Adenosine Triphosphate Release Research Articles

Role of an Aromatic-Aromatic Interaction in the Assembly and Trafficking of the Zebrafish Panx1a Membrane Channel.

Pannexin 1 (Panx1) is a ubiquitously expressed hexameric integral membrane protein known to function as an adenosine triphosphate (ATP) release channel. Panx1 proteins exist in unglycosylated core form (Gly0). They undergo critical post-translational modifications forming the high mannose glycosylation state (Gly1) in the endoplasmic reticulum (ER) and the complex glycosylation state (Gly2) in the Golgi apparatus. The regulation of transition from the ER to the cell membrane is not fully understood. Using site-specific mutagenesis, dye uptake assays, and interaction testing, we identified two conserved aromatic residues, Trp123 and Tyr205, in the transmembrane domains 2 and 3 of the zebrafish panx1a protein. Results suggest that both residues primarily govern the assembly of panx1a subunits into channels, with mutant proteins failing to interact. The results provide insight into a mechanism enabling regulation of Panx1 oligomerization, glycosylation, and trafficking.

ATP-P2X7–Induced Inflammasome Activation Contributes to Melanocyte Death and CD8+ T-Cell Trafficking to the Skin in Vitiligo

Extracellular adenosine 5'-triphosphate (ATP) is a well-known inflammasome-activating signal. Emerging evidence demonstrates a critical role for inflammasome activation in vitiligo pathogenesis. However, the specific molecular mechanism of inflammasome-dependent melanocyte degeneration in vitiligo is still not clear. This study presents how extracellular ATP, released from keratinocytes by oxidative stress, affects melanocyte survival in vitiligo skin. H2O2-induced oxidative injury increased ATP release from keratinocytes and skin tissues. The high concentration of extracellular ATP induced both ROS production and cell death in melanocytes. Treatment with ATP caused the activation of caspase-1 as well as the production of active forms of IL-1β and IL-18 via P2X7 receptor in keratinocytes and melanocytes. Lesional and perilesional skin of vitiligo showed higher levels of ATP as well as upregulation of active caspase-1 compared with nonlesional skin, suggesting its possible role in inflammasome activation in vitiligo. Moreover, the elevated expression of CXCL9 in keratinocytes, mediated through ATP/P2X7 receptor-dependent inflammasome activation, was responsible for CLA+CD8+ T-cell chemotaxis into the skin. These results demonstrate that extracellular ATP as a danger signal activates the inflammasome pathway and increases cutaneous chemotaxis of CD8+ T cells via CXCL9 in vitiligo. Therefore, targeting ATP-P2X7 signaling may be a potential strategy for vitiligo treatment.

A subset of taste receptor cells express biocytin‐permeable channels activated by reducing extracellular Ca2+ concentration

Taste receptor cells (type II cells) transmit taste information to taste nerve fibres via ATP-permeable channels, including calcium homeostasis modulator (CALHM), connexin and/or pannexin1 channels, via the paracrine release of adenosine triphosphate (ATP) as a predominant transmitter. In the present study, we demonstrate that extracellular Ca2+ -dependent biocytin-permeable channels are present in a subset of type II cells in mouse fungiform taste buds using biocytin uptake, immunohistochemistry and in situ whole-cell recordings. Type II cells were labelled with biocytin in an extracellular Ca2+ concentration ([Ca2+ ]out )-sensitive manner. We found that the ratio of biocytin-labelled type II cells to type II cells per taste bud was approximately 20% in 2mM Ca2+ saline, and this ratio increased to approximately 50% in nominally Ca2+ -free saline. The addition of 300µM GdCl3 , which inhibits various channels including CALHM1 channels, significantly inhibited biocytin labelling in nominally Ca2+ -free saline, whereas the addition of 20µM ruthenium red did not. Moreover, Cs+ -insensitive currents increased in nominally Ca2+ -free saline in approximately 40% of type II cells. These increased currents appeared at a potential of above -35mV, reversed at approximately +10mV and increased with depolarization. These results suggest that biocytin labels type II cells via ion channels activated by [Ca2+ ]out reduction, probably "CALHM-like" channels, on the basolateral membrane and that taste receptor cells can be categorized into two groups based on differences in the expression levels of [Ca2+ ]out -dependent biocytin-permeable channels. These data indicate electrophysiological and pharmacologically relevant properties of biocytin-permeable channels and suggest their contributions to taste signal transduction.

High shear stress amplitude in combination with prolonged stimulus duration determine induction of osteoclast formation by hematopoietic progenitor cells

To date, it is unclear how fluid dynamics stimulate mechanosensory cells to induce an osteoprotective or osteodestructive response. We investigated how murine hematopoietic progenitor cells respond to 2 minutes of dynamic fluid flow stimulation with a precisely controlled sequence of fluid shear stresses. The response was quantified by measuring extracellular adenosine triphosphate (ATP), immunocytochemistry of Piezo1, and sarcoplasmic/endoplasmic Ca2+ reticulum ATPase 2 (SERCA2), and by the ability of soluble factors produced by mechanically stimulated cells to modulate osteoclast differentiation. We rejected our initial hypothesis that peak wall shear stress rate determines the response of hematopoietic progenitor cells to dynamic fluid shear stress, as it had only a minor correlation with the abovementioned parameters. Low stimulus amplitudes corresponded to activation of Piezo1, SERCA2, low concentrations of extracellular ATP, and inhibition of osteoclastogenesis and resorption area, while high amplitudes generally corresponded to osteodestructive responses. At a given amplitude (3 Pa) and waveform (square), the duration of individual stimuli (duty cycle) showed a strong correlation with the release of ATP and osteoclast number and resorption area. Collectively, our data suggest that hematopoietic progenitor cells respond in a viscoelastic manner to loading, since a combination of high shear stress amplitude and prolonged duty cycle is needed to trigger an osteodestructive response. PLAIN LANGUAGE SUMMARY: In case of painful joints or missing teeth, the current intervention is to replace them with an implant to keep a high-quality lifestyle. When exercising or chewing, the cells in the bone around the implant experience mechanical loading. This loading generally supports bone formation to strengthen the bone and prevent breaking, but can also stimulate bone loss when the mechanical loading becomes too high around orthopedic and dental implants. We still do not fully understand how cells in the bone can distinguish between mechanical loading that strengthens or weakens the bone. We cultured cells derived from the bone marrow in the laboratory to test whether the bone loss response depends on (i) how fast a mechanical load is applied (rate), (ii) how intense the mechanical load is (amplitude), or (iii) how long each individual loading stimulus is applied (duration). We mimicked mechanical loading as it occurs in the body, by applying very precisely controlled flow of fluid over the cells. We found that a mechanosensitive receptor Piezo1 was activated by a low amplitude stimulus, which usually strengthens the bone. The potential inhibitor of Piezo1, namely SERCA2, was only activated by a low amplitude stimulus. This happened regardless of the rate of application. At a constant high amplitude, a longer duration of the stimulus enhanced the bone-weakening response. Based on these results we deduce that a high loading amplitude tends to be bone weakening, and the longer this high amplitude persists, the worse it is for the bone.

Shockwaves Suppress Adipocyte Differentiation via Decrease in PPARγ

Adipogenesis is a crucial cellular process that contributes to the expansion of adipose tissue in obesity. Shockwaves are mechanical stimuli that transmit signals to cause biological responses. The purpose of this study is to evaluate the effects of shockwaves on adipogenesis. We treated 3T3L-1 cells and human primary preadipocytes for differentiation with or without shockwaves. Western blots and quantitative real-time reverse transcriptase PCR (qRT-PCR) for adipocyte markers including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding proteins (C/EBPα) were performed. Extracellular adenosine triphosphate (ATP) and intracellular cyclic adenosine monophosphate (cAMP) levels, which are known to affect adipocyte differentiation, were measured. Shockwave treatment decreased intracellular lipid droplet accumulation in primary human preadipocytes and 3T3-L1 cells after 11–12 days of differentiation. Levels of key adipogenic transcriptional factors PPARγ and/or C/EBPα were lower in shockwave-treated human primary preadipocytes and 3T3L-1 cells after 12–13 days of differentiation than in shockwave-untreated cells. Shockwave treatment induced release of extracellular ATP from preadipocytes and decreased intracellular cAMP levels. Shockwave-treated preadipocytes showed a higher level of β-catenin and less PPARγ expression than shockwave-untreated cells. Supplementation with 8-bromo-cAMP analog after shockwave treatment rescued adipocyte differentiation by preventing the effect of shockwaves on β-catenin, Wnt10b mRNA, and PPARγ expression. Low-energy shockwaves suppressed adipocyte differentiation by decreasing PPARγ. Our study suggests an insight into potential uses of shockwave-treatment for obesity.

Examining Local Cell-to-Cell Signalling in the Kidney Using ATP Biosensing.

Cell-to-cell communication is an essential process for the efficient function of cells and tissues. Central to this is the purinergic transmission of purines, with ligands such as adenosine triphosphate (ATP). Altered cell-to-cell communication, and in particular changes in the paracrine release of extracellular ATP, plays crucial roles in pathophysiological conditions, such as diabetes. ATP biosensing provides a reliable, real-time measurement of local extracellular ATP concentrations. This allows the detection of altered ATP release, which underlies the progression of inflammation and fibrosis and is a potential therapeutic target. Here we describe in a step-by-step basis how to utilize sensitive microelectrode biosensors to detect low, real-time concentrations of ATP, in vitro.

The CD40-ATP-P2X 7 Receptor Pathway: Cell to Cell Cross-Talk to Promote Inflammation and Programmed Cell Death of Endothelial Cells.

Extracellular adenosine 5′-triphosphate (ATP) functions not only as a neurotransmitter but is also released by non-excitable cells and mediates cell–cell communication involving glia. In pathological conditions, extracellular ATP released by astrocytes may act as a “danger” signal that activates microglia and promotes neuroinflammation. This review summarizes in vitro and in vivo studies that identified CD40 as a novel trigger of ATP release and purinergic-induced inflammation. The use of transgenic mice with expression of CD40 restricted to retinal Müller glia and a model of diabetic retinopathy (a disease where the CD40 pathway is activated) established that CD40 induces release of ATP in Müller glia and triggers in microglia/macrophages purinergic receptor-dependent inflammatory responses that drive the development of retinopathy. The CD40-ATP-P2X7 pathway not only amplifies inflammation but also induces death of retinal endothelial cells, an event key to the development of capillary degeneration and retinal ischemia. Taken together, CD40 expressed in non-hematopoietic cells is sufficient to mediate inflammation and tissue pathology as well as cause death of retinal endothelial cells. This process likely contributes to development of degenerate capillaries, a hallmark of diabetic and ischemic retinopathies. Blockade of signaling pathways downstream of CD40 operative in non-hematopoietic cells may offer a novel means of treating diabetic and ischemic retinopathies.

ATP mediates the interaction between human blastocyst and endometrium.

ObjectiveEmbryo implantation needs a reciprocal interaction between competent embryo and receptive endometrium. Adenosine triphosphate (ATP) produced by stressed or injured cells acts as an important signalling molecule. This study aims to investigate whether adenosine triphosphate (ATP) plays an important role in the dialogue of human blastocyst‐endometrium.Materials and methodsThe concentration of lactate was analysed in culture medium from human embryos collected from in vitro fertilization patients. Extracellular ATP was measured by ATP Bioluminescent Assay Kit. Ishikawa cells and T‐HESCs were treated with ATP, ATP receptor antagonist, ATP hydrolysis enzyme or inhibitors of ATP metabolic enzymes. The levels of gene expression were evaluated by real‐time PCR and immunoassay.ResultsWe showed that injured human endometrial epithelial cells could rapidly release ATP into the extracellular environment as an important signalling molecule. In addition, blastocyst‐derived lactate induces the release of non‐lytic ATP from human endometrial receptive epithelial cells via connexins. Extracellular ATP stimulates the secretion of IL8 from epithelial cells to promote the process of in vitro decidualization. Extracellular ATP could also directly promote the decidualization of human endometrial stromal cells via P2Y‐purinoceptors. More importantly, the supernatants of injured epithelial cells clearly induce the decidualization of stromal cells in time‐dependent manner.ConclusionOur results suggest that ATP should play an important role in human blastocyst‐endometrium dialogue for the initiation of decidualization.

Studies on APP metabolism related to age-associated mitochondrial dysfunction in APP/PS1 transgenic mice.

The aging brain with mitochondrial dysfunction and a reduced adenosine 5’-triphosphate (ATP) has been implicated in the onset and progression of β-Amyloid (Aβ)-induced neuronal toxicity in AD. To unravel the function of ATP and the underlying mechanisms on AD development, APP/PS1 double transgenic mice and wild-type (WT) C57 mice at 6 and 10 months of age were studied. We demonstrated a decreased ATP release in the hippocampus and platelet of APP/PS1 mice, comparing to C57 mice at a relatively early age. Levels of Aβ were raised in both hippocampus and platelet of APP/PS1 mice, accompanied by a decrease of α-secretase activity and an increase of β-secretase activity. Moreover, our results presented an age-dependent rise in mitochondrial vulnerability to oxidation in APP/PS1 mice. In addition, we found decreased pSer473-Akt levels, increased GSK3β activity by inhibiting phosphorylation at Ser9 in aged APP/PS1 mice and these dysfunctions probably due to down-regulation of Bcl-2 and up-regulation of cleaved caspase-3. Therefore, we demonstrate that PI3K/Akt/GSK3β signaling pathway could be involved in Aβ-associated mitochondrial dysfunction of APP/PS1 mice and APP abnormal metabolism in platelet might provide potential biomarkers for early diagnosis of AD.

New targets for overactive bladder-ICI-RS 2109.

To review evidence for novel drug targets that can manage overactive bladder (OAB) symptoms. A think tank considered evidence from the literature and their own research experience to propose new drug targets in the urinary bladder to characterize their use to treat OAB. Five classes of agents or cellular pathways were considered. (a) Cyclic nucleotide-dependent (cyclic adenosine monophosphate and cyclic guanosine monophosphate) pathways that modulate adenosine triphosphate release from motor nerves and urothelium. (b) Novel targets for β3 agonists, including the bladder wall vasculature and muscularis mucosa. (c) Several TRP channels (TRPV1 , TRPV4 , TRPA1 , and TRPM4 ) and their modulators in affecting detrusor overactivity. (d) Small conductance Ca2+ -activated K+ channels and their influence on spontaneous contractions. (e) Antifibrosis agents that act to modulate directly or indirectly the TGF-β pathway-the canonical fibrosis pathway. The specificity of action remains a consideration if particular classes of agents can be considered for future development as receptors or pathways that mediate actions of the abovementioned potential agents are distributed among most organ systems. The tasks are to determine more detail of the pathological changes that occur in the OAB and how the specificity of potential drugs may be directed to bladder pathological changes. An important conclusion was that the storage, not the voiding, phase in the micturition cycle should be investigated and potential targets lie in the whole range of tissue in the bladder wall and not just detrusor.

Novel Evidence That the Pannexin 1 Channel Is Involved in Adenosine Triphosphate (ATP) Release from Cells for Optimal Mobilization of Hematopoietic Stem Progenitor Cells, and the Pannexin 1 SNP 5 (Rs3020015) T/C Polymorphism Characterizes Poor Mobilizer Status in Patients

Background . Pannexins have been shown to act predominantly as large transmembrane channels connecting the intracellular and extracellular space, allowing the passage of ions and small molecules, such as adenosine triphosphate (ATP), between these compartments. Pannexin 1 channels are involved in the release of ATP from cells and have been shown to be involved in the early stages of the innate immune response through an interaction with the P2X7 purinergic receptor. Recently, we provided evidence that activation of innate immunity in BM after administration of G-CSF produces "sterile inflammation" in the BM microenvironment and activates the pannexin channel to release ATP, which through binding to the P2X7 receptor leads to the mobilization of HSPCs (Leukemia 2018, 32:1920-1931). Corroborating our observation, it has been recently reported that the presence of the Gln460Arg SNP polymorphism within the P2X7 receptor gene in HSPCs, which is always co-inherited with Ala348Thr to form the gain-of-function haplotype 4, resulted in a significant increase in CD34+ HSPC mobilization (Leukemia 2018; 32:2724-2726). To shed more light on the novel concept that pannexin-1-released ATP, which interacts with P2X7, plays a crucial role in the egress of HSPCs from BM into peripheral blood (PB), we focused on the role of pannexin 1 in the mobilization process. Hypothesis. We hypothesized that pannexin 1 deficiency would negatively impact mobilization of HSPCs. Materials and Methods. First, we mobilized mice with G-CSF or AMD3100 in the presence of pannexin-1-blocking peptide. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, we analyzed five types of polymorphisms in the human pannexin 1 gene (SNP1-Rs1138800 A/C, SNP2-Rs7928030 G/C, SNP3-Rs12294985 C/T, SNP4-Rs127933348 A/G, and SNP5-Rs3020015 T/C) and correlated them with good or poor mobilization status of the patients. Patients in our studies were mobilized in the Bone Marrow Transplant Unit, Warsaw Medical University with G-CSF using a standard mobilization protocol, and poor mobilizers were identified as patients who were not able to mobilize the required number of CD34+ cells according to standard criteria. In our studies DNA from patient blood samples was isolated and fragments of DNA amplified and subsequently sequenced. Our patients in the good- and poor-mobilizer groups were matched for age, sex, and basic disorders. Results. We found that mice with blocked pannexin 1 channels mobilized HSPCs significantly less efficiently. More importantly, our patient data revealed that ~60% of patients that turned out to be poor HSPC mobilizers (n=20) displayed the pannexin 1 polymorphism SNP5 (Rs3020015) T/C. This polymorphism was observed in only 1 out of 26 good-mobilizer patients. Conclusions. Our results further support an important role for ATP-mediated purinergic signaling in the mobilization of HSPCs. Moreover, the pannexin 1 SNP5Rs3020015 T/C polymorphism may serve as a diagnostic tool to identify poor mobilizers. This investigation shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in the case of poor mobilizers. Disclosures No relevant conflicts of interest to declare.

Novel Evidence That the Nlrp3 Inflammasome Plays a Role in Bone Marrow As a “Cogwheel” Connecting Purinergic Signaling with Activation of the Complement Cascade to Induce “Sterile Inflammation”, Which Is Required for Optimal Mobilization of Hematopoietic Stem/Progenitor Cells

Background . In order to develop more efficient mobilization strategies, we have to better understand the mobilization process at the molecular and cellular levels. We envision pharmacological mobilization of HSPCs as the result of "sterile inflammation" in the bone marrow (BM) microenvironment induced by pro-mobilizing agents (e.g., G-CSF or AMD3100). This leads to activation of the complement cascade (ComC), which is required for egress of HSPCs from BM into peripheral blood (PB) (Leukemia 2018; 32:1116-1123). In support of this hypothesis, we reported recently that BM-residing innate immunity cells (e.g., monocytes, granulocytes and dendritic cells) release adenosine triphosphate (ATP) into the extracellular space during the initiation phase of mobilization. ATP released from the cells is an important signaling molecule that activates purinergic signaling receptors on the surface of hematopoietic cells (Leukemia 2018, 32:1920-1931) and is released mainly by pannexin 1 channels. Inhibition of this channel significantly decreases mobilization efficacy (Leukemia 2018, 32:1920-1931). In addition, ATP in the extracellular BM space is processed by the CD39 and CD73 ectonucleotidases to adenosine (Ado), which inhibits mobilization. To shed more light on the molecular mechanism governing mobilization of HSPCs, we focused on the role of the Nlrp3 inflammasome, which is highly expressed in hematopoietic cells. Moreover, while ATP binds to the P2X7 and P2X4 purinergic receptors and strongly activates Nlrp3, Ado inhibits migration of HSPCs in a heme oxygenase 1 (HO-1)-dependent manner.Hypothesis. We hypothesized that the Nlrp3 inflammasome is a link or "cogwheel" between purinergic signaling and the ComC that orchestrates optimal mobilization of HSPCs. Materials and Methods. To test this hypothesis we first mobilized wild type mice by G-CSF and AMD3100 administration in the presence of the small-molecule Nlrp3 inhibitor MCC950 or the Nlrp3 inflammasome activator nigericin, and the results were subsequently compared with results for Nlrp3-KO mice. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. The secretion of ATP from BM cells was inhibited by employing the pannexin-1-blocking drug probenecid or a synthetic pannexin-1-blocking peptide (Panx-1). Nlrp3 inflammasome mRNA expression was measured in BM innate immunity cells, CD11b/Gr-1+ cells, and CD11b/Gr-1+ cells isolated under steady-state conditions from mobilized mice stimulated with ATP or Ado. Elements of the activated inflammasome, such as caspase-1, IL-1b, and IL-18, were measured at the mRNA (RT-PCR) and protein (ELISA) levels. In parallel, we evaluated i) the expression of HO-1 in HSPCs by employing RQ-PCR and western blot analysis and ii) activation of the ComC by C5a ELISA. Results. While mobilization of HSPCs was significantly decreased in P2X7-KO mice and mice with Nlrp3 inflammasome deficiency, it was significantly increased in the presence of the Nlrp3 inflammasome activator nigericin. Proper activation of the inflammasome required ATP release in a pannexin-1-dependent manner and stimulation of hematopoietic cells in a P2X7 purinergic receptor-dependent manner. Moreover, Ado inhibited the mobilization process by upregulating HO-1 in HSPCs, which, as we observed, inhibits the Nlrp3 inflammasome in HSPCs. Finally, while activation of the Nlrp3 inflammasome was positively correlated with activation of the ComC, inhibition of the Nlrp3 inflammasome had the opposite effect. Conclusions. We demonstrate for the first time that purinergic signaling involving ATP and its metabolite adenosine regulate the mobilization of HSPCs in an Nlrp3 inflammasome-dependent manner. While ATP triggers and promotes this process via activation of the Nlrp3 inflammasome, adenosine has an inhibitory effect by upregulating HO-1 (Figure 1). Based on these findings, stimulation of the Nlrp3 inflammasome by ATP, inhibiting the Ado level by small-molecule inhibitors of the CD39 or CD73 enzymes involved in generation of extracellular Ado, or direct inhibition of HO-1 in HSPCs by employing small-molecule inhibitors may provide the basis for more efficient mobilization strategies. Figure 1 Disclosures No relevant conflicts of interest to declare.

Vesicular nucleotide transporter mediates adenosine triphosphate release in compressed human periodontal ligament fibroblast cells and participates in tooth movement-induced nociception in rats

ObjectivePain control is imperative in orthodontic treatment. Adenosine triphosphate (ATP) is a key mediator released from periodontal ligament cells that excites nociceptive nerve endings. Vesicular nucleotide transporter (VNUT), encoded by the Solute carrier family 17 member 9 (SLC17A9) gene, participates in ATP uptake into secretory vesicles; thus, it may mediate tooth movement-induced pain. In the present study, we examined whether VNUT in periodontal ligament cells participates in tooth movement-induced nociception. DesignExpression levels of SLC17A9, connexin 43, and pannexin 1 in human periodontal ligament fibroblasts (HPDLFs) were examined by quantitative reverse transcription-polymerase chain reaction. Mechanical force via centrifugation-induced ATP release was measured using an ATP bioluminescence assay. Inhibitors were used to evaluate the role of ATP transporters. Face-grooming behaviors were assessed as indicators of nociceptive responses after experimental tooth movement in rats, as well as the effects of drugs for the pain-like behavior. ResultsAfter HPDLFs underwent mechanical stimulation by centrifugation, SLC17A9 mRNA expression in the cells was significantly upregulated. Increased ATP release from HPDLFs after mechanical stimulation was suppressed by treatment with clodronic acid, a VNUT inhibitor, at concentrations of 0.1 and 1.0 μM. In rats, face-grooming behaviors (indicators of nociception) were significantly increased on day 1 after experimental tooth movement. Increased face-grooming behaviors were suppressed by systemic administration of clodronic acid (0.1 mg/kg). ConclusionsThese results indicate that release of ATP from periodontal ligament cells via VNUT is important for nociceptive transduction during orthodontic treatment. Thus, VNUT may provide a novel drug target for tooth movement-induced pain.

Danger-associated extracellular ATP counters MDSC therapeutic efficacy in acute GVHD

AbstractMyeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD), donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer, resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen–induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival, indicating in vivo biologic effects, no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1β (IL-1β), we expected that IL-1β KO donor MDSCs would be superior in subverting GVHD, but such MDSCs proved inferior relative to WT. IL-1β release and IL-1 receptor expression was required for optimal MDSC function, and exogenous IL-1β added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1β could diminish survival in GVHD. However, loss of inflammasome activation and IL-1β release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1β signaling, improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum, the ATP release site, synergized with WT MDSCs, as did regulatory T-cell infusion, which we showed reduced but did not eliminate MDSC inflammasome activation, as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments.

Adenosine Triphosphate Release and P2 Receptor Signaling in Piezo1 Channel-Dependent Mechanoregulation.

Organs and tissues and their constituent cells are physiologically submitted to diverse types of mechanical forces or stress, one common sequence of which is release of intracellular ATP into extracellular space. Extracellular ATP is a well-established autocrine or paracrine signaling molecule that regulates multiple cell functions and mediates cell-to-cell communications via activating the purinergic P2 receptors, more specifically, ligand-gated ion channel P2X receptors and some of the G-protein-coupled P2Y receptors. The molecular mechanisms that sense mechanical and transduce forces to trigger ATP release are poorly understood. The Piezo1, a newly identified mechanosensing ion channel, shows widespread expression and confers mechanosensitivity in many different types of cells. In this mini-review, we briefly introduce the Piezo1 channel and discuss the evidence that supports its important role in the mechanoregulation of diverse cell functions and, more specifically, critical engagement of ATP release and subsequent P2 receptor activation in Piezo1 channel-dependent mechanoregulation. Such ATP release-mediated coupling of the Piezo1 channel and P2 receptors may serve a signaling mechanism that is more common than we currently understand in transducing mechanical information to regulation of the attendant cell functions in various organs and tissues.

Extracellular nucleotide signaling in solid organ transplantation.

The role of extracellular purine nucleotides, including adenosine triphosphate (ATP) and adenosine, as modulators of posttransplantation outcome and ischemia-reperfusion injury is becoming increasingly evident. Upon pathological release of ATP, binding and activation of P2 purinergic surface receptors promote tissue injury and inflammation, while the expression and activation of P1 receptors for adenosine have been shown to attenuate inflammation and limit ischemia-induced damage, which are central to the viability and long-term success of allografts. Here we review the current state of the transplant field with respect to the role of extracellular nucleotide signaling, with a focus on the sources and functions of extracellular ATP. The connection between ischemia reperfusion, purinergic signaling, and graft preservation, as well as the role of ATP and adenosine as driving factors in the promotion and suppression of posttransplant inflammation and allograft rejection, are discussed. We also examine novel therapeutic approaches that take advantage of the ischemia-reperfusion-responsive and immunomodulatory roles for purinergic signaling with the goal of enhancing graft viability, attenuating posttransplant inflammation, and minimizing complications including rejection, graft failure, and associated comorbidities.

Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging.

Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.

Targeting chronic lymphocytic leukemia with N-methylated thrombospondin-1–derived peptides overcomes drug resistance

AbstractChronic lymphocytic leukemia (CLL), the most common adulthood leukemia in Western countries, is a very heterogeneous disease characterized by a peripheral accumulation of abnormal CD5+ B lymphocytes in the immune system. Despite new therapeutic developments, there remains an unmet medical need for CLL. Here, we demonstrate that the use of N-methylated thrombospondin-1 (TSP-1)–derived peptides is an efficient way to kill the malignant CLL cells, including those from high-risk individuals with poor clinical prognosis, del11q, del17p, 2p gain, or complex karyotype. PKT16, our hit N-methylated peptide, triggers the elimination of the leukemic cells, sparing the nontumor cells, including the hematopoietic precursors, and reduces the in vivo tumor burden of a CLL-xenograft mice model. A complementary analysis underscores the improved cytotoxic efficiency of PKT16 compared with the previously described TSP-1–derived probes, such as PKHB1. PKT16 elicits an original caspase-independent programmed necrotic mode of cell death, different from necroptosis or ferroptosis, implicating an intracellular Ca2+ deregulation that provokes mitochondrial damage, cell cycle arrest, and the specific death of the malignant CLL cells. The activation of the Gαi proteins and the subsequent drop of cyclic adenosine monophosphate levels and protein kinase A activity regulate this cytotoxic cascade. Remarkably, PKT16 induces the molecular hallmarks of immunogenic cell death, as defined by the calreticulin plasma membrane exposure and the release of adenosine triphosphate and high-mobility group box 1 protein from the dying CLL cells. Thus, PKT16 appears to be able to stimulate an anticancer in vivo immune response. Collectively, our results pave the way toward the development of an efficient strategy against CLL.

Optimized bioluminescence analysis of adenosine triphosphate (ATP) released by platelets and its application in the high throughput screening of platelet inhibitors.

Activated platelets release adenosine trisphosphate (ATP) and bioluminescence analysis of ATP release is usually used to monitor activation of platelets induced by various stimulants. However, bioluminescence analysis of ATP possesses poor linearity, the signal is quickly attenuated, and the accuracy of ATP release from platelets is hard to determine accurately enough to be used in a high throughput screening of platelet inhibitors. The present study was designed to optimize bioluminescence analysis of ATP released by platelets and expand its application in high throughput screening of platelet inhibitors. The results showed that accuracy of ATP analysis was significantly improved by adding coenzyme A (CoA) and signal attenuation of ATP analysis was greatly postponed by adding bovine serum albumin (BSA) both in Hank’s balanced salt solution (HBSS) and Tyrode’s buffer. Furthermore, ATP release of activated platelets and inhibitory effects of Ly294002 and Staurosporine on platelet activation were accurately determined by our optimized bioluminescence analysis of ATP. Thus, we have successfully constructed an optimized bioluminescence analysis of ATP which can be used in high throughput screening of platelet inhibitors.

Chemotherapy-Induced Tumor Cell Death at the Crossroads Between Immunogenicity and Immunotolerance: Focus on Acute Myeloid Leukemia.

In solid tumors and hematological malignancies, including acute myeloid leukemia, some chemotherapeutic agents, such as anthracyclines, have proven to activate an immune response via dendritic cell-based cross-priming of anti-tumor T lymphocytes. This process, known as immunogenic cell death, is characterized by a variety of tumor cell modifications, i.e., cell surface translocation of calreticulin, extracellular release of adenosine triphosphate and pro-inflammatory factors, such as high mobility group box 1 proteins. However, in addition to with immunogenic cell death, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, such as the overexpression of indoleamine 2,3-dioxygensase 1, which may ultimately hamper anti-tumor T-cells via the induction of T regulatory cells. The aim of this review is to summarize the current knowledge about the mechanisms and effects by which chemotherapy results in both activation and suppression of anti-tumor immune response. Indeed, a better understanding of the whole process underlying chemotherapy-induced alterations of the immunological tumor microenvironment has important clinical implications to fully exploit the immunogenic potential of anti-leukemia agents and tune their application.