Adenosine A2A Receptor Expression Research Articles

Salidroside attenuates chronic hypoxia-induced pulmonary hypertension via adenosine A2a receptor related mitochondria-dependent apoptosis pathway

Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial remodeling mainly due to excess cellular proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Salidroside, an active ingredient isolated from Rhodiola rosea is proposed to exert protective effects against PAH. However, the function of salidroside in PAH has not been investigated systematically and the underlying mechanisms are not clear. To investigate the effects of salidroside on PAH, the mice in chronic hypoxia model of PAH were given by an increasing concentration of salidroside (0, 16mg/kg, 32mg/kg, and 64mg/kg). After salidroside treatment, the chronic hypoxia-induced right ventricular hypertrophy and pulmonary arterial remodeling were attenuated, suggesting a protective role played by salidroside in PAH. To explore the potential mechanisms, the apoptosis of PASMCs after salidroside treatment under hypoxia conditions were determined in vivo and in vitro, and also the mitochondria-dependent apoptosis factors, Bax, Bcl-2, cytochrome C, and caspase 9 were examined. The results revealed that salidroside reversed hypoxia-induced cell apoptosis resistance at least partially via a mitochondria-dependent pathway. In addition, salidroside upregulated the expression of adenosine A2a receptor (A2aR) in lung tissues of mice and in PASMCs in vitro after hypoxia exposure. Combined the evidence above, we conclude that salidroside can attenuate chronic hypoxia-induced PAH by promoting PASMCs apoptosis via an A2aR related mitochondria dependent pathway.

Placental expression of adenosine A2A receptor and hypoxia inducible factor-1 alpha in early pregnancy, term and pre-eclamptic pregnancies: Interactions with placental renin-angiotensin system

Hypoxia-inducible factors (HIFs), adenosine and tissue renin-angiotensin-system (RAS) promote angiogenesis and vascularisation. We investigated the temporal expression placental adenosine A2AR receptor and HIF-1α in early pregnancy and at delivery in normotensive (NT) and pre-eclamptic (PE) women. Results were compared to our previously reported angiotensin receptor data.Expression of A2AR and HIF-1α was highest at ≤10 weeks, positively correlated through pregnancy and was higher in PE than NT at delivery. The A2AR associated with the AT4R only in early pregnancy. We suggest adenosine and RAS may interact to promote placentation with a potential adaptation to poor placental perfusion in PE.

Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment.

High concentrations of adenosine in tumor microenvironments inhibit antitumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2AR) that suppress their activation and inhibit immune killing of tumors, a role for myeloid cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study, we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2a(f/f)-LysMCre(+/-) mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL12 expression in tumor-associated macrophages and with >90% reductions in IL10 expression in tumor-associated macrophages, dendritic cells (DC), and Ly6C(+) or Ly6G(+) myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and natural killer (NK) cells. Depletion of CD8(+) T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8(+) T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen-specific CD8(+) T cells in lung infiltrates. Overall, the findings indicate that myeloid cell A2ARs have direct myelosuppressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs, and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors.

Activated Human iNKT Cells in Pediatric Sickle Cell Disease Patients and in Culture Upregulate Ectonucleotidase CD39 and Adenosine a2A Receptor

Background: Sickle cell disease (SCD) is a chronic degenerative disease punctuated by periodic painful vaso-occlusive crises (pVOC). When sickle hemoglobin polymerizes, it causes erythrocytes to become rigid and sticky and produces multi-cellular aggregates that can occlude venules. We have shown that invariant natural killer T (iNKT) cells are pivotal in causing inflammation and injury in sickle cell disease (Wallace KL, Marshall MA, et al. Blood. 2009; 114(3):667-76). Activation of iNKT cells causes an induction of anti-inflammatory adenosine A2A receptors (A2ARs) that have a negative feedback role to limit inflammation. We found that an A2AR activation inhibits iNKT cell activation and protects SCD mice from tissue injury (Wallace KL, Linden J. Blood 2010; 116(23):5010-20). We have also seen in adult SCD patients that the expression of the A2AR is significantly higher in iNKT cells during pVOC than at steady state (Field JJ, Lin G, et al. Blood 2013; 121(17):3329-42). In addition, CD39 and CD73, ectonucleotidases that generate adenosine from ATP, reduce inflammation by activating the A2AR (Crikis S, Lu B, et al. Am J Transplant 2010; 10(12):2586-95).Hypothesis and Aims: We hypothesized that pediatric SCD patients will have elevated A2AR and CD39 expression on iNKT cells during pVOC compared to steady state. We also hypothesized that the elevated A2AR immunoreactivity on activated iNKT cells is associated with enhanced A2AR signaling and reduced iNKT cell activation and inflammation. Our aims were: 1) to measure A2ARs and CD39 on iNKT cells of pediatric SCD patients at steady state and during pVOC, and also on cultured human iNKT cells in vitro with and without activation; and 2) to measure immunoreactive A2AR protein on activated cultured iNKT cells and correlate it with functional A2AR signaling.Methods: We collected blood from pediatric SCD patients at steady state and during pVOC. We used fluorescence-activated cell sorting (FACS) assays to evaluate the level of A2AR and CD39 immunofluorescence. For cultured human iNKT cell studies, human iNKT cells were prepared from healthy donor whole blood and expanded in culture. iNKT cells were activated through engagement of TCRs (with anti-CD3 and anti-CD28 Abs), cytokines (IL-12 and IL-18), or a combination of TCR engagement and cytokines. We treated the activated iNKT cells with vehicle or the A2AR agonist, regadenoson. iNKT cells were analyzed at 48 hours by FACS for A2AR and CD39. To determine if induced A2ARs are functional we utilized cAMP assays. A2AR is a Gs coupled receptor, which when activated, activates adenylyl cyclase to generate cAMP.Results: iNKT cells from pediatric sickle cell patients have higher A2AR and CD39 immunofluorescence during pVOC than at steady state. In cultured iNKT cells we found both A2AR and CD39 immunofluorescence are higher in activated iNKT cells than in non-activated cells. We discovered that iNKT cell activation through TCRs and cytokines is significantly stronger than either activation method alone. We also found both in vivo and in vitro that high A2AR expression on iNKT cells is associated with high CD39 expression. The A2ARs induced on activated iNKT cells are functional by showing significantly higher cAMP from activated iNKT cells exposed to Regadenoson.Conclusions: Our findings indicate that CD39 and the A2AR function together to provide a mechanism to gradually turn off activated iNKT cells and modulate the inflammatory response. Understanding this negative feedback system will help us to better understand the control of inflammation in SCD during vaso-occlusive crises and to identify novel therapies to treat and prevent the complications of SCD. Further studies may include determining if A2AR activation decreases iNKT cell activation, and if blockade of CD39 enhances iNKT cell activation. DisclosuresNo relevant conflicts of interest to declare.

Effects of adolescent caffeine consumption on cocaine sensitivity.

Caffeine is the most commonly used psychoactive substance, and consumption by adolescents has risen markedly in recent years. We identified the effects of adolescent caffeine consumption on cocaine sensitivity and determined neurobiological changes within the nucleus accumbens (NAc) that may underlie caffeine-induced hypersensitivity to cocaine. Male Sprague-Dawley rats consumed caffeine (0.3 g/l) or water for 28 days during adolescence (postnatal day 28-55; P28-P55) or adulthood (P67-P94). Testing occurred in the absence of caffeine during adulthood (P62-82 or P101-121). Cocaine-induced and quinpirole (D2 receptor agonist)-induced locomotion was enhanced in rats that consumed caffeine during adolescence. Adolescent consumption of caffeine also enhanced the development of a conditioned place preference at a sub-threshold dose of cocaine (7.5 mg/kg, i.p.). These behavioral changes were not observed in adults consuming caffeine for an equivalent period of time. Sucrose preferences were not altered in rats that consumed caffeine during adolescence, suggesting there are no differences in natural reward. Caffeine consumption during adolescence reduced basal dopamine levels and augmented dopamine release in the NAc in response to cocaine (5 mg/kg, i.p.). Caffeine consumption during adolescence also increased the expression of the dopamine D2 receptor, dopamine transporter, and adenosine A1 receptor and decreased adenosine A2A receptor expression in the NAc. Consumption of caffeine during adulthood increased adenosine A1 receptor expression in the NAc, but no other protein expression changes were observed. Together these findings suggest that caffeine consumption during adolescence produced changes in the NAc that are evident in adulthood and may contribute to increases in cocaine-mediated behaviors.

Role of adenosine receptor subtypes in methamphetamine reward and reinforcement

The neurobiology of methamphetamine (MA) remains largely unknown despite its high abuse liability. The present series of studies explored the role of adenosine receptors on MA reward and reinforcement and identified alterations in the expression of adenosine receptors in dopamine terminal areas following MA administration in rats. We tested whether stimulating adenosine A1 or A2A receptor subtypes would influence MA-induced place preference or MA self-administration on fixed and progressive ratio schedules in male Sprague–Dawley rats. Stimulation of either adenosine A1 or A2A receptors significantly reduced the development of MA-induced place preference. Stimulating adenosine A1, but not A2A, receptors reduced MA self-administration responding. We next tested whether repeated experimenter-delivered MA administration would alter the expression of adenosine receptors in the striatal areas using immunoblotting. We observed no change in the expression of adenosine receptors. Lastly, rats were trained to self-administer MA or saline for 14 days and we detected changes in adenosine A1 and A2A receptor expression using immunoblotting. MA self-administration significantly increased adenosine A1 in the nucleus accumbens shell, caudate-putamen and prefrontal cortex. MA self-administration significantly decreased adenosine A2A receptor expression in the nucleus accumbens shell, but increased A2A receptor expression in the amygdala. These findings demonstrate that MA self-administration produces selective alterations in adenosine receptor expression in the nucleus accumbens shell and that stimulation of adenosine receptors reduces several behavioral indices of MA addiction. Together, these studies shed light onto the neurobiological alterations incurred through chronic MA use that may aid in the development of treatments for MA addiction.

Adenosine A3 receptors negatively regulate the engulfment-dependent apoptotic cell suppression of inflammation

Timed initiation of apoptotic cell death followed by efficient removal mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Besides phagocytosis, clearance of apoptotic cells also involves suppression of inflammatory responses by apoptotic cells mediated by both direct inhibition of pro-inflammatory cytokine production and release of soluble anti-inflammatory factors, which act in a paracrine or autocrine fashion to amplify or sustain the anti-inflammatory response. Previous work has demonstrated that during engulfment of apoptotic cells adenosine is produced in sufficient amounts to trigger both adenosine A2A receptors (A2ARs) and A3 receptors (A3Rs). Adenosine bound to A2ARs of macrophages activated the adenylate cyclase pathway to suppress the apoptotic-cell induced, NO-dependent formation of neutrophil migration factors. Here we show by using A3R null engulfing macrophages that the adenosine produced triggers the A3Rs as well, which attenuate the A2AR signaling by inhibiting adenylate cyclase. As a result, the balance in the activation of A2ARs and A3Rs determines the amounts of NO and consequently the levels of neutrophil chemoattractants formed. Since during phagocytosis of apoptotic cells the expression of A2ARs increases, while that of A3Rs decreases, on long term adenosine suppresses the proinflammatory responses in engulfing macrophages.

Striatal adenosine A2A receptor expression is controlled by S-adenosyl-L-methionine-mediated methylation.

Adenosine A2A receptor (A2AR) is a G protein-coupled receptor enriched in the striatum for which an increased expression has been demonstrated in certain neurological diseases. Interestingly, previous in vitro studies demonstrated that A2AR expression levels are reduced after treatment with S-adenosyl-L-methionine (SAM), a methyl donor molecule involved in the methylation of important biological structures such as DNA, proteins, and lipids. However, the in vivo effects of SAM treatment on A2AR expression are still obscure. Here, we demonstrated that 2weeks of SAM treatment produced a significant reduction in the rat striatal A2AR messenger RNA (mRNA) and protein content as well as A2AR-mediated signaling. Furthermore, when the content of 5-methylcytosine levels in the 5'UTR region of ADORA2A was analyzed, this was significantly increased in the striatum of SAM-treated animals; thus, an unambiguous correlation between SAM-mediated methylation and striatal A2AR expression could be established. Overall, we concluded that striatal A2AR functionality can be controlled by SAM treatment, an issue that might be relevant for the management of these neurological conditions that course with increased A2AR expression.

Increased striatal adenosine A2A receptor levels is an early event in Parkinson’s disease-related pathology and it is potentially regulated by miR-34b

Adenosine A2A receptor (A2AR) is a G-protein coupled receptor that stimulates adenylyl cyclase activity. In the brain, A2ARs are found highly enriched in striatal GABAergic medium spiny neurons, related to the control of voluntary movement. Pharmacological modulation of A2ARs is particularly useful in Parkinson’s disease (PD) due to their property of antagonizing dopamine D2 receptor activity. Increases in A2AR levels have been described in PD patients showing an important loss of dopaminergic denervation markers, but no data have been reported about A2AR levels in incidental PD brains. In the present report, we show that increased A2ARs protein levels were also detected in the putamen of incidental PD cases (Braak PD stages 1–2) with respect to age-matched controls. By contrast, A2ARs mRNA levels remained unchanged, suggesting that posttranslational mechanisms could be involved in the regulation of A2ARs. It has been described how miR-34b/c downregulation is an early event in PD cases. We found that miR-34b levels are also significantly reduced in the putamen of incidental PD cases and along disease progression. Given that 3′UTR of A2AR contains a predicted target site for miR-34b, the potential role of this miRNA in protein A2AR levels was assessed. In vitro studies revealed that endogenous A2AR protein levels increased when miR-34b function was blocked using a specific anti-miR-34b. Moreover, using a luciferase reporter assay with point mutations in a miR-34b predicted binding site within the 3'UTR region of A2AR mRNA abolished the effect of the miRNA using a miR-34b mimic. In addition, we showed a reduced percentage of DNA methylation in the 5'UTR region of ADORA2A in advanced PD cases. Overall, these findings reveal that increased A2AR protein levels occur in asymptomatic PD patients and provide new insights into the molecular mechanisms underlying A2AR expression levels along the progression of this neurodegenerative disease.

Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells.

Adenosine is a purine metabolite that can mediate anti-inflammatory responses in the digestive tract through the A(2A) adenosine receptor (A(2A)AR). We examined the role of this receptor in the control of inflammation in the adoptive transfer model of colitis. Infection of A(2A)AR(-/-) mice with Helicobacter hepaticus increased colonic inflammation scores compared with uninfected A(2A)AR controls. Comparison of T cell subsets in wild-type and A(2A)AR(-/-) mice revealed differences in markers associated with activated helper T (Th) cells and regulatory T (Treg) cells. Previous studies showed that expression of A(2A)AR on CD45RB(HI) and CD45RB(LO) Th cells is essential for the proper regulation of colonic inflammation. Adoptive transfer of CD45RB(HI) with CD45RB(LO) from wild-type mice into RAG1(-/-)/A(2A)AR(-/-) mice induced severe disease within 3 wk, although transfer of the same subsets into RAG1(-/-) mice does not induce colitis. This suggests that the presence of A(2A)AR on recipient cells is also important for controlling colitis. To investigate the role of A(2A)AR in myeloid cells, chimeric recipients were generated by injection of bone marrow from RAG1(-/-) or RAG1(-/-)/A(2A)AR(-/-) mice into irradiated RAG1(-/-) mice. After adoptive transfer, these recipients did not develop colitis, regardless of A(2A)AR expression by the donor. Together, our results suggest that the control of inflammation in vivo is dependent on A(2A)AR signaling through multiple cell types that collaborate in the regulation of colitis by responding to extracellular adenosine.

Different Adenosine A2A Receptor Expression in Peripheral Cells from Elderly Patients with Vascular Dementia and Alzheimer's Disease

The line between vascular dementia (VaD) and Alzheimer's disease (AD) is often blurred. In this study we investigated whether adenosine A2A receptor (A2AR) expression can be used to differentiate between VaD and AD. We evaluated the expression of this receptor in the peripheral blood mononuclear cells of patients with VaD, mild cognitive impairment, AD, and controls. We found statistically significant lower levels of A2AR mRNA in VaD compared to AD subjects. These data suggest that A2AR expression may help in the differential diagnosis between VaD and AD.

NF-κB enhances hypoxia-driven T-cell immunosuppression via upregulation of adenosine A2A receptors

Hypoxia affects inflammation by modulating T-cell activation via the adenosinergic system. We supposed that, in turn, inflammation influences cell hypoxic behavior and that stimulation of T-cells in inflammatory conditions involves the concerted action of the nuclear factor κB (NF-κB) and the related hypoxia-inducible factor 1α (HIF-1α) on the adenosinergic system. We addressed this hypothesis by monitoring both transcription factors and four adenosinergic signaling parameters – namely adenosine, adenosine deaminase (ADA), adenosine A2A receptor (A2AR) and cAMP – in T-cells stimulated using phorbol myristate acetate and phytohemagglutinin and submitted to hypoxic conditions which were mimicked using CoCl2 treatment. We found that cell viability was more altered in stimulated than in resting cells under hypoxia. Detailed analysis showed that: i) NF-κB activation remained at basal level in resting hypoxic cells but greatly increased following stimulation, stimulated hypoxic cells exhibiting the higher level; ii) HIF-1α production induced by hypoxia was boosted via NF-κB activation in stimulated cells whereas hypoxia increased HIF-1α production in resting cells without further activating NF-κB; iii) A2AR expression and cAMP production increased in stimulated hypoxic cells whereas adenosine level remained unchanged due to ADA regulation; and iv) the presence of H2S, an endogenous signaling molecule in inflammation, reversed the effect of stimulation on cell viability by down-regulating the activity of transcription factors and adenosinergic immunosuppression. We also found that: i) the specific A2AR agonist CGS-21680 increased the suppressive effect of hypoxia on stimulated T-cells, the antagonist ZM-241385 exhibiting the opposite effect; and ii) Rolipram, a selective inhibitor of cAMP-specific phosphodiesterase 4, and 8-Br-cAMP, a cAMP analog which preferentially activates cAMP-dependent protein kinase A (PKA), increased T-cell immunosuppression whereas H-89, a potent and selective inhibitor of cAMP-dependent PKA, restored cell viability. Together, these data indicate that inflammation enhances T-cell sensitivity to hypoxia via NF-κB activation. This process upregulates A2AR expression and enhances cAMP production and PKA activation, resulting in adenosinergic T-cell immunosuppression that can be modulated via H2S.

Therapeutic implications of estrogen for cerebral vasospasm and delayed cerebral ischemia induced by aneurysmal subarachnoid hemorrhage.

Cerebral vasospasm (CV) remains the leading cause of delayed morbidity and mortality following aneurysmal subarachnoid hemorrhage (SAH). However, increasing evidence supports etiologies of delayed cerebral ischemia (DCI) other than CV. Estrogen, specifically 17β-estradiol (E2), has potential therapeutic implications for ameliorating the delayed neurological deterioration which follows aneurysmal SAH. We review the causes of CV and DCI and examine the evidence for E2-mediated vasodilation and neuroprotection. E2 potentiates vasodilation by activating endothelial nitric oxide synthase (eNOS), preventing increased inducible NOS (iNOS) activity caused by SAH, and decreasing endothelin-1 production. E2 provides neuroprotection by increasing thioredoxin expression, decreasing c-Jun N-terminal kinase activity, increasing neuroglobin levels, preventing SAH-induced suppression of the Akt signaling pathway, and upregulating the expression of adenosine A2a receptor. The net effect of E2 modulation of these various effectors is the promotion of neuronal survival, inhibition of apoptosis, and decreased oxidative damage and inflammation. E2 is a potentially potent therapeutic tool for improving outcomes related to post-SAH CV and DCI. However, clinical evidence supporting its benefits remains lacking. Given the promising preclinical data available, further studies utilizing E2 for the treatment of patients with ruptured intracranial aneurysms appear warranted.

Chapter Five - Adenosine Receptors and Dyskinesia in Pathophysiology

First, the recent progress in the pathogenesis of levodopa-induced dyskinesia was described. Serotonin neurons play an important role in conversion from levodopa to dopamine and in the release of converted dopamine into the striatum in the Parkinsonian state. Since serotonin neurons lack buffering effects on synaptic dopamine concentration, the synaptic dopamine markedly fluctuates depending on the fluctuating levodopa concentration in the serum after taking levodopa. The resultant pulsatile stimulation makes the striatal direct-pathway neurons get potential that releases excessive GABA into the output nuclei of the basal ganglia. When levodopa is administered, the stored GABA is released, the output nuclei become hypoactive, and then dyskinesias emerge. Second, effects of adenosine A2A receptor antagonists on dyskinesia were described. It has been demonstrated that the expression of adenosine A2A receptors is increased in Parkinson's disease (PD) patients with dyskinesias, suggesting that blockade of A2A receptors is beneficial for dyskinesias. Preclinical studies have shown that A2A receptor antagonists reduce liability of dyskinesias in PD models. Clinical trials have demonstrated that A2A antagonists increase functional ON-time (ON without troublesome dyskinesia) in PD patients suffering from wearing-off phenomenon, although they may increase dyskinesia in patients with advanced PD.

Adenosine A2A receptor and retinal pathological neovascularization in mice

Objective To investigate the role of adenosine A2A receptor plays in retinal pathological neovascularization in mice.Methods A total of 202 mice were divided into room-air group (n =66) and oxygen induced retinopathy (OIR) group (n=136).Among room-air group,there were 18 A2A knock-out (KO) mice (KO subgroup) and 24 C57BL/6 mice as wide type (wide type subgroup).OIR group were divided into OIR control subgroup (n =48),A2A-OIR subgroup (n =24) and Caffeine-OIR subgroup (n =64).The retinal neovascularization of OIR group was induced by oxygen.The pathological neovascularization was determined by retinal sections.Fluorescent quantitative polymerase chain reaction (PCR) was used to measure the mRNA expression of A2A and vascular endothelial growth factor (VEGF).0.1,0.3,1.0 g/L Caffeine was dissolve in drinking water of lactating females in Caffeine-OIR subgroup,non-perfusion areas of retina in mice at the age of 0-17,0-7,7-17,7-12,and 12-17 days were analyzed in different dosage and when the dosage as 1.0 g/L.Results Compared with OIR control subgroup,the retinal non-perfusion areas and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in A2A-OIR subgroup were reduced significantly (t =7.694,7.747; P ＜0.001).Compared with wide type subgroup,the level of A2A and VEGF mRNA in OIR control subgroup increased significantly (t=4.036,2.230; P＜0.05).Compared with OIR control subgroup,the level of VEGF mRNA in A2A-OIR subgroup decreased significantly (t=3.122,P＜0.01).Compared with OIR control subgroup,the retinal non perfusion areas in mice at the dosage of 0.1 and 1.0 g/L (t=2.397,4.533) and at the age of 0-17,0-7 days when the dosage as 1.0 g/L (t=4.070,2.399) were reduced significantly (P＜0.05).Conclusions The expression of adenosine A2A receptor increases in oxygeninduced retinal pathological neovascularization..Adenosine A2A receptor may regulate the expression of VEGF.A2A receptor inactivation can inhibit oxygen-induced retinal pathological neovascularization. Key words: Retinal neovascularization/physiopathology; Receptor, adenosine A2A; Caffeine

NF-κB Activation Mediates Induction Of Anti-Inflammatory Adenosine A2A Receptors In iNKT Cells Of Sickle Cell Patients During Vaso-Occlusive Episodes and Upon Activation Of Cultured Human iNKT Cells

iNKT cells are activated in mice and people with vaso-occlusive sickle cell disease (SCD) and play a pivotal role in initiating an inflammatory cascade. In mice, iNKT cell activation requires NF-κB signaling [Stanic, AK et al. J Immunol. 172:4667, 2004]. In mice with SCD, lung inflammation is associated with induction in iNKT cells of IFN-γ and anti-inflammatory adenosine A2A receptor (A2AR) mRNAs [Wallace, KL et al. Blood 116:5010, 2010]. The A2AR is one of four G protein coupled adenosine receptors (A1, A2A, A2B and A3) and it is the most abundant adenosine receptor expressed in T cells. In patients with SCD, painful vaso-occlusive crisis (pVOC) is associated with NF-κB activation and increased expression of A2AR immunoreactivity detected with the monoclonal Ab, 7F6-G5-A2, in circulating iNKT cells. Infusion of a moderate dose (6 nM) of the A2AR agonist, regadenoson during pVOC reduced iNKT cell activation over time [Field, J. et al. Blood 121:3329, 2013]. Since A2AR activation inhibits iNKT cell activation, we reasoned that iNKT cell activation during pVOC might occur preferentially in iNKT cells with low A2AR expression. However, in the current study we show that activated human Vα24+ CD4+ iNKT cells concordantly (i.e. on the same cells) expressed Ser-536 phosphorylated/activated NF-κB (p-NFκB), cytokines and A2ARs. These findings suggest that NF-κB signaling increases A2AR expression as a counter-regulatory mechanism to render activated iNKT cells highly sensitive to inhibition by A2AR activation. Spare receptor theory predicts that enhanced receptor expression will increase the functional potency of agonists. To test this hypothesis we infused into stable SCD patients at steady state a low dose of regadenoson (2 nM) well below its receptor binding KD that was measured by competition for radioligand binding to be 12 nM. Low-dose regadenoson infusion reduced the percentage of p-NF-κB-high/activated iNKT cells by an average of 69% over 12 hours (P = 0.005). These findings suggest that A2AR induction is a counter-regulatory mechanism that limits iNKT cell activation by increasing the sensitivity of activated iNKT cells to inhibition by adenosine. In order to further test the hypothesis that NF-κB activation stimulates A2AR transcription, we examined cultured human iNKT cells. Blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of cytokine and A2AR mRNA and protein upon iNKT activation by plate-bound anti-CD3 antibodies. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells serves as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. We conclude that A2AR agonists have therapeutic potential for treating inflammatory diseases associated with iNKT cell activation, including SCD. Disclosures:Off Label Use: Ragadenoson is used to inhibit activation of iNKT cells.

Extracellular adenosine regulates naive T cell development and peripheral maintenance

Adenosine produced as a byproduct of metabolic activity is present in all tissues and produces dose-dependent suppression of TCR signaling. Naive T cell maintenance depends on inhibition of TCR signals by environmental sensors, which are yet to be fully defined. We produced mice with a floxed adenosine A2A receptor (A2AR) gene, Adora2a, and show that either global A2AR deletion or cre-mediated T cell deletion elicits a decline in the number of naive but not memory T cells. A2AR signaling maintains naive T cells in a quiescent state by inhibiting TCR-induced activation of the phosphatidylinositide 3-kinase (PI3K)-AKT pathway, thereby reducing IL-7Rα down-regulation and naive T cell apoptosis. Patterns of IL-7Rα expression on T cells in chimeric mice reconstituted with Adora2a(+/+) and Adora2a(-/-) bone marrow cells suggest that decreased IL-7Rα in naive T cells is a cell-intrinsic consequence of Adora2a deletion. In addition, A2AR expression increases in early thymic T cell development and contributes to progression of double-negative thymic precursors to single-positive thymocytes with increased IL-7Rα expression. Therefore, A2AR signaling regulates T cell development and maintenance to sustain normal numbers of naive T cells in the periphery.

Purinergic signaling gene network expression in bovine polymorphonuclear neutrophils during the peripartal period

An effective immune response relies on efficient activation of polymorphonuclear neutrophilic leukocytes (PMNL). The PMNL release cellular ATP in response to inflammatory mediators. Although extracellular ATP is rapidly degraded to adenosine, both compounds can readily bind to either the purinergic receptor P1 (adenosine) or P2 (ATP). The P1 and P2 receptors are members of the G-protein-coupled receptor family. The peripartal period is characterized by marked changes in metabolic and inflammatory status that are functionally related with immune responses in the cow. We evaluated the mRNA expression of genes associated with purinergic signaling in PMNL during the peripartal period. Seven multiparous Holstein cows were dried off at d −50 relative to expected parturition and fed a controlled-energy diet (net energy for lactation=1.24Mcal/kg of dry matter) for ad libitum intake during the entire dry period. After calving, all cows were fed a common lactation diet (net energy for lactation=1.65Mcal/kg of dry matter) until 30d in milk. Blood PMNL collected at −10, 3, and 21d in milk were used to study the expression of 22 genes associated with adhesion to endothelium, chemoattractant binding at the plasma membrane, and purinergic signaling. Other blood samples around calving were used to analyze concentrations of insulin, metabolites, and whole-blood phagocytosis. The expression of purinergic receptor P2Y, G-protein coupled, 2 (P2RY2) increased on d 3 and then decreased on d 21. This response suggested that ATP could play a role in the amplification of chemotactic signals. In contrast, the expression of genes encoding cell adhesion [selectin L (SELL) and selectin P ligand (SELPLG)], chemoattractant receptors [complement component 5a receptor 1 (C5AR1), IL-8 receptor α (CXCR1), IL-8 receptor β (CXCR2), and platelet-activating factor receptor (PTAFR)], and adenosine receptors [adenosine A1 receptor (ADORA1) and adenosine A3 receptor (ADORA3)] decreased between −10 and 3d. The decrease coincided with a marked increase in blood nonesterified fatty acids and hydroxybutyrate concentrations, and a decrease in glucose and insulin concentrations. The increase in metabolites also was associated with greater expression of leukotriene B4 receptor (LTB4R) on d 3 and 21 compared with d −10, which is involved in inflammatory prostaglandin synthesis. Most chemoattractant receptors increased by 21d, but cell adhesion genes and blood leukocyte phagocytosis was lower. The expression of adenosine A2a receptor (ADORA2A), which is associated with immunosuppression of PMNL and that of adenosine uptake channels [solute carrier family 29 (nucleoside transporters), member 1 (SLC29A1) and member 2 (SLC29A2)] and the nucleotidase adenosine deaminase (ADA) was greater at 3 and 21d compared with −10d. The reduction in key immune responses, such as cell adhesion and chemotaxis, by bovine PMNL could partly be a function of changes in mRNA expression of genes associated with purinergic signaling.

The Presence of Adenosine A2a Receptor in Thyrocytes and Its Involvement in Graves' IgG-Induced VEGF Expression

Goitrogenesis in Graves' disease (GD) has been attributed to anti-TSH receptor antibody stimulation. Recently, a role for adenosine A2a receptor (A2aR) in goiter formation was reported in the thyroglobulin-A2aR transgenic mice. However, it is unclear whether A2aR is expressed in the thyroid and whether it is associated with the pathogenesis of goiter in GD. Here, we confirmed the expression of A2aR in FRTL-5 cells, primary normal human thyrocytes (both sexes were used without regard to sex), and thyroid tissue (both sexes were used without regard to sex) by PCR, Western blotting, immunohistochemistry, and immunofluorescence. After treatments with A2aR-specific agonist 2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine or GD IgG, the mRNA and protein levels of vascular endothelial growth factor (VEGF), a growth factor related to goitrogenesis, were evaluated along with upstream signaling pathways. A2aR activation and GD IgG promoted the expression of VEGF in thyrocytes, which was accompanied by the activation of cAMP/protein kinase A/phosphorylated-cAMP-response element-binding protein, peroxisome proliferator-activated receptor γ coactivator-1α, and hypoxia-inducible factor-1α. The changes induced by GD IgG were partially abrogated by A2aR small interfering RNA and an A2aR antagonist. These results were supported by data on the goiter samples from the thyrotropin receptor adenovirus-induced GD mouse model (female). These data demonstrate that GD IgG could up-regulate the VEGF expression through A2aR, indicating a potential mechanism for goitrogenesis in GD.

A feedback loop in PPARγ–adenosine A2A receptor signaling inhibits inflammation and attenuates lung damages in a mouse model of LPS-induced acute lung injury

Although peroxisome proliferator-activated receptor-γ (PPARγ) and adenosine A2A receptor (A2AR) are reported to be anti-inflammatory factors in acute lung injury (ALI), their internal link and synergic or antagonistic effect after activation are poorly understood. Here, we found that PPARγ and A2AR could upregulate the mRNA and protein expressions of each other in lung tissues of LPS-induced mouse ALI model and murine macrophages. Further investigation demonstrated that PPARγ upregulated A2AR expression by directly binding to a DR10 response element (−218 to −197) within A2AR gene promoter region. Instead of directly interacting with PPARγ, A2AR stimulated PPARγ expression via protein kinase A (PKA)–cAMP response element binding protein (CREB) signaling by provoking the binding of CREB to a cAMP responsive element (CRE)-like site in PPARγ gene promoter region. In addition, combination of PPARγ and A2AR agonists was found to exert obviously better effect on suppressing neutrophil infiltration and inflammatory cytokine expressions, attenuating lung edema, pathological changes and improving lung function of blood gas exchange than their single application. These findings reveal a novel functional positive feedback loop between PPARγ and A2AR signaling to potentialize their effect on inhibiting inflammation and attenuating lung damages in ALI. It suggests that targeting this PPARγ–A2AR signaling rather than PPARγ or A2AR alone may be a more attractive and efficient potential therapeutic strategy for ALI.