P2X7 Receptor Activation Increases Expression of Caveolin-1 And

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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

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(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 after treatment with nystatin or MβCD for plasma membrane domains increasing the generation of ceramide 10 min (P<0.05), irrespectively of stimulation with LPS and ATP from sphingomyelin in a reaction catalyzed by acid (Fig. 1A). In addition, treatment with imipramine – which inhibits sphingomyelinase (Lu et al., 2012). The increase in ceramide acidic sphingomyelinase activity – attenuated the increase in CD39 levels is accompanied by an increased expression of TLR4 in lipid enzyme activity induced by treatment with LPS+ATP (P<0.05; raft domains (Lu et al., 2012; Tawadros et al., 2015). Interestingly, Fig. 1A).

Fig. 1. P2X7-dependent increase in CD39 activity depends on lipid raft integrity in LPS-primed macrophages. (A,B) ATP hydrolysis (A) and AMP hydrolysis (B) in LPS-primed macrophages (1 µg/ml LPS for 4 h), stimulated with 500 µM ATP for 3 h that were then treated with 10 mM methyl-β-cyclodextrin (MβCD) or 50 mg/ml nystatin, for 10 min or benzyl alcohol (20 µM) for 30 min. One set of data is from cells treated with imipramine (30 µM) for 30 min before priming. Data are expressed as mean± s.e.m. of three independent experiments performed in triplicates. *P<0.05 when compared to unstimulated and untreated groups. #P<0.05 when non-treated groups were compared to drug-treated groups. Journal of Cell Science

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Next, to confirm that the increase in CD39 activity induced by in Fiji/ImageJ software (Fig. 3F), and Pearson correlation LPS+ATP depends on raft formation during which the enzyme coefficient values were calculated for double-labeled points becomes more stable and active, we treated the cells with benzyl (Fig. 3F and G). ATP+LPS treatment significantly increased the R alcohol to increase membrane fluidity. After treatment with benzyl value for colocalization, whereas pretreatment with P2X7 receptor alcohol, there was no increase in ATP hydrolysis in cells stimulated antagonists or imipramine inhibited this effect (Fig. 3G). with LPS+ATP (P>0.05; Fig. 1A), confirming that changes in plasma membrane fluidity and composition can interfere with the Caveolin-1 is important for P2X7 receptor-mediated increase activity of CD39 enzyme. When evaluating CD73 functionality, we in STAT3 activation observed increased activity after priming cells with LPS (Fig. 1B). Finally, we evaluated the relevance of caveolin-rich membrane Nevertheless, CD73 activity was not affected by lipid raft disruption domains in macrophages regarding the activation of the (Fig. 1B). transcription factor STAT3, which is involved in the induction of CD39 expression in immune cells (Chalmin et al., 2012). LPS ATP triggers movement of CD39 to lipid rafts in LPS-primed increases STAT-3 phosphorylation in wild type (WT) macrophages, macrophages and P2X7 receptor activation in response to Bz-ATP significantly To confirm that CD39 is located within these membrane domains in potentiates the activation of this transcription factor, whereas macrophages, we double-labeled LPS- and ATP-treated peritoneal treatment with 500 µM ATP did not increase levels of macrophages with antibodies recognizing CD39 and flotillin-2 phosphorylated STAT3 (p-STAT3) (Fig. 4A-B). This is possibly (used as a general marker of lipid rafts) (Zhao et al., 2011). In due to the fact that Bz-ATP is 10× more potent than ATP in control cells and cells treated with ATP only, CD39 and flotillin-2 activating the P2X7 receptor. Nevertheless, we do not exclude colocalized in a less-structured pattern, with some (but not all) participation of other mechanisms and, potentially, other P2 receptors flotillin-2-positive spots observed in cells stained for CD39 in the regulation of STAT3 activation in these settings. However, (Fig. 2A). In contrast, cells treated with both LPS and ATP when we used macrophages from caveolin-1-deficient mice, levels of displayed numerous and more-conspicuous spots of fluorescence that p-STAT3 were significantly diminished in both LPS- and were consistently double-stained for CD39 and flotillin-2 (Fig. 2A); LPS+BzATP-treated groups when compared with WT groups this suggests a high CD39 concentration within membrane domains (Fig. 4A,B). To test for the putative contribution of P2X7 receptor and is likely to represent lipid rafts. Treatment with MβCD to STAT3 activation, we further pre-treated WT cells with P2X7 diminished the double-labeled spots of fluorescence (Fig. 2A). receptor inhibitors, i.e. A740003 and oxidized-ATP (oATP), and then These observations were confirmed by plug-in Coloc2 analysis exposed these cells to LPS or LPS+BzATP. We found that either performed with software Fiji/ImageJ (Fig. 2B-F), generating the inhibitor significantly decreased p-STAT3 levels in cells treated with Pearson correlation coefficient of the pixel-intensity correlation for LPS or LPS+Bz-ATP (data not shown). These data suggest that P2X7 double-labeled points that indicates colocalization. receptor-dependent STAT3 phosphorylation in LPS-primed As depicted in Fig. 2G, ATP+LPS treatment significantly macrophages depends on caveolin-rich membrane domains. increased the R value for colocalization, whereas MβCD treatment dissipated this effect, i.e. reducing the R value significantly when DISCUSSION compared to both control and LPS+ATP groups (Fig. 2G). Purinergic signaling modulates macrophage activity and immune responses. Here, we show that the P2X7 receptor activates a lipid P2X7 receptor activation triggers CD39 to caveolae of raft-dependent regulatory mechanism that modulates macrophage macrophages CD39 activity. P2X7 receptor activation increases activity of CD39 Given that CD39 has palmitoylation sites and has previously been and targets this protein to lipid rafts, whereas drugs that disrupt reported to be located in caveolae of certain cell types (Koziak et al., cholesterol-enriched domains decreased its activity. P2X7 receptor 2000), we evaluated whether this enzyme can also be located in inhibition blocked increases in caveolin-1 expression and inhibited caveolae within the membrane of peritoneal macrophages treated colocalization with CD39. Interestingly, the formation of membrane with LPS+ATP. For this, we double-labeled LPS- and ATP-treated rafts is linked to the functionality of various components of the peritoneal macrophages (pre-treated or not with the P2X7 receptor purinergic signaling, such as the P2X7 receptor and CD39. The inhibitor A740003 or imipramine) with antibodies recognizing latter is anchored to the plasma membrane by two transmembrane CD39 and caveolin-1 (a marker of caveolar domains). domains. Drug- or detergent-induced disruption of these domains We verified that treatment with LPS+ATP induced a clear significantly decreases the activity of the CD39, further indicating increase in caveolin-1 expression, showing some points consistently that its activity requires the integrity of both transmembrane double labeled, suggesting colocalization of caveolin-1 and CD39 domains in the plasma membrane (Papanikolaou et al., 2005). (Fig. 3A). Furthermore, this effect was prevented by P2X7 receptor Grinthal and Guidotti (2006) have proposed that changes in the blockade or the treatment with imipramine. mechanical properties (dependent on lipid composition) of the As we have previously shown, LPS and LPS+ATP treatment plasma membrane domain where CD39 molecules are inserted boost CD39 activity (Savio et al., 2017a), and these inhibitory modulate CD39 activity, by modifying the stability of its effects are blocked by P2X7 receptor inhibitors (A740003 and transmembrane domains. This is in agreement with our data, oxidized-ATP) or imipramine (Fig. 3B). In addition, MFI analysis showing that drugs that disrupt lipid raft membrane domains (i.e. for CD39 and caveolin-1 showed that treatment with ATP+LPS nystatin or MβCD) abolish the ATP-induced increase in CD39 significantly increases the cell surface levels of these proteins, activity in LPS-primed macrophages and decreases the CD39 whereas inhibition of P2X7 receptor blocks these effects (Fig. 3C colocalization with flotillin-2, suggesting that CD39 activity is and D). The increased expression of caveolin-1 in peritoneal dependent on its localization in specific lipid microdomains. macrophages after treatment with LPS+ATP or LPS+BzATP Moreover, treating cells with benzyl alcohol, which acts by was confirmed by western blot experiments (Fig. 3E). increasing membrane fluidity (Nagy et al., 2007), reverses

Immunofluorescence images were also analyzed by Coloc2 plugin increase in ATP hydrolysis after treatment with LPS+ATP, Journal of Cell Science

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Fig. 2. ATP treatment increases colocalization of CD39 and flotillin-2 in LPS-primed peritoneal macrophages. Mouse peritoneal macrophages from 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 stain the 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 LPS and ATP (fourth row), or with ATP, LPS and 10 mM methyl-β-cyclodextrin (MβCD) (fifth row). CD39 and flotillin-2 colocalized in ‘spots’ when cells were treated with ATP 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<0.05 compared with unstimulated control or with the LPS+ATP group, respectively. Journal of Cell Science

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Fig. 3. P2X7 receptor activation increases CD39 expression in caveolae of macrophages. LPS-primed peritoneal macrophages (1 µg/ml for 4 h) stimulated with 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 antibodies recognizing CD39 (green) and the lipid raft marker caveolin-1 (red), as well as with Hoechst 33258 (blue). (A) Representative immunofluorescence images of cells stained 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 (500µMATP or 100 µM Bz-ATP). (F,G) Representative intensity histogram output (F) of Coloc2 analysis performed with Fiji/ImageJ and Pearson correlation coefficient of the pixel-intensity correlation (G) indicating colocalization. Data are expressed as mean±s.e.m. of three independent experiments. *,#P<0.05 compared with the unstimulated control or compared to the LPS+ATP group, respectively. Journal of Cell Science

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through a mechanism that, at least in part, is dependent upon formation of lipid membrane domains. Our immunofluorescence experiments suggested that CD39 is located within lipid rafts in peritoneal murine macrophages, and that treatment with ATP induces aggregation of this protein and its association with other lipid raft markers, such as flotillin-2 or caveolin-1, although no coimmunoprecipitation experiments have been carried out to show physical interactions between these proteins. These membrane structures probably represent larger ceramide-enriched membrane domains, whose formation might be directly mediated through P2X7 receptor activation in response to treatment with ATP. Accordingly, this effect was prevented by either blockage of P2X7 receptor or treatment with the sphingomyelinase inhibitor imipramine. In the same line of evidence, the presence of palmitoylation sites in CD39 which colocalize with caveolin-1 from canine kidney cells has been reported (Koziak et al., 2000). Therefore, we hypothesize that P2X7 receptor activation promotes the formation of CD39-expressing caveolae. In summary, our study here demonstrated an increase in CD39 activity in response to stimulation of TLR4 (4 h) and P2X7 receptor. This increase was dependent on the integrity of membrane rafts, in which the CD39 is located after cells had been stimulated with LPS and ATP. The P2X7 receptor seems to modulate the functionality of CD39 by facilitating the formation of membrane domains, in which it becomes more stable and active (Fig. 5). To understand the mechanisms that modulate CD39 activity is crucial, given the immunoregulatory roles of this enzyme, as well as its impact on macrophage activation and proinflammatory Fig. 4. Caveolin-1 is important for STAT3 activation in LPS-primed responses mediated upon activation of the P2X7 receptor (Savio macrophages. (A,B) Representative western blot (A) and its densitometric analysis (B) of levels of active, i.e. phosphorylated STAT3 (p-STAT3) in et al., 2017a). Our results support the involvement of purinergic untreated and LPS-primed macrophages derived from wild-type (WT) or signaling and lipid raft formation in cellular responses during caveolin-1−/− mice stimulated with P2X7 receptor agonists (500 µM inflammation and infection. They also suggest therapeutic ATP or 100 µM Bz-ATP). Data are expressed as mean±s.e.m. of three approaches, including the administration of drugs that modulate independent experiments. *P<0.05 compared with the unstimulated control the formation of lipid raft or of soluble apyrases that mimic the # −/− group. P<0.05 when comparing WT with caveolin-1 animals. ND, action of CD39. not detectable.

MATERIALS AND METHODS thereby demonstrating that changes in the composition and fluidity Animals and general reagents of the plasma membrane can modulate CD39 activity in We used male wild-type C57BL/6 mice (8–10 weeks old) in this study. In macrophages. some experiments caveolin-1 null (caveolin-1−/−) mice (B6/129SJ) mice Others and we infer that P2X7 receptor participates in lipid purchased from The Jackson Laboratory (Bar Harbor, ME) were used. metabolism and formation of lipid rafts. In fact, the activation of this Animals were housed at a ratio of five mice per cage, with water and food ad receptor stimulates the sphingomyelinase activity that convert libitum, on a 12 h light/dark cycle (lights on at 7:00 am), and at a temperature sphingomyelin into ceramide promoting the formation of ceramide- of 22±1°C. The procedures for the care and use of animals were according to enriched membrane domains (Garcia-Marcos et al., 2006; Lepine the guidelines of the Brazilian College of Animal Experimentation (COBEA) and to the Guide for the Care and Use of Laboratory Animals et al., 2006). In this context, it is possible that the P2X7 receptor (National Research Council, USA). All experiments were approved by the stimulates the formation of stable lipid membrane domains in which Commission for the Ethical Use of Research Animals (CEUA) from the CD39 becomes more stable and active. Federal University of Rio de Janeiro (UFRJ) (approved protocol number: The results obtained in this study are in keeping with this IBCCF138) and by the Institutional Animal Care and Use Committees hypothesis, since treatment with ATP induced an increase in the (IACUC) of Beth Israel Deaconess Medical Center (approved protocol activity of CD39. In this regard, we have already shown that P2X7 number: 019-2015). ATP, ADP, AMP, oxidized-ATP, benzyl alcohol and receptor activation boosts CD39 expression and activity imipramine were obtained from Sigma-Aldrich, MO. A740003 were in peritoneal macrophages (Savio et al., 2017a). In addition, we purchased from Tocris Inc, Ellsville, MO. have reported that P2X7 receptor activation is important for activation of STAT3 (Savio et al., 2017a), a transcription factor Peritoneal macrophages that promotes CD39 expression in immune cells (Chalmin et al., Murine macrophages were harvested from the peritoneal cavity of adult 2012). Moreover, De Marchi et al. (2019) have recently shown mice. Cells obtained from the peritoneal cavity were plated in suspension into 24- or 96-well tissue culture plates (TPP AG, Switzerland) at a density that P2X7 receptor blockade reduces CD39 and CD73 expression in of 2×105 cells per well and incubated in non-supplemented Gibco® DMEM T effector and dendritic cells. Here, we found that caveolin-1 (Thermo Fisher Scientific, Rockford, IL) for 1 h, at 37°C, 5% CO2 expression is key to P2X7 receptor-mediated phosphorylation of atmosphere. Non-adherent cells were removed by washing three times with STAT3 in LPS-primed macrophages. Thus, the P2X7 receptor might PBS, and adherent cells were cultured overnight in Gibco® DMEM (Thermo modulate the enzymatic activity of CD39 at transcriptional level Fisher Scientific) complete medium before use in experiments. Journal of Cell Science

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

Isolation, characterization and differentiation of bone marrow- through a filter for cell culture (Cell Strainer; 40 µm) and centrifuged for derived macrophages 10 min at 300 g at room temperature. Pellets were resuspended in red blood Medullar cells obtained from mouse tibias and femurs were resuspended in cells lysis buffer (cat. no. A10492-01, Thermo Fisher Scientific). ® non-supplemented DMEM Gibco (Thermo Fisher Scientific), passed Subsequently, cells were centrifuged for 10 min at 300 g and washed Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237560. doi:10.1242/jcs.237560 twice with PBS. Mononuclear bone marrow-derived macrophages cat. no. H3569, Life Technologies, Eugene, OR), and then mounted and (BMDMs) were then plated at a density of 1×108 cells in polystyrene examined in a fluorescence microscope Zeiss AxioVert 200M and the three- flasks and cultured in DMEM Gibco® supplemented with 20% fetal bovine dimensional images (z-stack) in a Spinning Disk Confocal Microscope serum (Sigma-Aldrich), antibiotics (100 IU penicillin/ml and 100 mg ZEISS Cell Observer SD (Peabody, MA). streptomycin/ml; Gibco®) and 10 ng/ml macrophage colony-stimulating factor (M-CSF) (cat. no. 315-02, Peprotech, NJ). Cells were maintained at Cell microscopy analysis 37°C in an atmosphere of 5% CO2. After 4 days, culture medium was Mean fluorescence intensity (MFI) was measured in Zen Lite Blue software replaced, and non-adherent cells removed and discarded. After reaching (Carl Zeiss). For this quantification, the background was initially subtracted 80% confluence, cells were detached from polystyrene plates upon addition and the region of interest (ROI) was selected in individual cells by using the of 0.25% trypsin-EDTA (Sigma-Aldrich) and plated again for subsequent freehand selection tool of the software to calculate the ratios of caveolin-1 experiments. The phenotype of BMDMs was determined by flow cytometry and CD39 MFI in response to different treatments. This is provided by the using the following antibodies: APC labeled anti-CD11b APC (cat. no. software based on the intensity of the related pixels. 101226, BioLegend, San Diego, CA) at 1:100 dilution and FITC labeled Colocalization analyses of the ratios of CD39 to flotillin-2 and of CD39 to anti-F4/80 (cat. no. MCA497FT, AbD Serotec®, Bio-Rad, Hercules, CA) caveolin-1 were performed using Coloc-2 Fiji-ImageJ plugin version 3.0. used at 1:100 dilution. Isotypes were used as negative control. For specific analysis, different image channels (green and red) were overlaid in the same z-plane, generating yellow spots where the two molecules LPS priming and pharmacological treatments studied were present at the same pixel locations. Ten fields per condition per For in vitro experiments, macrophages were left untreated or were primed experiment were randomly chosen and measured by an investigator, who with 1 µg/ml bacterial lipopolysaccharide (LPS) for 4 h to induce an was blind to the experimental conditions. The point spread function (PSF) inflammatory response, and then stimulated with 500 µM ATP or 100 µM was calculated and set to 3.0, whereas randomizations were set to 10.0. The BzATP to activate P2X7 receptors for 1 h or 3 h. Alternatively, cells were Pearson correlation coefficient was calculated as indicative of colocalization primed with 1 µg/ml LPS for 4 h, stimulated with 500 µM ATP for 3 h, and (Bolte and Cordelieres,̀ 2006; Adler and Parmryd, 2010; McCuaig et al., then treated with 50 mg/ml nystatin or 10 mM methyl-β-cyclodextrin 2015; Schindelin et al., 2012). (MβCD) for 10 min before, or with benzyl alcohol (to increase membrane fluidity; 20 mM) 30 min before ectonucleotidase assays (Nagy et al., 2007). Western blotting In some wells, cells were pretreated with P2X7 receptor antagonists (300 µM Macrophages were lysed in ice-cold modified RIPA buffer (50 mM Tris-HCl oATP for 2 h or 100 nM A740003 for 30 min) or the sphingomyelinase pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl) inhibitor imipramine (30 µM for 30 min) before priming with LPS (Bai et al., supplemented with Complete Proteinase Inhibitor Cocktails (Roche 2014). All reagents were purchased from Sigma-Aldrich. Diagnostics) and Phosphatase Inhibitor Cocktails (Sigma-Aldrich, MO). The lysates were sonicated briefly on ice and centrifuged at 18,000 g for Ectonucleotidase activity assays 10 min at 4°C. Protein concentrations were determined by Bio-Rad DC Activities of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and protein assay reagent (Bio-Rad Laboratories), using bovine serum albumin as 5′-nucleotidase (CD73) were estimated in a reaction medium consisting of the standard. Proteins (10 µg per lane) were boiled in XT Sample Buffer (cat. 20 mM HEPES buffer (pH 7.5) containing 1 mM CaCl2 (for ATP) or no. 161-0791, Bio-Rad Laboratories), separated using 4–12% Criterion XT MgCl2 (for AMP), 120 mM NaCl, 5 mM KCl, 60 mM glucose, 1 mM Bis-Tris SDS-PAGE (Bio-Rad Laboratories) and transferred to PVDF sodium azide and 0.1% mM albumin (all reagents from Sigma-Aldrich). membranes (cat. no. IPVH00010, Millipore) by semi-dry electroblotting. Peritoneal resident macrophages (2×105) were used in a final volume of The latter were then probed with specific antibodies against proteins of 200 µl reaction medium, and enzymatic reactions were started by addition of interest. Bands were visualized using HRP-conjugated goat anti-mouse, ATP or AMP to a final concentration of 2 mM, followed by incubation for donkey anti-rabbit or donkey anti-sheep IgG and the SuperSignal West 30 min at 37°C. Reactions were stopped by addition of 200 µl of 10% Femto Maximum Sensitivity Substrate reagents applied (cat. no. PI-34096, trichloroacetic acid (TCA) (Sigma-Aldrich). Incubation times, protein Thermo Scientific) according to the manufacturer’s instructions (Savio concentrations, reaction mixtures and substrate concentrations were chosen et al., 2017a). according to a previous study (Vuaden et al., 2011). The amount of inorganic phosphate (Pi) released was measured using the colorimetric method Statistical analysis described by Chan et al. (1986). Controls to correct for non-enzymatic Pi in Results are expressed as mean±standard error of mean (±s.e.m.). Statistical samples were performed adding the nucleotides (ATP or AMP) after the analysis was performed by one-way analysis of variance (ANOVA), reactions had been stopped with TCA. All reactions were performed in followed by Tukey’s multiple range tests. Differences between groups were triplicates, and enzyme activities were expressed in nmol Pi released per considered statistically significant when P<0.05. minute per number of cells. Competing interests Immunocytochemistry The authors declare no competing or financial interests. After LPS priming and pharmacological treatments described above, samples were fixed with 4% paraformaldehyde and 4% sucrose for 15 min at Author contributions Conceptualization: L.E.B.S., M.S.L., S.C.R., R.C.; Methodology: L.E.B.S., Y.W., room temperature, and blocked with 10% horse serum and 1% BSA in PBS R.C.S.; Software: L.E.B.S.; Validation: L.E.B.S.; Formal analysis: L.E.B.S., for 30 min at room temperature. Samples were then incubated (for 3 h at P.d.A.M., S.A.C.S.S., J.C.d.S., S.D.S.O., R.D.M., E.K., M.S.L.; Investigation: room temperature) with the following primary antibodies (in 0.1% BSA in L.E.B.S., Y.W., M.S.L., S.C.R., R.C.S.; Resources: S.C.R., R.C.S.; Data curation: PBS): C42A3 rabbit anti-flotillin-2 mAb (cat. no. 3436, Cell Signaling L.E.B.S., P.d.A.M., S.A.C.S.S., J.C.d.S., S.D.S.O.; Writing - original draft: L.E.B.S.; Technology, Danvers, MA) diluted 1:50 and goat anti-CD39 (cat. no. Writing - review & editing: L.E.B.S., P.d.A.M., S.D.S.O., R.D.M., E.K., M.S.L., S.C.R., AF4398 (R&D Systems, Minneapolis, MN) diluted 1:200; or mice anti- R.C.S.; Supervision: S.C.R., R.C.S.; Project administration: R.C.; Funding caveolin-1 (7C8) (cat. no. NB100-615, Novus Biologicals, Littleton, CO) acquisition: S.C.R., R.C.S. diluted 1:400 and goat anti-CD39 (cat. no. AF4398, R&D Systems) diluted 1:200. Cells were then washed and incubated at room temperature for 1 h Funding with the following secondary antibodies (diluted 1:300, in 0.1% BSA in This work was supported by funds from the Conselho Nacional de Desenvolvimento ® Cientıficó e Tecnológico do Brasil (CNPq), Coordenação de Aperfeiçoamento de PBS): anti-rabbit IgG (H+L)-Alexa Fluor 488 (Cell Signaling Technology) ı́ ̀ ™ Pessoal de N vel Superior (CAPES), Fundação de Amparo a Pesquisa do Estado do and anti-sheep IgG (H+L) Cy 5 (Jackson ImmunoResearch Laboratories, Rio de Janeiro (FAPERJ), National Institutes of Health (NIH) (grant no. ® West Grove, PA); or anti-goat IgG (H+L)-Alexa Fluor 488 and anti-mice 5R01DK108894 to M.S.L. and grant no. R21CA221702 to S.C.R.) and U.S. ® IgG (H+L)-Alexa Fluor 597 (Life Technologies, Eugene, OR). Finally, Department of Defense (DOD) (grant no. W81XWH-16-0464 to S.C.R.). Deposited samples were stained with DAPI or Hoechst 33258 nuclear dye (1:10,000, in PMC for release after 12 months. Journal of Cell Science

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