Murine and human MSCs were isolated from either the bone marrow of C57BL/6 mice (MSC) or the human umbilical cord (UC-MSC) of newborns and characterized as previously reported [26,27,28]. When indicated, murine MSCs or UC-MSCs were stimulated with 1 μg/mL of oligomycin (OLN; Calbiochem, Merck, Darmstadt, Germany) for 24 h (MSCOLN). LDHA activity was inhibited with 50 µM galloflavin (Cayman Chemical, Ann Arbor, Michigan, USA) for 24 h (MSCGF or MSCOLN+GF) or with 50 pmol LDHA-siRNA (Ambion Inc., Austin, Texas, USA) for 4 h (MSCsiLDHA or MSCOLN+siLDHA or UC-MSCsiDLHA) with the Fugene transfection reagent (Promega, Madison, Wisconsin, USA) following the manufacturer’s instructions. The validation of LDHA inhibition was performed by qPCR, lactate production measurement and by western blot analysis.
Th1 and Th17 differentiation and immunosuppressive assay
Naïve CD4+ T cells were obtained using the MojoSort Mouse CD4 T Cell Isolation Kit (Biolegend, San Diego, California, USA) following the manufacturer’s instructions. Naïve CD4+ T cells were then stained with cell trace violet (CTV) (Life-Technology, Thermo Fisher, Carlsbad, California, USA) and cultured in a specific lymphocyte medium (MLR) in the presence of specific culture conditions to induce Th1 or Th17 cells as we previously described . To test the immunosuppressive effect of lactate release by MSCs, naive CD4+ T cells prompted to differentiate into Th1 or Th17 were cultured alone or with 10, 25, or 50 mM L-Lactate or in the presence of MSCs under the different experimental conditions described above at a cell ratio of one MSCs per 12.5 lymphocytes in MLR medium. After 72 h, CD4+ T cell proliferation and differentiation were analyzed by flow cytometry.
Flow cytometry analysis
Murine CD4+ T cells were stimulated for 4 h at 37 ºC with 50 ng/mL of phorbol myristate acetate (PMA) (Merck, Darmstadt, Germany), 1 mg/mL of ionomycin (Merck, Darmstadt, Germany) and 10 mg/mL of brefeldin A (Sigma-Aldrich, Merck, Darmstadt, Germany) in MLR medium as described . Then, CD4+ T cells were stained with anti-CD25 antibodies (BD Pharmingen, San Diego, California, USA) in the presence of the LIVE/DEAD Fixable near-IR stain (Invitrogen, Thermo Fisher, Carlsbad, California, USA) and fixed with the FOXP3 Cytofix/Cytoperm buffer (eBioscience, San Diego, California, USA). Finally, the cells were labeled with intracellular fluorochrome-conjugated antibodies against IFNγ, IL17 (BD Pharmingen, USA) and FOXP3 (eBioscience, San Diego, California, USA), as previously described . Flow cytometry was conducted using the BD FACS Canto II (BD Biosciences, San Diego, California, USA), and the data were analyzed with the FlowJo software (BD Biosciences, San Diego, California, USA).
Measurement of MSC lactate production
MSCs were cultured in supplemented Dulbecco’s Modified Eagle Medium (DMEM) without phenol red and fetal bovine serum (FBS) to assess inhibition of lactate production in MSCs after treatments. After 24 h, the supernatant was collected, and concentration of L-Lactate was measured with the Colorimetric Lactate Assay Kit (BioVision, Abcam, UK) according to the manufacturer’s instructions. Briefly, a standard curve was prepared with 0, 2, 4, 6, 8 and 10 nmol/well of lactate standard in a 96-well plate. Then, 1 µL of each sample and the standard curve wells were incubated for 30 min at room temperature and protected from light in a 50 µL reaction volume containing 1 µL of Lactate Enzyme Mix, 1 µL of probe, and Lactate Assay Buffer. Finally, absorbance was measured (OD, 570 nm), and lactate concentration was determined by plotting and replacing the lactate standard curve.
The metabolism of murine MSC was determined in real time by measuring the Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR), using the XF96 analyzer (Seahorse Biosciences, North Billerica, Massachusetts, USA) as previously described .
MCT and HCAR1 expression by quantitative real-time PCR (qRT-PCR)
RNA of Th1, Th17, and naive CD4+ T cells were extracted using TRIzol Reagent (Invitrogen, Thermo Fisher, Carlsbad, California, USA) and then treated with DNAase I (Invitrogen, Thermo Fisher, Carlsbad, California, USA) to remove genomic DNA contamination. The purity and quantification of total RNA was measured with a NanoDrop 2000 (Thermo Scientific). Reverse transcription was performed with iScript™ cDNA Synthesis Kit (Bio-Rad, Los Angeles, California, USA) according to the manufacturer’s instructions. For qRT-PCR, 2 µL of HOT FIREPol ® EvaGreen ® qPCR Mix Plus (ROX) (Solis Biodyne, Tartu, Estonia) was used in a final volume of 10 µL containing 2 µL of cDNA diluted 1:1, 1 µM of the following sets of primers: 18S, sense 5′-GCCCGAAGCGTTTACTTTGA-3′ and antisense 5′-TTGCGCCGGTCCAAGAATTT-3′; MCT1, sense 5′-TGCAACGACCAGTGAAGTATC-3′ and antisense 5′-CAAGCCCAAGACCTCCAATAA-3′; MCT2, sense 5′- ATATTCAACACCACCTCCAGTC-3′ and antisense 5′-TGAAGCCAACGGTGAGATAAA-3′; MCT4, sense 5′- ATGAGTTTGGGATTGGCTACA-3′ and antisense 5′-GTGGTGAGGTAGATCTGGATAATG-3′; HCAR1, sense 5′-ATCCTGGTCTTCGTGCTTGG-3′ and antisense 5′- CTGTCCGAAGGGGTAAGCAG-3′. The reaction was carried out in an Mx3000P QPCR System (Agilent Technologies, USA). The relative amount of mRNA of each gene was calculated with the relative quantification method (2-ΔΔCt) and normalized according to the expression of naive CD4+ T cells in basal conditions.
DTH mouse model
A DTH murine model was used to evaluate the effect of lactate inhibition in MSCs on their anti-inflammatory capacity. For that purpose, 1 mg/mL of albumin from chicken egg white (OVA) (Sigma-Aldrich, Merck, Darmstadt, Germany) in Complete Freund’s Adjuvant were intradermic injected into the lower back of C57BL/6 mice. After 5 days, paw swelling was measured and a boost of OVA in saline solution (control group) or in combination with MSCs under the different experimental conditions were injected in the hindlimb paws. After 24 h, paw thickness was measured again, and euthanasia was performed. Subpopulations of anti- or proinflammatory CD4+ T cells in the draining lymph nodes were determined by flow cytometry using a FACS CANTO II flow cytometer.
Freshly isolated naïve murine CD4+ T cells or human peripheral blood mononuclear cells (PBMCs) were cultured in the presence or absence of 10 mM, 20 mM or 50 mM L-Lactate for 3 days. Apoptosis was measured using Anexinn V (Invitrogen, Thermo Fisher, Carlsbad, California, USA) and propidium iodide (PI) kits (eBioscience, San Diego, California, USA), following manufacturer’s instructions, and flow cytometry using a FACS CANTO II flow cytometer.
PBMCs from healthy donors (HD) were labeled with CellTrace™ Violet (Life Technologies, Thermo Fisher, Carlsbad, California, USA) and co-cultured with UC-MSCs under the different mentioned conditions in the presence of phytohemagglutinin (PHA) (5 µg/mL) (Sigma-Aldrich, Merck, Darmstadt, Germany) in MLR media. The proliferation was evaluated by FACS using a FACS CANTO II flow cytometer.
Data are shown as the mean ± SD. All in vitro experiments were performed at least three times independently using two different biological samples each time. For the DTH murine model, twelve to eighteen mice per experimental group were used. The experiment was repeated three times as follows: 1st DTH experiment consisting in six animals per experimental group, including the non-treated mice induced with the DTH model, treated with murine MSCs and with murine MSCs pretreated with galloflavin. The second DTH murine model had seven animals per experimental group in the non-treated mice induced with the DTH model, treated with murine MSCs, with murine MSCs pretreated with galloflavin and murine MSCs pretreated with a siRNA against LDH. Finally, the third experiment included six animals in the non-treated DTH mice, five animals treated with murine MSCs, and eight animals treated with murine MSCs pretreated with a siRNA against LDH. Data that were identified as outlawyers were excluded. Also, 4 popliteal lymph nodes from the in vivo experiments (one non-treated DTH, one treated with murine MSC, one with siRNA against LDH and one with galloflavin) were lost during the experimental processing due to poor lymph nodes cells recovery. The p-values were generated by non-parametric analysis using the Kruskal–Wallis test for multiple comparisons and the Mann–Whitney test to compare two groups for all the data that did not fit with normal Gaussian distribution. Two-ways ANOVA and t tests were used for normal distribution. p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) were considered statistically significant. Data were analyzed with the GraphPad Prism TM 6 software (GraphPad Software, San Diego, California, USA).