P2X7 Antagonist Research Articles

Current treatment of Psoriasis triggered by Cytokine Storm and future immunomodulation strategies.

Psoriasis is a chronic condition caused by an inflammation mediated mainly by cytokines and T cells. In COVID-19, the same type of imbalance is common, generating the Cytokine Storm and promoting a worsening in the skin conditions of patients with autoimmune disorders, such as Psoriasis. In this context, one of the main mediators of immune responses presented by SARS-CoV-2 infected patients is the Purinergic System. This immunological resource is capable of stimulating the hyperinflammatory state presented by infected individuals, mainly by the activity of the P2X7 receptor, culminating in the Cytokine Storm and consequently in the Psoriasis crisis. Currently, different drugs are used for patients with Psoriasis, such as immunosuppressants and small molecules; however, the safety of these drugs in infected patients has not been analyzed yet. In this context, studies are being developed to evaluate the possible administration of these traditional drugs to COVID-19 patients with Psoriasis crisis. Along with that, researchers must evaluate the potential of administrating P2X7 antagonists to these patients as well, improving both the systemic and the dermatological prognostics of patients, by reducing the Cytokine Storm and its general effects, but also avoiding the provocation of Psoriasis crisis.

A Second Drug Binding Site in P2X3.

Purinergic P2X3 receptors form trimeric cation-gated channels, which are activated by extracellular ATP. P2X3 plays a crucial role in chronic cough and affects over 10% of the population. Despite considerable efforts to develop drugs targeting P2X3, the highly conserved structure within the P2X receptor family presents obstacles for achieving selectivity. Camlipixant, a potent and selective P2X3 antagonist, is currently in phase III clinical trials. However, the mechanisms underlying receptor desensitization, ion permeation, principles governing antagonism, and the structure of P2X3 when bound to camlipixant remain elusive. In this study, we established a stable cell line expressing homotrimeric P2X3 and utilized a peptide scaffold to purify the complex and determine its structure using cryo-electron microscopy (cryo-EM). P2X3 binds to camlipixant at a previously unidentified drug-binding site and functions as an allosteric inhibitor. Structure-activity studies combined with modeling and simulations have shed light on the mechanisms underlying the selective targeting and inhibition of P2X3 by camlipixant, distinguishing it from other members of the P2X receptor family.

A Hybrid Approach Combining Shape-Based and Docking Methods to Identify Novel Potential P2X7 Antagonists from Natural Product Databases.

P2X7 is an ATP-activated purinergic receptor implicated in pro-inflammatory responses. It is associated with the development of several diseases, including inflammatory and neurodegenerative conditions. Although several P2X7 receptor antagonists have recently been reported in the literature, none of them is approved for clinical use. However, the structure of the known antagonists can serve as a scaffold for discovering effective compounds in clinical therapy. This study aimed to propose an improved virtual screening methodology for the identification of novel potential P2X7 receptor antagonists from natural products through the combination of shape-based and docking approaches. First, a shape-based screening was performed based on the structure of JNJ-47965567, a P2X7 antagonist, using two natural product compound databases, MEGx (~5.8 × 103 compounds) and NATx (~32 × 103 compounds). Then, the compounds selected by the proposed shape-based model, with Shape-Tanimoto score values ranging between 0.624 and 0.799, were filtered for drug-like properties. Finally, the compounds that met the drug-like filter criteria were docked into the P2X7 allosteric binding site, using the docking programs GOLD and DockThor. The docking poses with the best score values were submitted to careful visual inspection of the P2X7 allosteric binding site. Based on our established visual inspection criteria, four compounds from the MEGx database and four from the NATx database were finally selected as potential P2X7 receptor antagonists. The selected compounds are structurally different from known P2X7 antagonists, have drug-like properties, and are predicted to interact with key P2X7 allosteric binding pocket residues, including F88, F92, F95, F103, M105, F108, Y295, Y298, and I310. Therefore, the combination of shape-based screening and docking approaches proposed in our study has proven useful in selecting potential novel P2X7 antagonist candidates from natural-product-derived compounds databases. This approach could also be useful for selecting potential inhibitors/antagonists of other receptors and/or biological targets.

Single- and Multiple-Dose Pharmacokinetics of Gefapixant (MK-7264), a P2X3 Receptor Antagonist, in Healthy Adults.

Gefapixant (MK-7264, RO4926219, AF-219) is a first-in-class P2X3 antagonists being developed to treat refractory or unexplained chronic cough. The initial single- and multiple-dose safety, tolerability, and pharmacokinetics of gefapixant at doses ranging from 7.5 to 1800 mg were assessed in four clinical trials. Following single-dose administration of 10-450 mg, the pharmacokinetic (PK) profile of gefapixant in plasma and urine demonstrated low inter-subject variability and a dose-proportional exposure. Following administration of multiple doses twice daily, the plasma exposures were dose-proportional at doses ranging from 7.5 to 50 mg and less than dose-proportional at doses ranging from 100 to 1800 mg. The time to mean peak drug concentration ranged from 2 to 3 h post-dose, and steady state was achieved by 7 days after dosing, with an accumulation ratio of approximately 2, comparing data from day 1 to steady state. The mean apparent terminal half-life ranged from 8.2 to 9.6 h. Gefapixant was primarily excreted unmodified in urine. Gefapixant was well tolerated following single-dose administration up to 1800 mg and multiple doses up to 1800 mg twice daily; there were no serious adverse events (AEs) reported. The most common AE reported was dysgeusia. The PK profile supports a twice-daily dosing regimen.

Implications of COVID-19 in Parkinson's disease: the purinergic system in a therapeutic-target perspective to diminish neurodegeneration.

The pathophysiology of Parkinson's disease (PD) is marked by degeneration of dopaminergic neurons in the substantia nigra. With advent of COVID-19, which is closely associated with generalized inflammation and multiple organ dysfunctions, the PD patients may develop severe conditions of disease leading to exacerbated degeneration. This condition is caused by the excessive release of pro-inflammatory markers, called cytokine storm, that is capable of triggering neurodegenerative conditions by affecting the blood-brain barrier (BBB). A possible SARS-CoV-2 infection, in serious cases, may compromise the immune system by triggering a hyperstimulation of the neuroimmune response, similar to the pathological processes found in PD. From this perspective, the inflammatory scenario triggers oxidative stress and, consequently, cellular dysfunction in the nervous tissue. The P2X7R seems to be the key mediator of the neuroinflammatory process, as it acts by increasing the concentration of ATP, allowing the influx of Ca2+ and the occurrence of mutations in the α-synuclein protein, causing activation of this receptor. Thus, modulation of the purinergic system may have therapeutic potential on the effects of PD, as well as on the damage caused by inflammation of the BBB, which may be able to mitigate the neurodegeneration caused by diseases. Considering all the processes of neuroinflammation, oxidative stress, and mitochondrial dysfunction that PD propose, we can conclude that the P2X7 antagonist acts in the prevention of viral diseases, and it also controls purinergic receptors formed by multi-target compounds directed to self-amplification circuits and, therefore, may be a viable strategy to obtain the desired disease-modifying effect. Thus, purinergic system receptor modulations have a high therapeutic potential for neurodegenerative diseases such as PD.

Inflammasome modulation with P2X7 inhibitor A438079-loaded dressings for diabetic wound healing.

The inflammasome is a multiprotein complex critical for the innate immune response to injury. Inflammasome activation initiates healthy wound healing, but comorbidities with poor healing, including diabetes, exhibit pathologic, sustained activation with delayed resolution that prevents healing progression. In prior work, we reported the allosteric P2X7 antagonist A438079 inhibits extracellular ATP-evoked NLRP3 signaling by preventing ion flux, mitochondrial reactive oxygen species generation, NLRP3 assembly, mature IL-1β release, and pyroptosis. However, the short half-life in vivo limits clinical translation of this promising molecule. Here, we develop a controlled release scaffold to deliver A438079 as an inflammasome-modulating wound dressing for applications in poorly healing wounds. We fabricated and characterized tunable thickness, long-lasting silk fibroin dressings and evaluated A438079 loading and release kinetics. We characterized A438079-loaded silk dressings in vitro by measuring IL-1β release and inflammasome assembly by perinuclear ASC speck formation. We further evaluated the performance of A438079-loaded silk dressings in a full-thickness model of wound healing in genetically diabetic mice and observed acceleration of wound closure by 10 days post-wounding with reduced levels of IL-1β at the wound edge. This work provides a proof-of-principle for translating pharmacologic inhibition of ATP-induced inflammation in diabetic wounds and represents a novel approach to therapeutically targeting a dysregulated mechanism in diabetic wound impairment.

Molecular insights into P2X signalling cascades in acute kidney injury.

Acute kidney injury (AKI) is a critical health issuewith high mortality and morbidity rates in hospitalized individuals. The complex pathophysiology and underlying health conditions further complicate AKI management. Growing evidence suggests the pivotal role of ion channels in AKI progression, through promoting tubular cell death and altering immune cell functions. Among these channels, P2X purinergic receptors emerge as key players in AKI pathophysiology. P2X receptors gated by adenosine triphosphate (ATP), exhibit increased extracellular levels of ATP during AKI episodes. More importantly, certain P2X receptor subtypes upon activation exacerbate the situation by promoting the release of extracellular ATP. While therapeutic investigations have primarily focused on P2X4 and P2X7 subtypes in the context of AKI, while understandingabout other subtypes still remains limited. Whilst some P2X antagonists show promising results against different types of kidney diseases, their role in managing AKI remains unexplored. Henceforth, understanding the intricate interplay between P2X receptors and AKI is crucial for developing targeted interventions. This review elucidates the functional alterations of all P2X receptors during normal kidney function and AKI, offering insights into their involvement in AKI. Notably, we have highlighted the current knowledge of P2X receptor antagonists and the possibilities to use them against AKI in the future. Furthermore, the review delves into the pathways influenced by activated P2X receptors during AKI, presenting potential targets for future therapeutic interventions against this critical condition.

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 μg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.

Acute pulpitis promotes purinergic signaling to induce pain in rats via P38MAPK/NF-κB signaling pathway.

Toothache is one of the most common types of pain, but the mechanisms underlying pulpitis-induced pain remain unknown. The ionotropic purinergic receptor family (P2X) is reported to mediate nociception in the nervous system. This study aims to investigate the involvement of P2X3 in the sensitisation of the trigeminal ganglion (TG) and the inflammation caused by acute pulpitis. An acute tooth inflammation model was established by applying LPS to the pulp of SD rats. We found that the increased expression of P2X3 was induced by acute pulpitis. A selective P2X3 inhibitor (A-317491) reduced pain-like behavior in the maxillofacial region of rats and depressed the activation of neurons in the trigeminal ganglion induced by pulpitis. The upregulated MAPK signaling (p-p38, p-ERK1/2) expression in the ipsilateral TG induced by pulpitis could also be depressed by the application of the P2X3 inhibitor. Furthermore, the expression of markers of inflammatory processes, such as NF-κB, TNF-α and IL-1β, could be induced by acute pulpitis and deduced by the intraperitoneal injection of P2X3 antagonists. Our findings demonstrate that purinergic P2X3 receptor signaling in TG neurons contributes to pulpitis-induced pain in rats and that P2X3 signaling may be a potential therapeutic target for tooth pain.

A shared mechanism for TNP-ATP recognition by members of the P2X receptor family

P2X receptors (P2X1–7) are non-selective cation channels involved in many physiological activities such as synaptic transmission, immunological modulation, and cardiovascular function. These receptors share a conserved mechanism to sense extracellular ATP. TNP-ATP is an ATP derivative acting as a nonselective competitive P2X antagonist. Understanding how it occupies the orthosteric site in the absence of agonism may help reveal the key allostery during P2X gating. However, TNP-ATP/P2X complexes (TNP-ATP/human P2X3 (hP2X3) and TNP-ATP/chicken P2X7 (ckP2X7)) with distinct conformations and different mechanisms of action have been proposed. Whether these represent species and subtype variations or experimental differences remains unclear. Here, we show that a common mechanism of TNP-ATP recognition exists for the P2X family members by combining enhanced conformation sampling, engineered disulfide bond analysis, and covalent occupancy. In this model, the polar triphosphate moiety of TNP-ATP interacts with the orthosteric site, while its TNP-moiety is deeply embedded in the head and dorsal fin (DF) interface, creating a restrictive allostery in these two domains that results in a partly enlarged yet ion-impermeable pore. Similar results were obtained from multiple P2X subtypes of different species, including ckP2X7, hP2X3, rat P2X2 (rP2X2), and human P2X1 (hP2X1). Thus, TNP-ATP uses a common mechanism for P2X recognition and modulation by restricting the movements of the head and DF domains which are essential for P2X activation. This knowledge is applicable to the development of new P2X inhibitors.

Blockage of the fractalkine pathway reduces hyperalgesia and prevents morphological glial alterations-Comparison between inflammatory and neuropathic orofacial pain in male rats.

This study aimed to evaluate the effects of inhibitors of the fractalkine pathway in hyperalgesia in inflammatory and neuropathic orofacial pain in male rats and the morphological changes in microglia and satellite glial cells (SGCs). Rats were submitted to zymosan-induced arthritis of the temporomandibular joint or infraorbital nerve constriction, and treated intrathecally with a P2 X7 antagonist, a cathepsin S inhibitor or a p-38 mitogen-activated protein kinase (MAPK) inhibitor. Mechanical hyperalgesia was evaluated 4 and 6 h following arthritis induction or 7 and 14 days following nerve ligation. The expression of the receptor CX3 CR1 , phospho-p-38 MAPK, ionized calcium-binding adapter molecule-1 (Iba-1), and glutamine synthetase and the morphological changes in microglia and SGCs were evaluated by confocal microscopy. In both inflammatory and neuropathic models, untreated animals presented a higher expression of CX3 CR1 and developed hyperalgesia and up-regulation of phospho-p-38 MAPK, which was prevented by all drugs (p < .05). The number of microglial processes endpoints and the total branch length were lower in the untreated animals, but the overall immunolabeling of Iba-1 was altered only in neuropathic rats (p < .05). The mean area of SGCs per neuron was significantly altered only in the inflammatory model (p < .05). All morphological alterations were reverted by modulating the fractalkine pathway (p < .05). In conclusion, the blockage of the fractalkine pathway seemed to be a possible therapeutic strategy for inflammatory and neuropathic orofacial pain, reducing mechanical hyperalgesia by impairing the phosphorylation of p-38 MAPK and reverting morphological alterations in microglia and SGCs.

Increased Purinergic Signaling in Human Dental Pulps With Inflammatory Pain is Sex-Dependent

An enhanced understanding of neurotransmitter systems contributing to pain transmission aids in drug development, while the identification of biological variables like age and sex helps in the development of personalized pain management and effective clinical trial design. This study identified enhanced expression of purinergic signaling components specifically in painful inflammation, with levels increased more in women as compared to men. Inflammatory dental pain is common and potentially debilitating; as inflammation of the dental pulp can occur with or without pain, it provides a powerful model to examine distinct pain pathways in humans. In control tissues, P2X3 and P2X2 receptors colocalized with PGP9.5-positive nerves. Expression of the ecto-nucleotidase NTPDase1 (CD39) increased with exposure to extracellular adenosine triphosphate (ATP), implying CD39 acted as a marker for sustained elevation of extracellular ATP. Both immunohistochemistry and immunoblots showed P2X2, P2X3, and CD39 increased in symptomatic pulpitis, suggesting receptors and the ATP agonist were elevated in patients with increased pain. The increased expression of P2X3 and CD39 was more frequently observed in women than men. In summary, this study identifies CD39 as a marker for chronic elevation of extracellular ATP in fixed human tissue. It supports a role for increased purinergic signaling in humans with inflammatory dental pain and suggests the contribution of purines shows sexual dimorphism. This highlights the potential for P2X antagonists to treat pain in humans and stresses the need to consider sex in clinical trials that target pain and purinergic pathways. PerspectiveThis article demonstrates an elevation of ATP-marker CD39 and of ATP receptors P2X2 and P2X3 with inflammatory pain and suggests the rise is greater in women. This highlights the potential for P2X antagonists to treat pain and stresses the consideration of sexual dimorphism in studies of purines and pain.

Antagonism of the brain P2X7 ion channel attenuates repeated social defeat induced microglia reactivity, monocyte recruitment and anxiety-like behavior in male mice

Chronic stress is linked to increased anxiety. Repeated social defeat (RSD) in mice causes anxiety that is dependent on activated neurons, reactive microglia, and accumulation of monocytes in the brain. This response requires interactions between the immune system and central nervous system (CNS). Neuronal activation within threat appraisal regions is a key response to RSD, however, it is unclear how microglia become activated. One potential explanation is that microglia express a purinergic non-selective ligand gated adenosine-triphosphate (ATP) receptor 7 (P2X7). Activation of P2X7 promotes the release of chemokines and cytokines, and recruitment of monocytes to the brain. Thus, the purpose of this study was to determine if a novel P2X7 antagonist blocked neuronal and microglia interactions and the corresponding anxiety following RSD. Male mice were administered (i.p.) a P2X7 antagonist, JNJ-54471300, prior to each cycle of RSD. Fourteen hours after RSD, behavioral deficits including social avoidance and anxiety-like were determined. Moreover, several immune parameters were assessed. RSD caused neuronal activation in stress-responsive regions, monocyte production and release, splenomegaly, and social avoidance. These parameters were unaffected by P2X7 antagonism. RSD-associated proportional area of Iba-1+ microglia, monocyte accumulation in the brain, IL-1β mRNA expression in enriched myeloid cells, plasma IL-6, and anxiety-like behavior were ameliorated by P2X7 antagonism. Gene expression analysis in the hippocampus and amygdala showed regional specific responses to RSD and some were reversed with P2X7 antagonism. Overall, blocking P2X7 activation attenuated RSD-induced microglia reactivity with corresponding reduction in neuroinflammation, monocyte accumulation, and anxiety-like behavior in male mice.

Extracellular ATP (eATP) inhibits the progression of endometriosis and enhances the immune function of macrophages

BackgroundExtracellular adenosine triphosphate (eATP) is an important inflammatory mediator that can boost the antitumour immune response, but its role in endometriosis remains unknown. We hypothesized that eATP could inhibit endometriosis cell function both directly and indirectly through macrophages. MethodsPeritoneal and cyst fluid from endometriosis patients and non-endometriosis controls was collected to measure eATP levels. The addition of eATP was performed to explore its effects on endometriotic cell and macrophage functions, including cell proliferation, apoptosis, pyroptosis, mitochondrial membrane potential, phagocytosis, and the production of inflammatory cytokines and reactive oxygen species. A coculture of endometriotic epithelial cells and U937 macrophages was established, followed by P2X7 antagonist and eATP treatment. Endometriosis model eATP-treated rats were used to evaluate in situ cell death and macrophage marker expression. ResultsThe pelvic microenvironment of endometriosis patients shows high eATP levels, which could induce endometriotic epithelial cell apoptosis and pyroptosis and significantly inhibit cell growth via the MAPK/JNK/Akt pathway. eATP treatment ameliorated endometriosis-related macrophage dysfunction and promoted macrophage recruitment. eATP treatment in the presence of macrophages exerted a stronger cytotoxic effect on endometriotic epithelial cells by regulating P2X7. eATP treatment effectively induced cell death in an endometriosis rat model and prominently increased the macrophage number without affecting the eutopic endometrium. ConclusioneATP induces endometriotic epithelial cell death and enhances the immune function of macrophages to inhibit the progression of endometriosis, while eutopic endometrium is not affected. eATP treatment may serve as a nonhormonal therapeutic strategy for endometriosis.

Efficacy and safety of pharmacotherapy for refractory or unexplained chronic cough: a systematic review and network meta-analysis

Refractory chronic cough (RCC) has a significant impact on patient's health-related quality of life and represents a challenge in clinical management. However, the optimal treatment for RCC remains controversial. This study aimed to investigate and compare the efficacy and safety of the current pharmacological therapeutic options for RCC. A systematic review was performed by searching PubMed, Web of Science, Embase, and Ovid databases from January 1, 2008 to March 1, 2023. All randomised control trials (RCTs) reporting outcomes of efficacy or/and safety were included in the Bayesian network meta-analysis. Here, we compared the effects on Leicester Cough Questionnaire (LCQ), Visual Analogue Scale (VAS), and objective cough frequency of patients with RCC. Besides, we also compared the incidence of adverse events (AEs) for analysis of safety. PROSPERO registration: CRD42022345940. 19 eligible RCTs included 3326 patients and 7 medication categories: P2X3 antagonist, GABA modulator, Transient Receptor Potential (TRP) modulator, NK-1 agonist, opioid analgesic, macrolide, and sodium cromoglicate. Compared with placebo, mean difference (MD) of LCQ and 24 h cough frequency for P2X3 antagonist relief were 1.637 (95% CI: 0.887-2.387) and-11.042 (P=0.035). Compared with placebo, effect sizes (MD for LCQ and cough severity VAS) for GABA modulator were 1.347 (P=0.003) and-7.843 (P=0.003). In the network meta-analysis, gefapixant is the most effective treatment for patients with RCC (The Surface Under the Cumulative Ranking Curves (SUCRA) is 0.711 in LCQ, 0.983 in 24 h cough frequency, and 0.786 in cough severity VAS). Lesogaberan had better efficacy than placebo (SUCRA: 0.632 vs. 0.472) in 24 h cough frequency. Eliapixant and lesogaberan had better efficacy than placebo in cough severity VAS. However, TRP modulator had worse efficacy than placebo. In the meta-analysis of AEs, the present study found P2X3 antagonist had a significant correlation to AEs (RR: 1.129, 95% CI: 1.012-1.259), especially taste-related AEs (RR: 6.216, P<0.05). In this network meta-analysis, P2X3 antagonist showing advantages in terms of efficacy is currently the most promising medication for treatment of RCC. GABA modulator also showed potential efficacy for RCC but with AEs of the central system. Nevertheless, the role of TRP modulator needed to be revisited. Further research is needed to determine the potential beneficiary population for optimizing the pharmacological management of chronic cough. National Natural Science Foundation of China (81870079), Guangdong Science and Technology Project (2021A050520012), Incubation Program of National Science Foundation for Distinguished Young Scholars (GMU2020-207).

Attenuation of Microglial Activation and Pyroptosis by Inhibition of P2X7 Pathway Promotes Photoreceptor Survival in Experimental Retinal Detachment.

Photoreceptor (PR) death is the ultimate cause of irreversible vision loss in retinal detachment (RD). Although microglial infiltration in the subretinal space (SRS) was observed after RD, the molecular mechanism underlying microglial activation and the outcomes of infiltrating microglia remain unclear. We aimed to uncover the mechanism of initiation of microglial activation to help explore potential therapy to promote PR survival. An RD model was conducted by injecting sodium hyaluronate into SRS of C57BL/6J wild type mice. Adenosine triphosphate (ATP) was measured by a ATP Microplate Assay Kit. Bioinformatics analysis was used to evaluate the upregulated receptor relating to ATP binding in human datasets and mouse transcriptomes of RD. Expression of P2X7, its downstream signaling pathways, and microglial pyroptosis were confirmed by qPCR, WB, and immunofluorescence in vivo and in vitro. The cell viability of PR was measured by cell counting kit-8. Brilliant Blue G, a P2X7 antagonist, was subretinally or intraperitoneally injected to inhibit microglial activation in vivo and was applied for microglia cell line treatment in vitro. The decrease in microglial activation and pyroptosis was detected by immunofluorescence and WB. The protective effect on PR was measured by hematoxylin and eosin staining, TUNEL assay, and electroretinogram analysis. The results showed that extracellular ATP released in the SRS after RD triggered P2X7 activation and attracted microglia. The downstream cascade of inflammasome activation induced by P2X7 activation contributed to microglial pyroptosis and then to PR death. ATP-activated microglia led to PR death in vitro. P2X7 blockade rescued PR morphologically and functionally by inhibiting microglial activation and pyroptosis. These results elucidate that ATP-induced P2X7-mediated microglial activation leads to microglial pyroptosis, contributing to PR death. Appropriate inhibition of microglial pyroptosis might serve as a pharmacotherapeutic strategy for decreasing PR death in RD.

Mechanistic insights into severe pulmonary inflammation caused by silica stimulation: The role of macrophage pyroptosis

Respirable silica dust is a common hazard faced by occupational workers and prolonged exposure to this dust can lead to pulmonary inflammation, fibrosis and, in severe cases, silicosis. However, the underlying mechanism by which silica exposure causes these physical disorders is not yet understood. In this study, we aimed to shed light on this mechanism by establishing in vitro and in vivo silica exposure models from the perspective of macrophages. Our results showed that compared to the control group, silica exposure resulted in an upregulation of the pulmonary expression of P2X7 and Pannexin-1, but this effect was suppressed by treatment with MCC950, a specific inhibitor of NLRP3. Our in vitro studies showed that silica exposure induced mitochondrial depolarization in macrophages, which led to a reduction of intracellular ATP and an influx of Ca2+. Furthermore, we found that creating an extracellular high potassium environment by adding KCl to the macrophage medium inhibited the expression of pyroptotic biomarkers and pro-inflammatory cytokines such as NLRP3 and IL-1β. Treatment with BBG, a P2X7 antagonist, also effectively inhibited the expression of P2X7, NLRP3, and IL-1β. On the other hand, treatment with FCF, a Pannexin-1 inhibitor, suppressed the expression of Pannexin-1 but had no effect on the expression of pyroptotic biomarkers such as P2X7, NLRP3, and IL-1β. In conclusion, our findings suggest that silica exposure triggers the opening of P2X7 ion channels, resulting in intracellular K+ efflux, extracellular Ca2+ influx, and the assembly of the NLRP3 inflammasome, ultimately leading to macrophage pyroptosis and pulmonary inflammation.

Animal Models for the Investigation of P2X7 Receptors.

The P2X7 receptor is a trimeric ligand-gated cation channel activated by extracellular adenosine 5'-triphosphate. The study of animals has greatly advanced the investigation of P2X7 and helped to establish the numerous physiological and pathophysiological roles of this receptor in human health and disease. Following a short overview of the P2X7 distribution, roles and functional properties, this article discusses how animal models have contributed to the generation of P2X7-specific antibodies and nanobodies (including biologics), recombinant receptors and radioligands to study P2X7 as well as to the pharmacokinetic testing of P2X7 antagonists. This article then outlines how mouse and rat models have been used to study P2X7. These sections include discussions on preclinical disease models, polymorphic P2X7 variants, P2X7 knockout mice (including bone marrow chimeras and conditional knockouts), P2X7 reporter mice, humanized P2X7 mice and P2X7 knockout rats. Finally, this article reviews the limited number of studies involving guinea pigs, rabbits, monkeys (rhesus macaques), dogs, cats, zebrafish, and other fish species (seabream, ayu sweetfish, rainbow trout and Japanese flounder) to study P2X7.

Pathological ventricular remodelling and purinergic signaling: Emerging roles of the ATP-gated P2X4 receptor

Pathological ventricular remodelling is an abnormal healing process following myocardial infarction (MI), which mechanisms remain debated. Several hypotheses have been proposed to explain the links between inflammatory response secondary to MI and the evolution towards pathological remodelling, including the involvement of ATP and its cellular receptors: the purinergic receptors. Our working hypothesis is that there is a maintenance/resurgence of a tissue inflammatory response in MI patients with pathological remodelling that would hinder the healing process and may be associated with a specific purinergic profile. The objective of the project is to study the impact of ischemia-reperfusion on cardiac fibroblast's’ profiles and to identify an associated purinergic signature. First, we studied the effect of ischaemia (5 h) and reperfusion (24, 48 h) on human primary cardiac fibroblasts (HCF) phenotype by studying the expression of markers associated with the activated state: periostin and the differentiated state: smooth muscle alpha-actin (αSMA) by Western blot. Simultaneously, the expression level of P2 purinergic receptors (P2R) was quantified by RT-qPCR and WB over time. In a second step, we chose to investigate the involvement of P2X4 in the activation and differentiation of cardiac fibroblasts. Therefore, we blocked its activity during reperfusion with its specific antagonist 5-BDBD (5 μM). In addition, wound healing studies were conducted with and without P2X4's antagonist to investigate its involvement in the migratory capacity of cardiac fibroblasts. Ischaemia-reperfusion (IR) appears to induce an initial increase in periostin expression at 24 h followed by a decrease at 48 h as compared to normoxia. This may be related to a more important secretion of periostin at 48 h, as suggested by its increase in supernatants. Concerning αSMA, IR induces a decrease in its expression in a sustained manner over time. Regarding P2X4, there is a decrease in its mRNA and protein levels. The use of its antagonist in normoxia and IR decreases αSMA expression but does not affect periostin expression. Similarly, its blockade decreases the basal migratory capacity of fibroblasts. IR seems to prevent fibroblast differentiation by maintaining them into an activated state. P2X4 could potentially be involved by decreasing fibroblast differentiation, thus impacting αSMA expression. Yet, other purinergic receptors whose expression levels vary following MI might also be involved.

P2X and P2Y receptor antagonists reduce inflammation in ATP-induced microglia.

P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuro-excitatory substances in the microglia. The P2X4, P2X7 and P2Y12 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known if blocking P2X4, P2X7 and P2Y12 receptors is associated with the expression and the release of interleukin-1B (IL-1β), interleukin-6 (IL-6), or tumor necrosis factor-α (TNF-α) in cultured neonatal spinal cord microglia. For this reason, we examined the effects of P2X4, P2X7 and P2Y12 antagonists on the expression and the release of IL-1β, IL-6, and TNF-α in ATP-stimulated microglia. In this study, we observed the effect of A-740003, PSB-12062 and MRS 2395 (P2X4, P2X7 and P2Y12 receptors antagonist, respectively), on the expression and release of IL-1β, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ATP induced the increased expression of IL-1β, IL-6 and TNF-α at the level of messenger RNA (mRNA). ATP-evoked increase in IL-1β, IL-6 and TNF-α mRNA expression was inhibited by the P2X4 receptor antagonist A-740003 or P2X7 receptor antagonist PSB-12062, respectively. Similarly, ATP-evoked release of IL-1β, IL-6 and TNF-α was inhibited by A-740003 and PSB-12062. Furthermore, ATP-evoked increased expression of Iba-1, IL-1β, IL-6 and TNF-α mRNA, and release of IL-1β, IL-6 and TNF-α were nearly all blocked after co-administration of A-740003 plus PSB-12062. Finally, ATP-evoked increased gene expression and release of IL-1β, IL-6 and TNF-α were also inhibited by MRS 2395 (P2Y12 antagonist). These observations suggest a new clue for therapeutic strategies to treat the neuro-inflammation.