Activating A1 adenosine receptor signaling boosts early pulmonary neutrophil recruitment in aged mice in response to Streptococcus pneumoniae infection.

BACKGROUND: Streptococcus pneumoniae (pneumococcus) is a leading cause of pneumonia in older adults. Successful control of pneumococci requires robust pulmonary neutrophil influx early in infection. However, aging is associated with aberrant neutrophil recruitment and the mechanisms behind that are not understood. Here we explored how neutrophil recruitment following pneumococcal infection changes with age and the host pathways regulating this. RESULTS: Following pneumococcal infection there was a significant delay in early neutrophil recruitment to the lungs of aged mice. Neutrophils from aged mice showed defects in trans-endothelial migration in vitro compared to young controls. To understand the pathways involved, we examined immune modulatory extracellular adenosine (EAD) signaling, that is activated upon cellular damage. Signaling through the lower affinity A2A and A2B adenosine receptors had no effect on neutrophil recruitment to infected lungs. In contrast, inhibition of the high affinity A1 receptor in young mice blunted neutrophil recruitment to the lungs following infection. A1 receptor inhibition decreased expression of CXCR2 on circulating neutrophils, which is required for trans-endothelial migration. Indeed, A1 receptor signaling on neutrophils was required for their ability to migrate across endothelial cells in response to infection. Aging was not associated with defects in EAD production or receptor expression on neutrophils. However, agonism of A1 receptor in aged mice rescued the early defect in neutrophil migration to the lungs and improved control of bacterial burden. CONCLUSIONS: This study suggests age-driven defects in EAD damage signaling can be targeted to rescue the delay in pulmonary neutrophil migration in response to bacterial pneumonia.

Extracellular adenosine 5'-diphosphate promotes MCP-1/CCL2 expression via the P2Y13 purinergic receptor/ERK signaling axis in temporomandibular joint-derived mouse fibroblast-like synoviocytes.

BACKGROUND: Temporomandibular joint osteoarthritis (TMJ-OA) causes cartilage degeneration, bone cavitation, and fibrosis of the TMJ. However, the mechanisms underlying the fibroblast-like synoviocyte (FLS)-mediated inflammatory activity in TMJ-OA remain unclear. METHODS AND RESULTS: Reverse transcription-quantitative polymerase chain reaction analysis revealed that the P2Y1, P2Y12, and P2Y13 purinergic receptor agonist adenosine 5'-diphosphate (ADP) significantly induces monocyte chemotactic protein 1 (MCP-1)/ C-C motif chemokine ligand 2 (CCL2) expression in the FLS1 synovial cell line. In contrast, the uracil nucleotide UTP, which is a P2Y2 and P2Y4 agonist, has no significant effect on MCP-1/CCL2 production in FLS1 cells. In addition, the P2Y13 antagonist MRS 2211 considerably decreases the expression of ADP-induced MCP-1/CCL2, whereas ADP stimulation enhances extracellular signal-regulated kinase (ERK) phosphorylation. Moreover, it was found that the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 reduces ADP-induced MCP-1/CCL2 expression. CONCLUSION: ADP enhances MCP-1/CCL2 expression in TMJ FLSs via P2Y13 receptors in an MEK/ERK-dependent manner, thus resulting in inflammatory cell infiltration in the TMJ. Collectively, the findings of this study contribute to a partial clarification of the signaling pathway underlying the development of inflammation in TMJ-OA and can help identify potential therapeutic targets for suppressing ADP-mediated purinergic signaling in this disease.

Extracellular ATP and adenosine in tumor microenvironment: Roles in epithelial-mesenchymal transition, cell migration, and invasion.

Under nonpathological conditions, the extracellular nucleotide concentration remains constant and low (nM range) because of a close balance between ATP release and ATP consumption. This balance is completely altered in cancer disease. Adenine and uridine nucleotides are found in the extracellular space of tumors in high millimolar (mM) concentrations acting as extracellular signaling molecules. In general, although uridine nucleotides may be involved in different tumor cell responses, purinergic signaling in cancer is preferentially focused on adenine nucleotides and nucleosides. Extracellular ATP can bind to specific receptors (P receptors) triggering different responses, or it can be hydrolyzed by ectoenzymes bound to cell membranes to render the final product adenosine. The latter pathway plays an important role in the increase of adenosine in tumor microenvironment. In this study, we will focus on extracellular ATP and adenosine, their effects acting as ligands of specific receptors, activating ectoenzymes, and promoting epithelial-mesenchymal transition, migration, and invasion in cancer cells. Finding the roles that these nucleotides play in tumor microenvironment may be important to design new intervention strategies in cancer therapies.

Extracellular adenosine triphosphate induces IDO and IFNγ expression of human periodontal ligament cells through P2 X7 receptor signaling.

BACKGROUND: Mechanical stimuli induce the release of adenosine triphosphate into the extracellular environment by human periodontal ligament cells (hPDLCs). Extracellular adenosine triphosphate (eATP) plays the role in both inflammation and osteogenic differentiation. eATP involves in immunosuppressive action by increasing immunosuppressive molecules IDO and IFNγ expression on immune cells. However, the role of eATP on the immunomodulation of hPDLCs remains unclear. This study aimed to examine the effects of eATP on the IDO and IFNγ expression of hPDLCs and the participation of purinergic P2 receptors in this phenomenon. METHODS: hPDLCs were treated with eATP. The mRNA and protein expression of indoleamine-pyrrole 2,3-dioxygenase (IDO) and interferon-gamma (IFNγ) were determined. The role of the purinergic P2 receptor was determined using calcium chelator (EGTA) and PKC inhibitor (PKCi). Chemical inhibitors (KN62 and BBG), small interfering RNA (siRNA), and P2 X7 receptor agonist (BzATP) were used to confirm the involvement of P2 X7 receptors on IDO and IFNγ induction by hPDLCs. RESULTS: eATP significantly enhanced mRNA expression of IDO and IFNγ. Moreover, eATP increased kynurenine which is the active metabolite of tryptophan breakdown catalyzed by the IDO enzyme and significantly induced IFNγ protein expression. EGTA and PKCi reduced eATP-induced IDO and IFNγ expressions by hPDLCs, confirming the role of calcium signaling. Chemical P2 X7 inhibitors (KN62 and BBG) and siRNA targeting the P2 X7 receptor significantly inhibited the eATP-induced IDO and IFNγ production. Correspondingly, BzATP markedly increased IDO and IFNγ expression. CONCLUSION: eATP induced immunosuppressive function of hPDLCs by promoting IDO and IFNγ production via P2 X7 receptor signaling. eATP may become a promising target for periodontal regeneration by modulating immune response and further triggering tissue healing.

Transcriptome Profiling Reveals CD73 and Age-Driven Changes in Neutrophil Responses against Streptococcus pneumoniae.

Neutrophils are required for host resistance against Streptococcus pneumoniae, but their function declines with age. We previously found that CD73, an enzyme required for antimicrobial activity, is downregulated in neutrophils (also known as polymorphonuclear leukocytes [PMNs]) from aged mice. This study explored transcriptional changes in neutrophils induced by S. pneumoniae to identify pathways controlled by CD73 and dysregulated with age. Pure bone marrow-derived neutrophils isolated from wild-type (WT) young and old and CD73 knockout (CD73KO) young mice were mock challenged or infected with S. pneumoniae ex vivo. RNA sequencing (RNA-Seq) was performed to identify differentially expressed genes (DEGs). We found that infection triggered distinct global transcriptional changes across hosts that were strongest in CD73KO neutrophils. Surprisingly, there were more downregulated than upregulated genes in all groups upon infection. Downregulated DEGs indicated a dampening of immune responses in old and CD73KO hosts. Further analysis revealed that CD73KO neutrophils expressed higher numbers of long noncoding RNAs (lncRNAs) than those in WT controls. Predicted network analysis indicated that CD73KO-specific lncRNAs control several signaling pathways. We found that genes in the c-Jun N-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) pathway were upregulated upon infection in CD73KO mice and in WT old mice, but not in WT young mice. This corresponded to functional differences, as phosphorylation of the downstream AP-1 transcription factor component c-Jun was significantly higher in neutrophils from infected CD73KO mice and old mice. Importantly, inhibition of JNK/AP-1 rescued the ability of these neutrophils to kill S. pneumoniae. Together, our findings revealed that the ability of neutrophils to modify their gene expression to better adapt to bacterial infection is in part regulated by CD73 and declines with age.

A1 adenosine receptor signaling reduces Streptococcus pneumoniae adherence to pulmonary epithelial cells by targeting expression of platelet-activating factor receptor.

Extracellular adenosine production is crucial for host resistance against Streptococcus pneumoniae (pneumococcus) and is thought to affect antibacterial immune responses by neutrophils. However, whether extracellular adenosine alters direct host-pathogen interaction remains unexplored. An important determinant for lung infection by S. pneumoniae is its ability to adhere to the pulmonary epithelium. Here we explored whether extracellular adenosine can directly impact bacterial adherence to lung epithelial cells. We found that signaling via A1 adenosine receptor significantly reduced the ability of pneumococci to bind human pulmonary epithelial cells. A1 receptor signaling blocked bacterial binding by reducing the expression of platelet-activating factor receptor, a host protein used by S. pneumoniae to adhere to host cells. In vivo, A1 was required for control of pneumococcal pneumonia as inhibiting it resulted in increased host susceptibility. As S. pneumoniae remain a leading cause of community-acquired pneumonia in the elderly, we explored the role of A1 in the age-driven susceptibility to infection. We found no difference in A1 pulmonary expression in young versus old mice. Strikingly, triggering A1 signaling boosted host resistance of old mice to S. pneumoniae pulmonary infection. This study demonstrates a novel mechanism by which extracellular adenosine modulates resistance to lung infection by targeting bacterial-host interactions.

A Body of Circumstantial Evidence for the Irreversible Ectonucleotidase Inhibitory Action of FSCPX, an Agent Known as a Selective Irreversible A1 Adenosine Receptor Antagonist So Far.

In previous studies using isolated, paced guinea pig left atria, we observed that FSCPX, known as a selective A1 adenosine receptor antagonist, paradoxically increased the direct negative inotropic response to A1 adenosine receptor agonists (determined using concentration/effect (E/c) curves) if NBTI, a nucleoside transport inhibitor, was present. Based on mathematical modeling, we hypothesized that FSCPX blunted the cardiac interstitial adenosine accumulation in response to nucleoside transport blockade, probably by inhibiting CD39 and/or CD73, which are the two main enzymes of the interstitial adenosine production in the heart. The goal of the present study was to test this hypothesis. In vitro CD39 and CD73 inhibitor assays were carried out; furthermore, E/c curves were constructed in isolated, paced rat and guinea pig left atria using adenosine, CHA and CPA (two A1 adenosine receptor agonists), FSCPX, NBTI and NBMPR (two nucleoside transport inhibitors), and PSB-12379 (a CD73 inhibitor), measuring the contractile force. We found that FSCPX did not show any inhibitory effect during the in vitro enzyme assays. However, we successfully reproduced the paradox effect of FSCPX in the rat model, mimicked the "paradox" effect of FSCPX with PSB-12379, and demonstrated the lipophilia of FSCPX, which could explain the negative outcome of inhibitor assays with CD39 and CD73 dissolved in a water-based solution. Taken together, these three pieces of indirect evidence are strong enough to indicate that FSCPX possesses an additional action besides the A1 adenosine receptor antagonism, which action may be the inhibition of an ectonucleotidase. Incidentally, we found that POM-1 inhibited CD73, in addition to CD39.

CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A2B Adenosine Receptor Signaling.

Glioblastoma (GB) is one of the deadliest brain cancers to afflict humans, and it has a very poor survival rate even with treatment. The extracellular adenosine-generating enzyme CD73 is involved in many cellular functions that can be usurped by tumors, including cell adhesion, proliferation, invasion, and angiogenesis. We set out to determine the role of CD73 in GB pathogenesis. To do this, we established a unique GB mouse model (CD73-FLK) in which we spatially expressed CD73 on endothelial cells in CD73-/- mice. This allowed us to elucidate the mechanism of host CD73 versus GB-expressed CD73 by comparing GB pathogenesis in WT, CD73-/-, and CD73-FLK mice. GB in CD73-/- mice had decreased tumor size, decreased tumor vessel density, and reduced tumor invasiveness compared with GB in WT mice. Interestingly, GBs in CD73-FLK mice were much more invasive and caused complete distortion of the brain morphology. We showed a 20-fold upregulation of A2B AR on GB compared with sham, and its activation induced matrix metalloproteinase-2, which enhanced GB pathogenesis. Inhibition of A2B AR signaling decreased multidrug resistance transporter protein expression, including permeability glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). Further, we showed that blockade of A2B AR signaling potently increased GB cell death induced by the chemotherapeutic drug temozolomide. Together, these findings suggest that CD73 and A2B AR play a multifaceted role in GB pathogenesis and progression and that targeting the CD73-A2B AR axis can benefit GB patients and inform new approaches for therapy to treat GB patients.SIGNIFICANCE STATEMENT Glioblastoma (GB) is the most devastating primary brain tumor. GB patients' median survival is 16 months even with treatment. It is critical that we develop prophylaxes to advance GB treatment and improve patient survival. CD73-generated adenosine has been implicated in cancer pathogenesis, but its role in GB was not ascertained. Here, we demonstrated that host CD73 plays a prominent role in multiple areas of glioblastoma pathogenesis, including promoting GB growth, its angiogenesis, and its invasiveness. We found a 20-fold increase in A2B adenosine receptor (AR) expression on GB compared with sham, and its inhibition increased GB chemosensitivity to temozolomide. These findings strongly indicate that blockade or inhibition of CD73 and the A2B AR are prime targets for future GB therapy.

The yin and yang functions of extracellular ATP and adenosine in tumor immunity.

Extracellular adenosine triphosphate (eATP) and its main metabolite adenosine (ADO) constitute an intrinsic part of immunological network in tumor immunity. The concentrations of eATP and ADO in tumor microenvironment (TME) are controlled by ectonucleotidases, such as CD39 and CD73, the major ecto-enzymes expressed on immune cells, endothelial cells and cancer cells. Once accumulated in TME, eATP boosts antitumor immune responses, while ADO attenuates immunity against tumors. eATP and ADO, like yin and yang, represent two opposite aspects from immune-activating to immune-suppressive signals. Here we reviewed the functions of eATP and ADO in tumor immunity and attempt to block eATP hydrolysis, ADO formation and their contradictory effects in tumor models, allowing the induction of effective anti-tumor immune responses in TME. These attempts documented that therapeutic approaches targeting eATP/ADO metabolism and function may be effective methods in cancer therapy.

Radiolabeled Human Monoclonal Antibody 067-213 has the Potential for Noninvasive Quantification of CD73 Expression.

BACKGROUND: CD73 is an ectonucleotidase regulating extracellular adenosine concentration and plays an important role in adenosine-mediated immunosuppressive pathways. The efficacy of CD73-targeted therapy depends on the expression levels of CD73; therefore, monitoring CD73 status in cancer patients would provide helpful information for selection of patients who would benefit from CD73-targeted therapy. Here, we evaluated the ability of 111In-labeled antibody 067-213, which has high affinity for human CD73, to act as a noninvasive imaging probe. METHODS: Cell binding and competitive inhibition assays for 111In-labeled 067-213 were conducted using MIAPaCa-2 (high CD73 expression) and A431 (low CD73 expression) cells. For in vivo assessments, biodistribution and SPECT/CT studies were conducted in MIAPaCa-2 and A431 tumor-bearing mice. To estimate the absorbed dose in humans, biodistribution and SPECT/CT studies were conducted in healthy rats. RESULTS: 111In-labeled 067-213 bound to MIAPaCa-2 and A431 cells in a CD73-dependent manner and the affinity loss after 111In-labeling was limited. Biodistribution and SPECT/CT studies with 111In-labeled 067-213 in mice showed high uptake in MIAPaCa-2 tumors and lower uptake in A431 tumors. In rats, the probe did not show high uptake in normal organs, including endogenously CD73-expressing organs. The estimated absorbed doses in humans were reasonably low. CONCLUSIONS: 111In-labeled 067-213 showed CD73-expression-dependent tumor uptake and low uptake in normal organs and tissues. Radiolabeled 067-213 holds promise as an imaging probe for noninvasive evaluation of CD73 expression levels in patients. Our data encourage further clinical studies to clarify a role for CD73 monitoring in patients receiving CD73-targeted immune therapy.

Cell type- and tissue-specific functions of ecto-5'-nucleotidase (CD73).

Ecto-5'-nucleotidase [cluster of differentiation 73 (CD73)] is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, many protective physiological functions of CD73 need to be taken into account for new targeted therapies. This review examines CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene NT5E cause the rare disease "arterial calcifications due to deficiency of CD73." Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. In the context of the central nervous system, CD73 is antinociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, a role that is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia-reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury is less clear. For example, CD73 has a promoting role in liver fibrosis but is protective in lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.

Extracellular adenosine: a critical signal in liver fibrosis.

Extracellular adenosine nucleoside is a potent, endogenous mediator that signals through specific G protein-coupled receptors, and exerts pleiotropic effects on liver physiology, in health and disease. Particularly, adenosinergic or adenosine-mediated signaling pathways impact the progression of hepatic fibrosis, a common feature of chronic liver diseases, through regulation of matrix deposition by liver myofibroblasts. This review examines the current lines of evidence on adenosinergic regulation of liver fibrosis and myofibroblasts, identifies unanswered research questions, and proposes important future areas of investigation.

Activation of NLRP3 signalling accelerates skin wound healing.

The process of skin wound healing involves the following three steps: inflammation, tissue formation and tissue remodelling. These optimal steps are required for the development of normal wound healing. Recent reports demonstrated that inflammasomes are involved in the innate immune response. In the present study, we examined whether the activation of inflammasomes affects the process of skin wound repair. The skin wound repair model was established using wild-type (WT), NACHT, LRR and PYD domains-containing protein 3 (NALP3) knockout (KO) and ASC-KO mice. The wounds were observed every other day, and changes in wound size over time were calculated using photography. Wound repair in NALP3-KO and ASC-KO mice was significantly impaired compared with WT mice. Isoliquiritigenin, an inhibitor of NALP3, decreased the rate of wound repair in WT mice. mRNA expression of pro-inflammatory cytokines in the wound sites of NALP3-KO mice was markedly decreased compared with WT mice. Treatment with adenosine triphosphate (ATP), a ligand of NALP3, upregulated the mRNA expression of pro-inflammatory cytokines at the wound site and accelerated wound healing in the WT mice. Scratch assay revealed that ATP accelerated wound closure in mouse embryonic fibroblasts from WT mice but not from NALP3-KO mice. In conclusion, the present study demonstrated that NALP3 pathway activation is involved in wound repair, and the topical use of ATP may be useful as an effective treatment for accelerating wound healing.

Sera from Septic Patients Contain the Inhibiting Activity of the Extracellular ATP-Dependent Inflammasome Pathway.

Immunoparalysis is a common cause of death for critical care patients with sepsis, during which comprehensive suppression of innate and adaptive immunity plays a significant pathophysiological role. Although the underlying mechanisms are unknown, damage-associated molecular patterns (DAMPs) from septic tissues might be involved. Therefore, we surveyed sera from septic patients for factors that suppress the innate immune response to DAMPs, including adenosine triphosphate (ATP), monosodium urate, and high mobility group box-1. Macrophages, derived from THP-1 human acute monocytic leukemia cells, were incubated with each DAMP, in the presence or absence of sera that were collected from critically ill patients. Secreted cytokines were then quantified, and cell lysates were assayed for relevant intracellular signaling mediators. Sera from septic patients who ultimately did not survive significantly suppressed IL-1ß production only in response to extracellular ATP. This effect was most pronounced with sera collected on day 3, and persisted with sera collected on day 7. However, this effect was not observed when THP-1 cells were treated with sera from survivors of sepsis. Septic sera collected at the time of admission (day 1) also diminished intracellular levels of inositol 1,4,5-triphosphate and cytosolic calcium (P < 0.01), both of which are essential for ATP signaling. Finally, activated caspase-1 was significantly diminished in cells exposed to sera collected on day 7 (P < 0.05). In conclusion, the sera of septic patients contain certain factors that persistently suppress the immune response to extracellular ATP, thereby leading to adverse clinical outcomes.

P2X receptors: Insights from the study of the domestic dog.

Fifty years ago, the late Geoffrey Burnstock described the concept of purinergic nerves and transmission bringing into existence the broader concepts of purinergic signaling including P2X receptors. These receptors are trimeric ligand-gated cation channels activated by extracellular adenosine 5'-triphosphate (ATP). P2X receptors have important roles in health and disease and continue to gain interest as potential therapeutic targets in inflammatory, neurological, cardiovascular and many other disorders including cancer. Current understanding of P2X receptors has largely arisen from the study of these receptors in humans and rodents, but additional insights have been obtained from the study of P2X receptors in the domestic dog, Canis familiaris. This review article will briefly introduce purinergic signaling and P2X receptors, before detailing the pharmacological profiles of the two recombinant canine P2X receptors studied to date, P2X7 and P2X4. The article will then describe the current state of knowledge concerning the distribution and function of the P2X receptor family in dogs. The article will also discuss the characterization of single nucleotide polymorphisms in the canine P2RX7 gene, and contrast this variation to the canine P2RX4 gene, which is largely conserved between dogs. Finally, this article will outline published examples of the use of dogs to study the pharmacokinetics of P2X7 and P2X3 antagonists, and how they have contributed to the preclinical testing of antagonists to human P2X7, CE-224,535, and human P2X3, Gefapixant (AF-219, MK-7264) and Eliapixant (BAY, 1817080), with Gefapixant gaining recent approval for use in the treatment of refractory chronic cough in humans. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.

Adeno-Associated Viral Vector-Delivered Pannexin-1 Mimetic Peptide Alleviates Airway Inflammation in an Allergen-Sensitized Mouse Model.

Asthma is a chronic inflammatory disease around the world. Extracellular adenosine triphosphate works as a dangerous signal in responding to cellular stress, irritation, or inflammation. It has also been reported its association with the pathogenicity in asthma, with increased level in lungs of asthmatics. Pannexin-1 is one of the routes that contributes to the release of adenosine triphosphate form intracellular to extracellular. The aim of this study was to apply pannexin-1 peptide antagonist 10Panx1 into adeno-associated viral (AAV) vectors on ovalbumin (OVA)-induced asthmatic mouse model. The results demonstrated that this treatment was able to reduce the adenosine triphosphate level in bronchoalveolar lavage fluid and downregulate the major relevant to the symptom of asthma attack, airway hyperresponsiveness to methacholine. The histological data also gave a positive support with decreased tissue remodeling and mucus deposition. Other asthmatic related features, including eosinophilic inflammation and OVA-specific T helper type 2 responses, were also decreased by the treatment. Beyond the index of inflammation, the proportion of effector and regulatory T cells was examined to survey the potential mechanism behind. The data provided a slightly downregulated pattern in lung GATA3+ CD4 T cells. However, an upregulated population of CD25+FoxP3+ CD4 T cells was seen in spleens. These data suggested that exogeneous expression of 10Panx1 peptide was potential to alleviated asthmatic airway inflammation, and this therapeutic effect might be from 10Panx1-mediated disruption of T cell activation or differentiation. Collectively, AAV vector-mediated 10Panx1 expression could be a naval therapy option to develop.

Increased Expression of Mitochondrial UQCRC1 in Pancreatic Cancer Impairs Antitumor Immunity of Natural Killer Cells via Elevating Extracellular ATP.

Pancreatic cancer (PC) is one of the most lethal malignancies characterized by a highly immunosuppressive tumor microenvironment (TME). Previously, we have reported that ubiquinol-cytochrome c reductase core protein I (UQCRC1), a key component of mitochondrial complex III, is generally upregulated in PC and produces extracellular ATP (eATP) to promote PC progression. Here, we sought to investigate whether the oncogenic property of UQCRC1 is generated through its effects on natural killer (NK) cells in the TME. We found that UQCRC1 overexpression in PC cells inhibited cytotoxicity of NK cells, as well as the infiltration of NK cells toward PC, whereas knockdown of UQCRC1 enhanced the cytotoxicity and chemotaxis of NK cells. Adoptive NK cell therapy in the subcutaneous mouse model and CIBERSORTx analysis with human PC specimens confirmed UQCRC1 elicited immunosuppressive effects on NK cells. Such UQCRC1-induced impairment of NK cells was mediated by eATP and its metabolite adenosine via P2Y11R and A2AR, respectively. Mechanistically, we found the UQCRC1/eATP axis reduced the expression of chemokine CCL5 in cancer cells and altered the balance of activating receptor DNAM-1 and inhibitory receptor CD96 on NK-92MI cells, resulting in decreased chemotaxis and exhausted phenotype of NK-92MI cells. Taken together, our study provides the evidence to support a novel mechanism by which energy metabolism change in cancer cells remodels the TME and impedes NK cell surveillance. It also suggests that targeting UQCRC1 may be a potential combined strategy for PC immunotherapy.

The Potential of Purinergic Signaling to Thwart Viruses Including SARS-CoV-2.

A long-shared evolutionary history is congruent with the multiple roles played by purinergic signaling in viral infection, replication and host responses that can assist or hinder viral functions. An overview of the involvement of purinergic signaling among a range of viruses is compared and contrasted with what is currently understood for SARS-CoV-2. In particular, we focus on the inflammatory and antiviral responses of infected cells mediated by purinergic receptor activation. Although there is considerable variation in a patient's response to SARS-CoV-2 infection, a principle immediate concern in Coronavirus disease (COVID-19) is the possibility of an aberrant inflammatory activation causing diffuse lung oedema and respiratory failure. We discuss the most promising potential interventions modulating purinergic signaling that may attenuate the more serious repercussions of SARS-CoV-2 infection and aspects of their implementation.

Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

BACKGROUND & AIMS: Although cancer immunotherapies are effective for advanced-stage cancers, there are no clinically approved immunotherapies for colon cancers (CRCs). Therefore, there is a high demand for the development of novel therapies. Extracellular adenosine-mediated signaling is considered a promising target for advanced-stage cancers that are nonresponsive to programmed death 1 (PD-1)-/programmed death-ligand 1 (PD-L1)-targeted immunotherapies. In this study, we aimed to elucidate novel tumorigenic mechanisms of extracellular adenosine. METHODS: To investigate the effects of extracellular adenosine on tumor-associated macrophages, peripheral blood-derived human macrophages were treated with adenosine and analyzed using flow cytometry and Western blot. Changes in adenosine-treated macrophages were further assessed using multi-omics analysis, including total RNA sequencing and proteomics. Colon cancer mouse models were used to measure the therapeutic efficacy of AB680 and palbociclib. We also used tissue microarrays of patients with CRC, to evaluate their clinical relevance. RESULTS: Extracellular adenosine-mediated reduction of cyclin D1 (CCND1) was found to be critical for the regulation of immune checkpoint molecules and PD-L1 levels in human macrophages, indicating that post-translational modification of PD-L1 is affected by adenosine. A potent CD73 selective inhibitor, AB680, reversed the effects of adenosine on CCND1 and PD-L1. This result strongly suggests that AB680 is a combinatory therapeutic option to overcome the undesired side effects of the cyclin-dependent kinase 4/6 inhibitor, palbociclib, which increases PD-L1 expression in tumors. Because palbociclib is undergoing clinical trials for metastatic CRC in combination with cetuximab (clinical trial number: NCT03446157), we validated that the combination of AB680 and palbociclib significantly improved anti-tumor efficacy in CRC animal models, thereby highlighting it as a novel immunotherapeutic strategy. We further assessed whether the level of CCND1 in tumor-associated macrophages was indeed reduced in tumor sections obtained from patients with CRC, for evaluating the clinical relevance of this strategy. CONCLUSIONS: In this study, we demonstrated that a novel combination therapy of AB680 and palbociclib may be advantageous for the treatment of CRC.

Muscle satellite cells are impaired in type 2 diabetic mice by elevated extracellular adenosine.

Muscle regeneration is known to be defective under diabetic conditions. However, the underlying mechanisms remain less clear. Adult quiescent muscle satellite cells (MuSCs) from leptin-receptor-deficient (i.e., db/db) diabetic mice are defective in early activation in vivo, but not in culture, suggesting the involvement of pathogenic niche factors. Elevated extracellular adenosine (eAdo) and AMP (eAMP) are detected under diabetic conditions. eAdo and eAMP potently inhibit cell cycle re-entry of quiescent MuSCs and injury-induced muscle regeneration. Mechanistically, eAdo and eAMP engage the equilibrative Ado transporters (ENTs)-Ado kinase (ADK)-AMPK signaling axis in MuSCs to inhibit the mTORC1-dependent cell growth checkpoint. eAdo and eAMP also inhibit early activation of quiescent fibroadipogenic progenitors and human MuSCs by the same mechanism. Treatment of db/db diabetic mice with an ADK inhibitor partially rescues the activation defects of MuSCs in vivo. Thus, both ADK and ENTs represent potential therapeutic targets for restoring the regenerative functions of tissue stem cells in patients with diabetes.