RESUMEN
Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.
Asunto(s)
Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Neoplasias/inmunología , Nucleótidos Cíclicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Tirosina Quinasa c-Mer/inmunología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Antígeno B7-H1/inmunología , Células Cultivadas , Femenino , Inmunidad Innata , Inmunoterapia , Interferón Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Nucleotidiltransferasas/deficiencia , Nucleotidiltransferasas/metabolismo , Fagocitosis , Receptor de Muerte Celular Programada 1/inmunología , Receptores Purinérgicos P2X7/deficiencia , Transducción de Señal/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa c-Mer/genéticaRESUMEN
Potassium (K+) efflux across the plasma membrane is thought to be an essential mechanism for ATP-induced NLRP3 inflammasome activation, yet the identity of the efflux channel has remained elusive. Here we identified the two-pore domain K+ channel (K2P) TWIK2 as the K+ efflux channel triggering NLRP3 inflammasome activation. Deletion of Kcnk6 (encoding TWIK2) prevented NLRP3 activation in macrophages and suppressed sepsis-induced lung inflammation. Adoptive transfer of Kcnk6-/- macrophages into mouse airways after macrophage depletion also prevented inflammatory lung injury. The K+ efflux channel TWIK2 in macrophages has a fundamental role in activating the NLRP3 inflammasome and consequently mediates inflammation, pointing to TWIK2 as a potential target for anti-inflammatory therapies.
Asunto(s)
Inflamasomas/metabolismo , Inflamación/fisiopatología , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Caspasa 1/deficiencia , Caspasa 1/metabolismo , Línea Celular , Inflamasomas/efectos de los fármacos , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Lesión Pulmonar/metabolismo , Lesión Pulmonar/fisiopatología , Macrófagos/trasplante , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/deficiencia , Quinina/farmacología , ARN Interferente Pequeño/farmacología , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/metabolismo , Sepsis/metabolismo , Sepsis/fisiopatología , Transducción de Señal/efectos de los fármacosRESUMEN
Extracellular ATP (eATP) is an ancient 'danger signal' used by eukaryotes to detect cellular damage1. In mice and humans, the release of eATP during inflammation or injury stimulates both innate immune activation and chronic pain through the purinergic receptor P2RX72-4. It is unclear, however, whether this pathway influences the generation of immunological memory, a hallmark of the adaptive immune system that constitutes the basis of vaccines and protective immunity against re-infection5,6. Here we show that P2RX7 is required for the establishment, maintenance and functionality of long-lived central and tissue-resident memory CD8+ T cell populations in mice. By contrast, P2RX7 is not required for the generation of short-lived effector CD8+ T cells. Mechanistically, P2RX7 promotes mitochondrial homeostasis and metabolic function in differentiating memory CD8+ T cells, at least in part by inducing AMP-activated protein kinase. Pharmacological inhibitors of P2RX7 provoked dysregulated metabolism and differentiation of activated mouse and human CD8+ T cells in vitro, and transient P2RX7 blockade in vivo ameliorated neuropathic pain but also compromised production of CD8+ memory T cells. These findings show that activation of P2RX7 by eATP provides a common currency that both alerts the nervous and immune system to tissue damage, and promotes the metabolic fitness and survival of the most durable and functionally relevant memory CD8+ T cell populations.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Memoria Inmunológica , Receptores Purinérgicos P2X7/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Activación Enzimática , Femenino , Homeostasis , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/fisiología , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genéticaRESUMEN
Severe urinary tract infections are commonly caused by sub-strains of Escherichia coli secreting the pore-forming virulence factor α-hemolysin (HlyA). Repeated or severe cases of pyelonephritis can cause renal scarring that subsequently can lead to progressive failure. We have previously demonstrated that HlyA releases cellular ATP directly through its membrane pore and that acute HlyA-induced cell damage is completely prevented by blocking ATP signaling. Local ATP signaling and P2X7 receptor activation play a key role in the development of tissue fibrosis. This study investigated the effect of P2X7 receptors on infection-induced renal scarring in a murine model of pyelonephritis. Pyelonephritis was induced by injecting 100 million HlyA-producing, uropathogenic E. coli into the urinary bladder of BALB/cJ mice. A similar degree of pyelonephritis and mortality was confirmed at day 5 after infection in P2X7+/+ and P2X7-/- mice. Fibrosis was first observed 2 weeks after infection, and the data clearly demonstrated that P2X7-/- mice and mice exposed to the P2X7 antagonist, brillian blue G, show markedly less renal fibrosis 14 days after infection compared with controls (P < 0.001). Immunohistochemistry revealed comparable early neutrophil infiltration in the renal cortex from P2X7+/+ and P2X7-/- mice. Interestingly, lack of P2X7 receptors resulted in diminished macrophage infiltration and reduced neutrophil clearance in the cortex of P2X7-/- mice. Hence, this study suggests the P2X7 receptor to be an appealing antifibrotic target after renal infections.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Riñón/metabolismo , Pielonefritis , Receptores Purinérgicos P2X7/deficiencia , Escherichia coli Uropatógena , Animales , Fibrosis , Riñón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Pielonefritis/genética , Pielonefritis/metabolismo , Pielonefritis/microbiología , Pielonefritis/prevención & control , Receptores Purinérgicos P2X7/metabolismo , Escherichia coli Uropatógena/metabolismo , Escherichia coli Uropatógena/patogenicidadRESUMEN
Previously we reported that the sensitivity of CD4+ T cells to ATP does not depend on P2X7 receptor (P2X7R) expression levels but on their activation and differentiation stages. Therefore, here we have investigated a potential relationship between the sensitivity of CD8+ T cells to ATP and their stages of differentiation. Thus, the CD8+ subpopulation exhibits a drastically reduced sensitivity to ATP with aging, which parallels the strong increase of an effector/memory CD8+ subset expressing high levels of CD44 cell adhesion molecule and CD45RB transmembrane phosphatase (CD44hiCD45RBhi). Using l-selectin/CD62L, CC-chemokine receptor 7, and CD127/IL-7 receptor-α markers, we showed that effector/memory CD8+ T cells belong to a central or effector memory subset. In contrast, the CD44hiCD45RBhi effector/memory subset is absent or poorly expressed in the CD4+ T subpopulation regardless of age. While ATP treatment can trigger channel and pore formation, CD62L shedding, phosphatidylserine exposure, and cell death in the CD44loCD45RBhi-naive CD8+ subset, it is unable to induce these cellular activities in the CD44hiCD45RBhi effector/memory CD8+ subset. Importantly, both CD44loCD45RBhi-naive and CD44hiCD45RBhi effector/memory subsets express similar low levels of P2X7R, demonstrating that the sensitivity of CD8+ T cells to ATP depends on the stage of differentiation instead of P2X7R expression levels.-Mellouk, A., Bobé, P. CD8+, but not CD4+ effector/memory T cells, express the CD44highCD45RBhigh phenotype with aging, which displays reduced expression levels of P2X7 receptor and ATP-induced cellular responses.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Envejecimiento/inmunología , Envejecimiento/metabolismo , Animales , Linfocitos T CD4-Positivos/clasificación , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/clasificación , Linfocitos T CD8-positivos/inmunología , Señalización del Calcio , Diferenciación Celular/inmunología , Receptores de Hialuranos/metabolismo , Memoria Inmunológica , Selectina L/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
ATP is a (co)transmitter and signaling molecule in the CNS. It acts at a multitude of ligand-gated cationic channels termed P2X to induce rapid depolarization of the cell membrane. Within this receptor-channel family, the P2X7 receptor (R) allows the transmembrane fluxes of Na+, Ca2+, and K+, but also allows the slow permeation of larger organic molecules. This is supposed to cause necrosis by excessive Ca2+ influx, as well as depletion of intracellular ions and metabolites. Cell death may also occur by apoptosis due to the activation of the caspase enzymatic cascade. Because P2X7Rs are localized in the CNS preferentially on microglia, but also at a lower density on neuroglia (astrocytes, oligodendrocytes) the stimulation of this receptor leads to the release of neurodegeneration-inducing bioactive molecules such as pro-inflammatory cytokines, chemokines, proteases, reactive oxygen and nitrogen molecules, and the excitotoxic glutamate/ATP. Various neurodegenerative reactions of the brain/spinal cord following acute harmful events (mechanical CNS damage, ischemia, status epilepticus) or chronic neurodegenerative diseases (neuropathic pain, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis) lead to a massive release of ATP via the leaky plasma membrane of neural tissue. This causes cellular damage superimposed on the original consequences of neurodegeneration. Hence, blood-brain-barrier permeable pharmacological antagonists of P2X7Rs with excellent bioavailability are possible therapeutic agents for these diseases. The aim of this review article is to summarize our present state of knowledge on the involvement of P2X7R-mediated events in neurodegenerative illnesses endangering especially the life quality and duration of the aged human population.
Asunto(s)
Enfermedades del Sistema Nervioso Central/patología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Receptores Purinérgicos P2X7/metabolismo , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/metabolismo , Dolor Crónico/metabolismo , Dolor Crónico/fisiopatología , Epilepsia/metabolismo , Epilepsia/fisiopatología , Humanos , Ratones Transgénicos , Neuralgia/metabolismo , Neuralgia/patología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/deficienciaRESUMEN
Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R-/-) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R-/- mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R-/- mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E2 (PGE2) release from primary parotid cells of wild-type but not P2X7R-/- mice. To attempt to uncover the mechanism underlying differential PGE2 release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE2 release in P2X7R-/- mice. Irradiated P2X7R-/- mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.
Asunto(s)
Rayos gamma , Glándula Parótida/metabolismo , Traumatismos por Radiación/prevención & control , Receptores Purinérgicos P2X7/deficiencia , Salivación , Xerostomía/prevención & control , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Ratones Endogámicos C57BL , Ratones Noqueados , Glándula Parótida/efectos de los fármacos , Glándula Parótida/fisiopatología , Prostaglandina-E Sintasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Traumatismos por Radiación/genética , Traumatismos por Radiación/metabolismo , Traumatismos por Radiación/fisiopatología , Receptores Purinérgicos P2X7/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Salivación/efectos de los fármacos , Xerostomía/genética , Xerostomía/metabolismo , Xerostomía/fisiopatologíaRESUMEN
The alveolar epithelial cells represent an important part of the alveolar barrier, which is maintained by tight junction proteins, particularly JAM-A, occludin, and claudin-18, which regulate paracellular permeability. In this study, we report on a strong increase in epithelial JAM-A expression in P2X7 receptor knockout mice when compared to the wildtype. Precision-cut lung slices of wildtype and knockout lungs and immortal epithelial lung E10 cells were treated with bleomycin, the P2X7 receptor inhibitor oxATP, and the agonist BzATP, respectively, to evaluate early changes in JAM-A expression. Biochemical and immunohistochemical data showed evidence for P2X7 receptor-dependent JAM-A expression in vitro. Inhibition of the P2X7 receptor using oxATP increased JAM-A, whereas activation of the receptor decreased the JAM-A protein level. In order to examine the role of GSK-3ß in the expression of JAM-A in alveolar epithelial cells, we used lithium chloride for GSK-3ß inhibiting experiments, which showed a modulating effect on bleomycin-induced alterations in JAM-A levels. Our data suggest that an increased constitutive JAM-A protein level in P2X7 receptor knockout mice may have a protective effect against bleomycin-induced lung injury. Bleomycin-treated precision-cut lung slices from P2X7 receptor knockout mice responded with a lower increase in mRNA expression of JAM-A than bleomycin-treated precision-cut lung slices from wildtype mice.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Células Epiteliales Alveolares/metabolismo , Animales , Bleomicina , Moléculas de Adhesión Celular/genética , Ratones , Agonistas del Receptor Purinérgico P2X/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores Purinérgicos P2X7/deficienciaRESUMEN
apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/agonistas , Apolipoproteínas E/agonistas , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Transportador 1 de Casete de Unión a ATP/metabolismo , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacología , Animales , Apolipoproteínas E/metabolismo , Aziridinas/química , Aziridinas/farmacología , Benzamidas/química , Benzamidas/farmacología , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Estructura Molecular , Naftoquinonas/química , Naftoquinonas/farmacología , Antagonistas del Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X7/deficiencia , Bibliotecas de Moléculas Pequeñas/química , Sulfonamidas/química , Sulfonamidas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Extracellular adenosine triphosphate (ATP) binds as a danger signal to purinergic receptor P2X7 and promotes inflammasome assembly and interleukin-1ß expression. We hypothesized a functional role of the signal axis ATP-P2X7 in inflammasome activation and the chronic inflammation driving atherosclerosis. METHODS: P2X7-competent and P2X7-deficient macrophages were isolated and stimulated with lipopolysaccharide, ATP, or both. To assess whether P2X7 may have a role in atherosclerosis, P2X7 expression was analyzed in aortic arches from low density lipoprotein receptor-/- mice consuming a high-cholesterol or chow diet. P2X7+/+ and P2X7-/- low density lipoprotein receptor-/- mice were fed a high-cholesterol diet to investigate the functional role of P2X7 knockout in atherosclerosis. Human plaques were derived from carotid endarterectomy and stained against P2X7. RESULTS: Lipopolysaccharide or ATP stimulation alone did not activate caspase 1 in isolated macrophages. However, priming with lipopolysaccharide, followed by stimulation with ATP, led to an activation of caspase 1 and interleukin-1ß in P2X7-competent macrophages. In contrast, P2X7-deficient macrophages showed no activation of caspase 1 after sequential stimulation while still expressing a basal amount of interleukin-1ß. P2X7 receptor was higher expressed in murine atherosclerotic lesions, particularly by lesional macrophages. After 16 weeks of a high-cholesterol diet, P2X7-deficient mice showed smaller atherosclerotic lesions than P2X7-competent mice (0.162 cm2±0.023 [n=9], P2X7-/- low density lipoprotein receptor-/- : 0.084 cm2±0.01 [n=11], P=0.004) with a reduced amount of lesional macrophages. In accord with our in vitro findings, lesional caspase 1 activity was abolished in P2X7-/- mice. In addition, intravital microscopy revealed reduced leukocyte rolling and adhesion in P2X7-deficient mice. Last, we observe increased P2X7 expression in human atherosclerotic lesions, suggesting that our findings in mice are relevant for human disease. CONCLUSIONS: P2X7 deficiency resolved plaque inflammation by inhibition of lesional inflammasome activation and reduced experimental atherosclerosis. Therefore, P2X7 represents an interesting potential new target to combat atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Inflamasomas/metabolismo , Receptores Purinérgicos P2X7/deficiencia , Adenosina Trifosfato/toxicidad , Animales , Aterosclerosis/inducido químicamente , Humanos , Inflamasomas/antagonistas & inhibidores , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/prevención & control , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The expression of aquaporin 5 in alveolar epithelial type I cells under conditions of cadmium-induced injury has not yet been discovered. We investigated the effect of the P2X7R agonist BzATP under this condition, since P2X7R is involved in altered regulation of aquaporin 5 in pulmonary fibrosis. CdCl2/TGF-ß1 treatment of lung epithelial MLE-12 cells was leading to increasing P2X7R, and aquaporin 5 protein levels. The aquaporin 5 expression was P2X7R-independent in MLE-12 cells under cadmium, as was shown in blocking experiments with oxATP. Further, the expression of both proteins increased after 24 h CdCl2/TGF-ß1 treatment of precision-cut lung slices, but decreased after 72 h. Using immunohistochemistry, the activation of the P2X7R with the agonist BzATP modulated the aquaporin 5 immunoreactivity in the alveolar epithelium of precision-cut lung slices from wild-type but not from P2X7R knockout mice. Similarly, aquaporin 5 protein was reduced in BzATP-treated immortal lung epithelial E10 cells. Surprisingly, untreated alveolar epithelial type II cells of P2X7R knockouts exhibited a pronounced apical immunoreactivity in addition to the remaining alveolar epithelial type I cells. BzATP exposure did not alter this distribution pattern, but increased the number of apoptotic alveolar epithelial type II cells in wild-type lung slices.
Asunto(s)
Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Acuaporina 5/biosíntesis , Cloruro de Cadmio/toxicidad , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Purinérgicos P2X7/deficienciaRESUMEN
Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X7 receptor deficiency, or treated with a P2X7 receptor inhibitor or anti-VCAM-1 Abs, were subjected to a clinically relevant two-hit LPS and mechanical ventilation-induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Ab neutralization of sVCAM-1 or deficiency or antagonism of P2X7R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X7 receptor-mediated activation of the metalloproteinase ADAM-17. In conclusion, sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses.
Asunto(s)
Lesión Pulmonar Aguda/inmunología , Neutrófilos/inmunología , Receptores Purinérgicos P2X7/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Lesión Pulmonar Aguda/patología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/patología , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/metabolismoRESUMEN
We recently demonstrated that ischemic tolerance was dependent on astrocytes, for which HIF-1α had an essential role. The mild ischemia (preconditioning; PC) increased HIF-1α in a biphasic pattern, that is, a quick and transient increase in neurons, followed by a slow and sustained increase in astrocytes. However, mechanisms underlying such temporal difference in HIF-1α increase remain totally unknown. Here, we show that unlike a hypoxia-dependent mechanism in neurons, astrocytes increase HIF-1α via a novel hypoxia-independent but P2X7-dependent mechanism. Using a middle cerebral artery occlusion (MCAO) model of mice, we found that the PC (a 15-min MCAO period)-evoked increase in HIF-1α in neurons was quick and transient (from 1 to 3 days after PC), but that in astrocytes was slow-onset and long-lasting (from 3 days to at least 2 weeks after PC). The neuronal HIF-1α increase was dependent on inhibition of PHD2, an oxygen-dependent HIF-1α degrading enzyme, whereas astrocytic one was independent of PHD2. Astrocytes even do not possess this enzyme. Instead, they produced a sustained increase in P2X7 receptors, activation of which resulted in HIF-1α increase. The hypoxia-independent but P2X7-receptor-dependent mechanism could allow astrocytes to cause long-lasting HIF-1α expression, thereby leading to induction of ischemic tolerance efficiently. GLIA 2017;65:523-530.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/patología , Regulación de la Expresión Génica/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Infarto de la Arteria Cerebral Media/patología , Precondicionamiento Isquémico/métodos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Lateralidad Funcional/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genéticaRESUMEN
BACKGROUND & AIMS: The severity of sepsis can be linked to excessive inflammatory responses resulting in hepatic injury. P2X7 receptor activation by extracellular ATP (eATP) exacerbates inflammation by augmenting cytokine production; while CD39 (ENTPD1) scavenges eATP to generate adenosine, thereby limiting P2X7 activation and resulting in A2A receptor stimulation. We aim to determine how the functional interaction of P2X7 receptor and CD39 control the macrophage response, and consequently impact on sepsis and liver injury. METHODS: Sepsis was induced by cecal ligation and puncture in C57BL/6 wild-type (WT) and CD39-/- mice. Several in vitro assays were performed using peritoneal or bone marrow derived macrophages to determine CD39 ectonucleotidase activity and its role in sepsis-induced liver injury. RESULTS: CD39 expression in macrophages limits ATP-P2X7 receptor pro-inflammatory signaling. P2X7 receptor paradoxically boosts CD39 activity. Inhibition and/or deletion of P2X7 receptor in LPS-primed macrophages attenuates cytokine production and inflammatory signaling as well as preventing ATP-induced increases in CD39 activity. Septic CD39-/- mice exhibit higher levels of inflammatory cytokines and show more pronounced liver injury than WT mice. Pharmacological P2X7 blockade largely prevents tissue damage, cell apoptosis, cytokine production, and the activation of inflammatory signaling pathways in the liver from septic WT, while only attenuating these outcomes in CD39-/- mice. Furthermore, the combination of P2X7 blockade with adenosine A2A receptor stimulation completely inhibits cytokine production, the activation of inflammatory signaling pathways, and protects septic CD39-/- mice against liver injury. CONCLUSIONS: CD39 attenuates sepsis-associated liver injury by scavenging eATP and ultimately generating adenosine. We propose boosting of CD39 would suppress P2X7 responses and trigger adenosinergic signaling to limit systemic inflammation and restore liver homeostasis during the acute phase of sepsis. Lay summary: CD39 expression in macrophages limits P2X7-mediated pro-inflammatory responses, scavenging extracellular ATP and ultimately generating adenosine. CD39 genetic deletion exacerbates sepsis-induced experimental liver injury. Combinations of a P2X7 antagonist and adenosine A2A receptor agonist are hepatoprotective during the acute phase of abdominal sepsis.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Hígado/inmunología , Hígado/lesiones , Receptores Purinérgicos P2X7/metabolismo , Sepsis/inmunología , Agonistas del Receptor de Adenosina A2/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/genética , Apirasa/deficiencia , Apirasa/genética , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Interleucina-1beta/biosíntesis , Hígado/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genética , Factor de Transcripción STAT3/metabolismo , Sepsis/terapia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
Infection and injury of the gut are associated with cell damage and release of molecules such as extracellular adenosine 5'-triphosphate (ATP), which is recognised by the purinergic P2X7 receptor (P2X7R). P2X7R is widely expressed in the gut by antigen-presenting cells (APCs) and epithelial cells, but the role of the P2X7R on epithelial cells is poorly understood. We investigated P2X7R in intestinal epithelium in vitro and in vivo using two model infections, Toxoplasma gondii and Trichinella spiralis. Lipopolysaccharide and ATP treatment of intestinal epithelial cells and infection with T. gondii in vitro did not promote inflammasome-associated interleukin-1ß (IL-1ß) or IL-18 secretion, but promoted C-C motif chemokine ligand 5 (CCL5), tumour necrosis factor-α and IL-6 production that were significantly reduced when the P2X7R was blocked. Similarly, in vivo, infection with either T. spiralis or T. gondii induced rapid upregulation of epithelial CCL5 in wild-type (wild-type (WT)) mice that was significantly reduced in P2X7R-/- littermate controls. The effects of reduced epithelial CCL5 were assayed by investigating recruitment of dendritic cells (DCs) to the epithelium. Infection induced a rapid recruitment of CD11c+CD103+ DC subsets into the epithelial layer of WT mice but not P2X7R-/- mice. In vitro chemotaxis assays and bone marrow chimeras demonstrated the importance of epithelial P2X7R in DC recruitment. P2X7R signalling in epithelial cells mediates chemokine responses to promote initiation of host immunity to infection.
Asunto(s)
Tracto Gastrointestinal/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/parasitología , Receptores Purinérgicos P2X7/metabolismo , Inmunidad Adaptativa , Animales , Quimiocina CCL5/biosíntesis , Quimiotaxis , Células Dendríticas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Carga de Parásitos , Receptores Purinérgicos P2X7/deficiencia , Linfocitos T/inmunología , Toxoplasma , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Toxoplasmosis/patologíaRESUMEN
OBJECTIVE: Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. APPROACH AND RESULTS: We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6(-/-) arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6(-/-) mice were protected against MT elevation in myocardial infarction-induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA-GTP binding, myosin light chain, P42-P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43(+/-) and P2rx7(-/-) mesenteric resistance arteries. CONCLUSIONS: Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.
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Arteriolas/metabolismo , Mesenterio/irrigación sanguínea , Receptores Purinérgicos P2/metabolismo , Vasoconstricción , Adenosina Trifosfatasas/metabolismo , Animales , Arteriolas/efectos de los fármacos , Arteriolas/fisiopatología , Presión Sanguínea , Señalización del Calcio , Células Cultivadas , Conexina 43/deficiencia , Conexina 43/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Genotipo , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Hidrólisis , Mecanotransducción Celular , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Infarto del Miocardio/complicaciones , Miocitos del Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fenotipo , Fosforilación , Agonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2/deficiencia , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/genética , Uridina Difosfato/farmacología , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
The purinergic receptor P2X7 represents an ATP-gated ionotropic receptor with a selective localization in alveolar epithelial type I cells of the lung. Despite the involvement of the receptor in inflammatory processes of the lung, it is not established whether this receptor plays a specific role in the alveolar epithelial cell biology. There is evidence that P2X7 receptor influences Wnt/ß-catenin signalling pathways in alveolar epithelial cells under conditions of injury. Here, we investigated the expression of GSK-3ß, a potent protein kinase involved in alveolar epithelial barrier functions, and of tight junction molecules occludin, claudin-4 and claudin-18 in wild-type and P2X7-/- mice. Western blot analysis, immunohistochemistry and quantitative real-time RT-PCR revealed a remarkable increase in claudin-18 mRNA and protein in lungs of P2X7-/- mice animals. Furthermore, alveolar epithelial cells from P2X7-/- animals showed decreased levels of GSK-3ß protein and its inactive form GSK-3ß (pS9). Conversely, claudin-18 knockout mice exhibited decreased P2X7 mRNA transcript abundance as measured by mRNA expression microarray and quantitative PCR. Our data are consistent with the hypothesis that P2X7R contributes to alveolar epithelial barrier function through effects on GSK-3ß. Furthermore, these data suggest a potential reciprocal regulation of claudin-18 and P2X7R in the alveolar epithelium.
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Células Epiteliales Alveolares/metabolismo , Claudinas/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Pulmón/citología , Receptores Purinérgicos P2X7/metabolismo , Animales , Claudinas/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Purinérgicos P2X7/deficienciaRESUMEN
Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-1(2+) uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.
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Proteínas ADAM/metabolismo , Adenosina Trifosfato/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Linfocitos B/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de IgE/metabolismo , Receptores Purinérgicos P2X7/genética , Bazo/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Benzoxazoles , Dipéptidos/farmacología , Colorantes Fluorescentes , Regulación de la Expresión Génica , Humanos , Ácidos Hidroxámicos/farmacología , Activación de Linfocitos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Microscopía Fluorescente , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Cultivo Primario de Células , Inhibidores de Proteasas/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Compuestos de Quinolinio , Receptores de IgE/genética , Receptores Purinérgicos P2X7/deficiencia , Transducción de Señal , Bazo/citología , Bazo/efectos de los fármacos , Tiofenos/farmacologíaRESUMEN
Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus, and NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells, and may participate in organ damage through incompletely characterized pathways. To better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-1ß and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2X7 receptor-mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines that further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/fisiología , Proteínas Portadoras/fisiología , Inflamasomas/fisiología , Inflamación/inmunología , Lupus Eritematoso Sistémico/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Activación Neutrófila , Neutrófilos/inmunología , Adulto , Animales , Comunicación Autocrina , Proteínas Portadoras/genética , Caspasa 1/fisiología , Activación Enzimática , Femenino , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Transporte Iónico , Lupus Eritematoso Sistémico/patología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Neutrófilos/metabolismo , Potasio/metabolismo , Receptores Purinérgicos P2X7/deficiencia , Receptores Purinérgicos P2X7/metabolismo , Organismos Libres de Patógenos Específicos , CatelicidinasRESUMEN
On the basis of studies in mouse macrophages, activation of the nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain-containing 3 (Nlrp3) inflammasome is thought to require two signals. The first signal is provided by TLR stimulation and triggers the synthesis of the IL-1ß precursor and Nlrp3. The second signal can be mediated by stimulation of the purinergic receptor P2X ligand-gated ion channel 7 (P2X7) by millimolar concentrations of ATP. However, these high concentrations of ATP are not found normally in the in vivo extracellular milieu, raising concern about the physiological relevance of the ATP-P2X7 pathway of inflammasome activation. In this article, we show that unlike macrophages, murine bone marrow-derived and splenic dendritic cells (DCs) can secrete substantial amounts of mature IL-1ß upon stimulation with TLR ligands in the absence of ATP stimulation. The differential ability of DCs to release IL-1ß and activate caspase-1 was associated with increased expression of Nlrp3 under steady-state conditions and of pro-IL-1ß and Nlrp3 after stimulation with TLR agonists. IL-1ß secretion from stimulated DCs was largely dependent on the Nlrp3 inflammasome, but independent of P2X7 and unaffected by incubation with apyrase. More importantly, i.p. administration of LPS induced IL-1ß production in serum, which was abrogated in Nlrp3-null mice but was unaffected in P2X7-deficient mice. These results demonstrate differential regulation of the Nlrp3 inflammasome in macrophages and DCs. Furthermore, they challenge the idea that the ATP-P2X7 axis is critical for TLR-induced IL-1ß production via the Nlrp3 inflammasome in vivo.