BackgroundThe ATP-gated ionotropic P2X7 receptor (P2X7R) has the unusual ability to function as a small cation channel and a trigger for permeabilization of plasmalemmal membranes. In murine microglia, P2X7R-mediated permeabilization is fundamental to microglial activation, proliferation, and IL-1β release. However, the role of the P2X7R in primary adult human microglia is poorly understood.MethodsWe used patch-clamp electrophysiology to record ATP-gated current in cultured primary human microglia; confocal microscopy to measure membrane blebbing; fluorescence microscopy to demonstrate membrane permeabilization, caspase-1 activation, phosphatidylserine translocation, and phagocytosis; and kit-based assays to measure cytokine levels.ResultsWe found that ATP-gated inward currents facilitated with repetitive applications of ATP as expected for current through P2X7Rs and that P2X7R antagonists inhibited these currents. P2X7R antagonists also prevented the ATP-induced uptake of large cationic fluorescent dyes whereas drugs that target pannexin-1 channels had no effect. In contrast, ATP did not induce uptake of anionic dyes. The uptake of cationic dyes was blocked by drugs that target Cl− channels. Finally, we found that ATP activates caspase-1 and inhibits phagocytosis, and these effects are blocked by both P2X7R and Cl− channel antagonists.ConclusionsOur results demonstrate that primary human microglia in culture express functional P2X7Rs that stimulate both ATP-gated cationic currents and uptake of large molecular weight cationic dyes. Importantly, our data demonstrate that hypotheses drawn from work on murine immune cells accurately predict the essential role of P2X7Rs in a number of human innate immune functions such as phagocytosis and caspase-1 activation. Therefore, the P2X7R represents an attractive target for therapeutic intervention in human neuroinflammatory disorders.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1353-8) contains supplementary material, which is available to authorized users.

Immune cells of myeloid origin show robust expression of ATP-gated P2X7 receptors, two-transmembrane ion channels permeable to Na + , K + , and Ca 2+. Receptor activation promotes inflammasome activation and release of the proinflammatory cytokines IL-1b and IL-18. In this study, we show that ATP generates facilitating cationic currents in monocyte-derived human macrophages and permeabilizes the plasma membrane to polyatomic cationic dyes. We find that antagonists of PLA 2 and Cl 2 channels abolish P2X7 receptor-mediated current facilitation, membrane permeabilization, blebbing, phospholipid scrambling, inflammasome activation, and IL-1b release. Our data demonstrate significant differences in the actions of ATP in murine and human macrophages and suggest that PLA 2 and Cl 2 channels mediate innate immunity downstream of P2X7 receptors in human macrophages.

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Within the human immune system, the expression of GPR56, an adhesion G-protein couple receptor, is restricted to cytotoxic Natural Killer (NK) and T lymphocytes, including T effector memory re-expressing CD45RA (TEMRA) cells. In primary NK cells, GPR56 acts as an inhibitory receptor, as it diminishes their ability to eliminate target cells. Overexpression of GPR56 in immortal NK-92 cells also leads to robust inhibition of cell chemotaxis. GPR56 is speculated to promote retention of cytotoxic lymphocytes in inflamed peripheral tissues. Thus, GPR56 has the potential to regulate immune responses that may lead to excessive inflammation and autoimmunity. The aim of the present study was, therefore, to examine whether GPR56 mediates cytotoxic immune cell functions and whether GPR56 expression is altered in autoimmune diseases. To tackle these questions, we first compared expression of GPR56 in healthy and autoimmune disease patient tissues using scRNA-seq datasets. Next, we tested whether stimulation of GPR56 with a monoclonal antibody modulated the migratory capacity of NK and CD8+ TEMRA cells. We found that chemotaxis toward CXCL12, IL-8, and CX3CL1 was strikingly inhibited by GPR56. Using bulk RNA-seq, we found GPR56-stimulation impacted a multitude of signaling cascades that regulate cell chemotaxis. Taken together, our results confirm that GPR56 is a marker for NK and TEMRA cells in healthy and autoimmune patient tissues and establish GPR56 as a key regulator of lymphocyte chemotaxis.

P2X receptors (P2XRs) belong to the family of ligand-gated ion channels and are activated by ATP. They are expressed throughout the human body and mediate crucial physiological roles, such as modulation of synaptic transmission, inflammation and neuropathic pain. Despite recent breakthroughs in our understanding of the P2XR structure through the crystallization of P2XRs, many molecular aspects of ATP binding remain unclear. Here we combined whole cell and single channel electrophysiological recordings using a range of ATP analogs with an extensive mutagenesis screen to elucidate 1) whether the chemical nature or conformational flexibility of agonist affects the channel open state and 2) if the three agonist binding sites are functionally coupled. Our data show that other nucleotide triphosphates, such as CTP, GTP and UTP, act as partial agonists that lead to an open state identical to ATP with regards to single channel conductance and flickering behavior. Subtle ATP analogues with either increased or decreased conformational flexibility of the ribose ring also show identical open channel properties, suggesting that chemical nature and binding pose of the agonist have little effect on the conformation of the open pore. Further, we show that ATP-evoked responses are potentiated by co-application of partial agonists at sub-saturating concentrations. This phenomenon is present also in the absence of divalent cations and when mutations are introduced within the binding site or at the subunit interface. These data shed new light on how P2XRs can be modulated in a presence of multiple potential agonists, e.g. in a synaptic environment.