Extracellular Adenosine Levels Research Articles

ADPORT-601: First-in-human study of adenosine 2A (A2A) and adenosine 2B (A2B) receptor antagonists in patients with select advanced solid tumors.

e14681 Background: Hypoxic tumors possess high levels of extracellular adenosine within the tumor microenvironment (TME) which suppresses both innate and adaptive immune responses by binding the adenosine receptors A2AR and A2BR. TT-10 (PORT-6) and TT-4 (PORT-7) are potent and selective antagonists of A2AR and A2BR, respectively. Both agents have shown monotherapy activity in both non-immunogenic (4T-1) and immunogenic (CT-26) tumor mouse models. Optimizing inhibition of A2AR and A2BR separately and in combination provides an opportunity to dissect the role of each signaling pathway within the TME. Methods: ADPORT-601 is a multi-center, open-label Phase 1 study, evaluating the safety, efficacy, PK and pharmacodynamics of PORT-6 in participants with mCRPC, RCC, or other selected solid tumors which are refractory to standard therapy. A subsequent arm will similarly test PORT-7 in participants with selected solid tumors. The Phase 1a cohorts utilize a standard 3+3 design. Phase 1b will be initiated once recommended doses have been established and will consist of monotherapy expansion cohorts for each drug, as well as both drugs combined at the optimal doses, and with other immunotherapy agents. A novel IHC-based assay will be implemented to select for patients with high A2AR expression. Metabolomic and gene expression profiling were performed on previously acquired bone biopsy and blood samples, respectively, to characterize the role of adenosine in CRPC. Results: 10 patients with mCRPC (n=6), RCC (n=3) or NSCLC (n=1) have been enrolled at escalating doses. Treatment-related AEs have been G1-2, with fatigue (29%), nausea (29%) and vomiting (14%) being the most reported. No related SAEs or dose limiting toxicities (DLTs) have been observed. Higher adenosine precursor metabolite (e.g., AMP, ADP, ATP, ADPR, inosine) levels were observed in image-guide bone metastatic lesions relative to non-tumor bearing bone controls (P<0.05; n=10/group). A2AR and A2BR mRNA levels were enriched (1.3 and 1.4 log2 fold change, p<0.05, respectively) in the plasma of mCRPC patients compared to those with localized disease. Updated safety, efficacy, PK and adenosine expression data will be reported at the meeting. Conclusions: PORT-6 is well tolerated to date. The TME within mCRPC bone metastases provides a unique target for A2AR and A2BR antagonism. Longitudinal blood and tissue samples, including bone biopsies, are being collected to enable biomarker analyses, allowing for elucidation of the relative contribution of A2AR and A2BR in suppressing anti-tumor immunity. Multi-omic translational analyses of tissue are planned to determine whether potent adenosine receptor antagonism will have an anti-tumor effect and convert the TME from immunosuppressive to proinflammatory. Clinical trial information: NCT04969315 .

Agrin-Lrp4 pathway in hippocampal astrocytes restrains development of temporal lobe epilepsy through adenosine signaling

BackgroundHuman patients often experience an episode of serious seizure activity, such as status epilepticus (SE), prior to the onset of temporal lobe epilepsy (TLE), suggesting that SE can trigger the development of epilepsy. Yet, the underlying mechanisms are not fully understood. The low-density lipoprotein receptor related protein (Lrp4), a receptor for proteoglycan-agrin, has been indicated to modulate seizure susceptibility. However, whether agrin-Lrp4 pathway also plays a role in the development of SE-induced TLE is not clear.MethodsLrp4f/f mice were crossed with hGFAP-Cre and Nex-Cre mice to generate brain conditional Lrp4 knockout mice (hGFAP-Lrp4−/−) and pyramidal neuron specific knockout mice (Nex-Lrp4−/−). Lrp4 was specifically knocked down in hippocampal astrocytes by injecting AAV virus carrying hGFAP-Cre into the hippocampus. The effects of agrin-Lrp4 pathway on the development of SE-induced TLE were evaluated on the chronic seizure model generated by injecting kainic acid (KA) into the amygdala. The spontaneous recurrent seizures (SRS) in mice were video monitored.ResultsWe found that Lrp4 deletion from the brain but not from the pyramidal neurons elevated the seizure threshold and reduced SRS numbers, with no change in the stage or duration of SRS. More importantly, knockdown of Lrp4 in the hippocampal astrocytes after SE induction decreased SRS numbers. In accord, direct injection of agrin into the lateral ventricle of control mice but not mice with Lrp4 deletion in hippocampal astrocytes also increased the SRS numbers. These results indicate a promoting effect of agrin-Lrp4 signaling in hippocampal astrocytes on the development of SE-induced TLE. Last, we observed that knockdown of Lrp4 in hippocampal astrocytes increased the extracellular adenosine levels in the hippocampus 2 weeks after SE induction. Blockade of adenosine A1 receptor in the hippocampus by DPCPX after SE induction diminished the effects of Lrp4 on the development of SE-induced TLE.ConclusionThese results demonstrate a promoting role of agrin-Lrp4 signaling in hippocampal astrocytes in the development of SE-induced development of epilepsy through elevating adenosine levels. Targeting agrin-Lrp4 signaling may serve as a potential therapeutic intervention strategy to treat TLE.

CD73 polymorphisms are associated with schizophrenia.

Ecto-5'-nucleotidase/CD73 enzyme plays a key role in the regulation of extracellular adenosine levels, thereby exerting influence on adenosine homeostasis. Emerging evidence suggests that perturbations in purines and ecto-5'-nucleotidase activity are associated with an augmented susceptibility to schizophrenia. However, the precise impact of genetic variations in CD73 on individuals with schizophrenia remains poorly understood. Here, our study demonstrated that rs3734442 allele and rs4431401 heterozygote were conferred a significant risk of schizophrenia disease (rs3734442: odds ratio, 0.556; 95% CI, 0.375 to 0.825; p = 0.004; rs4431401: odds ratio, 1.881, 95% CI, 1.117 to 3.166; p = 0.020). Comparing different genders, we observed a significant association between rs3734442 genotypes and male cases (rs3734442: odds ratio, 0.452; 95% CI, 0.257 to 0.796; p = 0.007). Likewise, there was a significant association between rs4431401 genotypes and male patients (rs4431401: odds ratio, 2.570; 95% CI, 1.196 to 5.522; p = 0.015). Based on family history and antipsychotics medication usage, our data reveals that the rs9444348 allele exhibits the most significant association with familial susceptibility to schizophrenia (odds ratio, 1.541; 95% CI, 1.009 to 2.353; p = 0.048 for A vs G). Moreover, individuals carrying variants of rs6922, rs2229523, and rs2065114 while being treated with clozapine demonstrate a higher frequency proportion compared to those receiving risperidone treatment (p = 0.035; p = 0.049; p = 0.027 respectively). Additionally, our results indicate that patients with GG genotype of rs9444348 had significantly higher likelihood of using clozapine instead of sulpiride (p = 0.048). Overall, our data strongly suggest that genetic variations in CD73 are significantly associated with schizophrenia risk and may serve as valuable resources for identifying therapeutic targets.

Exogenous Ketone Supplement Administration Abrogated Isoflurane-Anesthesia-Induced Increase in Blood Glucose Level in Female WAG/Rij Rats.

It has been demonstrated that isoflurane-induced anesthesia can increase the blood glucose level, leading to hyperglycemia and several adverse effects. The administration of a mix of ketone diester (KE) and medium-chain triglyceride (MCT) oil, named KEMCT, abolished the isoflurane-anesthesia-induced increase in blood glucose level and prolonged the recovery time from isoflurane anesthesia in a male preclinical rodent model, Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. While most preclinical studies use exclusively male animals, our previous study on blood glucose changes in response to KEMCT administration showed that the results can be sex-dependent. Thus, in this study, we investigated female WAG/Rij rats, whether KEMCT gavage (3 g/kg/day for 7 days) can change the isoflurane (3%)-anesthesia-induced increase in blood glucose level and the recovery time from isoflurane-evoked anesthesia using the righting reflex. Moreover, KEMCT-induced ketosis may enhance both the extracellular level of adenosine and the activity of adenosine A1 receptors (A1Rs). To obtain information on the putative A1R mechanism of action, the effects of an A1R antagonist, DPCPX (1,3-dipropyl-8-cyclopentylxanthine; intraperitoneal/i.p. 0.2 mg/kg), on KEMCT-generated influences were also investigated. Our results show that KEMCT supplementation abolished the isoflurane-anesthesia-induced increase in blood glucose level, and this was abrogated by the co-administration of DPCPX. Nevertheless, KEMCT gavage did not change the recovery time from isoflurane-induced anesthesia. We can conclude that intragastric gavage of exogenous ketone supplements (EKSs), such as KEMCT, can abolish the isoflurane-anesthesia-induced increase in blood glucose level in both sexes likely through A1Rs in WAG/Rij rats, while recovery time was not affected in females, unlike in males. These results suggest that the administration of EKSs as an adjuvant therapy may be effective in mitigating metabolic side effects of isoflurane, such as hyperglycemia, in both sexes.

The Impact of A3AR Antagonism on the Differential Expression of Chemoresistance-Related Genes in Glioblastoma Stem-like Cells.

Glioblastoma (GB) is the most aggressive and common primary malignant tumor of the brain and central nervous system. Without treatment, the average patient survival time is about six months, which can be extended to fifteen months with multimodal therapies. The chemoresistance observed in GB is, in part, attributed to the presence of a subpopulation of glioblastoma-like stem cells (GSCs) that are characterized by heightened tumorigenic capacity and chemoresistance. GSCs are situated in hypoxic tumor niches, where they sustain and promote the stem-like phenotype and have also been correlated with high chemoresistance. GSCs have the particularity of generating high levels of extracellular adenosine (ADO), which causes the activation of the A3 adenosine receptor (A3AR) with a consequent increase in the expression and activity of genes related to chemoresistance. Therefore, targeting its components is a promising alternative for treating GB. This analysis determined genes that were up- and downregulated due to A3AR blockades under both normoxic and hypoxic conditions. In addition, possible candidates associated with chemoresistance that were positively regulated by hypoxia and negatively regulated by A3AR blockades in the same condition were analyzed. We detected three potential candidate genes that were regulated by the A3AR antagonist MRS1220 under hypoxic conditions: LIMD1, TRIB2, and TGFB1. Finally, the selected markers were correlated with hypoxia-inducible genes and with the expression of adenosine-producing ectonucleotidases. In conclusion, we detected that hypoxic conditions generate extensive differential gene expression in GSCs, increasing the expression of genes associated with chemoresistance. Furthermore, we observed that MRS1220 could regulate the expression of LIMD1, TRIB2, and TGFB1, which are involved in chemoresistance and correlate with a poor prognosis, hypoxia, and purinergic signaling.

The impact of purine nucleosides on neuroplasticity in the adult brain.

Neuroplasticity refers to the nervous system's ability to adapt and reorganize its cell structures and neuronal networks in response to internal and external stimuli. In adults, this process involves neurogenesis, synaptogenesis, and synaptic and neurochemical plasticity. Several studies have reported the significant impact of the purinergic system on neuroplasticity modulation. And, there is considerable evidence supporting the role of purine nucleosides, such as adenosine, inosine, and guanosine, in this process. This review presents extensive research on how these nucleosides enhance the neuroplasticity of the adult central nervous system, particularly in response to damage. The mechanisms through which these nucleosides exert their effects involve complex interactions with various receptors and signaling pathways. Adenosine's influence on neurogenesis involves interactions with adenosine receptors, specifically A1R and A2AR. A1R activation appears to inhibit neuronal differentiation and promote astrogliogenesis, while A2AR activation supports neurogenesis, neuritogenesis, and synaptic plasticity. Inosine and guanosine positively impact cell proliferation, neurogenesis, and neuritogenesis. Inosine seems to modulate extracellular adenosine levels, and guanosine might act through interactions between purinergic and glutamatergic systems. Additionally, the review discusses the potential therapeutic implications of purinergic signaling in neurodegenerative and neuropsychiatric diseases, emphasizing the importance of these nucleosides in the neuroplasticity of brain function and recovery.

40 Hz light flickering promotes sleep through cortical adenosine signaling

Flickering light stimulation has emerged as a promising non-invasive neuromodulation strategy to alleviate neuropsychiatric disorders. However, the lack of a neurochemical underpinning has hampered its therapeutic development. Here, we demonstrate that light flickering triggered an immediate and sustained increase (up to 3 h after flickering) in extracellular adenosine levels in the primary visual cortex (V1) and other brain regions, as a function of light frequency and intensity, with maximal effects observed at 40 Hz frequency and 4000 lux. We uncovered cortical (glutamatergic and GABAergic) neurons, rather than astrocytes, as the cellular source, the intracellular adenosine generation from AMPK-associated energy metabolism pathways (but not SAM-transmethylation or salvage purine pathways), and adenosine efflux mediated by equilibrative nucleoside transporter-2 (ENT2) as the molecular pathway responsible for extracellular adenosine generation. Importantly, 40 Hz (but not 20 and 80 Hz) light flickering for 30 min enhanced non-rapid eye movement (non-REM) and REM sleep for 2–3 h in mice. This somnogenic effect was abolished by ablation of V1 (but not superior colliculus) neurons and by genetic deletion of the gene encoding ENT2 (but not ENT1), but recaptured by chemogenetic inhibition of V1 neurons and by focal infusion of adenosine into V1 in a dose-dependent manner. Lastly, 40 Hz light flickering for 30 min also promoted sleep in children with insomnia by decreasing sleep onset latency, increasing total sleep time, and reducing waking after sleep onset. Collectively, our findings establish the ENT2-mediated adenosine signaling in V1 as the neurochemical basis for 40 Hz flickering-induced sleep and unravel a novel and non-invasive treatment for insomnia, a condition that affects 20% of the world population.

ATP-adenosine axis regulation combined with microneedle assisted photoimmunotherapy to boost the immunotherapy efficiency

Immunogenic cell death (ICD) is associated with the release of damage-associated molecular patterns, including ATP, to promote an effective immune cycle against tumors. However, tumors have evolved an effective strategy for degrading extracellular immunostimulatory ATP via the ATP-adenosine axis, allowing the sequential action of the ectonucleotidases CD39 to degrade accumulated immunostimulatory ATP into pleiotropic immunosuppressive adenosine. Here, an ingenious dissolving microneedle patch (DMNs) is designed for the intralesional delivery of CD39 inhibitor (sodium polyoxotungstate, POM-1) and ICD inducer (IR780) co-encapsulated solid lipid nanoparticles (P/I SLNs) for antitumor therapy. Upon insertion into the tumor site, IR780 induces ICD modalities with the release of damage-associated molecular patterns from endogenous tissues, which activates the antitumor immune cycle. Simultaneously, POM-1 promotes the liberation of immunostimulatory ATP and lowers the level of immunosuppressive extracellular adenosine, which supported immune control of tumors via recruiting CD39-expressing immune cells. In vivo antitumor studies prove that this platform can effectively eliminate mice melanoma (tumor growth inhibitory rate of 96.5%) and colorectal adenocarcinoma (tumor growth inhibitory rate of 93.5%). Our results shed light on the immunological aspects of combinatorial phototherapy and ATP-adenosine regulation, which will broaden the scope of synergistic antitumor immunotherapy.

Caffeine-folic acid-loaded-chitosan nanoparticles combined with methotrexate as a novel HepG2 immunotherapy targeting adenosine A2A receptor downstream cascade

BackgroundMethotrexate (MTX) is a common chemotherapeutic drug that inhibits DNA synthesis and induces apoptosis. Treatment with MTX increased CD73 expression, which leads to higher levels of extracellular adenosine. Adenosine levels are also high in the tumor microenvironment through Cancer cells metabolism. That promotes the survival of cancer cells and contributes to tumor immune evasion through the Adenosine 2a Receptor. A2A receptor antagonists are an emerging class of agents that treat cancers by enhancing immunotherapy, both as monotherapy and in combination with other therapeutic agents. Caffeine is an adenosine receptor antagonist. Herein, we demonstrate the ability of a novel well prepared and characterized nano formula CAF-FA-CS-NPs (D4) for A2aR blockade when combination with MTX to improve its antitumor efficacy by enhancing the immune system and eliminating immune suppression.MethodsCAF-FA-CS-NPs (D4) were prepared and characterized for particle size, loading efficiency, and release profile. Molecular docking was used to validate the binding affinity of caffeine and folic acid to A2A receptor. The effects of the nano formula were evaluated on human liver cancer cells (HepG2), breast cancer cells (MCF-7), and MDA-MB-231, as well as normal human cells (WI-38). Different combination ratios of MTX and D4 were studied to identify the optimal combination for further genetic studies.ResultsMolecular docking results validated that caffeine and folic acid have binding affinity to A2A receptor. The CS-NPs were successfully prepared using ionic gelation method, with caffeine and folic acid being loaded and conjugated to the nanoparticles through electrostatic interactions. The CAF loading capacity in D4 was 77.9 ± 4.37% with an encapsulation efficiency of 98.5 ± 0.37. The particle size was optimized through ratio variations. The resulting nanoparticles were fully characterized. The results showed that (D4) had antioxidant activity and cytotoxicity against different cancer cells. The combination of D4 with MTX (IC50 D4 + 0.5 IC50 MTX) resulted in the downregulation of Bcl-2, FOXP3, CD39, and CD73 gene expression levels and upregulation of Bax and A2AR gene expression levels in HepG2 cells.ConclusionsThis study suggests that CAF-FA-CS-NPs (D4) in combination with MTX may be a promising candidate for cancer immunotherapy, by inhibiting A2aR signaling and leading to improved immune activation and anti-tumor activity of MTX.

Defective insulin-stimulated equilibrative nucleoside transporter-2 activity and altered subcellular transporter distribution drive the loss of adenosine homeostasis in diabetic kidney disease progression

AimProgression of diabetic nephropathy (DN) is linked to the dysregulated increase of adenosine and altered signaling properties. A major contribution to the maintenance of physiological extracellular adenosine levels relies on cellular uptake activity through plasma membrane nucleoside transporters. Because kidney cells are responsive to insulin, this study aims to determine how DN affects insulin regulation of the equilibrative nucleoside transporter-2 (ENT2). MethodsHuman Podocytes and rat glomeruli were used to study ENT2 regulation. The effects of diabetes and insulin on ENT2 mediated transport activity were determined measuring the fraction of total adenosine uptake in sodium-free medium which is inhibitable by hypoxanthine. Alterations in ENT2 subcellular distribution were assessed in the kidney of people affected with DN and diabetic rats. The consequences of impaired ENT2 activity on the kidney were evaluated using dipyridamole in an animal model. ResultsInsulin upregulates ENT2 uptake activity by increasing the Vmax, thus counteracting decreased adenosine uptake due to high d-glucose and achieving extracellular adenosine homeostasis. Insulin promoted ENT2 translocation to the plasma membrane dependent on PI3-kinase/Akt signaling and actin cytoskeleton integrity. However, in diabetic rats, the insulin-mediated induction of ENT2 activity was lost. Additionally, reduced Akt activation in response to insulin correlated with decreased ENT2 distribution at the plasma membrane. Kidney tissues from diabetic rats and human DN biopsies showed ENT2 redistribution to an intracellular pattern, evidencing dysfunctional adenosine uptake. Through ENT inhibition, we evidenced increased proteinuria and induced alpha-smooth muscle actin as a result of profibrotic activation of cells in the kidney. ConclusionDeficient insulin regulation of ENT2 activity contributes to chronically high adenosine levels and glomerular alterations that underline diabetic kidney disease progression.

Novel microRNAs modulating ecto-5'-nucleotidase expression.

The expression of immune checkpoint molecules (ICMs) by cancer cells is known to counteract tumor-reactive immune responses, thereby promoting tumor immune escape. For example, upregulated expression of ecto-5'-nucleotidase (NT5E), also designated as CD73, increases extracellular levels of immunosuppressive adenosine, which inhibits tumor attack by activated T cells. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Thus, the binding of miRNAs to the 3'-untranslated region of target mRNAs either blocks translation or induces degradation of the targeted mRNA. Cancer cells often exhibit aberrant miRNA expression profiles; hence, tumor-derived miRNAs have been used as biomarkers for early tumor detection. In this study, we screened a human miRNA library and identified miRNAs affecting the expression of ICMs NT5E, ENTPD1, and CD274 in the human tumor cell lines SK-Mel-28 (melanoma) and MDA-MB-231 (breast cancer). Thereby, a set of potential tumor-suppressor miRNAs that decreased ICM expression in these cell lines was defined. Notably, this study also introduces a group of potential oncogenic miRNAs that cause increased ICM expression and presents the possible underlying mechanisms. The results of high-throughput screening of miRNAs affecting NT5E expression were validated in vitro in 12 cell lines of various tumor entities. As result, miR-1285-5p, miR-155-5p, and miR-3134 were found to be the most potent inhibitors of NT5E expression, while miR-134-3p, miR-6859-3p, miR-6514-3p, and miR-224-3p were identified as miRNAs that strongly enhanced NT5E expression levels. The miRNAs identified might have clinical relevance as potential therapeutic agents and biomarkers or therapeutic targets, respectively.

0032 ADA Functional Polymorphism Modulates REM Sleep Following Total Sleep Deprivation

Abstract Introduction Adenosine Deaminase (ADA) regulates extracellular levels of adenosine, a brain correlate of sleep homeostasis. A single nucleotide polymorphism (SNP) of the ADA gene (rs73598374) is known to be associated with increased homeostatic pressure for NREM sleep after total sleep deprivation (TSD). Whether this genotype effect also impacts REM sleep after TSD has not been systematically investigated. The limit cycle reciprocal interaction model posits that NREM/REM cycles are regulated by cholinergic and GABAergic neurotransmitter systems. While NREM/REM regulation is also thought to be mediated by sleep homeostatic state, evidence of an association with adenosine levels has not been reported. Here we investigate the influence of ADA on REM sleep at baseline and following TSD. Methods N=48 healthy, normal sleepers (ages 27.7 + 5.3, 23 females) participated in one of three in-laboratory TSD studies. Following 10h baseline sleep (22:00–08:00), participants underwent 38h TSD, with subsequent 10h recovery sleep (22:00–08:00). Sleep periods were recorded polysomnographically and scored visually according to AASM criteria. Genomic DNA was extracted from whole blood, and the ADA SNP was assayed using real-time PCR. Sleep stages were analyzed using mixed-effect ANOVA with fixed effects of ADA genotype, night (baseline vs. recovery), and their interaction, controlling for study, with a random effect over subject on the intercept. Results The frequency of genotypes (G/G: 42, A/G: 6) did not vary from Hardy-Weinberg equilibrium. There was no genotype effect on total sleep time and no genotype by night interaction (p>0.7). However, A allele carriers spent more time in REM during baseline and less during recovery compared to G/G homozygotes (genotype by night interaction: F[1,46]=8.13, p=0.007). Conversely, while not statistically significant (p>0.1), A allele carriers spent less time in NREM during baseline and more during recovery compared to G/G homozygotes. Conclusion ADA genotype was associated with a shift in balance between REM and NREM at baseline, as well as changes therein after TSD, without concomitant effect on total sleep time. This suggests a role for adenosine in mediating REM sleep regulation, possibly through a sleep homeostatic effect on the reciprocal interaction between NREM and REM. Support (if any) CDMRP W81XWH-05-1-0099; ONR N00014-13-C-0063; NIH R21CA16769; ARO W911NF2210223.

Hypoxia-inducible factor orchestrates adenosine metabolism to promote liver cancer development

Hypoxia-induced adenosine creates an immunosuppressive tumor microenvironment (TME) and dampens the efficacy of immune checkpoint inhibitors (ICIs). We found that hypoxia-inducible factor 1 (HIF-1) orchestrates adenosine efflux through two steps in hepatocellular carcinoma (HCC). First, HIF-1 activates transcriptional repressor MXI1, which inhibits adenosine kinase (ADK), resulting in the failure of adenosine phosphorylation to adenosine monophosphate. This leads to adenosine accumulation in hypoxic cancer cells. Second, HIF-1 transcriptionally activates equilibrative nucleoside transporter 4, pumping adenosine into the interstitial space of HCC, elevating extracellular adenosine levels. Multiple in vitro assays demonstrated the immunosuppressive role of adenosine on T cells and myeloid cells. Knockout of ADK in vivo skewed intratumoral immune cells to protumorigenic and promoted tumor progression. Therapeutically, combination treatment of adenosine receptor antagonists and anti-PD-1 prolonged survival of HCC-bearing mice. We illustrated the dual role of hypoxia in establishing an adenosine-mediated immunosuppressive TME and offered a potential therapeutic approach that synergizes with ICIs in HCC.

Spinal adenosine 2A receptor inhibition restores phrenic long-term facilitation during mild systemic inflammation

Inflammation undermines several forms of neuroplasticity, including phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity elicited by moderate acute intermittent hypoxia (mAIH; 3, 5-min episodes, 5 min intervals; arterial PO2 ~ 40–50 mmHg). Mild inflammation elicited by a low dose of the TLR-4 receptor agonist lipopolysaccharide (LPS; 100 mg/kg, i.p.) abolishes mAIH-induced pLTF by a mechanism that requires spinal p38 MAP kinase and protein phosphatase activity. Neuroinflammation activates microglia and other cells in the CNS, elevating extracellular levels of adenosine—a purine nucleoside known to suppress mAIH-induced pLTF. We hypothesized basal adenosine levels in ventral spinal C 3 -C 5 segments are increased in adult male Sprague-Dawley rats 24 hours post-LPS administration, thereby undermining mAIH-induced pLTF via adenosine 2A receptor (A 2A ) activation. Thus, we predicted that cervical intrathecal administration of the A 2A receptor inhibitor MSX-3 (10 μM, 12 μL) would rescue mAIH-induced pLTF in rats pre-treated with LPS. As predicted, LPS significantly increased basal ventral C 3 -C 5 adenosine levels (p=0.010 vs controls; n=7 per group). In control rats (saline injection, i.p.), MSX-3 enhanced pLTF versus vehicle controls (LPS: 110 ± 16% baseline; controls: 53 ± 6%; p=0.002; each n=6). As expected, mAIH-induced pLTF was abolished in LPS-treated rats (4 ± 6% baseline; n=6). MSX-3 restored mAIH-induced pLTF to levels equivalent to MSX-3 treated control rats (120 ± 14%; p<0.001; n=6; versus LPS controls with MSX-3: p=0.539). Thus, mild LPS-induced inflammation abolishes mAIH-induced pLTF by a mechanism associated with increased spinal adenosine levels and A 2A receptor activation. As we move mAIH forward as a therapeutic modality to improve breathing and non-respiratory movements in people with spinal cord injury or ALS, pretreatment with A 2A receptor inhibitors may offset the undermining effects of systemic inflammation associated with these severe neuromuscular disorders. NIH HL147554, HL148030, T32HL134621 (ABM) and the UF McKnight Brain Institute This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 4135: A novel plasma cell-based mechanism of action of adenosine immunomodulation and A2AR antagonism

Abstract High levels of extracellular adenosine, often found in the tumor microenvironment (TME), promote immune suppression mainly through the A2A receptor (A2AR) expressed by tumor-infiltrating immune cells. Inupadenant (formerly known as EOS100850) is an oral, non-brain penetrant, potent and highly selective small molecule antagonist of A2AR. In a Phase I/Ib clinical trial (NCT02740985), inupadenant as monotherapy showed initial evidence of clinical benefit in subjects with advanced solid tumors. In this study, tumor biopsies with a high number of A2AR-expressing immune cells at baseline were associated with response or stable disease.We now report that infiltration of A2AR+ cells was strongly correlated with the expression of B- and, more specifically, antibody-secreting cell (ASC)-related genes, as assessed by gene expression (Nanostring) and immunohistochemistry (IHC), hinting at a potential novel role for A2AR in B cell biology. Therefore, we explored the expression and function of A2AR in human B cells. Immunocytochemistry staining of A2AR on sorted tonsillar B cell subsets showed that A2AR was predominantly expressed on ASCs, including plasma cells and plasma blasts versus naïve or memory B cells. The preferential expression of A2AR by ASCs was confirmed on NSCLC tissues by multiplex immunofluorescence. In addition to A2AR, ASCs expressed other adenosine pathway markers such as CD39, suggesting that the adenosine pathway is a key mechanism through which ASC functions may be modulated. Using B cells derived from peripheral blood, the A2AR agonist CGS-21680 was shown to inhibit the maturation of B cells into plasma cells, and that maturation could be fully restored by inupadenant. CGS-21680 did not affect B cell or plasma cell viability, indicating that the effect of A2AR signaling on plasma cell differentiation is not due to preferential plasma cell death in culture. Importantly, baseline expression of B cell- and ASC-related markers measured by Nanostring and IHC was associated with response or stable disease to inupadenant monotherapy, supporting the notion that these cells may be a novel target of inupadenant. This is in line with recent reports showing that B cells, plasma cells and tertiary lymphoid structures are associated with favorable responses to cancer immunotherapy. Interestingly, four out of the five non-progressors treated with inupadenant as monotherapy and with available biomarker data showed a reduction in ASC infiltration after inupadenant treatment, suggesting that inupadenant may promote terminal plasma cell differentiation and migration out of the tumor tissue and to the bone marrow.Altogether, these data support a novel plasma cell-centric mechanism of action of inupadenant, which may complement its reported T cell-mediated anti-tumor activity. Citation Format: Chiara Martinoli, Paola Tieppo, Marjorie Mercier, Hussein Shehade, Noemie Wald, Anais Vezzu, Annelise Hermant, Boris Pirlot, Stephanie Ma, Maurizio Ceppi, Yvonne McGrath, Maura Rossetti. A novel plasma cell-based mechanism of action of adenosine immunomodulation and A2AR antagonism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4135.

Adenosine-independent regulation of the sleep–wake cycle by astrocyte activity

Astrocytes play a crucial role in regulating sleep–wake behavior, and adenosine signaling is generally thought to be involved. Here we show multiple lines of evidence supporting that modulation of the sleep–wake behavior by astrocyte Ca2+ activity could occur without adenosine signaling. In the basal forebrain and the brainstem, two brain regions that are known to be essential for sleep–wake regulation, chemogenetically-induced astrocyte Ca2+ elevation significantly modulated the sleep–wake cycle. Although astrocyte Ca2+ level positively correlated with the amount of extracellular adenosine, as revealed by a genetically encoded adenosine sensor, we found no detectable change in adenosine level after suppressing astrocyte Ca2+ elevation, and transgenic mice lacking one of the major extracellular ATP-adenosine conversion enzymes showed similar extracellular adenosine level and astrocyte Ca2+-induced sleep modulation. Furthermore, astrocyte Ca2+ is dependent primarily on local neuronal activity, causing brain region-specific regulation of the sleep–wake cycle. Thus, neural activity-dependent astrocyte activity could regulate the sleep–wake behavior independent of adenosine signaling.

The impact of inosine on hippocampal synaptic transmission and plasticity involves the release of adenosine through equilibrative nucleoside transporters rather than the direct activation of adenosine receptors.

Inosine has robust neuroprotective effects, but it is unclear if inosine acts as direct ligand of adenosine receptors or if it triggers metabolic effects indirectly modifying the activity of adenosine receptors. We now combined radioligand binding studies with electrophysiological recordings in hippocampal slices to test how inosine controls synaptic transmission and plasticity. Inosine was without effect at 30μM and decreased field excitatory post-synaptic potentials by 14% and 33% at 100 and 300μM, respectively. These effects were prevented by the adenosine A1 receptor antagonist DPCPX. Inosine at 300 (but not 100) μM also decreased the magnitude of long-term potentiation (LTP), an effect prevented by DPCPX and by the adenosine A2A receptor antagonist SCH58261. Inosine showed low affinity towards human and rat adenosine receptor subtypes with Ki values of > 300µM; only at the human and rat A1 receptor slightly higher affinities with Ki values of around 100µM were observed. Affinity of inosine at the rat A3 receptor was higher (Ki of 1.37µM), while it showed no interaction with the human orthologue. Notably, the effects of inosine on synaptic transmission and plasticity were abrogated by adenosine deaminase and by inhibiting equilibrative nucleoside transporters (ENT) with dipyridamole and NBTI. This shows that the impact of inosine on hippocampal synaptic transmission and plasticity is not due to a direct activation of adenosine receptors but is instead due to an indirect modification of the tonic activation of these adenosine receptors through an ENT-mediated modification of the extracellular levels of adenosine.

A role for N-glycosylation in active adenosine deaminase 2 production

BackgroundAdenosine deaminase 2 (ADA2) regulates extracellular levels of adenosine and the optimal expression of ADA2 is essential for modulating the immune system. However, the mechanisms regulating the production of active ADA2 enzyme are not fully understood. In this study, we examined the role of N-glycosylation in the formation of functional structures and the secretory pathway of ADA2. MethodsWe investigated the roles of N-glycosylation in the activity, homodimerization, and secretion of ADA2 via site-directed mutagenesis and the application of N-glycosylation inhibitors. Subcellular localization of ADA2 along with the endoplasmic reticulum (ER) glucosidase inhibitor was observed under confocal fluorescence microscope. ResultsInhibiting the initial N-glycosylation of ADA2 in the ER via site-directed mutagenesis or treatment with N-glycosylation inhibitors reduced the intracellular ADA2 activity and secretion. At this time, decreases in the ADA2 homodimers and ADA2 aggregation were observed in the cells. Treating the cells with castanospermine, an inhibitor of N-glycan editing in the ER, resulted in a reduction of the localization rate to the Golgi and markedly suppressed the ADA2 secretion. ConclusionsThese data suggest that the initial N-glycosylation and N-glycan editing in the ER are essential for the production of an active ADA2 enzyme and proper trafficking to the extracellular space. General significanceWith sufficient N-glycosylation in the ER, ADA2 exerts its function and is secreted extracellularly.

The low affinity A2B adenosine receptor enhances migratory and invasive capacity in vitro and angiogenesis in vivo of glioblastoma stem-like cells.

Glioblastoma (GBM) is the most common and deadly malignant brain tumor, with a median survival of 15 to 17 months for a patient. GBM contains a cellular subpopulation known as GBM stem-like cells (GSCs) that persist in hypoxic niches and are capable of infiltrating into healthy brain tissue. For this reason, GSCs are considered one of the main culprits for GBM recurrence. A hypoxic microenvironment increases extracellular adenosine levels, activating the low affinity A2B adenosine receptor (A2BAR). Adenosine, through A2BAR, is capable of modulating invasiveness. However, its role in the invasion/migration of hypoxic-GSCs is still unknown. This study aims to understand the importance of A2BAR in modulating the migratory/invasive capacity of GSCs under hypoxia. Data analysis from The Cancer Genome Atlas (TCGA) program correlates A2BAR expression with high-grade glioma and hypoxic necrotic areas. U87MG and primary culture-derived GSCs under hypoxic conditions (0.5% O2) increased A2BAR mRNA and protein levels. As expected, the migratory and invasive capacity of GSCs increased under hypoxia, which was counteracted by blocking A2BAR, through the downregulation of MMP9 activity and epithelial–mesenchymal transition marker expression. Finally, in a xenograft mouse model, we demonstrate that treatment with MRS1754 did not affect the tumor volume but could decrease blood vessel formation and VEGF expression. Our results suggest that extracellular adenosine, through the activation of A2BAR, enhances the migratory and invasive capacity of GSCs in vitro under hypoxic conditions. Targeting A2BAR can be an effective therapy for GBM recurrence.

Alternative adenosine Receptor activation: The netrin-Adora2b link.

During hypoxia or inflammation, extracellular adenosine levels are elevated. Studies using pharmacologic approaches or genetic animal models pertinent to extracellular adenosine signaling implicate this pathway in attenuating hypoxia-associated inflammation. There are four distinct adenosine receptors. Of these, it is not surprising that the Adora2b adenosine receptor functions as an endogenous feedback loop to control hypoxia-associated inflammation. First, Adora2b activation requires higher adenosine concentrations compared to other adenosine receptors, similar to those achieved during hypoxic inflammation. Second, Adora2b is transcriptionally induced during hypoxia or inflammation by hypoxia-inducible transcription factor HIF1A. Studies seeking an alternative adenosine receptor activation mechanism have linked netrin-1 with Adora2b. Netrin-1 was originally discovered as a neuronal guidance molecule but also functions as an immune-modulatory signaling molecule. Similar to Adora2b, netrin-1 is induced by HIF1A, and has been shown to enhance Adora2b signaling. Studies of acute respiratory distress syndrome (ARDS), intestinal inflammation, myocardial or hepatic ischemia and reperfusion implicate the netrin-Adora2b link in tissue protection. In this review, we will discuss the potential molecular linkage between netrin-1 and Adora2b, and explore studies demonstrating interactions between netrin-1 and Adora2b in attenuating tissue inflammation.