RESEARCH ARTICLE SPECIAL ISSUE: CELL BIOLOGY OF THE IMMUNE SYSTEM

P2X7 receptor activation increases expression of caveolin-1 andformation of macrophage lipid rafts, thereby boosting CD39 activityLuiz 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, Yan Wu2,Maria Serena Longhi2, Simon C. Robson2 and Robson Coutinho-Silva1,*

ABSTRACTMacrophages are tissue-resident immune cells that are crucial for theinitiation and maintenance of immune responses. Purinergicsignaling modulates macrophage activity and impacts cellularplasticity. The ATP-activated purinergic receptor P2X7 (also knownas P2RX7) has pro-inflammatory properties, which contribute tomacrophage activation. P2X7 receptor signaling is, in turn, modulatedby ectonucleotidases, such as CD39 (also known as ENTPD1),expressed in caveolae and lipid rafts. Here, we examined P2X7receptor activity and determined impacts on ectonucleotidaselocalization and function in macrophages primed withlipopolysaccharide (LPS). First, we verified that ATP boosts CD39activity and caveolin-1 protein expression in LPS-primedmacrophages. Drugs that disrupt cholesterol-enriched domains –such as nystatin and methyl-β-cyclodextrin – decreased CD39enzymatic activity in all circumstances. We noted that CD39colocalized with lipid raft markers (flotillin-2 and caveolin-1) inmacrophages that had been primed with LPS followed by treatmentwith ATP. P2X7 receptor inhibition blocked these ATP-mediatedincreases in caveolin-1 expression and inhibited the colocalizationwith CD39. Further, we found that STAT3 activation is significantlyattenuated caveolin-1-deficient macrophages treated with LPS orLPS+BzATP. Taken together, our data suggest that P2X7 receptortriggers the initiation of lipid raft-dependent mechanisms thatupregulates CD39 activity and could contribute to limit macrophageresponses restoring homeostasis.

KEY WORDS: Extracellular ATP, Ectonucleotidases, Purinergicsignaling, Lipid rafts, Macrophages, P2RX7

INTRODUCTIONMacrophages are monocyte-derived or tissue-resident immune cellscrucial for the initiation and maintenance of immune responses.These cells recognize pathogen-associated molecular pattern

molecules (PAMPs) and damage-associated molecular patternmolecules (DAMPs) through pathogen recognition receptors(PRRs), such as Toll-like receptors (TLRs), thereby promotinginflammatory responses (reviewed in Gong et al., 2019; Gordon andPlüddemann, 2019).

Extracellular adenosine triphosphate (eATP) is a well-characterized DAMP that modulates macrophage function andplasticity (Barberá-Cremades et al., 2016; Savio and Coutinho-Silva, 2019). This nucleotide can be released from stressed, injuredand dying cells or in response to TLR activation, reaching highconcentrations within the extracellular milieu (Cohen et al., 2013).Once outside the cells, eATP can activate type 2 purinergic (P2)receptors. The P2 receptor family comprises the P2Y G-protein-coupled receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12,P2Y13 and P2Y14) and the P2X ligand-gated ion channels (P2X1,P2X2, P2X3, P2X4, P2X5, P2X6 and P2X17) (Ralevic andBurnstock, 1998; Abbracchio et al., 2006).

Of the latter group, the P2X7 receptor (also known as P2RX7) is thesubtype that has been most-extensively studied during inflammationand was found to provide host defenses against parasites by inducingthe activation of several microbicidal mechanisms (reviewed in Savioet al., 2018, Savio and Coutinho-Silva, 2019). The P2X7 receptorinduces the production of reactive oxygen and nitrogen species, andthe release of inflammatory cytokines, such as IL-1β and IL-18, byacting as the second signal to activate the NLRP3 inflammasome(Ferrari et al., 2006; Cruz et al., 2007). Furthermore, the P2X7receptor is involved in the activation of signaling pathways, such asMyD88/NFκB, PI3K/Akt/mTOR and STAT3, and activation ofmitogen-activated protein kinase (MAPK) pathway proteins, such asMEKs and ERK1/2 (MAPK3/MAPK1) (Bradford and Soltoff, 2002;Skaper et al., 2010; Liu et al., 2011; Bian et al., 2013; Savio et al.,2017a; de Andrade Mello et al., 2017b). It is thought that eATP-mediated P2X7 receptor signaling is a key component of macrophageinflammatory machinery and associated intercellular signalingresponses (Savio et al., 2018; Zumerle et al., 2019).

eATP signaling is precisely regulated by nucleotide-metabolizingcell-surface enzymes, the so-called ectonucleotidases. The familyof ectonucleoside triphosphate diphosphohydrolases (E-NTPDases)and that of 5′-nucleotidase (CD73, also known as NT5E) are themost important ectonucleotidases expressed in immune cells.E-NTPDases hydrolyze extracellular tri- and diphosphonucleosidesto monophosphonucleosides. Ectonucleoside triphosphatediphosphohydrolase 1, 2 and 3 (ENTPD1, ENTPD2 and ENTPD3,respectively; hereafter referred to as CD39, CD39L1 and CD39L3,respectively) are ectoenzymes that are tightly bound to the plasmamembrane through two transmembrane domains; CD39 is thedominant ecto-enzyme expressed in macrophages (Lévesque et al.,2010; Savio et al., 2017a). CD73 is a glycosylphosphatidylinositol-anchored enzyme that catalyzes the hydrolysis of AMP to adenosineReceived 5 August 2019; Accepted 28 January 2020

1Laboratory of Immunophysiology, Biophysics Institute Carlos Chagas Filho,Federal University of Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil.2Departments of Medicine and Anesthesia, Beth Israel Deaconess Medical Center,Harvard Medical School, Harvard University, Boston, MA 02215, USA. 3Laboratoryof Molecular Biology and Biochemistry of Proteins, Biophysics Institute CarlosChagas Filho, Federal University of Rio de Janeiro, 21941-902 Rio de Janeiro,Brazil. 4Departments of Anesthesiology, University of Illinois at Chicago, Chicago,IL 60612, USA. 5Departments of Pharmacology, University of Illinois at Chicago,Chicago, IL 60612, USA.

*Authors for correspondence (savio@biof.ufrj.br; rcsilva@biof.ufrj.br)

L.E.B.S., 0000-0002-6712-6885; S.A.C.S.S., 0000-0003-3495-6302; S.D.S.O.,0000-0002-7654-1909; R.D.M., 0000-0003-3164-475X; M.S.L., 0000-0002-4510-1249; R.C., 0000-0002-7318-0204

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© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs237560. doi:10.1242/jcs.237560

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https://jcs.biologists.org/content/133/5mailto:savio@biof.ufrj.brmailto:rcsilva@biof.ufrj.brhttp://orcid.org/0000-0002-6712-6885http://orcid.org/0000-0003-3495-6302http://orcid.org/0000-0002-7654-1909http://orcid.org/0000-0003-3164-475Xhttp://orcid.org/0000-0002-4510-1249http://orcid.org/0000-0002-4510-1249http://orcid.org/0000-0002-7318-0204

(Zimmermann, 1996). These enzymes are important to regulatemacrophage activation by degrading eATP to yield adenosine thatgenerally has anti-inflammatory effects, acting mainly via A2A orA2B adenosine receptors (Lévesque et al., 2010; Cohen et al., 2013;Savio et al., 2017a). The expression of CD39 and CD73 is regulatedby the transcription factors STAT3 and GFI-1, amongst others(Chalmin et al., 2012; Savio et al., 2017a). Both enzymes playrelevant roles in the pathophysiology of several inflammatorydiseases, including cancer, atherosclerosis, sepsis, autoimmune andneurological diseases (Cognato et al., 2011; Savio et al., 2017a,b; deAndrade Mello et al., 2017a; De Giorgi et al., 2017; Allard et al.,2017; Longhi et al., 2017; Vuerich et al., 2019; Takenaka et al.,2019). Nevertheless, cellular mechanisms that regulate thefunctionality of these enzymes in innate immune cells, such asmacrophages, remain poorly defined.Lipid rafts are membrane microdomains enriched in

sphingolipids and cholesterol, which serve as a platform for thedynamic assembly of signaling complexes, including TLR-dependent signaling pathways, during the inflammatory process(Płóciennikowska et al., 2015). TLR2 has been described to beassociated with lipid domains (Vieira et al., 2010; Fessler and Parks,2011). In addition, TLR4 migrates to lipid rafts after stimulationwith lipopolysaccharide (LPS) and forms complexes with othermolecules, including CD14 (Triantafilou et al., 2002, 2007; Vieiraet al., 2010; Fessler and Parks, 2011; Płóciennikowska et al., 2015).Furthermore, bacterial infections induce the formation of stableplasma membrane domains increasing the generation of ceramidefrom sphingomyelin in a reaction catalyzed by acidsphingomyelinase (Lu et al., 2012). The increase in ceramidelevels is accompanied by an increased expression of TLR4 in lipidraft domains (Lu et al., 2012; Tawadros et al., 2015). Interestingly,

P2X7 receptor activation can also stimulate sphingomyelinaseactivity, which promotes the formation of ceramide-enrichedmembrane domains (Garcia-Marcos et al., 2006; Lepine et al.,2006). Furthermore, both P2X7 receptor and CD39 have beendescribed to be palmitoylated at conserved clusters of cysteineresidues, and this post-translational modification targets theseproteins to lipid rafts (Koziak et al., 2000; Gonnord et al., 2009;Murrell-Lagnado, 2017). Gangadharan et al. (2015) showed thatcaveolin-1 attenuates P2X7 receptor-dependent signaling byinducing endocytosis following activation of this receptor inosteoblasts. This phenomenon controls the duration andmagnitude of P2X7 receptor activation, and can also remove othersignaling proteins from the membrane. Therefore, we hypothesizedthat TLR activation and the eATP/P2X7 receptor proinflammatorysignaling response in macrophages modifies lipid and proteindynamics in the plasma membrane by modulating CD39 activity ina lipid raft-dependent manner.

RESULTSATP-induced CD39 activity in LPS-primed macrophages isdependent on organizational integrity of membrane raftsInitially, we evaluated whether treatment of peritoneal macrophageswith drugs that disrupt cholesterol-enriched domains – such asnystatin and methyl-β-cyclodextrin (MβCD) – can impact CD39 orCD73 GPI-linked activity. Indeed, CD39 activity was decreased inperitoneal macrophages after treatment with nystatin or MβCD for10 min (P

Next, to confirm that the increase in CD39 activity induced byLPS+ATP depends on raft formation during which the enzymebecomes more stable and active, we treated the cells with benzylalcohol to increase membrane fluidity. After treatment with benzylalcohol, there was no increase in ATP hydrolysis in cells stimulatedwith LPS+ATP (P>0.05; Fig. 1A), confirming that changes inplasma membrane fluidity and composition can interfere with theactivity of CD39 enzyme. When evaluating CD73 functionality, weobserved increased activity after priming cells with LPS (Fig. 1B).Nevertheless, CD73 activity was not affected by lipid raft disruption(Fig. 1B).

ATP triggers movement of CD39 to lipid rafts in LPS-primedmacrophagesTo confirm that CD39 is located within these membrane domains inmacrophages, we double-labeled LPS- and ATP-treated peritonealmacrophages with antibodies recognizing CD39 and flotillin-2(used as a general marker of lipid rafts) (Zhao et al., 2011). Incontrol cells and cells treated with ATP only, CD39 and flotillin-2colocalized in a less-structured pattern, with some (but not all)flotillin-2-positive spots observed in cells stained for CD39(Fig. 2A). In contrast, cells treated with both LPS and ATPdisplayed numerous andmore-conspicuous spots of fluorescence thatwere consistently double-stained for CD39 and flotillin-2 (Fig. 2A);this suggests a high CD39 concentration within membrane domainsand is likely to represent lipid rafts. Treatment with MβCDdiminished the double-labeled spots of fluorescence (Fig. 2A).These observations were confirmed by plug-in Coloc2 analysisperformed with software Fiji/ImageJ (Fig. 2B-F), generating thePearson correlation coefficient of the pixel-intensity correlation fordouble-labeled points that indicates colocalization.As depicted in Fig. 2G, ATP+LPS treatment significantly

increased the R value for colocalization, whereas MβCD treatmentdissipated this effect, i.e. reducing the R value significantly whencompared to both control and LPS+ATP groups (Fig. 2G).

P2X7 receptor activation triggers CD39 to caveolae ofmacrophagesGiven that CD39 has palmitoylation sites and has previously beenreported to be located in caveolae of certain cell types (Koziak et al.,2000), we evaluated whether this enzyme can also be located incaveolae within the membrane of peritoneal macrophages treatedwith LPS+ATP. For this, we double-labeled LPS- and ATP-treatedperitoneal macrophages (pre-treated or not with the P2X7 receptorinhibitor A740003 or imipramine) with antibodies recognizingCD39 and caveolin-1 (a marker of caveolar domains).We verified that treatment with LPS+ATP induced a clear

increase in caveolin-1 expression, showing some points consistentlydouble labeled, suggesting colocalization of caveolin-1 and CD39(Fig. 3A). Furthermore, this effect was prevented by P2X7 receptorblockade or the treatment with imipramine.As we have previously shown, LPS and LPS+ATP treatment

boost CD39 activity (Savio et al., 2017a), and these inhibitoryeffects are blocked by P2X7 receptor inhibitors (A740003 andoxidized-ATP) or imipramine (Fig. 3B). In addition, MFI analysisfor CD39 and caveolin-1 showed that treatment with ATP+LPSsignificantly increases the cell surface levels of these proteins,whereas inhibition of P2X7 receptor blocks these effects (Fig. 3Cand D). The increased expression of caveolin-1 in peritonealmacrophages after treatment with LPS+ATP or LPS+BzATPwas confirmed by western blot experiments (Fig. 3E).Immunofluorescence images were also analyzed by Coloc2 plugin

in Fiji/ImageJ software (Fig. 3F), and Pearson correlationcoefficient values were calculated for double-labeled points(Fig. 3F and G). ATP+LPS treatment significantly increased the Rvalue for colocalization, whereas pretreatment with P2X7 receptorantagonists or imipramine inhibited this effect (Fig. 3G).

Caveolin-1 is important for P2X7 receptor-mediated increasein STAT3 activationFinally, we evaluated the relevance of caveolin-rich membranedomains in macrophages regarding the activation of thetranscription factor STAT3, which is involved in the induction ofCD39 expression in immune cells (Chalmin et al., 2012). LPSincreases STAT-3 phosphorylation in wild type (WT) macrophages,and P2X7 receptor activation in response to Bz-ATP significantlypotentiates the activation of this transcription factor, whereastreatment with 500 µM ATP did not increase levels ofphosphorylated STAT3 (p-STAT3) (Fig. 4A-B). This is possiblydue to the fact that Bz-ATP is 10× more potent than ATP inactivating the P2X7 receptor. Nevertheless, we do not excludeparticipation of other mechanisms and, potentially, other P2 receptorsin the regulation of STAT3 activation in these settings. However,when we used macrophages from caveolin-1-deficient mice, levels ofp-STAT3 were significantly diminished in both LPS- andLPS+BzATP-treated groups when compared with WT groups(Fig. 4A,B). To test for the putative contribution of P2X7 receptorto STAT3 activation, we further pre-treated WT cells with P2X7receptor inhibitors, i.e. A740003 and oxidized-ATP (oATP), and thenexposed these cells to LPS or LPS+BzATP. We found that eitherinhibitor significantly decreased p-STAT3 levels in cells treated withLPS or LPS+Bz-ATP (data not shown). These data suggest that P2X7receptor-dependent STAT3 phosphorylation in LPS-primedmacrophages depends on caveolin-rich membrane domains.

DISCUSSIONPurinergic signaling modulates macrophage activity and immuneresponses. Here, we show that the P2X7 receptor activates a lipidraft-dependent regulatory mechanism that modulates macrophageCD39 activity. P2X7 receptor activation increases activity of CD39and targets this protein to lipid rafts, whereas drugs that disruptcholesterol-enriched domains decreased its activity. P2X7 receptorinhibition blocked increases in caveolin-1 expression and inhibitedcolocalization with CD39. Interestingly, the formation of membranerafts is linked to the functionality of various components of thepurinergic signaling, such as the P2X7 receptor and CD39. Thelatter is anchored to the plasma membrane by two transmembranedomains. Drug- or detergent-induced disruption of these domainssignificantly decreases the activity of the CD39, further indicatingthat its activity requires the integrity of both transmembranedomains in the plasma membrane (Papanikolaou et al., 2005).

Grinthal and Guidotti (2006) have proposed that changes in themechanical properties (dependent on lipid composition) of theplasma membrane domain where CD39 molecules are insertedmodulate CD39 activity, by modifying the stability of itstransmembrane domains. This is in agreement with our data,showing that drugs that disrupt lipid raft membrane domains (i.e.nystatin or MβCD) abolish the ATP-induced increase in CD39activity in LPS-primed macrophages and decreases the CD39colocalization with flotillin-2, suggesting that CD39 activity isdependent on its localization in specific lipid microdomains.Moreover, treating cells with benzyl alcohol, which acts byincreasing membrane fluidity (Nagy et al., 2007), reversesincrease in ATP hydrolysis after treatment with LPS+ATP,

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Fig. 2. ATP treatment increases colocalization of CD39 and flotillin-2 in LPS-primed peritoneal macrophages. Mouse peritoneal macrophagesfrom wild-type animals were labeled with antibodies recognizing CD39 (red) and the lipid raft marker protein flotillin-2 (green), as well as with DAPI (blue) to stainthe nucleus. (A) Cells were left untreated (Control; first row) or were treated with 1 µg/ml LPS for 4 h (second row), 500 µM ATP for 1 h (third row), with LPSand ATP (fourth row), or with ATP, LPS and 10 mMmethyl-β-cyclodextrin (MβCD) (fifth row). CD39 and flotillin-2 colocalized in ‘spots’when cells were treated withATP or with ATP and LPS. Scale bars: 2 µm. (B-F) Representative intensity histogram outputs of Coloc2 analysis performed with Fiji/ImageJ software,and (G) Pearson correlation coefficient of the pixel-intensity correlation indicating colocalization of CD39 and flotillin-2. Data are expressed as mean±s.e.m.of three independent experiments. *,#P

Fig. 3. P2X7 receptor activation increases CD39 expression in caveolae of macrophages. LPS-primed peritoneal macrophages (1 µg/ml for 4 h) stimulatedwith 500 µM ATP for 1 h in the absence or presence of pre-treatment with the P2X7 receptor inhibitors A740003 (0.1 µM 30 min before priming), oxidized-ATP(oATP; 200 µM for 2 h before priming) or the sphingomyelinase inhibitor imipramine (IMI; 30 µM for 30 min before priming) were labeled with antibodiesrecognizing CD39 (green) and the lipid raft marker caveolin-1 (red), as well as with Hoechst 33258 (blue). (A) Representative immunofluorescence images of cellsstained for CD39 and caveolin-1. (B-D) Enzymatic assay for ATP hydrolysis (B) and quantification of fluorescence intensity for CD39 (C) and caveolin-1 (D).(E) Representative western blot showing caveolin-1 expression in untreated and LPS-primed macrophages stimulated with P2X7 receptor agonists (500 µM ATPor 100 µM Bz-ATP). (F,G) Representative intensity histogram output (F) of Coloc2 analysis performed with Fiji/ImageJ and Pearson correlation coefficientof the pixel-intensity correlation (G) indicating colocalization. Data are expressed asmean±s.e.m. of three independent experiments. *,#P

thereby demonstrating that changes in the composition and fluidityof the plasma membrane can modulate CD39 activity inmacrophages.Others and we infer that P2X7 receptor participates in lipid

metabolism and formation of lipid rafts. In fact, the activation of thisreceptor stimulates the sphingomyelinase activity that convertsphingomyelin into ceramide promoting the formation of ceramide-enriched membrane domains (Garcia-Marcos et al., 2006; Lepineet al., 2006). In this context, it is possible that the P2X7 receptorstimulates the formation of stable lipid membrane domains in whichCD39 becomes more stable and active.The results obtained in this study are in keeping with this

hypothesis, since treatment with ATP induced an increase in theactivity of CD39. In this regard, we have already shown that P2X7receptor activation boosts CD39 expression and activityin peritoneal macrophages (Savio et al., 2017a). In addition, wehave reported that P2X7 receptor activation is important foractivation of STAT3 (Savio et al., 2017a), a transcription factorthat promotes CD39 expression in immune cells (Chalmin et al.,2012). Moreover, De Marchi et al. (2019) have recently shownthat P2X7 receptor blockade reduces CD39 and CD73 expression inT effector and dendritic cells. Here, we found that caveolin-1expression is key to P2X7 receptor-mediated phosphorylation ofSTAT3 in LPS-primed macrophages. Thus, the P2X7 receptor mightmodulate the enzymatic activity of CD39 at transcriptional level

through a mechanism that, at least in part, is dependent uponformation of lipid membrane domains.

Our immunofluorescence experiments suggested that CD39 islocated within lipid rafts in peritoneal murine macrophages, and thattreatment with ATP induces aggregation of this protein and itsassociation with other lipid raft markers, such as flotillin-2 orcaveolin-1, although no coimmunoprecipitation experiments havebeen carried out to show physical interactions between theseproteins. These membrane structures probably represent largerceramide-enriched membrane domains, whose formation might bedirectly mediated through P2X7 receptor activation in response totreatment with ATP. Accordingly, this effect was prevented by eitherblockage of P2X7 receptor or treatment with the sphingomyelinaseinhibitor imipramine. In the same line of evidence, the presence ofpalmitoylation sites in CD39 which colocalize with caveolin-1 fromcanine kidney cells has been reported (Koziak et al., 2000).Therefore, we hypothesize that P2X7 receptor activation promotesthe formation of CD39-expressing caveolae.

In summary, our study here demonstrated an increase in CD39activity in response to stimulation of TLR4 (4 h) and P2X7 receptor.This increase was dependent on the integrity of membrane rafts, inwhich the CD39 is located after cells had been stimulated with LPSand ATP. The P2X7 receptor seems to modulate the functionality ofCD39 by facilitating the formation of membrane domains, in whichit becomes more stable and active (Fig. 5).

To understand the mechanisms that modulate CD39 activity iscrucial, given the immunoregulatory roles of this enzyme, as wellas its impact on macrophage activation and proinflammatoryresponses mediated upon activation of the P2X7 receptor (Savioet al., 2017a). Our results support the involvement of purinergicsignaling and lipid raft formation in cellular responses duringinflammation and infection. They also suggest therapeuticapproaches, including the administration of drugs that modulatethe formation of lipid raft or of soluble apyrases that mimic theaction of CD39.

MATERIALS AND METHODSAnimals and general reagentsWe used male wild-type C57BL/6 mice (8–10 weeks old) in this study. Insome experiments caveolin-1 null (caveolin-1−/−) mice (B6/129SJ) micepurchased from The Jackson Laboratory (Bar Harbor, ME) were used.Animals were housed at a ratio of five mice per cage, with water and food adlibitum, on a 12 h light/dark cycle (lights on at 7:00 am), and at a temperatureof 22±1°C. The procedures for the care and use of animals were according tothe guidelines of the Brazilian College of Animal Experimentation(COBEA) and to the Guide for the Care and Use of Laboratory Animals(National Research Council, USA). All experiments were approved by theCommission for the Ethical Use of Research Animals (CEUA) from theFederal University of Rio de Janeiro (UFRJ) (approved protocol number:IBCCF138) and by the Institutional Animal Care and Use Committees(IACUC) of Beth Israel Deaconess Medical Center (approved protocolnumber: 019-2015). ATP, ADP, AMP, oxidized-ATP, benzyl alcohol andimipramine were obtained from Sigma-Aldrich, MO. A740003 werepurchased from Tocris Inc, Ellsville, MO.

Peritoneal macrophagesMurine macrophages were harvested from the peritoneal cavity of adultmice. Cells obtained from the peritoneal cavity were plated in suspensioninto 24- or 96-well tissue culture plates (TPP AG, Switzerland) at a densityof 2×105 cells per well and incubated in non-supplemented Gibco® DMEM(Thermo Fisher Scientific, Rockford, IL) for 1 h, at 37°C, 5% CO2atmosphere. Non-adherent cells were removed by washing three times withPBS, and adherent cells were cultured overnight in Gibco®DMEM (ThermoFisher Scientific) complete medium before use in experiments.

Fig. 4. Caveolin-1 is important for STAT3 activation in LPS-primedmacrophages. (A,B) Representative western blot (A) and its densitometricanalysis (B) of levels of active, i.e. phosphorylated STAT3 (p-STAT3) inuntreated and LPS-primed macrophages derived from wild-type (WT) orcaveolin-1−/− mice stimulated with P2X7 receptor agonists (500 µMATP or 100 µM Bz-ATP). Data are expressed as mean±s.e.m. of threeindependent experiments. *P

Isolation, characterization and differentiation of bone marrow-derived macrophagesMedullar cells obtained from mouse tibias and femurs were resuspended innon-supplemented DMEM Gibco® (Thermo Fisher Scientific), passed

through a filter for cell culture (Cell Strainer; 40 µm) and centrifuged for10 min at 300 g at room temperature. Pellets were resuspended in red bloodcells lysis buffer (cat. no. A10492-01, Thermo Fisher Scientific).Subsequently, cells were centrifuged for 10 min at 300 g and washed

Fig. 5. Schematic representation of the modulation of CD39 activity through P2X7 receptor signaling in activated macrophages. Under homeostaticconditions (upper panel) TLR2, the P2X7 receptor and CD39 are present in several lipid raft microdomains on the cell surface, while TLR4 is located outside therafts. In contrast, during inflammation (lower panel) (1) the recognition of PAMPs – such as bacterial lipopolysaccharide (LPS) – by PRRs can induces ATPrelease, thereby activating the P2X7 receptor. This, in turn, induces (2) an extensive release of ATP that occurs via the P2X7 receptor itself or via pannexinhemichannels, thereby increasing caveolin-1 expression and the formation of caveolar domains on the plasma membrane. (3) In these domains, CD39 mightbecomemore stable and active, favoring the process of ATP hydrolysis to ADP and its metabolites, which, in a purinergic regulatory mechanism, could contributeto limit macrophage activation and inflammation.

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twice with PBS. Mononuclear bone marrow-derived macrophages(BMDMs) were then plated at a density of 1×108 cells in polystyreneflasks and cultured in DMEM Gibco® supplemented with 20% fetal bovineserum (Sigma-Aldrich), antibiotics (100 IU penicillin/ml and 100 mgstreptomycin/ml; Gibco®) and 10 ng/ml macrophage colony-stimulatingfactor (M-CSF) (cat. no. 315-02, Peprotech, NJ). Cells were maintained at37°C in an atmosphere of 5% CO2. After 4 days, culture medium wasreplaced, and non-adherent cells removed and discarded. After reaching80% confluence, cells were detached from polystyrene plates upon additionof 0.25% trypsin-EDTA (Sigma-Aldrich) and plated again for subsequentexperiments. The phenotype of BMDMs was determined by flow cytometryusing the following antibodies: APC labeled anti-CD11b APC (cat. no.101226, BioLegend, San Diego, CA) at 1:100 dilution and FITC labeledanti-F4/80 (cat. no. MCA497FT, AbD Serotec®, Bio-Rad, Hercules, CA)used at 1:100 dilution. Isotypes were used as negative control.

LPS priming and pharmacological treatmentsFor in vitro experiments, macrophages were left untreated or were primedwith 1 µg/ml bacterial lipopolysaccharide (LPS) for 4 h to induce aninflammatory response, and then stimulated with 500 µM ATP or 100 µMBzATP to activate P2X7 receptors for 1 h or 3 h. Alternatively, cells wereprimed with 1 µg/ml LPS for 4 h, stimulated with 500 µM ATP for 3 h, andthen treated with 50 mg/ml nystatin or 10 mM methyl-β-cyclodextrin(MβCD) for 10 min before, or with benzyl alcohol (to increase membranefluidity; 20 mM) 30 min before ectonucleotidase assays (Nagy et al., 2007).In some wells, cells were pretreated with P2X7 receptor antagonists (300 µMoATP for 2 h or 100 nM A740003 for 30 min) or the sphingomyelinaseinhibitor imipramine (30 µM for 30 min) before priming with LPS (Bai et al.,2014). All reagents were purchased from Sigma-Aldrich.

Ectonucleotidase activity assaysActivities of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and5′-nucleotidase (CD73) were estimated in a reaction medium consisting of20 mM HEPES buffer (pH 7.5) containing 1 mM CaCl2 (for ATP) orMgCl2 (for AMP), 120 mM NaCl, 5 mM KCl, 60 mM glucose, 1 mMsodium azide and 0.1% mM albumin (all reagents from Sigma-Aldrich).Peritoneal resident macrophages (2×105) were used in a final volume of200 µl reaction medium, and enzymatic reactions were started by addition ofATP or AMP to a final concentration of 2 mM, followed by incubation for30 min at 37°C. Reactions were stopped by addition of 200 µl of 10%trichloroacetic acid (TCA) (Sigma-Aldrich). Incubation times, proteinconcentrations, reaction mixtures and substrate concentrations were chosenaccording to a previous study (Vuaden et al., 2011). The amount of inorganicphosphate (Pi) released was measured using the colorimetric methoddescribed by Chan et al. (1986). Controls to correct for non-enzymatic Pi insamples were performed adding the nucleotides (ATP or AMP) after thereactions had been stopped with TCA. All reactions were performed intriplicates, and enzyme activities were expressed in nmol Pi released perminute per number of cells.

ImmunocytochemistryAfter LPS priming and pharmacological treatments described above,samples were fixed with 4% paraformaldehyde and 4% sucrose for 15 min atroom temperature, and blocked with 10% horse serum and 1% BSA in PBSfor 30 min at room temperature. Samples were then incubated (for 3 h atroom temperature) with the following primary antibodies (in 0.1% BSA inPBS): C42A3 rabbit anti-flotillin-2 mAb (cat. no. 3436, Cell SignalingTechnology, Danvers, MA) diluted 1:50 and goat anti-CD39 (cat. no.AF4398 (R&D Systems, Minneapolis, MN) diluted 1:200; or mice anti-caveolin-1 (7C8) (cat. no. NB100-615, Novus Biologicals, Littleton, CO)diluted 1:400 and goat anti-CD39 (cat. no. AF4398, R&D Systems) diluted1:200. Cells were then washed and incubated at room temperature for 1 hwith the following secondary antibodies (diluted 1:300, in 0.1% BSA inPBS): anti-rabbit IgG (H+L)-Alexa Fluor® 488 (Cell Signaling Technology)and anti-sheep IgG (H+L) Cy™5 (Jackson ImmunoResearch Laboratories,West Grove, PA); or anti-goat IgG (H+L)-Alexa Fluor® 488 and anti-miceIgG (H+L)-Alexa Fluor® 597 (Life Technologies, Eugene, OR). Finally,samples were stained with DAPI or Hoechst 33258 nuclear dye (1:10,000,

cat. no. H3569, Life Technologies, Eugene, OR), and then mounted andexamined in a fluorescence microscope Zeiss AxioVert 200M and the three-dimensional images (z-stack) in a Spinning Disk Confocal MicroscopeZEISS Cell Observer SD (Peabody, MA).

Cell microscopy analysisMean fluorescence intensity (MFI) was measured in Zen Lite Blue software(Carl Zeiss). For this quantification, the background was initially subtractedand the region of interest (ROI) was selected in individual cells by using thefreehand selection tool of the software to calculate the ratios of caveolin-1and CD39 MFI in response to different treatments. This is provided by thesoftware based on the intensity of the related pixels.

Colocalization analyses of the ratios of CD39 to flotillin-2 and of CD39 tocaveolin-1 were performed using Coloc-2 Fiji-ImageJ plugin version 3.0.For specific analysis, different image channels (green and red) were overlaidin the same z-plane, generating yellow spots where the two moleculesstudied were present at the same pixel locations. Ten fields per condition perexperiment were randomly chosen and measured by an investigator, whowas blind to the experimental conditions. The point spread function (PSF)was calculated and set to 3.0, whereas randomizations were set to 10.0. ThePearson correlation coefficient was calculated as indicative of colocalization(Bolte and Cordelier̀es, 2006; Adler and Parmryd, 2010; McCuaig et al.,2015; Schindelin et al., 2012).

Western blottingMacrophages were lysed in ice-cold modified RIPA buffer (50 mM Tris-HClpH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl)supplemented with Complete Proteinase Inhibitor Cocktails (RocheDiagnostics) and Phosphatase Inhibitor Cocktails (Sigma-Aldrich, MO).The lysates were sonicated briefly on ice and centrifuged at 18,000 g for10 min at 4°C. Protein concentrations were determined by Bio-Rad DCprotein assay reagent (Bio-Rad Laboratories), using bovine serum albumin asthe standard. Proteins (10 µg per lane) were boiled in XT Sample Buffer (cat.no. 161-0791, Bio-Rad Laboratories), separated using 4–12% Criterion XTBis-Tris SDS-PAGE (Bio-Rad Laboratories) and transferred to PVDFmembranes (cat. no. IPVH00010, Millipore) by semi-dry electroblotting.The latter were then probed with specific antibodies against proteins ofinterest. Bands were visualized using HRP-conjugated goat anti-mouse,donkey anti-rabbit or donkey anti-sheep IgG and the SuperSignal WestFemtoMaximum Sensitivity Substrate reagents applied (cat. no. PI-34096,Thermo Scientific) according to the manufacturer’s instructions (Savioet al., 2017a).

Statistical analysisResults are expressed as mean±standard error of mean (±s.e.m.). Statisticalanalysis was performed by one-way analysis of variance (ANOVA),followed by Tukey’s multiple range tests. Differences between groups wereconsidered statistically significant when P

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RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237560. doi:10.1242/jcs.237560

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