α and could contribute to the protective role of A2bAR in a vascular injury model.
Asunto(s)
Metaloproteinasa 9 de la Matriz/biosíntesis , Receptor de Adenosina A2B/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Macrófagos/enzimología , Macrófagos/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Adenosina A2B/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.