Impaired ATP hydrolysis in blood plasma contributes to age-related neutrophil dysfunction.

BACKGROUND: The function of polymorphonuclear neutrophils (PMNs) decreases with age, which results in infectious and inflammatory complications in older individuals. The underlying causes are not fully understood. ATP release and autocrine stimulation of purinergic receptors help PMNs combat microbial invaders. Excessive extracellular ATP interferes with these mechanisms and promotes inflammatory PMN responses. Here, we studied whether dysregulated purinergic signaling in PMNs contributes to their dysfunction in older individuals. RESULTS: Bacterial infection of C57BL/6 mice resulted in exaggerated PMN activation that was significantly greater in old mice (64 weeks) than in young animals (10 weeks). In contrast to young animals, old mice were unable to prevent the systemic spread of bacteria, resulting in lethal sepsis and significantly greater mortality in old mice than in their younger counterparts. We found that the ATP levels in the plasma of mice increased with age and that, along with the extracellular accumulation of ATP, the PMNs of old mice became increasingly primed. Stimulation of the formyl peptide receptors of those primed PMNs triggered inflammatory responses that were significantly more pronounced in old mice than in young animals. However, bacterial phagocytosis and killing by PMNs of old mice were significantly lower than that of young mice. These age-dependent PMN dysfunctions correlated with a decrease in the enzymatic activity of plasma ATPases that convert extracellular ATP to adenosine. ATPases depend on divalent metal ions, including Ca2+, Mg2+, and Zn2+, and we found that depletion of these ions blocked the hydrolysis of ATP and the formation of adenosine in human blood, resulting in ATP accumulation and dysregulation of PMN functions equivalent to those observed in response to aging. CONCLUSIONS: Our findings suggest that impaired hydrolysis of plasma ATP dysregulates PMN function in older individuals. We conclude that strategies aimed at restoring plasma ATPase activity may offer novel therapeutic opportunities to reduce immune dysfunction, inflammation, and infectious complications in older patients.

Inhibition of Vascular Smooth Muscle Cell Proliferation by ENPP1: The Role of CD73 and the Adenosine Signaling Axis.

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.

ER and SOCE Ca2+ signals are not required for directed cell migration in human iPSC-derived microglia.

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or - following major injuries - by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca2+) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca2+ changes revealed that this phenomenon does not require Ca2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca2+-independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca2+ homeostasis.

Protection from environmental enteric dysfunction and growth improvement in malnourished newborns by amplification of secretory IgA.

Environmental enteric dysfunction (EED) is a condition associated with malnutrition that can progress to malabsorption and villous atrophy. Severe EED results in linear growth stunting, slowed neurocognitive development, and unresponsiveness to oral vaccines. Prenatal exposure to malnutrition and breast feeding by malnourished mothers replicates EED. Pups are characterized by deprivation of secretory IgA (SIgA) and altered development of the gut immune system and microbiota. Extracellular ATP (eATP) released by microbiota limits T follicular helper (Tfh) cell activity and SIgA generation in Peyer's patches (PPs). Administration of a live biotherapeutic releasing the ATP-degrading enzyme apyrase to malnourished pups restores SIgA levels and ameliorates stunted growth. SIgA is instrumental in improving the growth and intestinal immune competence of mice while they are continuously fed a malnourished diet. The analysis of microbiota composition suggests that amplification of endogenous SIgA may exert a dominant function in correcting malnourishment dysbiosis and its consequences on host organisms, irrespective of the actual microbial ecology.

Double-modified, thio and methylene ATP analogue facilitates wound healing in vitro and in vivo.

Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue-alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure-activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate SP and RP diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist-AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the SP-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial-mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the SP-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.

Potential of dapagliflozin to prevent vascular remodeling in the rat carotid artery following balloon injury.

BACKGROUND AND AIMS: Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been shown to reduce the risk of cardiovascular events independently of glycemic control. However, the possibility that SGLT2 inhibitors improve vascular restenosis is unknown. The aim of this study was to examine whether dapagliflozin could prevent neointima thickening following balloon injury and, if so, to determine the underlying mechanisms. METHODS: Saline, dapagliflozin (1.5 mg/kg/day), or losartan (30 mg/kg/day) was administered orally for five weeks to male Wistar rats. Balloon injury of the left carotid artery was performed a week after starting the treatment and rats were sacrificed 4 weeks later. The extent of neointima was assessed by histomorphometric and immunofluorescence staining analyses. Vascular reactivity was assessed on injured and non-injured carotid artery rings, changes of target factors by immunofluorescence, RT-qPCR, and histochemistry. RESULTS: Dapagliflozin and losartan treatments reduced neointima thickening by 32 % and 27 %, respectively. Blunted contractile responses to phenylephrine and relaxations to acetylcholine and down-regulation of eNOS were observed in the injured arteries. RT-qPCR investigations indicated an increased in gene expression of inflammatory (IL-1beta, VCAM-1), oxidative (p47phox, p22phox) and fibrotic (TGF-beta1) markers in the injured carotid. While these changes were not affected by dapagliflozin, increased levels of AT1R and NTPDase1 (CD39) and decreased levels of ENPP1 were observed in the restenotic carotid artery of the dapagliflozin group. CONCLUSIONS: Dapagliflozin effectively reduced neointimal thickening. The present data suggest that dapagliflozin prevents restenosis through interfering with angiotensin and/or extracellular nucleotides signaling. SGLT2 represents potential new target for limiting vascular restenosis.

Metabolic crosstalk: Extracellular ATP and the tumor microenvironment in cancer progression and therapy.

Adenosine 5'-triphosphate (ATP) is a vital element in energy information. It plays a critical role in transmitting signals inside the body, which is necessary for controlling the life activities of all cells, including tumor cells [1]. Its significance extends from intracellular signaling pathways to tumor regression. Purinergic signaling, a form of extracellular paracrine signaling, relies on purine nucleotides. Extracellular ectonucleotidases convert these purine nucleotides to their respective di and mono-phosphate nucleoside forms, contributing significantly to immune biology, cancer biology, and inflammation studies. ATP functions as a mighty damage-linked molecular pattern when released outside the cell, accumulating in inflammatory areas. In the tumor microenvironment (TME), purinergic receptors such as ATP-gated ion channels P2X1-5 and G protein-coupled receptors (GPCR) (P2Y) interact with ATP and other nucleotides, influencing diverse immune cell activities. CD39 and CD73-mediated extracellular ATP degradation contributes to immunosuppression by diminishing ATP-dependent activation and generating adenosine (ADO), potentially hindering antitumor immunity and promoting tumor development. Unraveling the complexities of extracellular ATP (e-ATP) and ADO effects on the TME poses challenges in identifying optimal treatment targets, yet ongoing investigations aim to devise strategies combating e-ATP/ADO-induced immunosuppression, ultimately enhancing anti-tumor immunity. This review explores e-ATP metabolism, its purinergic signaling, and therapeutic strategies targeting associated receptors and enzymes.

Microglial purinergic signaling in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disease. The prevalent features of AD pathogenesis are the appearance of ß-amyloid (Aß) plaques and neurofibrillary tangles, which cause microglial activation, synaptic deficiency, and neuronal loss. Microglia accompanies AD pathological processes and is also linked to cognitive deficits. Purinergic signaling has been shown to play a complex and tight interplay with the chemotaxis, phagocytosis, and production of pro-inflammatory factors in microglia, which is an important mechanism for regulating microglia activation. Here, we review recent evidence for interactions between AD, microglia, and purinergic signaling and find that the purinergic P2 receptors pertinently expressed on microglia are the ionotropic receptors P2X4 and P2X7, and the subtypes of P2YRs expressed by microglia are metabotropic receptors P2Y2, P2Y6, P2Y12, and P2Y13. The adenosine P1 receptors expressed in microglia include A1R, A2AR, and A2BR. Among them, the activation of P2X4, P2X7, and adenosine A1, A2A receptors expressed in microglia can aggravate the pathological process of AD, whereas P2Y2, P2Y6, P2Y12, and P2Y13 receptors expressed by microglia can induce neuroprotective effects. However, A1R activation also has a strong neuroprotective effect and has a significant anti-inflammatory effect in chronic neuroinflammation. These receptors regulate a variety of pathophysiological processes in AD, including APP processing, Aß production, tau phosphorylation, neuroinflammation, synaptic dysfunction, and mitochondrial dysfunction. This review also provides key pharmacological advances in purinergic signaling receptors.

Mesenchymal Stem Cells and Purinergic Signaling in Autism Spectrum Disorder: Bridging the Gap between Cell-Based Strategies and Neuro-Immune Modulation.

The prevalence of autism spectrum disorder (ASD) is still increasing, which means that this neurodevelopmental lifelong pathology requires special scientific attention and efforts focused on developing novel therapeutic approaches. It has become increasingly evident that neuroinflammation and dysregulation of neuro-immune cross-talk are specific hallmarks of ASD, offering the possibility to treat these disorders by factors modulating neuro-immunological interactions. Mesenchymal stem cell-based therapy has already been postulated as one of the therapeutic approaches for ASD; however, less is known about the molecular mechanisms of stem cell influence. One of the possibilities, although still underestimated, is the paracrine purinergic activity of MSCs, by which stem cells ameliorate inflammatory reactions. Modulation of adenosine signaling may help restore neurotransmitter balance, reduce neuroinflammation, and improve overall brain function in individuals with ASD. In our review article, we present a novel insight into purinergic signaling, including but not limited to the adenosinergic pathway and its role in neuroinflammation and neuro-immune cross-talk modulation. We anticipate that by achieving a greater understanding of the purinergic signaling contribution to ASD and related disorders, novel therapeutic strategies may be devised for patients with autism in the near future.

FERONIA orchestrates P2K1-driven purinergic signaling in plant roots.

Extracellular ATP (eATP) orchestrates vital processes in plants, akin to its role in animals. P2K1 is a crucial receptor mediating eATP effects. Immunoprecipitation tandem mass spectrometry data highlighted FERONIA's significant interaction with P2K1, driving us to explore its role in eATP signaling. Here, we investigated putative P2K1-interactor, FERONIA, which is a versatile receptor kinase pivotal in growth and stress responses. We employed a FERONIA loss-of-function mutant, fer-4, to dissect its effects on eATP signaling. Interestingly, fer-4 showed distinct calcium responses compared to wild type, while eATP-responsive genes were constitutively upregulated in fer-4. Additionally, fer-4 displayed insensitivity to eATP-regulated root growth and reduced cell wall accumulation. Together, these results uncover a role for FERONIA in regulating eATP signaling. Overall, our study deepens our understanding of eATP signaling, revealing the intricate interplay between P2K1 and FERONIA impacting the interface between growth and defense.

Global research status and trends of enteric glia: a bibliometric analysis.

Background: Enteric glia are essential components of the enteric nervous system. Previously believed to have a passive structural function, mounting evidence now suggests that these cells are indispensable for maintaining gastrointestinal homeostasis and exert pivotal influences on both wellbeing and pathological conditions. This study aimed to investigate the global status, research hotspots, and future directions of enteric glia. Methods: The literature on enteric glia research was acquired from the Web of Science Core Collection. VOSviewer software (v1.6.19) was employed to visually represent co-operation networks among countries, institutions, and authors. The co-occurrence analysis of keywords and co-citation analysis of references were conducted using CiteSpace (v6.1.R6). Simultaneously, cluster analysis and burst detection of keywords and references were performed. Results: A total of 514 publications from 36 countries were reviewed. The United States was identified as the most influential country. The top-ranked institutions were University of Nantes and Michigan State University. Michel Neunlist was the most cited author. "Purinergic signaling" was the largest co-cited reference cluster, while "enteric glial cells (EGCs)" was the cluster with the highest number of co-occurring keywords. As the keyword with the highest burst strength, Crohns disease was a hot topic in the early research on enteric glia. The burst detection of keywords revealed that inflammation, intestinal motility, and gut microbiota may be the research frontiers. Conclusion: This study provides a comprehensive bibliometric analysis of enteric glia research. EGCs have emerged as a crucial link between neurons and immune cells, attracting significant research attention in neurogastroenterology. Their fundamental and translational studies on inflammation, intestinal motility, and gut microbiota may promote the treatment of some gastrointestinal and parenteral disorders.

Purinergic regulation of pulmonary vascular tone.

Purinergic signaling is a crucial determinant in the regulation of pulmonary vascular physiology and presents a promising avenue for addressing lung diseases. This intricate signaling system encompasses two primary receptor classes: P1 and P2 receptors. P1 receptors selectively bind adenosine, while P2 receptors exhibit an affinity for ATP, ADP, UTP, and UDP. Functionally, P1 receptors are associated with vasodilation, while P2 receptors mediate vasoconstriction, particularly in basally relaxed vessels, through modulation of intracellular Ca2+ levels. The P2X subtype receptors facilitate extracellular Ca2+ influx, while the P2Y subtype receptors are linked to endoplasmic reticulum Ca2+ release. Notably, the primary receptor responsible for ATP-induced vasoconstriction is P2X1, with α,ß-meATP and UDP being identified as potent vasoconstrictor agonists. Interestingly, ATP has been shown to induce endothelium-dependent vasodilation in pre-constricted vessels, associated with nitric oxide (NO) release. In the context of P1 receptors, adenosine stimulation of pulmonary vessels has been unequivocally demonstrated to induce vasodilation, with a clear dependency on the A2B receptor, as evidenced in studies involving guinea pigs and rats. Importantly, evidence strongly suggests that this vasodilation occurs independently of endothelium-mediated mechanisms. Furthermore, studies have revealed variations in the expression of purinergic receptors across different vessel sizes, with reports indicating notably higher expression of P2Y1, P2Y2, and P2Y4 receptors in small pulmonary arteries. While the existing evidence in this area is still emerging, it underscores the urgent need for a comprehensive examination of the specific characteristics of purinergic signaling in the regulation of pulmonary vascular tone, particularly focusing on the disparities observed across different intrapulmonary vessel sizes. Consequently, this review aims to meticulously explore the current evidence regarding the role of purinergic signaling in pulmonary vascular tone regulation, with a specific emphasis on the variations observed in intrapulmonary vessel sizes. This endeavor is critical, as purinergic signaling holds substantial promise in the modulation of vascular tone and in the proactive prevention and treatment of pulmonary vascular diseases.

Curcumin modulates purinergic signaling and inflammatory response in cutaneous metastatic melanoma cells.

Cutaneous melanoma (CM) poses a therapeutic challenge due to its aggressive nature and often limited response to conventional treatments. Exploring novel therapeutic targets is essential, and natural compounds have emerged as potential candidates. This study aimed to elucidate the impact of curcumin, a natural compound known for its anti-inflammatory, antioxidant, and anti-tumor properties, on metastatic melanoma cells, focusing on the purinergic system and immune responses. Human melanoma cell line SK-Mel-28 were exposed to different curcumin concentrations for either 6 or 24 h, after which we assessed components related to the purinergic system and the inflammatory cascade. Using RT-qPCR, we assessed the gene expression of CD39 and CD73 ectonucleotidases, as well as adenosine deaminase (ADA). Curcumin effectively downregulated CD39, CD73, and ADA gene expression. Flow cytometry analysis revealed that curcumin significantly reduced CD39 and CD73 protein expression at specific concentrations. Moreover, the A2A receptor's protein expression decreased across all concentrations. Enzymatic activity assays demonstrated that curcumin modulated CD39, CD73, and ADA activities, with effects dependent on concentration and duration of treatment. Extracellular ATP levels increased after 24 h of curcumin treatment, emphasizing its role in modulating hydrolytic activity. Curcumin also displayed anti-inflammatory properties by reducing NLRP3 gene expression and impacting the levels of key inflammatory cytokines. In conclusion, this study unveils the potential of curcumin as a promising adjuvant in CM treatment. Curcumin modulates the expression and activity of crucial components of the purinergic system and exhibits anti-inflammatory effects, indicating its potential therapeutic role in combating CM. These findings underscore curcumin's promise and warrant further investigation in preclinical and clinical settings for melanoma management.

Interstitial Cells of Cajal and P2X3 Receptors at Ureteropelvic Junction Obstruction and Their Relationship with Pain Response.

Introduction: Etiopathogenesis and the symptomatology of ureteropelvic junction obstruction (UPJO) in the pediatric population has not yet been definitely clarified, suggesting a multifactorial nature of the condition. The aim was to analyze the association between the number of Interstitial Cells of Cajal (ICCs), as well as P2X3 receptors in ureteropelvic junction (UPJ) and the pain response in pediatric patients with hydronephrosis. Methods: 50 patients with congenital hydronephrosis underwent open or laparoscopic pyeloplasty at one of two departments of pediatric surgery and urology in Poland. Patients were divided into two groups according to the pain symptoms before surgery. A total of 50 samples of UPJ were obtained intraoperatively and underwent histopathological and immunohistochemical (IHC) analysis. Quantitative assessment of ICCs was based on the number of CD117(+) cells of adequate morphology in the subepithelial layer and the muscularis propria. Expression of P2X3 receptors was evaluated as the intensity of IHC staining. Results: Patients with hydronephrosis and accompanying pain were on average 60 months older (77 vs. 17 months) than children with asymptomatic hydronephrosis (p = 0.017). Symptomatic children revealed higher numbers of ICCs in both the subepithelial layer and in the lamina muscularis propria. In particular, symptomatic patients aged 2 years or more exhibited significantly higher numbers of ICCs in the subepithelial layer. Significant differences in the distribution of ICCs between the subepithelial layer and the lamina muscularis propria were observed in both groups. Expression of P2X3 receptors was limited to the urothelium and the muscle layer and correlated between these structures. There was no relationship between pain response and the expression of P2X3 receptors. Conclusions: ICCs and P2X3 receptors may participate in the pathogenesis of UPJO and in the modulation of pain response to a dilatation of the pyelocaliceal system. Explanation of the role of ICCs and P2X3 receptors in propagation of ureteral peristaltic wave and the modulation of pain stimuli requires further studies.

Microglia as integrators of brain-associated molecular patterns.

Microglia are brain-resident macrophages that play key roles in brain development and experience dependent plasticity. In this review we discuss recent findings regarding the molecular mechanisms through which mammalian microglia sense the unique molecular patterns of the homeostatic brain. We propose that microglial function is acutely controlled in response to 'brain-associated molecular patterns' (BAMPs) that function as indicators of neuronal activity and neural circuit remodeling. A further layer of regulation comes from instructive cytokine cues that define unique microglial functional states. A systematic investigation of the receptors and signaling pathways that mediate these two regulatory axes may begin to define a functional code for microglia-neuron interactions.

Purinergic enzymes on extracellular vesicles: immune modulation on the go.

An increase in the extracellular concentration of ATP as a consequence of cellular stress or cell death results in the activation of immune cells. To prevent inflammation, extracellular ATP is rapidly metabolized to adenosine, which deploys an anti-inflammatory signaling cascade upon binding to P1 receptors on immune cells. The ectonucleotidases necessary for the degradation of ATP and generation of adenosine are present on the cell membrane of many immune cells, and their expression is tightly regulated under conditions of inflammation. The discovery that extracellular vesicles (EVs) carry purinergic enzyme activity has brought forward the concept of EVs as a new player in immune regulation. Adenosine-generating EVs derived from cancer cells suppress the anti-tumor response, while EVs derived from immune or mesenchymal stem cells contribute to the restoration of homeostasis after infection. Here we will review the existing knowledge on EVs containing purinergic enzymes and molecules, and discuss the relevance of these EVs in immune modulation and their potential for therapy.

Purinergic signaling promotes premature senescence.

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.

ATP-P2X7 signaling mediates brain pathology while contributing to viral control in perinatal Zika virus infection.

Zika virus (ZIKV), the causative agent of Zika fever, is a flavivirus transmitted by mosquitoes of the Aedes genus. Zika virus infection has become an international concern due to its association with severe neurological complications such as fetal microcephaly. Viral infection can induce the release of ATP in the extracellular environment, activating receptors sensitized by extracellular nucleotides, such as the P2X7 receptor. This receptor is the primary purinergic receptor involved in neuroinflammation, neurodegeneration, and immunity. In this work, we investigated the role of ATP-P2X7 receptor signaling in Zika-related brain abnormalities. Wild-type mice (WT) and P2X7 receptor-deficient (P2X7-/-) C57BL/6 newborn mice were subcutaneously inoculated with 5 × 106plaque-forming units of ZIKV or mock solution. P2X7 receptor expression increased in the brain of Zika virus-infected mice compared to the mock group. Comparative analyses of the hippocampi from WT and P2X7-/-mice revealed that the P2X7 receptor increased hippocampal damage in CA1/CA2 and CA3 regions. Doublecortin expression decreased significantly in the brains of ZIKV-infected mice. WT ZIKV-infected mice showed impaired motor performance compared to P2X7-/- infected mice. WT ZIKV-infected animals showed increased expression of glial markers GFAP (astrocytes) and IBA-1 (microglia) compared to P2X7-/- infected mice. Although the P2X7 receptor contributes to neuronal loss and neuroinflammation, WT mice were more efficient in controlling the viral load in the brain than P2X7 receptor-deficient mice. This result was associated with higher induction of TNF-α, IFN-ß, and increased interferon-stimulated gene expression in WT mice than P2X7-/-ZIKV-infected. Finally, we found that the P2X7 receptor contributes to inhibiting the neuroprotective signaling pathway AKT/mTOR while stimulating the caspase-3 activation, possibly two distinct pathways contributing to neurodegeneration. These findings suggest that ATP-P2X7 receptor signaling contributes to the antiviral response in the brain of ZIKV-infected mice while increasing neuronal loss, neuroinflammation, and related brain abnormalities.

Extracellular Purine Metabolism-Potential Target in Multiple Sclerosis.

The purinergic signaling system comprises a complex network of extracellular purines and purine-metabolizing ectoenzymes, nucleotide and nucleoside receptors, ATP release channels, and nucleoside transporters. Because of its immunomodulatory function, this system is critically involved in the pathogenesis of multiple sclerosis (MS) and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). MS is a chronic neuroinflammatory demyelinating and neurodegenerative disease with autoimmune etiology and great heterogeneity, mostly affecting young adults and leading to permanent disability. In MS/EAE, alterations were detected in almost all components of the purinergic signaling system in both peripheral immune cells and central nervous system (CNS) glial cells, which play an important role in the pathogenesis of the disease. A decrease in extracellular ATP levels and an increase in its downstream metabolites, particularly adenosine and inosine, were frequently observed at MS, indicating a shift in metabolism toward an anti-inflammatory environment. Accordingly, upregulation of the major ectonucleotidase tandem CD39/CD73 was detected in the blood cells and CNS of relapsing-remitting MS patients. Based on the postulated role of A2A receptors in the transition from acute to chronic neuroinflammation, the association of variants of the adenosine deaminase gene with the severity of MS, and the beneficial effects of inosine treatment in EAE, the adenosinergic system emerged as a promising target in neuroinflammation. More recently, several publications have identified ADP-dependent P2Y12 receptors and the major extracellular ADP producing enzyme nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) as novel potential targets in MS.

P2X7 Receptor in Dendritic Cells and Macrophages: Implications in Antigen Presentation and T Lymphocyte Activation.

The P2X7 receptor, a member of the P2X purinergic receptor family, is a non-selective ion channel. Over the years, it has been associated with various biological functions, from modulating to regulating inflammation. However, its emerging role in antigen presentation has captured the scientific community's attention. This function is essential for the immune system to identify and respond to external threats, such as pathogens and tumor cells, through T lymphocytes. New studies show that the P2X7 receptor is crucial for controlling how antigens are presented and how T cells are activated. These studies focus on antigen-presenting cells, like dendritic cells and macrophages. This review examines how the P2X7 receptor interferes with effective antigen presentation and activates T cells and discusses the fundamental mechanisms that can affect the immune response. Understanding these P2X7-mediated processes in great detail opens up exciting opportunities to create new immunological therapies.