Mice
Six- to eight-week-old male C57BL/6 mice were purchased from Charles River (Quebec, Canada) and maintained under specific pathogen-free conditions in the animal facilities of the Université de Montréal. Experiments were performed according to state guidelines and approved by the Canadian Council on Animal Care.
BM cell culture
Single cell suspensions were prepared from BM of normal mice and cultured in 100-mm Petri dishes (Becton Dickinson, Quebec, Canada) in 10 ml of RPMI 1640 medium (Sigma, Ontario, Canada) supplemented with 10% (v/v) irradiated fetal bovine serum (Cedarlane, Ontario, Canada), 1 mM Sodium Pyruvate (Sigma), 50 μM β-mercaptoethanol (Sigma), 100 U/ml Penicillin, 150 U/ml Streptomycin (Cedarlane) and 2 mM L-glutamine (Cedarlane), in a 5% CO2 and 37 °C incubator. MDSCs were derived by treating BM cells with 40 ng/ml of GM-CSF and 40 ng/ml of IL-6 (both from Cedarlane) for 4 days as described by Marigo et al. [5].
When required, BM cells were treated for 15 min with 5 μM of Compound C (Comp-C, 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine, Sigma) dissolved in DMSO (1% v/v) to inhibit AMPK activity.
Prior to analysis, cells were detached using a phosphate buffer saline (PBS)-EDTA (2 mM) solution, rinsed with PBS and centrifuged for 6 min at 250 × g at 4 °C. Cells were counted using a hematocytometer and cell viability was determined by the Trypan Blue exclusion method.
MSC-1 cell culture
The generation, culture, and phenotype of the MSC-1 immortalized cell line has been described previously [8]. MSC-1 cells were grown in 75 cm2 T-flasks (VWR, Ontario, Canada) in RPMI 1640 medium (Sigma) supplemented with 10% (v/v) irradiated FBS (Cedarlane), 1 mM sodium pyruvate (Sigma), 100 U/ml Penicillin, 150 U/ml streptomycin (Cedarlane) and 2 mM L-glutamine (Cedarlane), in a 5% CO2 and 37 °C incubator. Cultures were inoculated at a cell density of 0.2 × 106 cells/ml and cells were passaged when they reached 80% confluence.
When required, iNOS and ARG1 activities were inhibited with 100 μM of 1400 W and 5 μM of BEC (both from Cedarlane), respectively.
Prior to analysis, cells were detached using 0.25% Trypsin and 1 mM EDTA (Invitrogen, Ontario, Canada), rinsed with PBS and centrifuged for 6 min at 250 × g at 4 °C. Cells were counted using a hemacytometer and viability was determined using the Trypan Blue exclusion method.
Assays
Glucose, lactate, glutamate and glutamine concentrations in supernatants were measured using a dual-channel immobilized oxidase enzyme biochemistry analyzer (2700 SELECT, YSI Inc. Life Sciences, Yellow Springs, OH, USA), using calibration buffers provided by the manufacturer.
Nitric oxide concentrations in supernatant were respectively assayed using a Nitrate/Nitrite Colorimetric Assay Kit (Cedarlane). Specific rates of nutrients consumption and metabolites production were calculated using the average method [45].
SDS-PAGE and western blot analyses
Detached cells were lysed in 100 μl of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% thesit, and 0.5% sodium deoxycholate), and insoluble material was removed by centrifugation at 10,000 × g for 5 min at 4°C. Samples were mixed 3:1 with sample buffer containing 0.6 mM DTT and boiled for 5 min. Proteins were separated by 10% SDS-PAGE at 200 V during 45 min. For Western blots, proteins were transferred to a PVDF membrane (Bio-Rad, Mississauga, Ontario, Canada) using Tris-glycine buffer for 1 h at 200 mA. The membrane was blocked with 5% skim milk/10 mM Tris–HCl, pH7.4, 150 mM NaCl, 0.1% Tween-20 and then probed with the appropriate primary antibodies (diluted 1:200) for AMPK α1/2 and p-AMPK α1/2 (Thr172) (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Specific antibody binding was detected using goat anti-rabbit IgG horseradish peroxidase (diluted 1:1000, R&D Systems, Minneapolis, MN) for 1 h at room temperature and visualized using an enhanced chemiluminescence detection reagent (Bio-Rad). Band intensities were compared with ImageJ software.
Determination of ARG 1 activity
Total cells were lysed with 50 μl of a lysis buffer containing 0.1% Triton X-100 (Sigma) and 100 μg/ml of pepstatin, antipain and aprotinin (all from EMD BioSciences, San Diego, CA). After 30 min in a thermomixer at 37 °C, cell debris was removed by centrifugation at 15,000 × g for 20 min and cell lysates were kept at −80 °C prior to analysis. ARG1 activity was quantified in cell lysates by urea determination with α-isonitrosopropiophenone as previously described by Munder et al., [46]. One unit of ARG1 activity is defined as the enzyme activity that catalyzes the production of 1 mol urea/min.
Cytotoxicity assay
The immunosuppressive activity of BM-derived MDSCs and MSC-1 cells was assessed by their ability to inhibit Jurkat cell growth (leukemic T-cells, clone E6-1, Cedarlane). Experiments were performed in 24-well tissue culture plates (VWR, Ontario, Canada) in a final volume of 1 ml. Jurkat cells were inoculated (500 μl at 0.2 × 106 cells/ml) in Millicell®PC 0.4 μm culture plate inserts (Millipore) and added to wells containing 500 μl of BM cell suspension (0.2 × 106 cells/ml) cultured in the presence of GM-CSF and IL-6 (40 ng/ml each) for 0, 24, 48, 72 and 96 h or to MSC-1 cells cultured in the presence of 1400 W and BEC (100 and 5 μM, respectively) for 16 h (500 μl at 0.2 × 106cells/ml).
To investigate the role of AMPK in the maintenance of BM-derived MDSCs immunosuppressive potential, Comp-C pre-treated BM cells cultured in the presence of GM-CSF and IL-6 (for 24, 48, 72 and 96 h) were rinsed with sterile PBS to remove Comp-C and re-suspended in GM-CSF and IL-6 (40 ng/ml each) enriched complemented culture medium. Mixed cultures were kept in a 5% CO2 and 37 °C incubator for 24 h for BM cells and BM-derived MDSCs, and for 32 h for MSC-1 cells. Jurkat cells were then counted using a hemacytometer and viability was determined using the Trypan Blue exclusion method. The small pore size of the culture inserts prevented Jurkat cells and detached BM-derived MDSCs and MSC-1 cells to cross-contaminate respective media/cell samples and so ensured the reliability of cell counts.
Respirometry test
Respirometry assays were performed as described by Lamboursain et al., [47]. Briefly, 3 ml of a 15 × 106 BM-derived MDSCs/ml suspension or 3 ml of a 5 × 106 MSC-1 cells/ml suspension were inoculated in a 10-ml borosilicate glass syringe (Sigma) in which the plunger was substituted by an Ingold pO2 probe (Mettler Toledo, Quebec, Canada). The respirometer was kept at 37 °C and magnetically agitated (60 RPM) to ensure the homogeneity of cell suspension. Dissolved oxygen was recorded by an acquisition system (Centris, Longueuil, Canada).
Metabolite extraction
The extraction protocol was based on the method developed by Kimball et al., [48]. Briefly, for each sample, 5 × 106 cells were extracted with 400 μl of 80% cold methanol in the presence of 0.2 g of sand (Sigma). After 10 min on dry ice, the mixture was vortexed and then sonicated in ice and water for 5 min. The samples were then centrifuged for 7 min at 21,000 × g and 4 °C to collect supernatants. The pellets were extracted a second and third time as described above with 200 μl of 50% cold methanol and 200 μl of cold water, respectively. Supernatants were mixed and stored at −80 °C prior to analysis. Extracts were filtered through 0.2 μm filters (Millipore, Ontario, Canada) before analysis.
Nucleotide concentrations
Nucleotides in BM cell and BM-derived MDSC extracts were analyzed using a 1290 UPLC system coupled to a 6460 triple quadruple mass spectrometer (both from Agilent Technologies, Quebec, Canada). Nucleotides were separated by a Symmetry C18 column (150*2.1 mm, 3.5 μm) (Waters) equipped with a Security C18 guard-column (Waters, 10*2.1 mm, 3.5 μm) by the ion-pair method. DMHA (N,N-dimethylhexylanine, Sigma) was used as an ion-pair reagent to improve the signal-to-noise ratio with positive ionization mode. The mobile phase consisted of Buffer A: 10 mM ammonium acetate, 15 mM DMHA at pH 7.0, and Buffer B: 50/50% (v/v) acetonitrile, 20 mM NH4OAc at pH 7.0. Mobile phase flow rate was set at 0.3 m/min with the following gradient: 0–10 min at 10% B, 10–20 min at linear gradient from 10 to 30% B, 20–21 min at linear gradient from 30 to 60% B, 21–26 min at 60% B, 26–27 min at linear gradient from 60 to 10% B and 27–35 min at 10% B.
Nucleotide concentrations in MSC-1 cell extracts were determined by ion-pairing liquid chromatography-electrospray ionization mass spectrometry (positive mode) using a HPLC-MS system (Waters, Milford, MA) equipped with a Symmetry C18 column (150*2.1 mm, 3.5 μm) (Waters) and a Security C18 guard-column (Waters, 10*2.1 mm, 3.5 μm). Likewise, DMHA was used as an ion-pair reagent. The mobile phase consisted of Buffer A, 10 mM ammonium acetate, 15 mM DMHA at pH 7.0, and Buffer B, 40% (v/v) acetonitrile in water. The flow rate was set at 0.3 ml/min using the following gradient: 0–10 min at 15% B, 10–12 min at linear gradient from 15 to 40% B, 12–30 min at linear gradient from 40 to 70% B, 30–35 min at 70% B, 35–37 min at linear gradient from 70% to 15% B and 37–45 min at 15% B. In both cases, quantification of metabolites (nucleotides and organic acids) was performed by integrating peak areas and using calibration curves. Cell specific concentrations in metabolites were calculated by normalizing the quantity of metabolites in cell extracts to the number of extracted cells.
Organic acid concentrations
Organic acids concentrations were assessed using the above-mentioned UPLC-MS/MS system using a Hypercarb column (100*2.1 mm, 5 μm) and a Hypercarb pre-column (2.1*10, 5 μm) (Thermo Fisher, Ontario, Canada). The mobile phase consisted of Buffer A, 20 mM ammonium acetate at pH 7.5, and Buffer B, 10% (v/v) methanol in water. The flow rate was set at 0.3 ml/min using the following gradient: 0–5 min at 10% A, 5–10 min at linear gradient from 10% to 20% A, 10–20 min at linear gradient from 20% to 100% A, 20–30 min at 100% A, 30–32 min at linear gradient from 100% to 10% A and 32–40 min at 10% A.
Statistical analysis
Data are shown as mean ± SEM (standard error of mean) of n = 3 independent experiments from 3 distinct cell cultures.