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Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor
Hayato Sakaki ,
Contributed equally to this work with: Hayato Sakaki, Mitsutoshi Tsukimoto
Affiliation Department of Radiation Biosciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda-shi Chiba, Japan
Mitsutoshi Tsukimoto ,
* E-mail: tsukim@rs.noda.tus.ac.jp
Hitoshi Harada,
Affiliation Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka-shi, Mie, Japan
Yoshinori Moriyama,
Affiliation Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
Shuji Kojima
Hayato Sakaki,
Mitsutoshi Tsukimoto,
Hayato Sakaki Mitsutoshi Tsukimoto ... Shuji Kojima
It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.
Citation: Sakaki H, Tsukimoto M, Harada H, Moriyama Y, Kojima S (2013) Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor. PLoS ONE 8(4): e59778. https://doi.org/10.1371/journal.pone.0059778
Editor: Yoshihiko Hoshino, National Institute of Infectious Diseases, Japan
Received: November 16, 2012; Accepted: February 18, 2013; Published: April 5, 2013
Copyright: © 2013 Sakaki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Parts of this work were supported by a Grant-in-Aid for Scientific Research (C) (No. 24590125) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
Sepsis is a systemic inflammatory response syndrome induced by microbial infection. It is generally caused by Gram-negative bacterial endotoxins or lipopolysaccharide (LPS), and is characterized by severe shock and multiple organ failure [1], [2]. Although the pathogenesis of sepsis-induced lethal shock and multiple organ injury leading to death is incompletely understood, current theory holds that a hyperactive and out-of-balance network of endogenous proinflammatory cytokines contributes to mortality and morbidity in sepsis [3], [4]. Indeed, dysregulation in cytokine networking is thought to play a fundamental role in the outcome of sepsis. The current therapeutic strategies for human septic shock are designed to neutralize one or more of the inflammatory mediators, yet effective treatment modalities remain elusive [3], [5]. In fact, the incidence of sepsis is currently estimated to be ∼750,000 annually in the United States alone, with a mortality rate of about 30%, and its incidence is continuing to increase [6], [7].
Blood monocytes and macrophages play a central role in complex innate immune responses to infections, which influence the development of Th1/Th2 adaptive immunity [8]. Macrophages are highly versatile phagocytic cells whose diverse effector functions can be selectively reprogrammed by an array of environmental signals [9], [10], [11]. The two major macrophage phenotypes are the classic pro-inflammatory M1 and the alternative anti-inflammatory M2 phenotypes [12], [13]. The M1 phenotype is induced in response to microbial products such as LPS or proinflammatory cytokines, including IFN-gamma and TNF-alpha. M1 macrophages up-regulate proinflammatory cytokines, including TNF-alpha, interleukin (IL)-6, and IL-12, and are associated with increased production of reactive oxygen species (ROS) and nitrogen intermediates [14]. It has been demonstrated that M1 phenotype induction is related to the severity of sepsis [15]. In patients with severe sepsis, high concentrations of circulating M1-type cytokines are strongly correlated with mortality [16]. Macrophages from these patients produce high levels of type 1 cytokines and chemokines that activate the endothelium and contribute to cardiac failure, loss of general organ perfusion, and death [17]. To establish novel therapies for inflammatory diseases including sepsis, it is important to understand the mechanisms that regulate macrophage activation. Although the mechanism of macrophage activation has been well investigated, an autocrine signaling in the activation process is not established.
Recently, extracellular nucleotides, such as ATP or UTP, have been identified as damage-associated molecular pattern molecules (DAMPs), which activate innate immune systems. Extracellular nucleotides activate P2 receptors, which are classified into two subfamilies, ionotropic P2X1-7 and G-protein coupled P2Y1-14 receptors [18]. Activation of P2 receptors is important for immune functions. For example, ATP-induced activation of P2X7 receptor plays a critical role in release of IL-1ß from LPS-primed macrophages [19]. ATP is released from macrophages stimulated with various pathogen-associated molecular patterns (PAMPs) and extracellular ATP is one of the most pleiotropic inflammasome activators [20], [21]. Although it is well known that large amounts of nucleotides are released from dead cells, living cells also release nucleotides via non-lytic mechanisms, such as maxianion channels, anion transporter, hemichannels, or exocytosis, in response to various stimuli [22]. Recently, we have shown that vesicular exocytosis of ATP and activation of P2X7 and P2Y6 receptors play an important role in activation of T cells [23]. However, although ATP release from macrophages has been reported [24], it has not yet been established whether this occurs via exocytosis. In this connection, SLC17A9 has been identified as a vesicular nucleotide transporter (VNUT), which plays a key role in vesicular storage of ATP [25]. It mediates the active accumulation of nucleotides, driven by an electrochemical gradient of protons across the membrane, generated by vacuolar proton-ATPase. SLC17A9 is responsible for exocytosis of ATP, in the form of ATP-containing vesicles, and is expressed in PC12 cells, type II taste cells, biliary epithelial cells, T lymphocytes, and lung cancer cells [25], [26], [27], [28], [29]. However, expression of SLC17A9 and vesicular exocytosis of ATP have not been reported in macrophages.
As we are interested in finding new therapeutic targets for treatment of inflammatory diseases such as sepsis, in this study we investigated the involvement of extracellular nucleotides-mediated autocrine signaling via exocytosis and activation of P2 receptors in macrophage activation. Our findings indicate that LPS-induced activation of macrophages is mediated, at least in part, by ATP release via exocytosis and activation of P2Y11 receptor, and this pathway is involved in the increased production of cytokines by activated macrophages. Therefore, we propose that P2Y11 antagonists may be candidate anti-inflammatory drugs for the treatment of sepsis or other inflammatory diseases.
Reagents and Antibody
RPMI1640 medium was purchased from Wako Pure Chemical. FBS was purchased from Biowest. Bafilomycin A, brefeldin A (BFA), carbenoxolone (CBX), clopidgrel, 18-glycyrrhetinic acid (18GA), flufenamic acid (FFA), glibenclamide, GdCl3, apyrase, PPADS, suramin, and LPS were purchased from Sigma-Aldrich. MRS2179, MRS2211, MRS2578, NF157, KH7 and A438079 were from Tocris Bioscience. NF449 was from Abcam. IFN-gamma was purchased from Roche Applied Science. Rabbit polyclonal anti-SLC17A9 antibody was generated by Prof. Moriyama (University of Okayama).
THP-1 human acute monocytic leukemia cells (ATCC) or mouse macrophage RAW264.7 cells (ATCC) were grown in RPMI1640 medium, supplemented with 10% fetal bovine serum, penicillin (100 units/mL) and streptomycin (100 µg/mL) in a humidified atmosphere of 5% CO2 in air at 37°C. THP-1 cells were cultured in RPMI 1640 media with 1 µg/ml LPS from Escherichia coli serotype 055:B5 (Sigma-Aldrich) and 1000 U/ml mouse IFN-gamma (R&D Systems) to generate M1 macrophages.
Determination of Extracellular ATP Concentration
THP-1 cells were washed and resuspended in RPMI1640-based buffer, containing 102 mM NaCl, 5 mM KCl, 0.4 mM CaCl2, 0.4 mM MgSO4, 23.8 mM NaHCO3, 5.6 mM Na2HPO4, 11.1 mM glucose and 10 mM HEPES-NaOH (pH 7.4), then 40 µL aliquots of THP-1 cells (1.0×106 cells/mL) were stimulated with LPS (10 µg/mL) and the extracellular ATP concentration was measured using ENLITEN® rLuciferase/Luciferin Reagent (Promega). At the indicated time points, each sample was centrifuged at 800×g for 1 min and 10 µL of the supernatant was collected for ATP determination. The luciferin-luciferase reagent (100 µL) was injected into the supernatant and chemiluminescence was measured with a WALLAC ARVO SX multilabel counter (PerkinElmer). ATP concentration in each sample was determined by comparing the luminescence of samples with those of standards in the range of 10−6 to 10−10 M.
Quantification of Lactate Dehydrogenase Release
Release of lactate dehydrogenase (LDH) into cell culture supernatant was quantified with a Cytotoxicity Detection Kit (Roche Applied Science), according to the supplied instructions. THP-1 cells (1.0×106 cells/mL) were incubated in a 96-well plate at 37°C for 30 min with LPS. At the end of incubation, supernatants were collected and the LDH content was measured. LDH release is expressed as a percentage of the total content determined by lysing an equal amount of cells with 1% Triton X-100.
Fluorescence Imaging
For staining of intracellular ATP, LPS- and IFN-gamma-treated THP-1 cells were incubated for 1 h with 50 µM 2′−/3′-O-(N′-methylanthraniloyl)-ATP (MANT-ATP) and 5 µM quinacrine dihydrochloride in RPMI1640-based buffer at 37°C. Stained cells were analyzed using a confocal laser scanning microscope (TCS SP2; Leica) equipped with a HCX PLApo 63×1.32 NA oil objective lens. Leica confocal software (TCS SP2, version 2.6.1) was used for image acquisition and processing. Fluorescence of MANT-ATP was detected at 430–480 nm with excitation at 364 nm, and fluorescence of quinacrine dihydrochloride was detected at 510–530 nm with excitation at 488 nm.
RT-PCR Analysis
Total RNA was isolated from THP-1 cells using a Fast Pure RNA kit (Takara Bio). The first-strand cDNA was synthesized from total RNA with PrimeScript Reverse Transcriptase (Takata Bio). Specific primers were designed with PrimerQuestSM (Integrated DNA Technologies, Inc.) and synthesized by Sigma Genosys (Sigma-Aldrich). The sequences of specific primers for human SLC17A9 were 5′-ACA CAC GAG CAG AGA GGA ACA CAA-3′ (sense) and 5′-TTT CTG GCT GTT GTC TGA CTG GGA-3′ (antisense). GAPDH mRNA was determined as a positive control. PCR was carried out by incubating each cDNA sample with the primers (0.5 µM each), PrimeSTAR® HS DNA Polymerase (0.625 U: Takara Bio), and deoxynucleotide mix (0.2 mM each: Takara Bio). Amplification was carried out for 35 cycles (94°C for 30 sec, annealing at 60°C for 30 sec). The products were then subjected to 2% agarose gel electrophoresis. Bands were stained with ethidium bromide (Sigma-Aldrich) and photographed.
Short Hairpin RNA Plasmid (shRNA) and siRNA Transfection for Knock-down
Transfection of THP-1 cells was performed using the SureSilencing™ shRNA plasmid Kit for human SLC17A9 (SABiosciences) or TriFECTa Kit for human P2Y11 receptor (Integrated DNA Technologies) and the Nucleofector System (Amaxa GmbH). Two different shRNA plasmids targeting SLC17A9, the negative control shRNA plasmid or the siRNA duplex oligonucleotides (10 nM) for reduction of human P2Y11 receptor were transfected by electroporation using the Amaxa system (Lonza) with Nucleofector solution V and Nucleofector program V-01. Seventy-two hours after transfection, reduction of expression of SLC17A9 and P2Y11 receptor was confirmed.
Real-time RT-PCR
Total RNA was extracted from THP-1 cells and first-strand cDNA was synthesized as described above. The cDNA was used as the template for real-time PCR analysis: reactions were performed in a Stratagene Mx3000P® QPCR system (Agilent Technologies). Specific primers were designed with PrimerQuestSM and synthesized by Sigma Genosys. The sequences of specific primers for human SLC17A9 were 5′-AGT CTG TGG TCT TTG CAT CAG CCT-3′ (sense) and 5′-TGT TGG CCA CAC CAA ACA GAA AGC-3′ (antisense). The sequences of specific primers for human P2Y11 were 5′-CGT GAG CTG AGC CAA TGA TGTG-3′ (sense) and 5′-GGG TGG GAA AGG CGA CTGC-3′ (antisense). RT2-qPCR primer assay for human IL-6 was purchased from Qiagen. GAPDH mRNA was determined as a positive control. Each sample was assayed in a 20 µl amplification reaction, containing cDNA, primer mixture (5 µM each of sense and antisense primers) and 2x KAPA SYBR® FAST qPCR Master Mix (Kapa Biosystems). The amplification program included 40 cycles of two steps, each comprising heating to 95°C and to 60°C, respectively. Fluorescent products were detected at the last step of each cycle. The obtained values were within the linear range of the standard curve and were normalized to yield the same amount of GAPDH mRNA expression.
Total cellular membrane protein was lysed in 30 µL of PBS containing 10 mM HEPES-NaOH, pH 7.4, 1% Triton X100, 5 mM EDTA, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1.04 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.8 µM aprotinin, 21 µM leupeptin, 36 µM bestatin, 15 µM pepstatin A and 14 µM E-64. The protein content in each sample was determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories). Equal amounts of protein lysate were dissolved in 2×sample buffer (50% glycerin, 2% SDS, 125 mM Tris, 10 mM DTT) and boiled for 10 min. Aliquots of samples containing 1 µg of protein were analyzed by means of 10% SDS-PAGE and bands were transferred onto a PVDF membrane. The blots were blocked overnight in TBST with 5% skim milk at 4°C, then incubated with rabbit polyclonal anti-SLC17A9 antibody (1∶5000) overnight at 4°C or with rabbit anti-rat P2X7 receptor antibody (1∶200) (Cell Signaling Technology, Inc.) for 90 min at room temperature as a loading control. After having been washed with TBST, blots were incubated with goat HRP-conjugated anti-rabbit IgG antibody (Cell Signaling Technology, Inc.) for 90 min at room temperature. The blots were again washed with TBST, and specific proteins were visualized by using ECL Western blotting detection reagents (GE Healthcare) according to the manufacturer's instructions.
Determination of Cytokines
THP-1 cells were stimulated with LPS (10 µg/mL). After incubation for 24 h, the culture supernatant was harvested for determination of IL-6. The concentration of IL-6 was measured by ELISA, as described below. A 96-well plate was coated with purified anti-human IL-6 mAb (1∶250) (eBioscience) and incubated overnight at 4°C, then the wells were washed with PBS containing 0.05% Tween-20, and nonspecific binding was blocked with PBS containing 1% BSA for 1 h at room temperature. The plate was washed, and the culture supernatant was added for 2 h at room temperature. The plate was washed again, and biotin-conjugated anti-human IL-6 mAb (1∶1000) (eBioscience) was added for 1 h at room temperature. The plate was further washed, and avidin-horseradish peroxidase (Sigma-Aldrich) was added. The plate was incubated for 30 min at room temperature, then washed, and 3,3′,5,5′-tetramethylbenzidine was added for a few minutes. The reaction was stopped by adding 5 N H2SO4. The absorbance at 450 nm was measured with an ImmunoReader NJ-2000 (Nihon InterMed). A standard curve was established with recombinant human IL-6 (15–1000 pg/mL), and the concentration of IL-6 was estimated from the standard curve.
Measurement of cAMP Level
The cAMP level was quantified with a DetectX High Sensitive Direct Cyclic AMP Chemiluminescent Immunoassay Kit (Arbor Assays), according to the supplied instructions. THP-1 cells or RAW264.7 cells (1.0×106 cells) were incubated in a 24 -well plate at 37°C for 10–30 min with ATP or LPS. At the end of incubation, cells were lyzed and the cAMP content was measured.
Preparation of Peritoneal Macrophages and in vitro Stimulation
Peritoneal cells were isolated by washing the peritoneal cavity with ice-cold PBS. The cells were cultured for 1 h and non-adherent cells were washed out with PBS. Adherent cells (peritoneal macrophages: 5×106) were cultured with LPS (1 µg/ml) in RPMI 1640 medium with 10% FBS for 24 h. Concentrations of IL-6 and TNF-alpha in the culture supernatants were measured by ELISA.
Flow Cytometric Analysis of Macrophage Polarization
THP-1 cells or mouse macrophages were incubated for 30 min at 37°C with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) against CCR7 in PBS. The cells were washed twice with PBS, and their fluorescence was assessed by flow cytometry.
Induction of LPS-induced Endotoxin Shock
Male 6-week-old C57BL/6 mice (20.1±0.77 g) were intraperitoneally (i.p.) injected with 400 µg of LPS from Escherichia coli serotype 055:B5 (Sigma-Aldrich). Several hours after the injection, concentrations of IL-1ß, IL-6, IL-12 and TNF-alpha in serum and peritoneal lavage fluid were measured by ELISA. The M1-type polarization of spleen and peritoneal macrophages was analyzed. The mice were treated and handled according to the Guide Principles for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society and with the approval of Tokyo University of Science's Institutional Animal Care and Use Committee (Permit Number: S12017).
Results are expressed as mean ± SE. The statistical significance of differences between control and other groups was calculated by using Dunnett's test with the Instat version 3.0 statistical package (GraphPad Software). The criterion of significance was set at P<0.05.
LPS-induced ATP Release via Exocytosis from THP-1 Cells
THP-1 human monocytes were primed with LPS and IFN-gamma for 24 h to induce differentiation into macrophages. Primed THP-1 cells were stimulated with 10 µg/mL of LPS, and the extracellular concentration of ATP was measured. As shown in Figure 1A, the extracellular concentration of ATP was increased soon after stimulation with LPS and peaked at 10 to 20 min. In this method, ATP released from cells is diluted in the culture medium and is rapidly metabolized by ecto-nucleotidases on the plasma membrane. Therefore, the detected concentration of ATP must be much lower than the real concentration at the cell surface. The peri-cellular concentration of ATP just after ATP release would be much higher than detected concentration of ATP in medium. We confirmed that treatment with LPS did not induce cell death within 30 min (Figure 1B), suggesting that LPS-induced ATP release would not be dependent on leakage of ATP from dead cells. In subsequent pharmacological experiments, we measured extracellular ATP at 10 min after stimulation with LPS.
Figure 1. LPS-induced ATP release from THP-1 cells.
(A) Cells were stimulated with LPS (10 µg/mL) and incubated for the indicated times, then the concentration of ATP in the culture medium was measured as described in Materials and Methods. (B) Cells were incubated with PBS or LPS for 30 min. At the end of incubation, supernatants were collected and LDH content was measured. The release of LDH is expressed as a percentage of total content determined by lysing an equal number of cells with 1% Triton X-100. (C) Cells were pretreated with GdCl3 (100 µM), FFA (50 µM), glibenclamide (100 µM), CBX (50 µM), 18GA (50 µM), BFA (10 µM) or bafilomycin A (50 nM) for 30 min. At 10 min after LPS stimulation, supernatants were collected and ATP contents were measured. (D–F) Cells were stained with MANT-ATP (50 µM, cyan, panel C) and quinacrine dihydrochloride (5 µM, magenta, panel D) for 1 h at 37°C. Then, stained cells were analyzed using a confocal laser scanning microscopy. The merged image is shown in panel E. Each value represents the mean ± SE (n = 4–8). Significant differences between the indicated groups and control group (LPS alone) are indicated by *(p<0.05) and **(P<0.01), respectively.
To identify the pathway of the ATP release, we examined the effects of several inhibitors. As shown in Figure 1C, pretreatment with CBX (inhibitor of pannexin hemichannel) blocked the release of ATP after LPS stimulation. Moreover, pretreatment with bafilomycin A (inhibitor of vesicular H+-ATPase) and BFA (inhibitor of vesicular trafficking), which are inhibitors of vesicular exocytosis of ATP, significantly blocked the release of ATP after LPS stimulation. However, pretreatment with 18GA (connexin hemichannel inhibitor), FFA (anion transporter inhibitor), glibenclamide (anion transporter inhibitor), or GdCl3 (maxi-anion channel inhibitor) did not block the ATP release. These results indicated that the release of ATP after LPS stimulation is regulated by plural mechanisms, including pannexin hemichannels and vesicular exocytosis.
To look for ATP-enriched vesicles in THP-1 cells, we stained intracellular ATP with the fluorescent ATP indicator MANT-ATP. Fluorescent vesicles were observed in the cytoplasm of primed THP-1 cells (Figure 1D). Protons are accumulated in ATP-enriched vesicles by vacuolar proton-ATPase, forming an electrochemical gradient of protons across the membrane that serves as the driving force for ATP accumulation. Staining with a low pH-sensitive fluorescent probe, quinacrine dihydrochloride, visualized the same population of vesicles as that stained by MANT-ATP (Figure 1E, F), indicating that ATP-enriched vesicles are present in primed THP-1 cells.
Next, to investigate whether exocytosis is involved in LPS-induced ATP release from THP-1 cells, we silenced the expression of SLC17A9 using two different shRNAs. We confirmed that mRNA and protein expression of SLC17A9 were suppressed by transfection with SLC17A9-shRNA (Figure 2A). The decreased SLC17A9 expression resulted in suppression of ATP release compared with that of scramble shRNA-transfected cells, in response to LPS stimulation (Figure 2B). Furthermore, we investigated the involvement of vesicular exocytosis of ATP in LPS-induced IL-6 production. LPS-induced IL-6 production was significantly suppressed in SLC17A9-knockdown cells (Figure 2C). These results indicated that LPS induces ATP release from macrophages via vesicular exocytosis, and this process is involved in LPS-induced IL-6 production.
Figure 2. SLC17A9 is involved in LPS-induced ATP release and IL-6 production in THP-1 cells.
(A) THP-1 cells were transfected with 2 µg of two different shRNAs targeting SLC17A9 (No. 1 or No. 2) or scramble shRNA. Seventy-two hours after transfection, cellular membrane proteins were extracted and the expression of SLC17A9 was detected by immunoblotting as described in Materials and Methods. (B) THP-1 cells transfected with two different shRNAs targeting SLC17A9 or the negative control shRNA were stimulated with LPS. At 10 min after LPS stimulation, the supernatants were collected and ATP content was measured (n = 8–12). (C) The shRNA-transfected cells were incubated with LPS for 24 h, and then the concentration of IL-6 in culture medium was measured by ELISA (n = 10–14). Each value represents the mean ± SE. Significant differences between the control group (scramble) and the indicated group are indicated by ***(p<0.001).
Role of P2Y11 Receptor in LPS-induced M1-type Polarization and IL-6 Production in Macrophages
M1 polarization supports resistance to intracellular bacteria and control of the acute phase of infection. First, we investigated how extracellular ATP affects M1-type macrophage polarization. When THP-1 cells were stimulated with LPS and IFN-gamma, THP-1 cells were polarized into M1-type macrophages. Addition of ATP (100 µM) significantly facilitated M1-polarization of THP-1 cells (Figure 3A), indicating that extracellular ATP contributes to effective polarization of M1-type macrophages.
Figure 3. Extracellular ATP contributes to M1 macrophage polarization via activation of P2 receptors.
(A–C) THP-1 cells were cultured for 24 h in medium or in medium supplemented with LPS and IFN-gamma in the absence or presence of ATP (A), or bafilomycin A (50 nM), BFA (10 µM) (B), apyrase (25 U/mL), PPADS (100 µM), A438079 (100 µM), suramin (100 µM), NF449 (10 µM), MRS2179 (100 µM), MRS2578 (10 µM), NF157 (50 µM), clopidogrel (30 µM), or MRS2211 (100 µM) (C). Expression of M1 macrophage marker CCR7 was measured by flow cytometry. The supernatants were assayed for IL-6 (n = 8–10). Each value represents the mean ± SE. A significant difference between the control group (non-treated or LPS alone) and a test group is indicated by ***(p<0.001).
To investigate the involvement of ATP release via exocytosis in M1-polarization, we examined the effect of bafilomycin A or BFA on M1-polarization of THP-1 cells. Pretreatment with these inhibitors significantly blocked M1-polarization (Figure 3B), suggesting an involvement of ATP release via exocytosis in M1-polarization of THP-1 cells.
Next, we examined the effects of P2 receptor antagonists on M1 macrophage polarization. It is reported that macrophages express P2 receptors such as P2X1, P2X7, P2Y1, P2Y2, P2Y6 and P2Y11 receptors [30], [31]. Pretreatment with apyrase (ecto-nucleotidase) significantly suppressed M1 macrophage polarization, suggesting the involvement of extracellular ATP. Although pretreatment with PPADS (P2X antagonist), A438079 (P2X7 antagonist), NF449 (P2X1 antagonist), MRS2179 (P2Y1 antagonist), MRS2578 (P2Y6 antagonist), clopidogrel (P2Y12 antagonist), or MRS2211 (P2Y13 antagonist) did not affect induction of M1 macrophage polarization, a broad P2Y receptor antagonist, suramin, significantly blocked induction of M1-type macrophage polarization (Figure 3C). Furthermore, a highly selective P2Y11 receptor antagonist, NF157, also significantly blocked the induction, indicating the involvement of P2Y11 receptor in M1 macrophage polarization.
We also examined the effect of NF157 on LPS-induced IL-6 production. Pretreatment with NF157 significantly blocked the production of IL-6 in THP-1 cells (Figure 4A). Although NF157 can inhibit not only P2Y11, but also P2X1 receptor, pretreatment with selective P2X1 antagonist NF449 was ineffective (Figure 4A), suggesting that the inhibitory effect of NF-157 on IL-6 production is dependent on the blockade of P2Y11 receptor in THP-1 cells.
Figure 4. Effect of P2Y11 receptor antagonist on LPS-induced IL-6 production in THP-1 cells.
(A) THP-1 cells were pretreated with NF449 (10 µM), NF157 (50 µM), or KH7 (100 µM) for 30 min. (B) Treatment with NF157 was initiated 30 min before to 180 min after the treatment with LPS (10 µg/mL). At 24 h after LPS stimulation, supernatants were collected and the concentration of IL-6 was measured by ELISA. (C) Cells were pre-treated with NF157 for 30 min, and then incubated with 100 µM ATP for 24 h. Supernatants were collected and the concentration of IL-6 was measured by ELISA. (D–E) Cells were incubated with LPS (10 µg/mL) for 1–6 h (D). Cells were pre-incubated with NF157, and incubated with LPS for 6 h (E). At the end of incubation, mRNA level of IL-6 was determined by RT2-PCR as described under Materials and Methods. Each value represents the mean ± SE (n = 4–11). Significant differences between a test group and control group (LPS or ATP alone) are indicated by **(p<0.01) and ***(p<0.001), respectively.
To further investigate the contribution of P2Y11 receptor to IL-6 production, we examined the effect of adenylate cyclase inhibitor KH7, because P2Y11 receptor is coupled with Gs-protein, which activates adenylate cyclase [32]. Although P2Y11 receptor is coupled with both Gs-protein and Gq-protein, it is the only Gs-coupled P2 receptor among the P2 receptor subtypes [32]. Pretreatment with KH7 completely blocked IL-6 production, indicating that increase of cAMP is critical for IL-6 production (Figure 4A). The inhibitory effect of NF157 on LPS-induced IL-6 production disappeared when NF157 treatment was done 30 min after LPS treatment, indicating that P2Y11 receptor is activated within 30 min after LPS stimulation (Figure 4B). Further, treatment with 100 µM ATP also causes IL-6 production, and ATP-induced IL-6 production was significantly inhibited by pretreatment with NF157 (Figure 4C). These results suggest that ATP-induced activation of P2Y11 receptor would be involved in LPS-induced IL-6 production.
We investigated the effect of NF157 on induction of IL-6 mRNA expression after LPS stimulation. The level of IL-6 mRNA was increased from 1 h to 6 h after LPS-stimulation (Figure 4D). The increase of IL-6 mRNA at 6 h was partially suppressed by pretreatment with NF157 (Figure 4E), suggesting that NF157 would suppress IL-6 production via both transcriptional and post-transcriptional levels.
To confirm ATP-induced activation of P2Y11 receptor in THP-1 cells, we measured a change in cAMP level after treatment with 10–1000 µM of ATP because P2Y11 receptor is Gs-protein coupled receptor [32]. At 10 minutes after treatment with ATP, cAMP level was significantly increased by 100 µM or 1 mM of ATP in THP-1 cells (Figure 5A). The increase of cAMP was inhibited by pretreatment with NF157 (Figure 5B), suggesting ATP-induced activation of P2Y11 receptor in THP-1 cells. Further, we investigated the activation of P2Y11 receptor after LPS stimulation in THP-1 cells. Treatment with LPS caused cAMP elevation in a time-dependent manner (Figure 5C), and the increase was blocked by NF157 (Figure 5D), indicating the activation of P2Y11 receptor after LPS stimulation.
Figure 5. P2Y11 antagonist NF157 blocked ATP or LPS-induced increase of cAMP levels in THP-1 cells.
(A) THP-1 cells were incubated with 10–100 µM of ATP for 10 min. (B) Cells were pre-incubated with NF157 (50 µM) for 30 min, and then incubated with 100 µM ATP for 10 min. (C) Cells were incubated with LPS (10 µg/mL) for indicated times. (D) Cells were pr)e-incubated with NF157 for 30 min, and then incubated with LPS. Intracellular cAMP level was measured as described in Materials and Methods. Data is expressed as fold increase in cAMP compared with control. Each value represents the mean ± SE (n = 4). Significant differences between the positive control group (ATP or LPS alone) and the indicated group are indicated by ***(p<0.001).
To confirm the involvement of P2Y11 receptor in IL-6 production and M1-polarization, P2Y11 receptor in THP-1 cells was knocked down by transfection with siRNA. The mRNA expression of P2Y11 receptor was decreased to 44.4% (siRNA-1) or 25.4% (siRNA-2) (Figure 6A). We confirmed that these transfections did not affect the expression of other receptors by detecting an expression of P2Y12 receptor mRNA (Figure 6B). Treatment of P2Y11-knockdown THP-1 cells with LPS failed to induce IL-6 production (Figure 6C) or M1 polarization (Figure 6D). These results indicate that P2Y11 receptor plays a major role in LPS-induced activation of macrophages.
Figure 6. P2Y11 receptor is involved in IL-6 production and M1 macrophage polarization in THP-1 cells.
(A–B) THP-1 cells were transfected with 3 µg of shRNA targeting P2Y11 (No.1 or No.2) or scramble shRNA. Twenty-four hours after transfection, total RNA was extracted and P2Y11 or P2Y12 gene expression was examined by assessing mRNA levels using real-time PCR. (C, D) Cells were transfected with siRNA targeting P2Y11 (No.1 or No.2) or scramble siRNA and incubated for 72 h. (C) The transfected cells were stimulated with LPS (10 µg/mL) and incubated for 24 h, and then the concentration of IL-6 in culture medium was measured by ELISA. (D) The transfected cells were cultured for 24 h in medium or in medium supplemented with LPS and IFN-gamma. Expression of M1 macrophage marker CCR7 was measured by flow cytometry. Each value represents the mean ± SE (n = 4–8). Significant differences between a test group and control group (scramble) are indicated by *(p<0.05) and ***(p<0.001), respectively.
P2Y11 Antagonist NF157 Attenuates the Elevation of Inflammatory Cytokines in Peritoneal Fluid and Serum of LPS-induced Septic Mice
We next investigated the effect of NF157 on activation of macrophages in vivo. However, among mammals, the expression of P2Y11 receptors has been found only in humans and canines, although the existence of a receptor with properties like those of the P2Y11 receptor in mice and rats has been suggested [33], [34], [35]. Since some papers have reported that NF157 inhibits murine P2Y11 like receptors [34], [35], we examined the effect of NF157 on P2Y11 like receptor in mouse macrophages. Treatment with 100 µM ATP caused increase of cAMP level, and NF157 partially suppressed ATP-induced cAMP elevation in mouse macrophage RAW264.7 cells (Figure S1). We examined the effect of NF157 on LPS-induced IL-6 production by isolated peritoneal macrophages. NF157, but not NF449, partially suppressed LPS- or ATP-induced IL-6 production from peritoneal macrophages (Figure S2). These results suggest that NF157 can suppress an activation of mouse P2Y11 like receptor partially.
It is well known that high doses of LPS induce excessive immune activation in vivo, and this resembles sepsis. We confirmed the elevation of pro-inflammatory cytokine levels in serum after administration of LPS to mice. The concentrations of IL-1ß, IL-6, IL-12, and TNF-alpha in serum were significantly increased at 3–24 h after administration of LPS (Figure 7A–D).
Figure 7. LPS treatment induced increased serum levels of cytokines.
(A–D) Male C57BL/6 mice at 6 weeks of age were given LPS (400 µg/head i.p.), and blood samples were collected at 3, 6, 12, and 24 h after LPS injection. Serum levels of IL-1ß, IL-6, IL-12 and TNF-alpha were determined as described in Materials and Methods.
To investigate whether P2Y11 receptor antagonist NF157 suppresses LPS-induced overproduction of cytokines in vivo, NF157 (100 µl at indicated concentration/head) was administered i.p. at 2 h before LPS injection. The cytokine levels in serum were measured at 6 h after treatment with LPS. As shown in Figure 8, treatment with NF157 significantly suppressed the increases of TNF-alpha, IL-1ß, IL-6 and IL-12 in serum. The inhibitory effects were dose-dependent, with a maximal effect at 500 µM. Even the lowest dose of NF157 examined (5 µM) was sufficient to suppress the production of cytokines. On the other hand, administration of P2X1 antagonist NF449 with LPS did not suppress increase of IL-1ß, IL-6, IL-12 and TNF-alpha in serum (Figure S3).
Figure 8. P2Y11 antagonist NF157 suppressed the increase in serum levels of cytokines in LPS-treated mice.
(A–D) Male C57BL/6 mice at 6 weeks of age were given LPS (400 µg/head i.p.). NF157 (100 µL) doses of 5, 50 and 500 µM were administered intraperitoneally at 2 h before LPS was injected. Blood samples were collected at 6 h after LPS injection. Serum levels of IL-1ß, IL-6, IL-12 and TNF-alpha were determined as described in Materials and Methods. Each value represents the mean ± SE (n = 4). Significant differences between a test group and control group (LPS alone) are indicated by *(p<0.05) and **(p<0.01), respectively.
We also measured the cytokine levels in peritoneal fluid and M1-type polarization of peritoneal and splenic macrophages. Administration of NF157 with LPS inhibited the LPS-induced increases of cytokine levels (IL-1ß, IL-6, IL-12 and TNF-alpha) in peritoneal lavage fluid at 6 h after LPS treatment; these increases would mainly reflect release from peritoneal macrophages stimulated by LPS (Figure 9A–D). The To determine whether NF157 treatment modulates LPS-induced M1 macrophage polarization in vivo, peritoneal and splenic macrophages were isolated from mice at 24 h after LPS injection, and the expression of CCR7 was examined. As shown in Figure 10A–B, treatment with NF157 at 2 h before LPS injection effectively blocked LPS-induced M1-type polarization of peritoneal and splenic macrophages. These results suggest that treatment with NF157 might be effective for suppression of innate inflammatory response in polymicrobial septic shock.
Figure 9. P2Y11 antagonist NF157 blocked the increase in peritoneal levels of cytokines in LPS-treated mice. (A
–D) Male C57BL/6 mice were injected intraperitoneally with LPS (400 µg/head i.p.). NF157 (100 µL of 500 µM) was administered i.p. at 2 h before LPS was injected. Peritoneal lavage fluid was collected 6 h after LPS injection for measurement of IL-1ß, IL-6, IL-12 and TNF-alpha. Each value represents the mean ± SE (n = 4). Significant differences between a test group and control group (LPS alone) are indicated by *(p<0.05) and ***(p<0.001), respectively.
Figure 10. NF157 blocked M1 polarization of macrophages in LPS-treated mice.
(A, B) Peritoneal and splenic macrophages were isolated from mice 24 h after LPS injection, and analyzed by flow cytometry for the expression of CCR7. Each value represents the mean ± SE. A significant difference between the positive control group (LPS alone) and the indicated group is indicated by **(p<0.01).
Finally, we assessed the inhibitory effect of post-treatment with NF157 on the onset of endotoxemia. NF157 was administered at 30–180 min after LPS, and serum cytokine levels were measured. Elevation of IL-6, IL-12, and TNF-alpha, but not IL-1ß, was significantly attenuated even when mice were post-treated with NF157 at 30–60 min after administration of LPS, indicating that treatment with NF157 might be effective in blocking the onset of endotoxemia even after activation of macrophages by LPS (Figure 11A–D).
Figure 11. Suppressive effect of post-treatment with NF157 on serum levels of cytokines in LPS-treated mice.
(A–D) Male C57BL/6 mice at 6 weeks of age were given LPS (400 µg/head i.p.). Treatment with NF157 was initiated 15 min before to 180 min after the onset of LPS injection. Blood samples were collected at 6 h after LPS injection. Serum levels of IL-1ß, IL-6, IL-12 and TNF-alpha were determined as described in Materials and Methods. Each value represents the mean ± SE (n = 4). Significant differences between a test group and control group (LPS alone) are indicated by *(p<0.05) and **(p<0.01), respectively.
Since activation of macrophages contributes to severe inflammation, such as occurs in sepsis, it is important to understand the process of macrophage activation. In this study, we investigated the involvement of extracellular ATP-mediated purinergic signaling in macrophage activation. The most striking conclusion of this work is that endogenous ATP, released via exocytosis, activates P2Y11 receptor as a crucial second signal for inducing M1 polarization of macrophages and production of pro-inflammatory cytokine IL-6. In accordance with this idea, we found that the P2Y11 antagonist NF157 effectively suppressed the LPS-induced innate inflammatory response in mice. This report is the first to identify vesicular exocytotic ATP release from macrophages, and we also established that this exocytotic ATP release plays a major role in macrophage activation and IL-6 production. Considering the results of our recent study on the involvement of vesicular exocytosis in T cell activation [28], autocrine regulation via exocytosis of ATP appears likely to play an important role in effective activation of immune cells. This hypothesis is also supported by our finding that ATP facilitated M1-type polarization of macrophages.
Before the identification of SLC17A9 as a vesicular nucleotide transporter [25], it had been difficult to establish the involvement of exocytosis in ATP release caused by various stimuli. Recently, by using transfecting human T cells with shRNA for SLC17A9, we have demonstrated the involvement of exocytosis in ATP release caused by activation of T cell receptor [28], as well as ATP release from lung cancer cells caused by TGF-ß1 [29]. Here, we used the same approach to show that vesicular exocytosis is also involved in LPS-induced macrophage activation. It has been reported that pannexin hemichannels and maxi-anion channels are also involved in ATP release during activation of T cells [36]. In the present study too, LPS-induced ATP release from macrophages was found to be reduced by treatment with an inhibitor of pannexin hemichannels. Thus, multiple pathways may contribute to ATP release from LPS-treated macrophages. Nevertheless, our data indicated that abrogation of exocytotic ATP release caused significant suppression of IL-6 production of macrophages, suggesting that activation of P2 receptors by exocytotically released ATP plays a major role in mediating IL-6 production induced by LPS. Since LPS-induced ATP release in mouse peritoneal fluid has been confirmed in vivo [37], it is very likely that ATP release has a substantial role in the LPS-induced innate immune response.
Our data indicate that extracellular ATP enhances LPS- and IFN-gamma-induced M1 polarization. In particular, treatment with 100 µM ATP, but not 1 mM ATP, caused facilitation of M1-type polarization. It is consistent with the data indicating cAMP elevation by 100 µM ATP. Though cAMP elevation was also observed by treatment with 1 mM ATP, the enhancement of M1 polarization was not observed in 1 mM ATP-treated cells. The reason why 1 mM ATP did not enhance M1 polarization would be that high dose of ATP negatively regulate M1 polarization via pyrophosphates, which is not dependent on purinergic receptor [38].
Since treatment with inhibitors of the exocytosis of ATP or ecto-nucleotidase significantly inhibited M1-type polarization, ATP release via exocytosis would play an important role in M1-type polarization. Treatment with P2Y11 receptor antagonist NF157 and knockdown of P2Y11 receptor both caused significant suppression of M1 polarization, indicating the involvement of P2Y11 receptor in the M1-type polarization. Since P2Y11 receptor is involved in differentiation of various cell types, such as monocyte-derived dendritic cells [39], the involvement of P2Y11 receptor in macrophage differentiation seems reasonable. Blockade of M1-type polarization must cause suppression of various inflammatory events associated with macrophages and other immune cells, which are stimulated by cytokines produced by M1-macrophages. Indeed, the LPS-induced increase of serum concentrations of inflammatory cytokines, which is a result of systemic immune response, was significantly suppressed by pretreatment of mice with NF157.
Treatment with ATP, even without LPS, induced IL-6 production, suggesting an essential role of extracellular ATP in IL-6 production in macrophages. LPS-induced IL-6 production was significantly suppressed by knockdown of the P2Y11 receptor. Since IL-6 production was also significantly suppressed by treatment with adenylate cyclase inhibitor, increase of cAMP appears to play a critical role in IL-6 production. The only P2 receptor coupled via Gs to adenylate cyclase is the P2Y11 receptor [32], so it is reasonable that induction of cAMP is involved in P2Y11-mediated IL-6 production. We also confirmed treatment with ATP causes cAMP elevation in THP-1 cells, and it was blocked by NF157, suggesting that extracellular ATP can induce the activation of P2Y11 receptor in THP-1 cells. Inhibition of IL-6 production was not observed in vitro when NF157 was applied 30 min after LPS, suggesting that activation of P2Y11 receptor occurs within 30 min after LPS stimulation to induce IL-6 production. It is consistent with the data indicating that LPS caused cAMP elevation within 30 min, which was inhibited by NF157. Since elevation of IL-6 level in serum is observed at an early phase of sepsis, blockade of P2Y11 receptor might be effective for suppression of IL-6 over-production in the early phase of sepsis. It has been reported that Gs-protein-coupled rat P2Y11-like receptor is involved in transcriptional regulation of IL-6 in bile duct epithelia [40], and this is consistent with our findings. Indeed, we found that injection of LPS into the peritoneal cavity of mice caused elevation of inflammatory cytokine levels in serum, and pretreatment with NF157 significantly suppressed the elevation of serum and peritoneal cytokine levels in these mice. Moreover, LPS-induced M1-polarization of splenic and peritoneal macrophages was suppressed by treatment with NF157. All these results suggest that blockade of P2Y11 by NF157 is effective for suppression of macrophage activation induced by LPS in vivo. Interestingly, this inhibitory effect was observed even at 30 min after LPS treatment, presumably before all the macrophages are activated.
Treatment with NF157 caused significant suppression of not only IL-6, but also IL-1ß, IL-12, and TNF-alpha elevation in serum and peritoneal fluid, presumably as a result of blockade of M1-polarization of macrophages. The effect of NF157 on peritoneal cytokine production may represent a direct effect on LPS-stimulated peritoneal macrophages. On the other hand, the elevation of cytokine levels in serum would be due to LPS-induced systemic immune response. In the peritoneal fluid, NF157 significantly diminished the production of IL-1ß, IL-12, TNF-alpha, but only partially suppressed IL-6 production. The inhibitory effect of NF157 on IL-6 production was lower in isolated mouse peritoneal macrophages than in human monocyte THP-1 cells, which is consistent with a partial inhibition of P2Y11 like receptor by NF157 in mouse macrophages. It has also been reported that P2Y11 agonist stimulates release of IL-8 from human monocyte-derived dendritic cells [41]. Overall, because NF157 blocks M1-type polarization of peritoneal and spleen macrophages, it seems reasonable that NF157 would effectively inhibit the whole-body immune response in humans. In any case, human P2Y11 receptor and mouse P2Y11 like receptor appear to have important roles in immune regulation.
P2X7 receptor is well known to induce IL-1ß release by activating caspase-1, which convert pro-IL-1ß to mature IL-1ß. On the other hand, blockade of P2Y11 receptor causes suppression of M1-type polarization, which would block pro-IL-1ß production. Thus, the mechanism of the blockade of IL-1ß release might be different between P2X7 receptor antagonists and P2Y11 receptor antagonists.
In conclusion, we have identified a novel mechanism of macrophage activation, which is mediated by autocrine regulation via exocytotic ATP release and activation of P2Y11 receptor. Our results suggest that P2Y11 receptor antagonists can block the activation of macrophages by LPS both in vitro and in vivo, and suppress the elevation of inflammatory cytokines, but further studies will be needed to investigate the effect of P2Y11 receptor antagonists on the pathological changes that occur in sepsis by using cecal ligation puncture model, which is a chronic sepsis model, or in other inflammatory diseases. Though blockade of mouse P2Y11 like receptor by NF157 was partially, significant suppression of immune response was observed in LPS-treated mice. Therefore, it is implicated that treatment of human with P2Y11 antagonist such as NF157 might be more effective in suppressing an immune response. We suggest that our findings would justify a detailed study of P2Y11 receptor antagonists as therapeutic candidates for treatment of inflammatory diseases including sepsis.
Figure S1.
P2Y11 antagonist NF157 blocked ATP-induced increase of cAMP levels in mouse macrophage RAW264.7 cells. (A) RAW264.7 cells were incubated with 10–100 µM of ATP for 10 min. (B) Cells were pre-incubated with NF157 (50 µM) for 30 min, and then incubated with 100 µM ATP for 10 min. Intracellular cAMP level was measured as described in Materials and Methods. Data is expressed as fold increase in cAMP compared with control. Each value represents the mean ± SE (n = 4). A significant difference between the positive control group (ATP) and the indicated group is indicated by *(p<0.05).
(TIF)
P2Y11 receptor antagonist NF157 suppressed production of IL-6 from peritoneal macrophages. Peritoneal macrophages were pretreated with NF449 (10 µM), NF157 (50 µM), or KH7 (100 µM) for 30 min. At 24 h after LPS (10 µg/mL) (A) or ATP (100 µM) (B) stimulation, supernatants were collected and the concentration of IL-6 was measured by ELISA (n = 5). Each value represents the mean ± SE. Significant differences between the positive control group (LPS) and the indicated group are indicated by *(p<0.05) and ***(p<0.001).
P2X1 antagonist NF449 did not suppressed the increase in serum levels of cytokines in LPS-treated mice. (A–D) Male C57BL/6 mice at 6 weeks of age were given LPS (400 µg/head i.p.). NF449 (100 µL of 100 µM) were administered intraperitoneally at 2 h before LPS was injected. Blood samples were collected at 6 h after LPS injection. Serum levels of IL-1ß, IL-6, IL-12 and TNF-alpha were determined as described in Materials and Methods. Each value represents the mean ± SE (n = 5).
Conceived and designed the experiments: MT HS. Performed the experiments: HS MT. Analyzed the data: HS MT HH YM SK. Contributed reagents/materials/analysis tools: YM. Wrote the paper: HS MT HH YM SK.
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Small interfering RNAs
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Nucleotide receptor signaling
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Inflammatory diseases
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A third of UK professionals are likely to consider risky behaviours that endanger or undermine data protection, according to research from Courion.
The research was conducted by OnePoll and based on surveying 1000 UK employees who have access to customer data at work. The results suggest a lack of awareness of basic data protection policies and worrying behaviours such as snooping on sensitive personal information and sharing work login details with colleagues.
While the majority of the respondents recognise that hackers are criminals, 1 in 5 believe hackers do a worthwhile job by exposing security defects that should have been fixed by organisations.
Younger employees hold this view more than their older counterparts with a quarter of 18-24 respondents supporting this view; 30% of this age group would snoop on sensitive customer data at work compared to only 12% of the 45-54 years old employees.
The research also revealed that men are more reckless than women when it comes to breaching their employer's data protection policies. Male employees are twice more likely to access the database of an old employer if they still have access rights and more likely to pass on confidential information for money or if they feel they have been treated unfairly (36% men vs. 21% women).


Echo Emerson used to be a golden girl: she went to all the right parties, had the best grades, the most popular boyfriend, a large group of friends and an older brother who adored her. Then, during her sophomore year, her brother Aires was killed in Afghanistan and, just months later, she was brutally attacked. Her arms are covered in ugly red scars, but even though Echo knows that her bipolar mother was somehow involved, that entire day is just a huge black hole in her memory.
Before his parents died in a fire, Noah Hutchinson used to be just like Echo, successful and popular. He played sports and dreamed about being accepted to some of the best colleges in the country. For the past two years, he and his two younger brothers have been separated and living in the foster system. The kids are together and with the same foster parents the entire time, but Noah has been moved around more times than he can count. Gradually, he gave up on his dreams, he stopped playing sports when he couldn't afford it anymore, and started experimenting with soft drugs.
Echo and Noah are united by a common goal: breaking into the school counselor's office and finding out what happened to Echo in her mother's apartment and where exactly Noah's baby brothers live.
Even though I had no trouble connecting with the characters (especially Echo), their relationship was very hard for me to swallow at first. McGarry kept telling me how they felt about each other and my mind registered it, but it took quite a while for me to actually feel it alongside them. This is my main grievance with this book: no matter how much I liked it, when I compare it to contemporary masterpieces written by Kirsty Eagar, Melina Marchetta, Cath Crowley, Jandy Nelson or Hannah Moskowitz, for example, somehow it loses some of its shine.
Another thing I just have to mention is Noah himself. It was impossible not to notice how truly wonderful he was, but instead of it being a good thing, it was a double-edged sword. Boys like Noah simply don't exist. Kids in his situation aren't just one deep conversation away from getting their act together, restoring their trust in humanity and setting their priorities straight. They don't just wake up that easily. I needed Noah to be damaged according to his circumstances, but he just looked like it on the surface and underneath he was perfect.
Now that I managed to get it all off my chest, please disregard everything I wrote and go grab a copy of this book. It is, after all, a very promising debut that will hopefully make you laugh and cry just as much as it did me. It is, perhaps, more suitable for teens and people in their early twenties, but I guess that's how it's supposed to be.
I've heard only gushing reviews about Pushing the Limits, so it's nice to see a review that isn't just like "OMG go buy nooowz, lol."
It sort of annoys me as well when a character just has a conversation with someone and then they're fine. Just...no.
Great review. Hopefully I can pick a copy of this up soon!
I finally broke down and requested this after reading all the glowing reviews. I wonder how I will see it....I will come back and compare reviews when I read it. I just checked my schedule and I was ahead until I found out I have three books coming in the mail that I have to read and review. That being said, I will be available to read The Prophet the week of August 5th if that works for you!
Like Pooja, I hadn't thought much about guys like Noah existing in the real world, but I think I do see your point. I think the best part of this book for me was Echo.
I get what you mean about books just not seeming realistic enough, and it seems to be very true in YA books these days when it comes to boy characters. They are often a bit TOO perfect. Howevever, this sounds like a really emotional book. I'm curious!
Great review, Maja. While I enjoyed a good chunk of this book, I have to say that I had reservations. And I agree with you about Noah. His awakening just seemed too easy.
I have seen this one around a lot. I don't read much contemporary, you know that (even Melina Marchetta, who is poor genius and has scored on the top 4 best books of my life, hasn't managed to pull me into the genre quite yet...) but this one particularly worries me--and you've picked out exactly why: perfect boys like Noah don't exist. When they are broken, they are broken.
I think that this whole being easy to fix might influence some younger minds to believe they can find their own Noah and "fix" him, which in turn could lead to a heap of trouble and a not-so-happy ending.
Great review! I have had so many people tell me (including several out of the blue) that this book is a must read. I don't really like contemporary, but their recommendations - and your review - have made me want to pick it up and at least consider it, just to get an idea of what's out there in the contemporary world. But yeah, I get the indecisive between ratings. Happens to me so often.
Maja, I find it really interesting that you mentioned the author "told" the reader of Echo and Noah's relationship, as opposed to showed. Do you think that is why you weren't entirely convinced of their feelings for each other?
I'm currently reading this one, and agree with much of what you said in your review. I love the realistic, damaged main characters, and the author really pulls on your emotional strings. Lovely review, Maja. :) You are so very eloquent.
This review had me laughing in the end when you told us to forget everything you said and pick up a copy. It was that, in the end, that made me buy it on my NOOK. I think the only thing so far that I read that will bother me is that apparently her mother is bipolar and it led her to do something to Echo. I think mental illness has ENOUGH stigma. I'm bipolar myself and I'm not sure how I feel about it being used as a plot device. We'll see though.
All in all Maja, this was an excellent review but, then again, I wouldn't expect any less.
"Kids in his situation aren't just one deep conversation away from getting their act together, restoring their trust in humanity and setting their priorities straight."
Exactly! I think that was my main problem too! Especially when Noah pulled out his all knowing, wisest one Yoda side. There were things about his wisdoms I didn't buy, but then again, stuff like that is easy to over look when he's pressing you up against a wall!


MADISON, WIS. – Sen. Bernie Sanders (I-Vt.) Walked onstage here Friday under a gray sky with occasional specks of snow and launched into a full-on attack against President Trump, marking him a "pathologic liar" in a state that was key to Trump's victory in 2016.
He repeated the epithet Saturday at a union hall in Michigan, another Trump state. Sanders said.
The blaster attacks on the president reflect Sanders's developing and arguably risky strategy for reaching out to Trump's voters – people the president has said would support him even if he shot someone. It's a sharp contrast with other Democratic candidates who are focused on mobilizing Trump opponents. Not incidentally, it's also a way to signal Democrats that Sanders is their best hope for knocking off Trump, at a time when many fear he is the opposite.
The most striking example of this strategy will play out Monday night when Sanders appears at a town hall meeting hosted by the Fox News Channel, an outlet many Democrats detest and one party blocked from hosting a debate.
Sanders's approach faces a significant test in Wisconsin, Michigan and Pennsylvania, all part of his current campaign swing, where many white, working-class voters were Sanders argues he can reclaim these voters by convincing them he can deliver the economic relief they were seeking all along.
But many Democrats across the country are not convinced, even though they increasingly fear the Sanders has a real shot at the nomination, given his solid support of support and the deeply fractured Democratic field, which has 18 candidates and counting.
Some worry that a national ticket led by seven-year-old democratic socialist, who wants to transform the government, will outnumber political center – not only helping help Trump win a second term but erasing recent gains by centrist Democratic lawmakers in suburban areas.
If Sanders "wins the nomination, Trump will be president again," said Rep. Kurt Schrader (D-Ore.). "I'll guarantee it."
Many of the Democratic gains in the 2018 election were made by the candidates who were not in Sanders' mold, including in the two of the states he visited in recent days.
In Wisconsin and Michigan Democrats nominated more traditional candidates for the governor and captured both seats from Republicans. Some Democrats say a similar model is the path to victory in 2020.
Framing Sanders as the face of the Democratic Party, as the Fox appearance could do, will help Trump rather than hurt him, they argue.
The divide Over Sanders reflects a dilemma at the heart of the Democratic primary: Should a party nominate a Democratic version of Trump who can match his combativeness, energizing the liberals and taking the fight with the president? Or should it embrace a consensus builder, one who can rise above the country's partisan grief and bring people together?
This question could take even more significance in the coming weeks with the expected former presidential candidate Joe Biden's candidacy. Biden's two-party approach would sharpen the contrast between the field's "unifiers" – Biden, Sen. Cory Booker (D-N.J.) and former congressman Beto O'Rourke – and its more aggressive partisans like Sanders and Sen. Elizabeth Warren (D-Mass.).
Democratic concerns about Sanders echo some of the attacks. Trump and his allies are deploying against the senator. The Republicans have signaled that they would love to run against him, saying they could paint his policies as extreme and link him to the Democrats up and down the ballot.
Trump komentārus par Sanders ir divpusēji, vienlaikus apvainojot viņu un veidojot viņu up Last year, Trump castigated the senator, whom he calls "Crazy Bernie," for opposing the skimpier health insurance policies backed by Trump, tweeting, "Crazy Bernie and his band of Congressional Demos will outlaw these plans. Disaster!
Then, as Sanders was entering the race earlier this year, Trump told reporters, "I like Bernie," stroking the complaints by Sanders loyalists that the Democratic Party mistreated him in 2016. "I think he was taken advantage of, "said Trump, at an Oval Office signing ceremony. "I thought what happened to Bernie Sanders four years ago was pretty sad."
Sanders' campaign manager, meanwhile, calls Trump a "faux Bernie Sanders" on trade, an area where both emphasize protecting American workers.
At his Michigan rally Saturday, Sanders got into Trump's reworked version of the North American Free Trade Agreement, calling it weak protection and urging the president to scrap it.
All this sets up an intriguing backdrop for Sanders's Fox appearance. Fox is Trump's favorite network, and he has a good relationship with some of its hosts. Sanders's appearance on Fox tell his viewers the president is a patologist who could provoke one of the president's outraged responses.
In his first two months as a candidate, Sanders has gotten off to a quick fundraising start, drawing large crowds and posting impressive Showings in early polls.
Still, Sanders's rallies of recent days have not attracted the same massive crowds as during his kickoff events in New York and Chicago earlier this year. The audiences have shown some signs of animosity towards current and former rivals; Mark Craig, 66, disparaged by Trump and Hillary Clinton with expletives as he cheered on Sanders during his speech in Saturday in Warren, Mich.
The next phase of his campaign, the officials said, will be focusing on activating a volunteer army of more than a million people and convincing voters that he is electable.
Campaign manager Faiz Shakir described this phase as "continuing to address what I think is one of the most critical issues in this race: Who is the best candidate to defeat Donald Trump? "He argues that Sanders is" uniquely positioned "to do so.
Sanders's supporters say voters are deeply frustrated and looking for a leader who will shake things up. They note that 11 percent of voters who picked up Sanders in the 2016 Democratic primary went on to vote for Trump in the general election, according to the American National Election Studies survey. Another 8 percent voted for minor-party candidates, and Sanders's camp argues he has a shot with them as well.
Anita Cox, a 52-year-old teacher who attended the Sanders meeting in Indiana Saturday wearing a "Feel the Bern "The button, she said, knows Trump voters who she says Sanders could win over. "Maybe they believed their lies," Cox said.
At a Sanders appearance in Michigan, Christina Fong, who supported Sanders in 2016, said she knew some people who voted for Sanders and then Trump. Sanders can win them back, she said.
"Fool, a 54-year-old musician from Grand Rapids, said," Unfortunately, a lot of times people are really angry, and sometimes they displace that grief. " "So they remain angry, which he gets into," she added, speaking of Sanders.
But many Democratic leaders are watching Sanders with concern, believing that better bet for beating Trump is less hostile messenger with more moderate message . Nominating someone who neither personifies diversity nor connects with centrists is one of the few ways Democrats could lose in 2020, they argue.
"I do not think anyone is basically socialism. I really do not, "said Sen. Joe Manchin III (DW.Va.), who won the reelection last year in a state Trump captured decisively in 2016.
Sanders's unapologetic embrace of a Medicare-for-all health plan that would effectively eliminate private insurance also worries some party workers . Democrats won in 2018 by depicting GOP health plans as extreme, they say, and they have no desire to see the tables turned in 2020.
"I think when people find out that certain candidates want to take away people's sponsored by employers health Ian Russell, former deputy executive executive of the Democratic Congressional Campaign Committee, which coordinates party's House campaigns.
Sanders campaigned for Democrats in the Midwest several times in the run-up to the 2018 midterm, but most of his endorsed candidates were lost. Kansas Brent Welder and Iowa's Pete D'Alessandro, for example, were defeated in their House by primaries by more moderate Democrats who went on to win in November.
Sen. Doug Jones (D-Ala.), Who faces reelection in a ruby red state, said his constituents want a consensus builder at the top of the ticket, "someone who can govern who can reach across not only across the corridor, but Within the various groups within the Democratic Party. "He declined to discuss whether Sanders qualifies.
In many ways, the current Democratic dynamics shows similarities with Republican landscapes in 2016, when Trump dominated: party unsure how to deal with a rowdy populist from outside its traditional ranks, a crowded field that plays to its advantage and unpredictable political climate.
"Speaking for North Carolina If America had a choice between a self-avowed socialist democrat and a free market capitalist, he would lose, "Sen said. Thom Tillis (R-N.C.), who is up for reelection. "Period, end of story."
As she left the Capitol on Wednesday, Warren, who is also seeking a nomination, agreed that Sanders has gotten off to a quick start. But when asked why, she was demurred.


Modify children's ride-on toy and increase the speed.
You can amp up your children's battery-powered ride on toys in a variety of ways that will make them feel like they are driving the real thing. You can double the battery power and increase the speed of a Power Wheels Jeep from five miles an hour to almost nine miles an hour. You can also add some traction and increase the life of the plastic wheels that come with most of these ride-on vehicles, making it easier to climb up and down hills without slipping.
Cut the power supply wires under the front hood of the Power Wheels vehicle.
Affix a red or white wire on the new 12-volt battery to denote positive with a female adaptor to the male, positive side of the battery. The new battery will have two male prongs, one positive (+) and one negative (-).
On the new battery, affix a black wire to denote negative with a female adaptor to the male, negative side of the battery.
Connect the positive wire from the original battery to the negative wire on the new battery, and crimp on a male adaptor.
Connect the negative wire from the original battery to the positive wire on the new battery and crimp on a female adaptor. You have effectively created two batteries in series, which has doubled the voltage while keeping the same amperage.
Crimp on a male adaptor to the vehicle's negative wire, which will be black. Crimp on a female adaptor to the positive wire which will be white or red.
Connect the male adaptor of the vehicle to the female adaptor of the batteries. Connect the female adaptor of the vehicle to male adaptor of the batteries.
Glue special bands on plastic toy tires for better traction.
Clean ride-on toy tires with soap and water where the traction bands will sit.
Put your hands inside the band, make an oval shape and stretch out each band.
Sit toy on the floor with wheels up and pull the band around each tire, centering it.
Pull up the band and put adhesive directly on the tire. Use about half an ounce per tire and secure bands on tires. The bands will increase traction and prolong wheel life.
Use silicone, 3M Super 77, Liquid Nails, or Permatex molding trim adhesive to glue traction bands onto toy tires.


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I wanted you all to know that I've now shared my book with a few friends.
I'm still in the early editing stages, but so much has changed already.
This is my very first opportunity to get some feedback on my story, and I'm now biting my nails as I wait for feedback.
I've tried to convey to them how much it means to me that they've "volunteered" to read this book, but I don't think they quite understand.
Allowing people to read it is scary.
I know, the book is indeed intended to be read, but still, it's a very humbling feeling to be compared to a world of excellent writers.
The fact they want to read MY book, baffles me.
Who am I to think I've got something good on my hands, right?
Anyway, I'm forever grateful to the handful few, the ones who are about to tell me if I'm on to something or just completely lost.
Such are the current feelings of an aspiring writer.
Love Comes Twice – OUT NOW!
Five days left – New chapter for you.
Can I gift you Chapter five?
Chapters one and two – as promised.


To make a video about an attraction that didn't exist, we needed the right people in it to help explain the locations of the park, and convincing renderings to make it work.
We knew this wasn't going to be another run-of-the-mill, boring pitch video.
We were tasked by Assiniboine Park Conservancy to come up with an treatment on a video to help them raise +$150 Million in capital funds for their amazingly beautiful attraction, Canada's Diversity Gardens.
This video had to say a lot about the project, while still being able to inspire and emotionally connect the viewer (and hopefully, the donor).
We pulled out all the stops, from writing, shooting, motion tracking, green-screening and compositing, and came up with this ambitious video that took you through all of the locations and kept you interested throughout.
Since the release of this video, they've almost reached their fundraising goal (in only 12 months!), and will continue to be a leading communication piece for the Assiniboine Park Conservancy.


One of the most sought after and distinguised tempura restaurants in Japan.
Two Star Michelin gourmet restaurant "Tempura Kondo" is one of the most sought after and distinguished tempura restaurants in Tokyo. With more than 50 years of professional artisanship, the owner at Tempura Kondo continues to push boundaries. The restaurant draws many gourmets, celebrities and master chefs from around the world who continue to be inspired and awed by the cuisine at Tempura Kondo.
Tempura Kondo redefines aesthetics, with ingredients feeling entirely different upon first sight.
The owner has said that it is essential that the ultimate potential and taste of every ingredient be maximized; including the umami in the batter, so that the luxurious tastes of the cuisine can truly be savored.
At Tempura Kondo, you'll find innovative creations, yet perfection in simplicity. Reservations are difficult to come by, however Tempura Kondo is definitely worth visiting, even if having to make a special trip to the area just to dine at the restaurant.
Lunch Open 12:00 p.m. - 3:00 p.m.
※Reservations for lunch taken at 12 p.m. or 1:30 p.m. only.
Dinner Open 5:00 p.m. - 8:30 p.m.
※Reservations for dinner accepted at 5:00 p.m., 6:00 p.m., 7:00 p.m., and 8:00 p.m. Seating for dinner is available at a time limit of 2 hours.
3 minute walk from Ginza Station using the subway.
Course includes: Tempura: Shrimp x 2、Fish x 3, Vegetable x 4. Rice, Pickled Vegetables, Miso Soup, and Fruit.
Tempura: Shrimp x 2、Fish x 3、Vegetable x 5. One of three choices below, and fruit.
1.	Tendon: Kakiage Tempura (mixed vegetable tempura) served over a bowl rice.
2.	Tencha: Kakiage Tempura served over a bowl of rice, with Japanese tea (to pour over rice) and pickles.
3.	Kakiage Tempura and rice served separately, along with miso soup and pickles.
※ Kaede and Yomogi includes one small side dish, and sashimi. Please note that the number of shrimp, fish, vegetables may vary.
All guests are required to order one course each.
※Prices in the parentheses is the price with tax included.
None (please use nearby coin-operated parking).
No particular dress code enforced. However we ask that no casual wear be worn (shorts, t-shirts, sandals, etc. ).
Please fill out the reservation form on this site.
※Payment for reserved courses with a credit card is requird.
※Any additional orders that you make for food or drink at the restaurant are to be paid for at the restaurant after your meal.


text:
Flight Lieutenant Paul Gordon Royle (17 January 1914 – 23 August 2015) was an Australian Royal Air Force pilot who was one of the last two survivors of the 76 men who were able to escape from the Stalag Luft III German prisoner-of-war camp in World War II in what became known as The Great Escape.

Early life
Royle was born in Perth, Western Australia and he attended Hale School and then worked in mining in Kalgoorlie. A Royal Air Force recruiting team visited Australia and he was selected for a short term commission and pilot training. He flew solo in a de Havilland Tiger Moth and then learnt to fly the Avro Anson twin engine trainer and the Bristol Blenheim aircraft at No.2 School of Army Cooperation at Andover, Hampshire. He was then posted to No. 53 Squadron RAF in Northern FrancePaul Royle at the Imperial War Museum. Retrieved 26 August 2014 as a pilot officer.

Captivity
On 17 May 1940, his aircraft crash landed after being attacked by Messerschmitt Bf 109 fighters whilst on a reconnaissance mission and he was taken prisoner by advancing German troops. After a year in Stalag Luft I he was transferred to Stalag Luft III Sagan, Germany (now Żagań, Poland) where he was one of the prisoners nicknamed "the penguins" who helped to dispose of sand from tunneling in their trousers and he started a tunnel under block 68.

The Great Escape
On the night of 24 March 1944, he was number 57 in the queue of war prisoners waiting to escape and teamed up with Flight Lieutenant Edgar Humphreys who was the next in line. After being pulled through the narrow tunnel on a trolley and climbing the exit ladder, Royle and Humphreys ran for the cover of pine trees and then set off in the direction of Switzerland. They evaded capture for two nights and crossed the Berlin to Breslau autobahn before they were arrested by home guard upon entering a village. The two men were interrogated by the Gestapo in Görlitz, Royle was then returned to solitary confinement in Stalag Luft III but Humphreys was one of the 50 escapers who were victims of the Stalag Luft III murders, having been selected for execution by SS-Gruppenführer Arthur Nebe on the orders of Adolf Hitler.

Later life
In January 1945 with Soviet forces only 16 miles away the camp was evacuated and the prisoners including Royle were marched west to Marlag und Milag Nord prisoner-of-war camp. He was finally liberated by British troops on 2 May 1945 and was flown to Britain where he was discharged from the RAF. He then attended the Royal School of Mines and worked in the mining and engineering industries upon his return to Australia. He married twice and celebrated his 100th birthday in January 2014.

Royle died, aged 101, on 23 August 2015, leaving Dick Churchill as the only remaining survivor of the 1944 escape from Stalag Luft III. Churchill himself died four years later.

References

Further reading
Brickhill, Paul. The Great Escape. New York: Norton, 1950.

External links
 Imperial War Museum Interview

1914 births
2015 deaths
Australian escapees
Australian prisoners of war
Australian World War II pilots
Participants in the Great Escape from Stalag Luft III
People educated at Hale School
People from Perth, Western Australia
Royal Air Force personnel of World War II
World War II prisoners of war held by Germany
Royal Air Force officers
Australian centenarians
Men centenarians

redpajama_set_name: RedPajamaWikipedia