The core structure of the ligand recognized by NOD-1 is the peptidoglycan-specific dipeptide, γ-D-glutamyl-meso-diaminopimelic Epacadostat acid (iE-DAP) and NOD-2 recognizes the muramyldipeptide (MDP), representing the minimal motif of bacterial peptidoglycan able of activating NOD2 . Given the significance of TLR and NLR in immunity and cell differentiation, in this study we explored the expression of NLR in MSC, the transcriptional response of MSC to NOD-1 and TLR-2 ligands and the ability of galectin-3, an identified candidate gene, to affect the inhibitory function of MSC on T-cell proliferation to alloantigens. The peptidoglycan-specific dipeptide, γ-D-glutamyl-meso-diaminopimelic acid
(iE-DAP, a NOD1 ligand) and control peptide (iE-Lys) were purchased from InvivoGen (Toulouse, France) Pam3CS(K)4, and a TLR2 ligand was purchased from Calbiochem (La Jolla, CA, USA). Conjugated anti-CD14, anti-CD4 were purchased from DakoCytomation (Copenhagen, Denmark). Conjugated anti-CD34, anti-CD105, anti-CD106 and anti-NOD2 monoclonal antibody (2D9) were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-NOD1 polyclonal antibodies were purchased from Cell Signalling (Danvers, MA, USA). Total RNA isolation kit Trizol and cDNA synthesis kit were purchased from Invitrogen (San Diego, CA, USA) and GE Healthcare AS (Oslo, Norway), respectively.
SYBR Green PCR Master Mix was purchased from www.selleckchem.com/products/z-vad-fmk.html Applied Biosystems (Foster City, CA, USA). An Illumina TotalPrep RNA Amplification Kit was purchased from Ambion (Austin, TX, USA). Expression arrays were purchased from Illumina (San Diego, CA, USA). Human VEGF monoclonal antibody (clone 26503, capture antibody), human VEGF 165 biotinylated affinity purified polyclonal antibodies (detection antibody) and the galectin ELISA kit were purchased from R&D systems (Abingdon, UK). MSC were isolated and expanded from bone marrow (BM) taken from iliac crest of adult volunteers with informed consent.
Heparinized BM was mixed with double volume of phosphate-buffered saline, and mononuclear cells were prepared by gradient centrifugation Thiamine-diphosphate kinase (Lymphoprep). Subsequently, the cells were cultured in 75-cm2 flask at a concentration of 30 × 106 per 20 ml Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2. After 48- to 72-h incubation, non-adherent cells were removed and adherent cells constituted the MSC cell population that was expanded. Cells were detached by a treatment with trypsin and EDTA (GibcoBRL, Grand Island, NY, USA) and replated at a density of 106 cells/75 cm2 flask. These cells were verified for positive staining for CD105 and CD106, and are negative for CD14, CD34 and CD4 markers. MSC were detached using trypsin/EDTA, resuspended in complete medium and placed at 37 °C for 2 h. Subsequently, cell aliquots (5 × 105) were incubated on ice with conjugated monoclonal antibodies against CD34, CD14, CD4, CD105 and CD106.