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Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm
Neurochemistry International xxx (2017) 1e9
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Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm , Pierluigi Navarra*, Lucia Lisi Emilia Laudati, Diego Curro Institute of Pharmacology, Catholic University Medical School, L.go F Vito 1, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2017 Received in revised form 24 February 2017 Accepted 3 March 2017 Available online xxx
Microglia express chemokines and their cognate receptors that were found to play important roles in many processes required for tumor development, such as tumor growth, proliferation, invasion, and angiogenesis. Among the chemokine receptor, CCR5 have been documented in different cancer models; in particular, CCR5 is highly expressed in human glioblastoma, where it is associated to poor prognosis. In the present study, we investigated the effect of CCR5 receptor blockade on a paradigm of microgliaglioma interaction; the CCR5 blocker maraviroc (MRV) was used as a pharmacological tool. We found that MVR is able to reduce the gene expression and function of the M2 markers ARG1 and IL-10 in presence of both basal glioma-released factors (C-CM) and activated glioma-released factors (LI-CM), but it up-regulates the M1 markers NO and IL-1b only if microglia is stimulated by LI-CM; the latter effect appears to be mediated by the inhibition of mTOR pathway. In addition, CCR5 blockade was associated to a significant reduction in microglia migration, an effect mediated through the inhibition of AKT pathway. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Microglia Glioblastoma iNOS Arginase AKT mTOR
1. Introduction Glioblastoma (GBM) is the most common and aggressive malignant primary brain cancer. Despite treatment with maximal surgical resection, radiotherapy and chemotherapy, patients diagnosed with GBM typically have short life expectancies with a median survival rate of 14e16 months (Buckner et al., 2007; Dello Russo et al., 2016). In most clinical trials, therapies targeting GBM cells have failed or resulted in limited clinical response, likely due to the acquired resistance mechanisms and significant molecular heterogeneity within the tumor (Osuka and Van Meir, 2017). Therefore, a need exists to discover novel cellular targets for the development of more effective therapies. Recently, the tumor microenvironment has emerged as an attractive additional target for possible therapeutic interventions. In fact, cells such as vascular cells, peripheral immune cells, microglia and neural precursor cells play an essential role in controlling the progression of the disease (Charles et al., 2012). Among these cells, microglia and macrophages appear to be the most common cell type in the GBM microenvironment, representing up to 30% of tumor tissue (Sliwa et al., 2007).
* Corresponding author. E-mail address: [email protected] (P. Navarra).
Interactions between microglia and GBM cells are bi-directional (Galv~ ao and Zong, 2013). Microglia express a variety of receptors, which can be activated by signals from glioma cells (Held-Feindt et al., 2010; Okada et al., 2009). For example glioma cells produce prostaglandin E2 and cytokines, such as interleukin (IL)-10, IL-4, IL6 and TGFb (Rolle et al., 2012), that are able to activate microglial cells; in particular, IL-4 causes alternative activation (M2) of microglia (Gadani et al., 2012). Conversely, under the influence of glioma, microglia release several classes of molecules, such as nitrites (NO), IL-10, IL1b, TNFa and urea, that promote glioma growth, progression and inflammatory activation (Li and Graeber, 2012). However, the role of microglia in GBM pathology is still unclear. In fact, in the last decade a large number of studies provided evidence for a tumor-supporting role of microglia; on the opposite, data in support of microglial anti-tumor properties have been also reported (Li and Graeber, 2012). Microglia express chemokines and their cognate receptors (Allen et al., 2007), which were found to play important roles in many processes required for tumor development, such as tumor growth, proliferation, invasion, and angiogenesis (Vakilian et al., 2017; Kakinuma and Hwang, 2006; Mantovani, 2010). Among the chemokine family, consisting of about 50 members that bind to 20 chemokine receptors, we focused our studies on CCR5 receptor. CCR5 is involved on HIV entry in host cells; for this reason, it is one of the most studied chemokine receptors (Hütter et al., 2015).
http://dx.doi.org/10.1016/j.neuint.2017.03.002 0197-0186/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Although the chemokine ligand-receptor systems, as mediators of inflammation, are inherent to the microglia-macrophage inflammatory cell lineage, it is worth of note that CCR5 is expressed in neoplastic mesenchymal cells that are considered the most invasive cell-type in GBM (Choi et al., 2015). Moreover, CCR5 and its ligand CCL5 have also been documented in different cancer models (Robinson et al., 2003; Soria and Ben-Baruch, 2008); in particular, CCR5 is highly expressed in human GBM, where it is associated to poor prognosis. High amounts of CCL5 are released by tumors showing a significantly shorter mean survival time compared to those releasing lower levels of CCL5. The National Cancer Institute (NCI) REMBRANDT data portal (Repository of Brain Neoplasia Data) reported a positive association between CCL5 expression and survival rates in glioma patients (Pham et al., 2012). In addition, CCL5/ CCR5 mediates the activation of Akt, and subsequently induces proliferation and invasive responses in U87 and U251 cells (Zhao et al., 2015). Recently, we used rat microglia to investigate the effects of different conditioned media obtained from glioma cells. We showed that microglia activation undergoes a different fate and express a distinctive phenotype, depending on the stimulus used. In particular, using control conditioned media (C-CM, i.e. a condition mimicking an initial stage of pathology), microglia tend toward a M2 polarization, whereas conditioned media obtained after glioma stimulation with LPS-IFNg (LI-CM) induced a mixed polarization profile, i.e. both M1 and M2 phenotypes (Lisi et al., 2014a). In the present study, we adopted the above experimental paradigm to investigate the effects of CCR5 blockade on primary rat microglia polarization profile as well as on the rate of cell migration. We used a pharmacological inhibitor, Maraviroc (MRV), which locks CCR5 conformation in an inactive state by allosteric inverse agonist activity (Lu and Wu, 2016); in particular, we compared the effects of MRV on primary microglia cells activated by C-CM versus the effects of MRV on microglia activated by LI-CM. 2. Materials and methods 2.1. Antibodies and reagents Materials Cell culture reagents [Dulbecco's modified Eagle's medium (DMEM), DMEM-F12 and fetal calf serum (FCS)] were from Invitrogen Corporation (Paisley, Scotland). Antibiotics were from Biochrom AG (Berlin, Germany). The rat recombinant proinflammatory cytokine, interferon-g (IFNg), was purchased by Endogen (Pierce Biotechnology, Rockford, IL, USA). Maraviroc was provided by Pfizer Italia SRL (Aprilia, RM, Italy). Primary antibodies: polyclonal rabbit phosphorylated-mTOR Ser2448 (Novus biological), monoclonal mouse b-actin (Sigma-Aldrich) polyclonal rabbit phospho IKb (Cell Signaling), polyclonal rabbit p-Akt S473, monoclonal mouse Akt, polyclonal mouse p70S6K (BD, Bioscience). Secondary antibody, anti-rabbit and anti-mouse immunoglobulin G(IgG)-horseradish peroxidase (HRP)-conjugated, were purchased from Sigma-Aldrich. 2.2. C6 glioma cells C6 glioma cells were passed once a week and were prepared as previously described (Lisi et al., 2011). Briefly, control-conditioned medium (C-CM) was obtained incubating C6 glioma cells for 4 h in plain medium in 1% FCS, and after three washes with PBS in fresh plain medium for an additional 24 h. After this second period of incubation, the CM was collected, centrifuged to remove cellular debris and stored as C-CM. Similarly, LPS/IFNg-conditioned medium (LI-CM) was prepared incubating C6 cells for 4 h with LPS 1 mg/ml and IFNg 10 Unit/ml, followed after three washes with PBS
by 24-h incubation in fresh plain medium. LI-CM was collected, centrifuged and stored after this second period of incubation, thus LI-CM did not contain the proinflammatory stimuli used to activate glioma cells. Both CM were stored at 80 C until the experiments on microglial cells were performed (Lisi et al., 2014a,b). 2.3. Cell proliferation assay Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation assay using the Delfia Cell Proliferation Kit (Perkin Elmer, Inc, Waltham MA, USA). Cells were seeded in 96-well plates with 10% FBS in culture medium, and treated with indicated agents for 48 h. BrdU was added during the last 16 h of incubation period. The assay was performed according to the manufacturer's instructions. 2.4. Cell viability measurement Cell viability was assessed by reduction of the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5- (4-sulfophenyl)-2Htetrazolium, inner salt; MTS) contained in the TACS® XTT Cell (Trevigen, Gaithersburg, MD, USA). For this assay cells were seeded in 96-well plates. At the end of the experimental procedure (46 h), 50 mL of MTS reagent were added to the cells that were further incubated for 2 h. Living cells bioreduce yellow MTS into a purple soluble formazan product with an absorbance peak at 492 nm, that was read in a spectrophotometric microplate reader (PerkinElmer Inc, MA, USA). 2.5. Microglia Primary enriched cultures of rat glial cells, microglia and astrocytes, were prepared as previously described (Dello Russo et al., 2009). In particular, microglial cells were obtained by mixed cultures of cortical glial cells (at in vitro day 14), by gentle shaking. Cells were plated with DMEM-F12, containing 10% FCS and antibiotics. Experiments were carried out in 10% FCS containing medium to reduce microglial cell death that we usually observe after the splitting from astrocytes (Vairano et al., 2002). Microglial cells were treated with soluble factors released by C6 glioma cells, as described above. The selective CCR5 receptor antagonist, Maraviroc, was administered in the concentration range of 1pM-1 mM. The use of animals for this experimental work has been approved by the Ethics Committee of the Catholic University Medical School. 2.6. Transwell cell migration assays Microglial cells were plated at density of 5 104 to the top chamber of 24-well Transwell units with 8.0 mm pores (Corning) in low-serum media containing the different treatments. The bottom chamber contained 10% FBS. Cells were allowed to migrate at 37 C in 5% CO2 for 16 h. Non-migrating cells were removed from the upper surface using a cotton swab. The lower surface was stained with Hema 3 (Fisher Scientific). Stained membranes were mounted on microscope slides and imaged using a Nikon Eclipse TE300 microscope. The number of migrated cells was determined in 4 representative fields, selected by a blinded investigator, using ImageJ software. Two separate membranes from 4 independent experiments were analyzed for each condition. 2.7. Nitrite assay The activity of the inducible form of nitric oxide synthase (iNOS or NOS2) was assessed indirectly by measuring nitrite accumulation in the incubation media. Briefly, an aliquot of the cell culture
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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media (80 mL) was mixed with 40 mL Griess Reagent (Sigma-Aldrich, St Louis, MO, USA) and the absorbance measured at 550 nm in a spectrophotometric microplate reader (PerkinElmer Inc. Waltham, MA, USA). A standard curve was generated during each assay in the range of concentrations 0e100 mM using NaNO2 (Sigma-Aldrich) as standard. In this range, standard detection resulted linear and the minimum detectable concentration of NaNO2 was z 3.12 mM. In the absence of stimuli, basal levels of nitrites were below the detection limit of the assay at all the time points studied. The levels of NO were normalized with the protein content determined by Bradford's method (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as standard.
2.8. Urea assay Urea levels in microglia cells were detected by the QuantiChrom Urea Assay kit (BIOassay System, Hayward, CA, USA), used according to the manufacturer's instructions. Briefly after incubation with the tested substances for 48 h, as indicated in figure legends, an aliquot of cell culture media (50 mL) was mixed with 200 mL Urea Reagent (Bioassay system) and the absorbance was measured at 430 nm in a spectrophotometric microplate reader (PerkinElmer Inc.). A standard curve was generated during each assay in a concentration range 0e100 mM using urea as standard. In this range, standard detection resulted in linear plots and the minimum detectable concentration of urea was 3.12 mM. The levels of urea were normalized with the protein content determined by Bradford's method (Bio-Rad, Hercules, CA, USA) using BSA as standard.
2.9. mRNA analysis in real-time PCR Total cytoplasmic RNA was extracted using the ReliaPrep RNA Tissue Miniprep Systemt (Promega, Madison, WI, USA), which included 15 min Dnase treatment. RNA concentration was measured using the Quant-iTTM RiboGreen RNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA). A standard curve in the range of 0e100 ng was run in each assay using 16S and 23S ribosomal RNA (rRNA) from E. coli as standard and provided by the kit. Aliquots (0.25 mg) of RNA were converted to cDNA using random hexamer primers. Quantitative changes in mRNA levels were estimated by real time PCR (Q-PCR) using the following cycling conditions: 35 cycles of denaturation at 95 C for 20 s; annealing and extension at 60 C for 20 s, using the Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Stratagene, La Jolla, CA, USA). PCR reactions were carried out in a 20 mL reaction volume in a MX3000P real time PCR machine (Stratagene). Primers used for the evaluation of gene expression are reported in Table 1. Relative mRNA concentrations were calculated from the take-off point of reactions (threshold cycle, Ct) using the comparative quantitation method performed by Stratagene software and based upon the -DDCt method. Ct values for GAPDH expression served as a normalizing signal. (Dello Russo et al., 2009).
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2.10. Western immunoblot Primary microglia cells were plated (5 105 cells/well) in 6 multiwell, treated for 24 h and then lysed in RIPA buffer (1 mM EDTA, 150 mMNaCl, 1% igepal, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 50 mM TriseHCl, pH 8.0) (Sigma-Aldrich) containing protease inhibitor cocktail diluted 1:250 (SigmaAldrich). The protein content in each sample was determined by Bradford's method (Bio-Rad, Hercules, CA, USA) using BSA as standard. An equivalent amount of cellular protein (10 or 20 mg per lane) was subjected was mixed 1:2 with 2$Laemmli Buffer (BioRad), boiled for 5 min at 95 C, and separated through 10% polyacrylamide sodium dodecyl sulfate gels. Apparent molecular weights were estimated by comparison to colored molecular weight markers (Sigma-Aldrich). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes by semidry electrophoretic transfer (Bio-Rad). The membranes were blocked with 10% (w/v) low-fat milk in TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) (Bio-Rad) for 1 h at room temperature (25 C), and incubated in the presence of the primary antibody overnight with gentle shaking at 4 C. Primary antibodies for Rabbit polyclonal phosphorylated-mTOR (Novus biological),b-actin (Sigma-Aldrich) anti-phospho IKKa/b (Cell Signaling), antiphospho AKT (RD system), anti-total AKT were used at the final concentration of 1:1000. Primary antibodies were removed, membranes washed three times in TBST, and further incubated for 1 h at 25 C in the presence of specific secondary antibody, antirabbit and anti-mouse immunoglobulin G(IgG)-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich), diluted 1:15,000 and 1:10,000 respectively. Following three washes in TBST, bands were visualized by incubation in ECL reagents (GE Healthcare, New York, NY, USA) and exposure to Hyperfilm ECL (GE Healthcare). The same membranes were washed three times in TBST, blocked with 10% (w/v) low-fat milk in TBST for 1 h at 25 C and used for b-actin immunoblot. 2.11. Immunocytochemistry For immunocytochemistry, microglia were plated at a density of 3.5 105 cells/well on coated glass coverslips and treated with PBS or MRV 1 mM (Lisi et al., 2014a,b). At this time, cultures were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min. After fixation, cultures were washed three times with PBS and permeabilized with 0.1% Triton X for 5 min; then washed and blocked with 1% BSA in PBS for 30 min. 1: 200 CCR5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS containing 0.1% BSA and 0.1% Triton X100 was added for 2 h. After incubation, 40,6-diamidino-2- phenylindole (DAPI) was added for 10 min and then washed twice with PBS. Coverslips were mounted using Vectashield mounting media. Images were obtained on a Nikon Eclipse TE300 inverted fluorescence microscope equipped with a Cool SNAP professional digital camera and LUCIAG/F imaging software.
Table 1 Primer sets used for Q-PCR analysis. Genes
Forward primers
Reverse primers
Product length
GAPDH iNOS IL-10 ARG1 IL-1b CCR5 IKBa
CCC TCG CCA TGG TAA ATA CAT CTG CAT GGA ACA GTA TAA GGC AAA C CAG CTG CGA CGC TGT CAT CGA TGC CCT CTG TCT TTT AGG GC GCA GGC CTG GGA CCT TGC TG AGC AGT GAT CCG AAA GGA GGG AAA GCC TGG CCA GTG TAG CAG TCT
ACT GGA TGG TAC GCT TGG TCT CAG ACA GTT TCT GGT CGA TGT CAT GA GCA GTC CAG TAG ATG CCG GGT G CCT CGA GGC TGT CCC TTA GA CGA CAG GGC CAA GCT CAC CG TAC TTG GAG TGC TGC CAG TGT CTT CAG CAC CCA AAC TCA CCA AGT
110 230 198 165 271 199 135
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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2.12. Data analysis All experiments were performed using 5e6 replicates per each experimental group, and repeated at least three times. For the RNA analysis, samples were assayed in triplicates, and the experiments were repeated at least twice. Data were analyzed by one way ANOVA followed by Bonferroni's post hoc test or by unpaired t-test. p values < 0.05 were considered significant. 3. Results First we looked at CCR5 gene expression in glioma C6 cells and checked whether it is a functional receptor. Fig. 1A shows that CCR5 receptor was expressed in C6 glioma cells. Under basal conditions, 100 and 1000 nM MRV was able to significant reduce both proliferation and viability of C6 cells after 3 days of treatment (Fig. 1B and C). Fig. 2 shows the expression and localization of CCR5 receptors in rat primary microglia cells. Concerning gene expression, MRV per se does not modify CCR5 expression (Fig. 2A). In normal conditions CCR5 receptor is localized at the level of microglia cell membranes (Fig 2B), whereas in the presence of 1 mM MRV the CCR5 receptor is internalized, as showed by the increase in fluorescence within the cytoplasmic compartment compared to basal conditions (Fig 2C). A second set of experiments were carried out to study microglia migration. Fig. 3 shows the results of transwell cell migration assays. Both types of stimuli, C-CM and LI-CM enhanced cell migration (Fig. 3B and C); 1 mM MRV given alone did not modify microglia migration (Fig. 3E), whereas it was able to inhibit microglia migration in the presence of glioma soluble factors (Fig. 3E and F). In particular, MRV was able to significantly reduce migration elicited by LI-CM whereas reduction exerted over the migration elicited by CCM did not attain statistical significance (Fig. 3). Subsequently we investigated the expression and phosphorylation degree of AKT as a possible mediator of the observed migration. As expected, both C-CM and LI-CM stimuli significantly
increase the phosphorylated portion of the AKT protein; such increase was completely reversed by 1 mM MRV, although the latter had no effect whatsoever on the total amount of AKT protein (Fig. 4). These data strongly suggest that MRV is able to reduce microglia cell migration through the block of AKT pathway. An additional series of experiments was carried out to investigate the effects of MRV on microglia inflammatory state elicited by glioma soluble factors. First we studied the levels of NO, taken as an M1 marker. After C-CM stimulation, MRV was able to reduce nitrite levels compared to vehicle, and the maximal effect was reached at MRV concentrations as low as 10 pM (Fig. 5A). On the opposite, when LI-CM was used as stimulus, 100 and 1000 nM MRV elicited a significantly increase, in NO release (Fig. 5B). All of the above changes in NO levels, either increases or reductions, were not associated to any variation in iNOS gene expression (Fig. 6A). Instead, a direct correlation was found between the changes in NO release and parallel changes in the levels of IKBa gene expression, with increases induced by MRV under C-CM stimulation and reductions after LI-CM exposure (Fig. 6B). These data were confirmed by the analysis of phospho-IKBa protein (Fig. 6CeD). Similar results were obtained with another M1 marker, the proinflammatory cytokine IL-1b; MRV increased the levels of IL-1b gene expression elicited by LI-CM but had no stimulatory effect, and even tended to decrease cytokine gene expression under C-CM stimulation (Fig. 7). Thus, MRV appears to consistently reduce the M1 phenotype in presence of C-CM while on the contrary it tends to up regulate M1 phenotype in presence of LI-CM. Looking at M2 markers, we investigated ARG1 and IL-10 gene expression. In particular, MRV was able to significant reduce ARG1 gene expression in presence of both C-CM and LI-CM (Fig. 8A). The reduction in ARG1 gene expression was associated to a significant reduction in urea production in presence of LI-CM (Fig. 8B) but not in presence of C-CM (Fig. 8C). Likewise, MRV was able to reduce IL10 gene expression in both experimental conditions (Fig. 9). Therefore, it appears that MVR altogether reduces M2 phenotype both in presence of CCM and LICM.
Fig. 1. CCR5 receptor in glioma c6 cells. A) CCR5 gene expression in C6 glioma cells. B-C) Effects of MRV on proliferation and viability of C6 cells. C6 glioma cells were treated with MRV for 3 days. Effects on cell proliferation were assessed by 5-bromo-2-deoxyuridine (BrDU) incorporation (B), while the effects on cell viability were assessed by the XTT reduction assay (C). Data were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. *P < 0.001, versus Control.
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Fig. 2. CCR5 receptor in microglia cells. (A) Microglial cells were cultured in presence with MRV (1 mM). Control cells were treated with 50% of complete medium with DMEM 1% of FBS. After 24 h, RNA was extracted and expression levels of CCR5 mRNA were quantified by q-PCR. Data represents mean ± SEM; n ¼ 3; one-way ANOVA followed by Dunnett's multiple comparison test. (B, C) After 48 h of culture in basal condition (B) or MRV 1 mM (C), microglia cultures were stained with the antibody for CCR5 (green). Nucleus is visualized using DAPI (blue). Magnification 20x. Images are representative of 3 independent experiments.
Fig. 3. Effects of MRV on microglia migration. Microglial cells were introduced into Transwell systems. Migration was allowed to proceed for 16 h. Representative photomicrographs of cells that migrated to the lower surfaces of Transwell membranes are shown. Images are representative of 3 independent experiments. Scale bar ¼ 1 mm. Data in A represents mean ± SEM; n ¼ 3; ***P < 0.001 versus Control, **P < 0.001, versus Control P < 0.001 versus LI-CM.; one-way ANOVA with Dunnett's post-hoc analysis. (B). Quantification of migration results.
Furthermore, we investigated the metabolic mTOR pathways as a possible downstream signaling mechanism involved in CCR5 blockade. A western blot analysis shows that MRV was able to
inhibit mTOR phosphorilation at Ser2448 in a 24-h treatment under LI-CM stimulation, whereas no effect was observed in presence of C-CM (Fig. 10A).
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Fig. 4. Effects of MRV on AKT pathway. Protein content were extracted from microglia after 24 h of treatment and subjected to western-blot to detect phospho-Akt and total Akt. Images are representative of 3 independent experiments.
Fig. 5. Effects of MRV on NO levels. Panel A shows microglia treated with C-CM alone or in combination with MRV; panel B shows microglia treated with LI-CM alone or in combination with MRV (B). After 48-h incubation, the medium was used for NO assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as mM of nitrites/mg of proteins; data are means ± SEM (n ¼ 6). **P < 0.05, ***P < 0.001.
4. Discussion In the present study, we investigated the effect of CCR5 receptor blockade on a paradigm of microglia-glioma interaction; the CCR5 blocker MVR was used as a pharmacological tool. We found that MVR is able to reduce the gene expression and function of the M2
markers ARG1 and IL-10 in presence of both C-CM and LI-CM stimuli, but it up-regulates the M1 markers NO and IL-1b only if microglia is stimulated by LI-CM; the latter effect appears to be mediated by the inhibition of mTOR pathway. In addition, CCR5 blockade was associated to a significant reduction in microglia migration, an effect mediated through the inhibition of AKT pathway. A well-known drug in HIV treatment, Maraviroc binds CCR5 and locks the receptor in the inactive state via an allosteric inverse agonist action (Lu and Wu, 2016). Our group and others have recently tested the hypothesis that MRV affect microglial activation during HIV-1 infection and/or in other diseases through the blockade of CCR5 receptor (Martin-Blondel et al., 2016). CCR5 is expressed both in microglia cells (Lisi et al., 2012) and in macrophages (Oppermann, 2004). Using primary microglial cultures activated by glycoprotein120 (Gp120) alone or in combination with IFNY, our group investigated the effects of MRV on the gene expression of pro-inflammatory cytokines. The drug showed opposite effects depending on whether microglia was pretreated with Gp120CN54 or in combination with IFNY; in fact we observed a down-regulation of pro-inflammatory cytokine gene expression after pretreatment with Gp120CN54 alone, whereas an increase was observed in the presence of INFY (Lisi et al., 2012). Maraviroc has also been shown to reduce neuropathic pain through the polarization of microglia and astroglia (Piotrowska et al., 2016). Current knowledge on the mechanisms of microglial activation shows a growing complexity compared to that originally described based on cell morphology only. Similar to macrophages, microglia shows different responses to various stimuli, adopting specific phenotypes of activation. In particular, two distinct pattern of activation are described: classically activated M1 cells endowed with cytotoxic properties, and alternatively activated M2 cells with phagocytic activities. We have recently shown that microglial cells present as a mixture of polarization phenotypes (M1 and M2) when exposed to activated glioma-derived factors, i.e. a condition
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Fig. 6. Effects of MRV on iNOS and IKBa gene expression. Total cytosolic RNA was prepared from control, or microglial cells treated with the described treatments for 24 h, and used for real time (Q)ePCR analysis of iNOS (A) and IKBa (B) gene expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± SEM of the mean, and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. (C) Protein content were extracted from microglia after 24 h of treatment and subjected to westernblot to detect IKBa and b-actin. Images are representative of 3 independent experiments.
Fig. 7. Effects of MRV on IL-1b gene expression. Total cytosolic RNA was prepared from control, or microglial cells treated with the described treatments for 24 h, and used for real time (Q)ePCR analysis of il-1b gene expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± SEM of the mean, and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test.
mimicking the late stage of pathology, with up-regulation of iNOS, ARG and IL-10. At variance, microglia exposed to basal gliomaderived factors, i.e. a condition resembling the early stage of pathology, shows a more specific pattern of activation, with increased M2 polarization status and up-regulation of IL-10 (Lisi et al., 2014a). In addition, we showed that the inhibition of mTOR polarizes glioma-activated microglial cells towards the M1 phenotype, preventing the induction of the M2 status (Lisi et al., 2014b). Other groups (Li et al., 2016; Paschoal et al., 2017; You et al., 2016) have also reported a polarization toward M1 in both microglia and macrophages subsequent to mTOR pathway modification. In the present study, we showed that MRV polarizes microglia cells
towards the M1 phenotype and prevents M2 status in presence of LI-CM, through a mechanism involving the inhibition of mTOR and NfKB-IKBa complex pathways. It should be pointed out that such effect of mTOR inhibition can only be observed under conditions of pro-inflammatory activation of the system. In fact, in presence of plain C-CM stimulus MVR fails to blunt mTOR activation, and the result of CCR5 blockade is rather a reduction of both M1 and M2 phenotypes. Having shown an effect of the chemokine-CCR5 pathway in the regulation of microglia cell migration, we further explored the involvement of AKT signaling. In fact, the role of this signaling pathway in GBM invasion has been thoroughly investigated using several Akt-targeted drugs as tools. A non-steroidal anti-inflammatory drug, sulindac and its metabolites are able to inhibit GBM invasion in vitro via de-phosphorylation of Akt at Ser473 (Lee et al., 2005). Likewise, the selective cyclooxygenase-2 inhibitor celecoxib is also able to inhibit GBM invasion, its effect involving the inhibition of Akt signaling activity (Paw et al., 2015). Morphine-induced microglial migration has also been shown to be mediated by the PI3K/Akt pathway (Horvath and DeLeo, 2009). In addition, Kubiatowski et al., (2001) reported that ligand-induced activation of the PI-3K/Akt signaling pathway is directly related to cell migration and invasiveness. Consistent with the present data showing a functional link among CCR5, AKT and microglia migration, Zhao et al., (2015) reported that CCL5eCCR5 mediates the activation of Akt, and subsequently induces proliferation and invasive responses in U87 and U251 cells. 5. Conclusion In conclusion, the main finding of this study is the functional relationship existing between the chemokine-CCR5 system and microglia polarization. Overall, the pharmacological blockade of CCR5 prevents the occurrence of a M2 phenotype; furthermore, under conditions mimicking the late stage of glioma pathology (i.e. LI-CM conditioning), CCR5 blockade also induces a prevailing M1 phenotype. Such changes in microglia polarization profile are
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Fig. 8. Effects of MRV on urea levels. Panel A shows microglia treated with C-CM alone or in combination with MRV; panel B shows microglia treated with LI-CM alone or in combination with MRV (B). After 48-h incubation, the medium was used for urea assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as mM of nitrites/mg of proteins; data are means ± SEM (n ¼ 6). **P < 0.05, ***P < 0.001. C) Total cytosolic RNA was prepared from control, or microglial cells treated with the described treatments for 24 h, and used for real time (Q)ePCR analysis of ARG-1 gene expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± SEM of the mean, and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test.
Fig. 9. Effects of MRV on IL-10 gene expression. Total cytosolic RNA was prepared from control, or microglial cells treated with the described treatments for 24 h, and used for real time (Q)ePCR analysis of il-10 gene expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± SEM of the mean, and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test.
potentially associated to cytotoxic and anti-tumor properties due to an induction of M1 profile, while in parallel a potential reduction in tumor growth is expected, because of the blockade of M2 phenotype; taken together, these changes suggest a possible clinical exploitation of CCR5 antagonists in the treatment of human GBM. Within this framework, the major limitation of these findings
Fig. 10. Effects of MRV on mTOR pathway. Protein content were extracted from microglia after 24 h of treatment and subjected to western-blot to detect phosphomTOR and b-actin. Images are representative of 3 independent experiments.
lies on the ‘distance’ existing between the nonclinical paradigm used here and the clinical setting; to mention but one huge difference between in-vitro modeling and human GBM, we consider that currently the capability to clearly distinguish between M1 and M2 phenotypes is currently questioned in the human pathology setting (Ransohoff, 2016). A side advantage in this scenario is the
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chance to think and go straightforward to off-label pilot studies with MRV in GBM patients, because of the well-established and positive benefit/risk ratio of the drug. Competing interest No conflict of interest to declare. Acknowledgement The authors would like to thank Gabriella Maria Pia Ciotti for kind supporting us with the assessment of CCR5 gene expression in C6 cells. References Allen, S.J., Crown, S.E., Handel, T.M., 2007. Chemokine: receptor structure, interactions, and antagonism. Annu. Rev. Immunol. 25, 787e820. Buckner, J.C., Brown, P.D., O'Neill, B.P., Meyer, F.B., Wetmore, C.J., Uhm, J.H., 2007. Central nervous system tumors. Mayo Clin. Proc. 82 (10), 1271e1286 (Review). Charles, N.A., Holland, E.C., Gilbertson, R., Glass, R., Kettenmann, H., 2012. The brain tumor microenvironment. Glia 60 (3), 502e514 (Review). Choi, S.A., Lee, J.Y., Kwon, S.E., Wang, K.C., Phi, J.H., Choi, J.W., Jin, X., Lim, J.Y., Kim, H., Kim, S.K., 2015. Human adipose tissue-derived mesenchymal stem cells target brain tumor-initiating cells. PLoS One 10 (6), e0129292. Dello Russo, C., Lisi, L., Tentori, L., Navarra, P., Graziani, G., Combs, C.K., 2016. Exploiting microglial functions for the treatment of glioblastoma. Curr. Cancer Drug Targets 95 (3), 480e488. Dello Russo, C., Lisi, L., Tringali, G., Navarra, P., 2009. Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation. Biochem. Pharmacol. 78 (9), 1242e1251. Gadani, S.P., Cronk, J.C., Norris, G.T., Kipnis, J., 2012. IL-4 in the brain: a cytokine to remember. J. Immunol. 189 (9), 4213e4219. ~o, R.P., Zong, H., 2013. Inflammation and gliomagenesis: Bi-Directional Galva communication at early and late stages of tumor progression. Curr. Pathobiol. Rep. 1 (1), 19e28. € ster, S.S., Wedderkopp, H., KnerlichHeld-Feindt, J., Hattermann, K., Müerko Lukoschus, F., Ungefroren, H., Mehdorn, H.M., Mentlein, R., 2010. CX3CR1 promotes recruitment of human glioma-infiltrating microglia/macrophages (GIMs). Exp. Cell Res. 316 (9), 1553e1566. Horvath, R.J., DeLeo, J.A., 2009. Morphine enhances microglial migration through modulation of P2X4 receptor signaling. J. Neurosci. 29 (4), 998e1005. Hütter, G., Bodor, J., Ledger, S., Boyd, M., Millington, M., Tsie, M., Symonds, G., 2015. CCR5 targeted cell therapy for HIV and prevention of viral escape, 27 Viruses 7 (8), 4186e4203. Kakinuma, T., Hwang, S.T., 2006. Chemokines, chemokine receptors, and cancer metastasis. J. Leukoc. Biol. 79 (4), 639e651. Kubiatowski, T., Jang, T., Lachyankar, M.B., Salmonsen, R., Nabi, R.R., Quesenberry, P.J., Litofsky, N.S., Ross, A.H., Recht, L.D., 2001. Association of increased phosphatidylinositol 3-kinase signaling with increased invasiveness and gelatinase activity in malignant gliomas. J. Neurosurg. 95 (3), 480e488. Lee, H.C., Park, I.C., Park, M.J., An, S., Woo, S.H., Jin, H.O., Chung, H.Y., Lee, S.J., Gwak, H.S., Hong, Y.J., Yoo, D.H., Rhee, C.H., Hong, S.I., 2005. Sulindac and its metabolites inhibit invasion of glioblastoma cells via down-regulation of Akt/ PKB and MMP-2. J. Cell Biochem. 94 (3), 597e610. Li, D., Wang, C., Yao, Y., Chen, L., Liu, G., Zhang, R., Liu, Q., Shi, F.D., Hao, J., 2016. mTORC1 pathway disruption ameliorates brain inflammation following stroke via a shift in microglia phenotype from M1 type to M2 type. FASEB J. 30 (10), 3388e3399. Li, W., Graeber, M.B., 2012. The molecular profile of microglia under the influence of glioma. Neuro Oncol. 14 (8), 958e978. Lisi, L., Laudati, E., Navarra, P., Dello Russo, C., 2014a. The mTOR kinase inhibitors
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polarize glioma-activated microglia to express a M1 phenotype. J. Neuroinflammation 11, 125. Lisi, L., Navarra, P., Feinstein, D.L., Dello Russo, C., 2011. The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes. J. Neuroinflammation 8 (1), 1. Lisi, L., Stigliano, E., Lauriola, L., Navarra, P., Dello Russo, C., 2014b. Proinflammatoryactivated glioma cells induce a switch in microglial polarization and activation status, from a predominant M2b phenotype to a mixture of M1 and M2a/B polarized cells. ASN Neuro 6 (3), 171e183. Lisi, L., Tramutola, A., De Luca, A., Navarra, P., Dello Russo, C., 2012. Modulatory effects of the CCR5 antagonist maraviroc on microglial pro-inflammatory activation elicited by gp120. J. Neurochem. 120 (1), 106e114. Lu, M., Wu, B., 2016. Structural studies of G protein-coupled receptors. IUBMB Life 68 (11), 894e903. Mantovani, A., 2010. The growing diversity and spectrum of action of myeloidderived suppressor cells. Eur. J. Immunol. 40 (12), 3317e3320. Martin-Blondel, G., Brassat, D., Bauer, J., Lassmann, H., Liblau, R.S., 2016. CCR5 blockade for neuroinflammatory diseasesebeyond control of HIV. Nat. Rev. Neurol. 12 (2), 95e105. Okada, H., Kohanbash, G., Zhu, X., Kastenhuber, E.R., Hoji, A., Ueda, R., Fujita, M., 2009. Immunotherapeutic approaches for glioma. Crit. Rev. Immunol. 29 (1), 1e42. Oppermann, M., 2004. Chemokine receptor CCR5: insights into structure, function, and regulation. Cell Signal 16 (11), 1201e1210. Osuka, S., Van Meir, E.G., 2017. Overcoming therapeutic resistance in glioblastoma: the way forward. J. Clin. Invest. 127 (2), 415e426. Paschoal, V.A., Amano, M.T., Belchior, T., Magdalon, J., Chimin, P., Andrade, M.L., Yamashita, A.S., Rosa Neto, J.C., C^ Ortiz-Silva, M., Castro, E., amara, N.O., Festuccia, W.T., 2017. mTORC1 inhibition with rapamycin exacerbates adipose tissue inflammation in obese mice and dissociates macrophage phenotype from function. Immunobiology 222 (2), 261e271. Paw, I., Carpenter, R.C., Watabe, K., Debinski, W., Lo, H.W., 2015. Mechanisms regulating glioma invasion. Cancer Lett. 362 (1), 1e7. Pham, K., Luo, D., Liu, C., Harrison, J.K., 2012. CCL5, CCR1 and CCR5 in murine glioblastoma: immune cell infiltration and survival rates are not dependent on individualexpression of either CCR1 or CCR5. J. Neuroimmunol. 246 (1e2), 10e17. Piotrowska, A., Kwiatkowski, K., Rojewska, E., Makuch, W., Mika, J., 2016. Maraviroc reduces neuropathic pain through polarization of microglia and astroglia evidence from in vivo and in vitro studies. Neuropharmacology 108, 207e219. Ransohoff, R.M.l., 2016. A polarizing question: do M1 and M2 microglia exist? Nat. Neurosci. 19 (8), 987e991. Robinson, S.C., Scott, K.A., Wilson, J.L., Thompson, R.G., Proudfoot, A.E., Balkwill, F.R., 2003. A chemokine receptor antagonist inhibits experimental breast tumor growth. Cancer Res. 63 (23), 8360e8365. Rolle, C.E., Sengupta, S., Lesniak, M.S., 2012. Mechanisms of immune evasion by gliomas. Adv. Exp. Med. Biol. 746, 53e76. Sliwa, M., Markovic, D., Gabrusiewicz, K., Synowitz, M., Glass, R., Zawadzka, M., Wesolowska, A., Kettenmann, H., Kaminska, B., 2007. The invasion promoting effect of microglia on glioblastoma cells is inhibited by cyclosporin A. Brain 130 (Pt 2), 476e489. Soria, G., Ben-Baruch, A., 2008. The inflammatory chemokines CCL2 and CCL5 in breast cancer. Cancer Lett. 267 (2), 271e285. Vairano, M., Dello Russo, C., Pozzoli, G., Battaglia, A., Scambia, G., Tringali, G., AloeSpiriti, M.A., Preziosi, P., Navarra, P., 2002. Erythropoietin exerts anti-apoptotic effects on rat microglial cells in vitro. Eur. J. Neurosci. 16 (4), 584e592. Vakilian, A., Khorramdelazad, H., Heidari, P., Sheikh Rezaei, Z., Hassanshahi, G., 2017. CCL2/CCR2 signaling pathway in glioblastoma multiforme. Neurochem. Int. 103, 1e7. You, W., Wang, Z., Li, H., Shen, H., Xu, X., Jia, G., Chen, G., 2016. Inhibition of mammalian target of rapamycin attenuates early brain injury through modulating microglial polarization after experimental subarachnoid hemorrhage in rats. J. Neurol. Sci. 367, 224e231. Zhao, L., Wang, Y., Xue, Y., Lv, W., Zhang, Y., He, S., 2015. Critical roles of chemokines receptor CCR5 in regulating glioblastoma proliferation and invasion. Acta Biochim. Biophys. Sin. (Shanghai) 47 (11), 890e898.
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Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm , Pierluigi Navarra*, Lucia Lisi Emilia Laudati, Diego Curro Institute of Pharmacology, Catholic University Medical School, L.go F Vito 1, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2017 Received in revised form 24 February 2017 Accepted 3 March 2017 Available online xxx
Microglia express chemokines and their cognate receptors that were found to play important roles in many processes required for tumor development, such as tumor growth, proliferation, invasion, and angiogenesis. Among the chemokine receptor, CCR5 have been documented in different cancer models; in particular, CCR5 is highly expressed in human glioblastoma, where it is associated to poor prognosis. In the present study, we investigated the effect of CCR5 receptor blockade on a paradigm of microgliaglioma interaction; the CCR5 blocker maraviroc (MRV) was used as a pharmacological tool. We found that MVR is able to reduce the gene expression and function of the M2 markers ARG1 and IL-10 in presence of both basal glioma-released factors (C-CM) and activated glioma-released factors (LI-CM), but it up-regulates the M1 markers NO and IL-1b only if microglia is stimulated by LI-CM; the latter effect appears to be mediated by the inhibition of mTOR pathway. In addition, CCR5 blockade was associated to a significant reduction in microglia migration, an effect mediated through the inhibition of AKT pathway. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Microglia Glioblastoma iNOS Arginase AKT mTOR
1. Introduction Glioblastoma (GBM) is the most common and aggressive malignant primary brain cancer. Despite treatment with maximal surgical resection, radiotherapy and chemotherapy, patients diagnosed with GBM typically have short life expectancies with a median survival rate of 14e16 months (Buckner et al., 2007; Dello Russo et al., 2016). In most clinical trials, therapies targeting GBM cells have failed or resulted in limited clinical response, likely due to the acquired resistance mechanisms and significant molecular heterogeneity within the tumor (Osuka and Van Meir, 2017). Therefore, a need exists to discover novel cellular targets for the development of more effective therapies. Recently, the tumor microenvironment has emerged as an attractive additional target for possible therapeutic interventions. In fact, cells such as vascular cells, peripheral immune cells, microglia and neural precursor cells play an essential role in controlling the progression of the disease (Charles et al., 2012). Among these cells, microglia and macrophages appear to be the most common cell type in the GBM microenvironment, representing up to 30% of tumor tissue (Sliwa et al., 2007).
* Corresponding author. E-mail address: [email protected] (P. Navarra).
Interactions between microglia and GBM cells are bi-directional (Galv~ ao and Zong, 2013). Microglia express a variety of receptors, which can be activated by signals from glioma cells (Held-Feindt et al., 2010; Okada et al., 2009). For example glioma cells produce prostaglandin E2 and cytokines, such as interleukin (IL)-10, IL-4, IL6 and TGFb (Rolle et al., 2012), that are able to activate microglial cells; in particular, IL-4 causes alternative activation (M2) of microglia (Gadani et al., 2012). Conversely, under the influence of glioma, microglia release several classes of molecules, such as nitrites (NO), IL-10, IL1b, TNFa and urea, that promote glioma growth, progression and inflammatory activation (Li and Graeber, 2012). However, the role of microglia in GBM pathology is still unclear. In fact, in the last decade a large number of studies provided evidence for a tumor-supporting role of microglia; on the opposite, data in support of microglial anti-tumor properties have been also reported (Li and Graeber, 2012). Microglia express chemokines and their cognate receptors (Allen et al., 2007), which were found to play important roles in many processes required for tumor development, such as tumor growth, proliferation, invasion, and angiogenesis (Vakilian et al., 2017; Kakinuma and Hwang, 2006; Mantovani, 2010). Among the chemokine family, consisting of about 50 members that bind to 20 chemokine receptors, we focused our studies on CCR5 receptor. CCR5 is involved on HIV entry in host cells; for this reason, it is one of the most studied chemokine receptors (Hütter et al., 2015).
http://dx.doi.org/10.1016/j.neuint.2017.03.002 0197-0186/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Although the chemokine ligand-receptor systems, as mediators of inflammation, are inherent to the microglia-macrophage inflammatory cell lineage, it is worth of note that CCR5 is expressed in neoplastic mesenchymal cells that are considered the most invasive cell-type in GBM (Choi et al., 2015). Moreover, CCR5 and its ligand CCL5 have also been documented in different cancer models (Robinson et al., 2003; Soria and Ben-Baruch, 2008); in particular, CCR5 is highly expressed in human GBM, where it is associated to poor prognosis. High amounts of CCL5 are released by tumors showing a significantly shorter mean survival time compared to those releasing lower levels of CCL5. The National Cancer Institute (NCI) REMBRANDT data portal (Repository of Brain Neoplasia Data) reported a positive association between CCL5 expression and survival rates in glioma patients (Pham et al., 2012). In addition, CCL5/ CCR5 mediates the activation of Akt, and subsequently induces proliferation and invasive responses in U87 and U251 cells (Zhao et al., 2015). Recently, we used rat microglia to investigate the effects of different conditioned media obtained from glioma cells. We showed that microglia activation undergoes a different fate and express a distinctive phenotype, depending on the stimulus used. In particular, using control conditioned media (C-CM, i.e. a condition mimicking an initial stage of pathology), microglia tend toward a M2 polarization, whereas conditioned media obtained after glioma stimulation with LPS-IFNg (LI-CM) induced a mixed polarization profile, i.e. both M1 and M2 phenotypes (Lisi et al., 2014a). In the present study, we adopted the above experimental paradigm to investigate the effects of CCR5 blockade on primary rat microglia polarization profile as well as on the rate of cell migration. We used a pharmacological inhibitor, Maraviroc (MRV), which locks CCR5 conformation in an inactive state by allosteric inverse agonist activity (Lu and Wu, 2016); in particular, we compared the effects of MRV on primary microglia cells activated by C-CM versus the effects of MRV on microglia activated by LI-CM. 2. Materials and methods 2.1. Antibodies and reagents Materials Cell culture reagents [Dulbecco's modified Eagle's medium (DMEM), DMEM-F12 and fetal calf serum (FCS)] were from Invitrogen Corporation (Paisley, Scotland). Antibiotics were from Biochrom AG (Berlin, Germany). The rat recombinant proinflammatory cytokine, interferon-g (IFNg), was purchased by Endogen (Pierce Biotechnology, Rockford, IL, USA). Maraviroc was provided by Pfizer Italia SRL (Aprilia, RM, Italy). Primary antibodies: polyclonal rabbit phosphorylated-mTOR Ser2448 (Novus biological), monoclonal mouse b-actin (Sigma-Aldrich) polyclonal rabbit phospho IKb (Cell Signaling), polyclonal rabbit p-Akt S473, monoclonal mouse Akt, polyclonal mouse p70S6K (BD, Bioscience). Secondary antibody, anti-rabbit and anti-mouse immunoglobulin G(IgG)-horseradish peroxidase (HRP)-conjugated, were purchased from Sigma-Aldrich. 2.2. C6 glioma cells C6 glioma cells were passed once a week and were prepared as previously described (Lisi et al., 2011). Briefly, control-conditioned medium (C-CM) was obtained incubating C6 glioma cells for 4 h in plain medium in 1% FCS, and after three washes with PBS in fresh plain medium for an additional 24 h. After this second period of incubation, the CM was collected, centrifuged to remove cellular debris and stored as C-CM. Similarly, LPS/IFNg-conditioned medium (LI-CM) was prepared incubating C6 cells for 4 h with LPS 1 mg/ml and IFNg 10 Unit/ml, followed after three washes with PBS
by 24-h incubation in fresh plain medium. LI-CM was collected, centrifuged and stored after this second period of incubation, thus LI-CM did not contain the proinflammatory stimuli used to activate glioma cells. Both CM were stored at 80 C until the experiments on microglial cells were performed (Lisi et al., 2014a,b). 2.3. Cell proliferation assay Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation assay using the Delfia Cell Proliferation Kit (Perkin Elmer, Inc, Waltham MA, USA). Cells were seeded in 96-well plates with 10% FBS in culture medium, and treated with indicated agents for 48 h. BrdU was added during the last 16 h of incubation period. The assay was performed according to the manufacturer's instructions. 2.4. Cell viability measurement Cell viability was assessed by reduction of the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5- (4-sulfophenyl)-2Htetrazolium, inner salt; MTS) contained in the TACS® XTT Cell (Trevigen, Gaithersburg, MD, USA). For this assay cells were seeded in 96-well plates. At the end of the experimental procedure (46 h), 50 mL of MTS reagent were added to the cells that were further incubated for 2 h. Living cells bioreduce yellow MTS into a purple soluble formazan product with an absorbance peak at 492 nm, that was read in a spectrophotometric microplate reader (PerkinElmer Inc, MA, USA). 2.5. Microglia Primary enriched cultures of rat glial cells, microglia and astrocytes, were prepared as previously described (Dello Russo et al., 2009). In particular, microglial cells were obtained by mixed cultures of cortical glial cells (at in vitro day 14), by gentle shaking. Cells were plated with DMEM-F12, containing 10% FCS and antibiotics. Experiments were carried out in 10% FCS containing medium to reduce microglial cell death that we usually observe after the splitting from astrocytes (Vairano et al., 2002). Microglial cells were treated with soluble factors released by C6 glioma cells, as described above. The selective CCR5 receptor antagonist, Maraviroc, was administered in the concentration range of 1pM-1 mM. The use of animals for this experimental work has been approved by the Ethics Committee of the Catholic University Medical School. 2.6. Transwell cell migration assays Microglial cells were plated at density of 5 104 to the top chamber of 24-well Transwell units with 8.0 mm pores (Corning) in low-serum media containing the different treatments. The bottom chamber contained 10% FBS. Cells were allowed to migrate at 37 C in 5% CO2 for 16 h. Non-migrating cells were removed from the upper surface using a cotton swab. The lower surface was stained with Hema 3 (Fisher Scientific). Stained membranes were mounted on microscope slides and imaged using a Nikon Eclipse TE300 microscope. The number of migrated cells was determined in 4 representative fields, selected by a blinded investigator, using ImageJ software. Two separate membranes from 4 independent experiments were analyzed for each condition. 2.7. Nitrite assay The activity of the inducible form of nitric oxide synthase (iNOS or NOS2) was assessed indirectly by measuring nitrite accumulation in the incubation media. Briefly, an aliquot of the cell culture
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media (80 mL) was mixed with 40 mL Griess Reagent (Sigma-Aldrich, St Louis, MO, USA) and the absorbance measured at 550 nm in a spectrophotometric microplate reader (PerkinElmer Inc. Waltham, MA, USA). A standard curve was generated during each assay in the range of concentrations 0e100 mM using NaNO2 (Sigma-Aldrich) as standard. In this range, standard detection resulted linear and the minimum detectable concentration of NaNO2 was z 3.12 mM. In the absence of stimuli, basal levels of nitrites were below the detection limit of the assay at all the time points studied. The levels of NO were normalized with the protein content determined by Bradford's method (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as standard.
2.8. Urea assay Urea levels in microglia cells were detected by the QuantiChrom Urea Assay kit (BIOassay System, Hayward, CA, USA), used according to the manufacturer's instructions. Briefly after incubation with the tested substances for 48 h, as indicated in figure legends, an aliquot of cell culture media (50 mL) was mixed with 200 mL Urea Reagent (Bioassay system) and the absorbance was measured at 430 nm in a spectrophotometric microplate reader (PerkinElmer Inc.). A standard curve was generated during each assay in a concentration range 0e100 mM using urea as standard. In this range, standard detection resulted in linear plots and the minimum detectable concentration of urea was 3.12 mM. The levels of urea were normalized with the protein content determined by Bradford's method (Bio-Rad, Hercules, CA, USA) using BSA as standard.
2.9. mRNA analysis in real-time PCR Total cytoplasmic RNA was extracted using the ReliaPrep RNA Tissue Miniprep Systemt (Promega, Madison, WI, USA), which included 15 min Dnase treatment. RNA concentration was measured using the Quant-iTTM RiboGreen RNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA). A standard curve in the range of 0e100 ng was run in each assay using 16S and 23S ribosomal RNA (rRNA) from E. coli as standard and provided by the kit. Aliquots (0.25 mg) of RNA were converted to cDNA using random hexamer primers. Quantitative changes in mRNA levels were estimated by real time PCR (Q-PCR) using the following cycling conditions: 35 cycles of denaturation at 95 C for 20 s; annealing and extension at 60 C for 20 s, using the Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Stratagene, La Jolla, CA, USA). PCR reactions were carried out in a 20 mL reaction volume in a MX3000P real time PCR machine (Stratagene). Primers used for the evaluation of gene expression are reported in Table 1. Relative mRNA concentrations were calculated from the take-off point of reactions (threshold cycle, Ct) using the comparative quantitation method performed by Stratagene software and based upon the -DDCt method. Ct values for GAPDH expression served as a normalizing signal. (Dello Russo et al., 2009).
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2.10. Western immunoblot Primary microglia cells were plated (5 105 cells/well) in 6 multiwell, treated for 24 h and then lysed in RIPA buffer (1 mM EDTA, 150 mMNaCl, 1% igepal, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 50 mM TriseHCl, pH 8.0) (Sigma-Aldrich) containing protease inhibitor cocktail diluted 1:250 (SigmaAldrich). The protein content in each sample was determined by Bradford's method (Bio-Rad, Hercules, CA, USA) using BSA as standard. An equivalent amount of cellular protein (10 or 20 mg per lane) was subjected was mixed 1:2 with 2$Laemmli Buffer (BioRad), boiled for 5 min at 95 C, and separated through 10% polyacrylamide sodium dodecyl sulfate gels. Apparent molecular weights were estimated by comparison to colored molecular weight markers (Sigma-Aldrich). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes by semidry electrophoretic transfer (Bio-Rad). The membranes were blocked with 10% (w/v) low-fat milk in TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) (Bio-Rad) for 1 h at room temperature (25 C), and incubated in the presence of the primary antibody overnight with gentle shaking at 4 C. Primary antibodies for Rabbit polyclonal phosphorylated-mTOR (Novus biological),b-actin (Sigma-Aldrich) anti-phospho IKKa/b (Cell Signaling), antiphospho AKT (RD system), anti-total AKT were used at the final concentration of 1:1000. Primary antibodies were removed, membranes washed three times in TBST, and further incubated for 1 h at 25 C in the presence of specific secondary antibody, antirabbit and anti-mouse immunoglobulin G(IgG)-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich), diluted 1:15,000 and 1:10,000 respectively. Following three washes in TBST, bands were visualized by incubation in ECL reagents (GE Healthcare, New York, NY, USA) and exposure to Hyperfilm ECL (GE Healthcare). The same membranes were washed three times in TBST, blocked with 10% (w/v) low-fat milk in TBST for 1 h at 25 C and used for b-actin immunoblot. 2.11. Immunocytochemistry For immunocytochemistry, microglia were plated at a density of 3.5 105 cells/well on coated glass coverslips and treated with PBS or MRV 1 mM (Lisi et al., 2014a,b). At this time, cultures were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min. After fixation, cultures were washed three times with PBS and permeabilized with 0.1% Triton X for 5 min; then washed and blocked with 1% BSA in PBS for 30 min. 1: 200 CCR5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS containing 0.1% BSA and 0.1% Triton X100 was added for 2 h. After incubation, 40,6-diamidino-2- phenylindole (DAPI) was added for 10 min and then washed twice with PBS. Coverslips were mounted using Vectashield mounting media. Images were obtained on a Nikon Eclipse TE300 inverted fluorescence microscope equipped with a Cool SNAP professional digital camera and LUCIAG/F imaging software.
Table 1 Primer sets used for Q-PCR analysis. Genes
Forward primers
Reverse primers
Product length
GAPDH iNOS IL-10 ARG1 IL-1b CCR5 IKBa
CCC TCG CCA TGG TAA ATA CAT CTG CAT GGA ACA GTA TAA GGC AAA C CAG CTG CGA CGC TGT CAT CGA TGC CCT CTG TCT TTT AGG GC GCA GGC CTG GGA CCT TGC TG AGC AGT GAT CCG AAA GGA GGG AAA GCC TGG CCA GTG TAG CAG TCT
ACT GGA TGG TAC GCT TGG TCT CAG ACA GTT TCT GGT CGA TGT CAT GA GCA GTC CAG TAG ATG CCG GGT G CCT CGA GGC TGT CCC TTA GA CGA CAG GGC CAA GCT CAC CG TAC TTG GAG TGC TGC CAG TGT CTT CAG CAC CCA AAC TCA CCA AGT
110 230 198 165 271 199 135
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2.12. Data analysis All experiments were performed using 5e6 replicates per each experimental group, and repeated at least three times. For the RNA analysis, samples were assayed in triplicates, and the experiments were repeated at least twice. Data were analyzed by one way ANOVA followed by Bonferroni's post hoc test or by unpaired t-test. p values < 0.05 were considered significant. 3. Results First we looked at CCR5 gene expression in glioma C6 cells and checked whether it is a functional receptor. Fig. 1A shows that CCR5 receptor was expressed in C6 glioma cells. Under basal conditions, 100 and 1000 nM MRV was able to significant reduce both proliferation and viability of C6 cells after 3 days of treatment (Fig. 1B and C). Fig. 2 shows the expression and localization of CCR5 receptors in rat primary microglia cells. Concerning gene expression, MRV per se does not modify CCR5 expression (Fig. 2A). In normal conditions CCR5 receptor is localized at the level of microglia cell membranes (Fig 2B), whereas in the presence of 1 mM MRV the CCR5 receptor is internalized, as showed by the increase in fluorescence within the cytoplasmic compartment compared to basal conditions (Fig 2C). A second set of experiments were carried out to study microglia migration. Fig. 3 shows the results of transwell cell migration assays. Both types of stimuli, C-CM and LI-CM enhanced cell migration (Fig. 3B and C); 1 mM MRV given alone did not modify microglia migration (Fig. 3E), whereas it was able to inhibit microglia migration in the presence of glioma soluble factors (Fig. 3E and F). In particular, MRV was able to significantly reduce migration elicited by LI-CM whereas reduction exerted over the migration elicited by CCM did not attain statistical significance (Fig. 3). Subsequently we investigated the expression and phosphorylation degree of AKT as a possible mediator of the observed migration. As expected, both C-CM and LI-CM stimuli significantly
increase the phosphorylated portion of the AKT protein; such increase was completely reversed by 1 mM MRV, although the latter had no effect whatsoever on the total amount of AKT protein (Fig. 4). These data strongly suggest that MRV is able to reduce microglia cell migration through the block of AKT pathway. An additional series of experiments was carried out to investigate the effects of MRV on microglia inflammatory state elicited by glioma soluble factors. First we studied the levels of NO, taken as an M1 marker. After C-CM stimulation, MRV was able to reduce nitrite levels compared to vehicle, and the maximal effect was reached at MRV concentrations as low as 10 pM (Fig. 5A). On the opposite, when LI-CM was used as stimulus, 100 and 1000 nM MRV elicited a significantly increase, in NO release (Fig. 5B). All of the above changes in NO levels, either increases or reductions, were not associated to any variation in iNOS gene expression (Fig. 6A). Instead, a direct correlation was found between the changes in NO release and parallel changes in the levels of IKBa gene expression, with increases induced by MRV under C-CM stimulation and reductions after LI-CM exposure (Fig. 6B). These data were confirmed by the analysis of phospho-IKBa protein (Fig. 6CeD). Similar results were obtained with another M1 marker, the proinflammatory cytokine IL-1b; MRV increased the levels of IL-1b gene expression elicited by LI-CM but had no stimulatory effect, and even tended to decrease cytokine gene expression under C-CM stimulation (Fig. 7). Thus, MRV appears to consistently reduce the M1 phenotype in presence of C-CM while on the contrary it tends to up regulate M1 phenotype in presence of LI-CM. Looking at M2 markers, we investigated ARG1 and IL-10 gene expression. In particular, MRV was able to significant reduce ARG1 gene expression in presence of both C-CM and LI-CM (Fig. 8A). The reduction in ARG1 gene expression was associated to a significant reduction in urea production in presence of LI-CM (Fig. 8B) but not in presence of C-CM (Fig. 8C). Likewise, MRV was able to reduce IL10 gene expression in both experimental conditions (Fig. 9). Therefore, it appears that MVR altogether reduces M2 phenotype both in presence of CCM and LICM.
Fig. 1. CCR5 receptor in glioma c6 cells. A) CCR5 gene expression in C6 glioma cells. B-C) Effects of MRV on proliferation and viability of C6 cells. C6 glioma cells were treated with MRV for 3 days. Effects on cell proliferation were assessed by 5-bromo-2-deoxyuridine (BrDU) incorporation (B), while the effects on cell viability were assessed by the XTT reduction assay (C). Data were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. *P < 0.001, versus Control.
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Fig. 2. CCR5 receptor in microglia cells. (A) Microglial cells were cultured in presence with MRV (1 mM). Control cells were treated with 50% of complete medium with DMEM 1% of FBS. After 24 h, RNA was extracted and expression levels of CCR5 mRNA were quantified by q-PCR. Data represents mean ± SEM; n ¼ 3; one-way ANOVA followed by Dunnett's multiple comparison test. (B, C) After 48 h of culture in basal condition (B) or MRV 1 mM (C), microglia cultures were stained with the antibody for CCR5 (green). Nucleus is visualized using DAPI (blue). Magnification 20x. Images are representative of 3 independent experiments.
Fig. 3. Effects of MRV on microglia migration. Microglial cells were introduced into Transwell systems. Migration was allowed to proceed for 16 h. Representative photomicrographs of cells that migrated to the lower surfaces of Transwell membranes are shown. Images are representative of 3 independent experiments. Scale bar ¼ 1 mm. Data in A represents mean ± SEM; n ¼ 3; ***P < 0.001 versus Control, **P < 0.001, versus Control P < 0.001 versus LI-CM.; one-way ANOVA with Dunnett's post-hoc analysis. (B). Quantification of migration results.
Furthermore, we investigated the metabolic mTOR pathways as a possible downstream signaling mechanism involved in CCR5 blockade. A western blot analysis shows that MRV was able to
inhibit mTOR phosphorilation at Ser2448 in a 24-h treatment under LI-CM stimulation, whereas no effect was observed in presence of C-CM (Fig. 10A).
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Fig. 4. Effects of MRV on AKT pathway. Protein content were extracted from microglia after 24 h of treatment and subjected to western-blot to detect phospho-Akt and total Akt. Images are representative of 3 independent experiments.
Fig. 5. Effects of MRV on NO levels. Panel A shows microglia treated with C-CM alone or in combination with MRV; panel B shows microglia treated with LI-CM alone or in combination with MRV (B). After 48-h incubation, the medium was used for NO assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as mM of nitrites/mg of proteins; data are means ± SEM (n ¼ 6). **P < 0.05, ***P < 0.001.
4. Discussion In the present study, we investigated the effect of CCR5 receptor blockade on a paradigm of microglia-glioma interaction; the CCR5 blocker MVR was used as a pharmacological tool. We found that MVR is able to reduce the gene expression and function of the M2
markers ARG1 and IL-10 in presence of both C-CM and LI-CM stimuli, but it up-regulates the M1 markers NO and IL-1b only if microglia is stimulated by LI-CM; the latter effect appears to be mediated by the inhibition of mTOR pathway. In addition, CCR5 blockade was associated to a significant reduction in microglia migration, an effect mediated through the inhibition of AKT pathway. A well-known drug in HIV treatment, Maraviroc binds CCR5 and locks the receptor in the inactive state via an allosteric inverse agonist action (Lu and Wu, 2016). Our group and others have recently tested the hypothesis that MRV affect microglial activation during HIV-1 infection and/or in other diseases through the blockade of CCR5 receptor (Martin-Blondel et al., 2016). CCR5 is expressed both in microglia cells (Lisi et al., 2012) and in macrophages (Oppermann, 2004). Using primary microglial cultures activated by glycoprotein120 (Gp120) alone or in combination with IFNY, our group investigated the effects of MRV on the gene expression of pro-inflammatory cytokines. The drug showed opposite effects depending on whether microglia was pretreated with Gp120CN54 or in combination with IFNY; in fact we observed a down-regulation of pro-inflammatory cytokine gene expression after pretreatment with Gp120CN54 alone, whereas an increase was observed in the presence of INFY (Lisi et al., 2012). Maraviroc has also been shown to reduce neuropathic pain through the polarization of microglia and astroglia (Piotrowska et al., 2016). Current knowledge on the mechanisms of microglial activation shows a growing complexity compared to that originally described based on cell morphology only. Similar to macrophages, microglia shows different responses to various stimuli, adopting specific phenotypes of activation. In particular, two distinct pattern of activation are described: classically activated M1 cells endowed with cytotoxic properties, and alternatively activated M2 cells with phagocytic activities. We have recently shown that microglial cells present as a mixture of polarization phenotypes (M1 and M2) when exposed to activated glioma-derived factors, i.e. a condition
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Fig. 6. Effects of MRV on iNOS and IKBa gene expression. Total cytosolic RNA was prepared from control, or microglial cells treated with the described treatments for 24 h, and used for real time (Q)ePCR analysis of iNOS (A) and IKBa (B) gene expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± SEM of the mean, and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. (C) Protein content were extracted from microglia after 24 h of treatment and subjected to westernblot to detect IKBa and b-actin. Images are representative of 3 independent experiments.
Fig. 7. Effects of MRV on IL-1b gene expression. Total cytosolic RNA was prepared from control, or microglial cells treated with the described treatments for 24 h, and used for real time (Q)ePCR analysis of il-1b gene expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± SEM of the mean, and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test.
mimicking the late stage of pathology, with up-regulation of iNOS, ARG and IL-10. At variance, microglia exposed to basal gliomaderived factors, i.e. a condition resembling the early stage of pathology, shows a more specific pattern of activation, with increased M2 polarization status and up-regulation of IL-10 (Lisi et al., 2014a). In addition, we showed that the inhibition of mTOR polarizes glioma-activated microglial cells towards the M1 phenotype, preventing the induction of the M2 status (Lisi et al., 2014b). Other groups (Li et al., 2016; Paschoal et al., 2017; You et al., 2016) have also reported a polarization toward M1 in both microglia and macrophages subsequent to mTOR pathway modification. In the present study, we showed that MRV polarizes microglia cells
towards the M1 phenotype and prevents M2 status in presence of LI-CM, through a mechanism involving the inhibition of mTOR and NfKB-IKBa complex pathways. It should be pointed out that such effect of mTOR inhibition can only be observed under conditions of pro-inflammatory activation of the system. In fact, in presence of plain C-CM stimulus MVR fails to blunt mTOR activation, and the result of CCR5 blockade is rather a reduction of both M1 and M2 phenotypes. Having shown an effect of the chemokine-CCR5 pathway in the regulation of microglia cell migration, we further explored the involvement of AKT signaling. In fact, the role of this signaling pathway in GBM invasion has been thoroughly investigated using several Akt-targeted drugs as tools. A non-steroidal anti-inflammatory drug, sulindac and its metabolites are able to inhibit GBM invasion in vitro via de-phosphorylation of Akt at Ser473 (Lee et al., 2005). Likewise, the selective cyclooxygenase-2 inhibitor celecoxib is also able to inhibit GBM invasion, its effect involving the inhibition of Akt signaling activity (Paw et al., 2015). Morphine-induced microglial migration has also been shown to be mediated by the PI3K/Akt pathway (Horvath and DeLeo, 2009). In addition, Kubiatowski et al., (2001) reported that ligand-induced activation of the PI-3K/Akt signaling pathway is directly related to cell migration and invasiveness. Consistent with the present data showing a functional link among CCR5, AKT and microglia migration, Zhao et al., (2015) reported that CCL5eCCR5 mediates the activation of Akt, and subsequently induces proliferation and invasive responses in U87 and U251 cells. 5. Conclusion In conclusion, the main finding of this study is the functional relationship existing between the chemokine-CCR5 system and microglia polarization. Overall, the pharmacological blockade of CCR5 prevents the occurrence of a M2 phenotype; furthermore, under conditions mimicking the late stage of glioma pathology (i.e. LI-CM conditioning), CCR5 blockade also induces a prevailing M1 phenotype. Such changes in microglia polarization profile are
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Fig. 8. Effects of MRV on urea levels. Panel A shows microglia treated with C-CM alone or in combination with MRV; panel B shows microglia treated with LI-CM alone or in combination with MRV (B). After 48-h incubation, the medium was used for urea assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as mM of nitrites/mg of proteins; data are means ± SEM (n ¼ 6). **P < 0.05, ***P < 0.001. C) Total cytosolic RNA was prepared from control, or microglial cells treated with the described treatments for 24 h, and used for real time (Q)ePCR analysis of ARG-1 gene expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± SEM of the mean, and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test.
Fig. 9. Effects of MRV on IL-10 gene expression. Total cytosolic RNA was prepared from control, or microglial cells treated with the described treatments for 24 h, and used for real time (Q)ePCR analysis of il-10 gene expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± SEM of the mean, and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test.
potentially associated to cytotoxic and anti-tumor properties due to an induction of M1 profile, while in parallel a potential reduction in tumor growth is expected, because of the blockade of M2 phenotype; taken together, these changes suggest a possible clinical exploitation of CCR5 antagonists in the treatment of human GBM. Within this framework, the major limitation of these findings
Fig. 10. Effects of MRV on mTOR pathway. Protein content were extracted from microglia after 24 h of treatment and subjected to western-blot to detect phosphomTOR and b-actin. Images are representative of 3 independent experiments.
lies on the ‘distance’ existing between the nonclinical paradigm used here and the clinical setting; to mention but one huge difference between in-vitro modeling and human GBM, we consider that currently the capability to clearly distinguish between M1 and M2 phenotypes is currently questioned in the human pathology setting (Ransohoff, 2016). A side advantage in this scenario is the
Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002
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Please cite this article in press as: Laudati, E., et al., Blockade of CCR5 receptor prevents M2 microglia phenotype in a microglia-glioma paradigm, Neurochemistry International (2017), http://dx.doi.org/10.1016/j.neuint.2017.03.002