© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs237560. doi:10.1242/jcs.237560 RESEARCH ARTICLE SPECIAL ISSUE: CELL BIOLOGY OF THE IMMUNE SYSTEM P2X7 receptor activation increases expression of caveolin-1 and formation of macrophage lipid rafts, thereby boosting CD39 activity Luiz Eduardo Baggio Savio1,*, Paola de Andrade Mello2, Stephanie Alexia Cristina Silva Santos3, Júlia Costa de Sousa3, Suellen D. S. Oliveira4, Richard D. Minshall4,5, Eleonora Kurtenbach3,YanWu2, Maria Serena Longhi2, Simon C. Robson2 and Robson Coutinho-Silva1,* ABSTRACT molecules (PAMPs) and damage-associated molecular pattern Macrophages are tissue-resident immune cells that are crucial for the molecules (DAMPs) through pathogen recognition receptors initiation and maintenance of immune responses. Purinergic (PRRs), such as Toll-like receptors (TLRs), thereby promoting signaling modulates macrophage activity and impacts cellular inflammatory responses (reviewed in Gong et al., 2019; Gordon and plasticity. The ATP-activated purinergic receptor P2X7 (also known Plüddemann, 2019). as P2RX7) has pro-inflammatory properties, which contribute to Extracellular adenosine triphosphate (eATP) is a well- macrophage activation. P2X7 receptor signaling is, in turn, modulated characterized DAMP that modulates macrophage function and by ectonucleotidases, such as CD39 (also known as ENTPD1), plasticity (Barberá-Cremades et al., 2016; Savio and Coutinho- expressed in caveolae and lipid rafts. Here, we examined P2X7 Silva, 2019). This nucleotide can be released from stressed, injured receptor activity and determined impacts on ectonucleotidase and dying cells or in response to TLR activation, reaching high localization and function in macrophages primed with concentrations within the extracellular milieu (Cohen et al., 2013). lipopolysaccharide (LPS). First, we verified that ATP boosts CD39 Once outside the cells, eATP can activate type 2 purinergic (P2) activity and caveolin-1 protein expression in LPS-primed receptors. The P2 receptor family comprises the P2Y G-protein- macrophages. Drugs that disrupt cholesterol-enriched domains – coupled receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, such as nystatin and methyl-β-cyclodextrin – decreased CD39 P2Y13 and P2Y14) and the P2X ligand-gated ion channels (P2X1, enzymatic activity in all circumstances. We noted that CD39 P2X2, P2X3, P2X4, P2X5, P2X6 and P2X17) (Ralevic and colocalized with lipid raft markers (flotillin-2 and caveolin-1) in Burnstock, 1998; Abbracchio et al., 2006). macrophages that had been primed with LPS followed by treatment Of the latter group, the P2X7 receptor (also known as P2RX7) is the with ATP. P2X7 receptor inhibition blocked these ATP-mediated subtype that has been most-extensively studied during inflammation increases in caveolin-1 expression and inhibited the colocalization and was found to provide host defenses against parasites by inducing with CD39. Further, we found that STAT3 activation is significantly the activation of several microbicidal mechanisms (reviewed in Savio attenuated caveolin-1-deficient macrophages treated with LPS or et al., 2018, Savio and Coutinho-Silva, 2019). The P2X7 receptor LPS+BzATP. Taken together, our data suggest that P2X7 receptor induces the production of reactive oxygen and nitrogen species, and β triggers the initiation of lipid raft-dependent mechanisms that the release of inflammatory cytokines, such as IL-1 and IL-18, by upregulates CD39 activity and could contribute to limit macrophage acting as the second signal to activate the NLRP3 inflammasome responses restoring homeostasis. (Ferrari et al., 2006; Cruz et al., 2007). Furthermore, the P2X7 receptor is involved in the activation of signaling pathways, such as KEY WORDS: Extracellular ATP, Ectonucleotidases, Purinergic MyD88/NFκB, PI3K/Akt/mTOR and STAT3, and activation of signaling, Lipid rafts, Macrophages, P2RX7 mitogen-activated protein kinase (MAPK) pathway proteins, such as MEKs and ERK1/2 (MAPK3/MAPK1) (Bradford and Soltoff, 2002; INTRODUCTION Skaper et al., 2010; Liu et al., 2011; Bian et al., 2013; Savio et al., Macrophages are monocyte-derived or tissue-resident immune cells 2017a; de Andrade Mello et al., 2017b). It is thought that eATP- crucial for the initiation and maintenance of immune responses. mediated P2X7 receptor signaling is a key component of macrophage These cells recognize pathogen-associated molecular pattern inflammatory machinery and associated intercellular signaling responses (Savio et al., 2018; Zumerle et al., 2019). eATP signaling is precisely regulated by nucleotide-metabolizing 1Laboratory of Immunophysiology, Biophysics Institute Carlos Chagas Filho, cell-surface enzymes, the so-called ectonucleotidases. The family Federal University of Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil. 2Departments of Medicine and Anesthesia, Beth Israel Deaconess Medical Center, of ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) Harvard Medical School, Harvard University, Boston, MA 02215, USA. 3Laboratory and that of 5′-nucleotidase (CD73, also known as NT5E) are the of Molecular Biology and Biochemistry of Proteins, Biophysics Institute Carlos most important ectonucleotidases expressed in immune cells. Chagas Filho, Federal University of Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil. 4Departments of Anesthesiology, University of Illinois at Chicago, Chicago, E-NTPDases hydrolyze extracellular tri- and diphosphonucleosides IL 60612, USA. 5Departments of Pharmacology, University of Illinois at Chicago, to monophosphonucleosides. Ectonucleoside triphosphate Chicago, IL 60612, USA. diphosphohydrolase 1, 2 and 3 (ENTPD1, ENTPD2 and ENTPD3, *Authors for correspondence ([email protected]; [email protected]) respectively; hereafter referred to as CD39, CD39L1 and CD39L3, respectively) are ectoenzymes that are tightly bound to the plasma L.E.B.S., 0000-0002-6712-6885; S.A.C.S.S., 0000-0003-3495-6302; S.D.S.O., membrane through two transmembrane domains; CD39 is the 0000-0002-7654-1909; R.D.M., 0000-0003-3164-475X; M.S.L., 0000-0002-4510- 1249; R.C., 0000-0002-7318-0204 dominant ecto-enzyme expressed in macrophages (Lévesque et al., 2010; Savio et al., 2017a). CD73 is a glycosylphosphatidylinositol- Received 5 August 2019; Accepted 28 January 2020 anchored enzyme that catalyzes the hydrolysis of AMP to adenosine Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237560. doi:10.1242/jcs.237560 (Zimmermann, 1996). These enzymes are important to regulate P2X7 receptor activation can also stimulate sphingomyelinase macrophage activation by degrading eATP to yield adenosine that activity, which promotes the formation of ceramide-enriched generally has anti-inflammatory effects, acting mainly via A2A or membrane domains (Garcia-Marcos et al., 2006; Lepine et al., A2B adenosine receptors (Lévesque et al., 2010; Cohen et al., 2013; 2006). Furthermore, both P2X7 receptor and CD39 have been Savio et al., 2017a). The expression of CD39 and CD73 is regulated described to be palmitoylated at conserved clusters of cysteine by the transcription factors STAT3 and GFI-1, amongst others residues, and this post-translational modification targets these (Chalmin et al., 2012; Savio et al., 2017a). Both enzymes play proteins to lipid rafts (Koziak et al., 2000; Gonnord et al., 2009; relevant roles in the pathophysiology of several inflammatory Murrell-Lagnado, 2017). Gangadharan et al. (2015) showed that diseases, including cancer, atherosclerosis, sepsis, autoimmune and caveolin-1 attenuates P2X7 receptor-dependent signaling by neurological diseases (Cognato et al., 2011; Savio et al., 2017a,b; de inducing endocytosis following activation of this receptor in Andrade Mello et al., 2017a; De Giorgi et al., 2017; Allard et al., osteoblasts. This phenomenon controls the duration and 2017; Longhi et al., 2017; Vuerich et al., 2019; Takenaka et al., magnitude of P2X7 receptor activation, and can also remove other 2019). Nevertheless, cellular mechanisms that regulate the signaling proteins from the membrane. Therefore, we hypothesized functionality of these enzymes in innate immune cells, such as that TLR activation and the eATP/P2X7 receptor proinflammatory macrophages, remain poorly defined. signaling response in macrophages modifies lipid and protein Lipid rafts are membrane microdomains enriched in dynamics in the plasma membrane by modulating CD39 activity in sphingolipids and cholesterol, which serve as a platform for the a lipid raft-dependent manner. dynamic assembly of signaling complexes, including TLR- dependent signaling pathways, during the inflammatory process RESULTS (Płóciennikowska et al., 2015). TLR2 has been described to be ATP-induced CD39 activity in LPS-primed macrophages is associated with lipid domains (Vieira et al., 2010; Fessler and Parks, dependent on organizational integrity of membrane rafts 2011). In addition, TLR4 migrates to lipid rafts after stimulation Initially, we evaluated whether treatment of peritoneal macrophages with lipopolysaccharide (LPS) and forms complexes with other with drugs that disrupt cholesterol-enriched domains – such as molecules, including CD14 (Triantafilou et al., 2002, 2007; Vieira nystatin and methyl-β-cyclodextrin (MβCD) – can impact CD39 or et al., 2010; Fessler and Parks, 2011; Płóciennikowska et al., 2015). CD73 GPI-linked activity. Indeed, CD39 activity was decreased in Furthermore, bacterial infections induce the formation of stable peritoneal macrophages