Figure 13. Summary of the effects of NFkB and HIF-1a on DC maturation. Ligation of TLRs leads to NFkB activation and subsequent upregulation of HIF-1a under normoxic conditions. HIF-1a contributes to NFkB driven phenotypic DC maturation and cytokine production. The effects were investigated using an inhibitor of NFkB/p65 nuclear translocation (YC-1), inhibition of HIF-1a nuclear translocation (cheto- min), and inhibition of HIF-1a protein accumulation (digoxin). CoCl2 and hypoxia served as controls. doi:10.1371/journal.pone.0010983.g013 Materials and Methods Generation and stimulation of human monocyte-derived DC (MoDC) Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats obtained from healthy blood donors (Regional Red Cross Blood Donation Center, Bern, Switzerland) by density gradient centrifugation over Ficoll-Paque (Amersham, Uppsala, Sweden). Monocytes were isolated from PBMC as previously described [45–47] or by CD14 positive selection by MACS (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according the manufacturer’s protocol (for experiments with digoxin). Purity of isolated monocytes was characterized by high expression of CD14 (.96% positive cells) and absent expression of CD1a and DC-SIGN. Monocytes were incubated for 6 days in RPMI 1640 medium (Invitrogen Life Technologies, Basel, Switzerland) containing 10% fetal calf serum ((FCS, Amimed / BioConcept, Allschwil, Switzerland), 1% [2 mM] L-Glutamine (Invitrogen), 1% [100 U/ml] Penicillin/Streptomycin (Invitrogen), 10 ng/ml GM-CSF (R&D Systems Europe Ltd, Abingdon, Oxon, UK), and 10 ng/ml IL-4 (R&D) to generate MoDC as described initially by Sallusto and Lanzavecchia [48]. Immature MoDC were characterized by absent expression of CD14 and high expression of CD1a, HLA-DR, DC-SIGN and phagocytic activity. Maturation of these cells leads to a massive up-regulation of CD40, CD80, CD83, CD86, ICAM-1, CCR7 and HLA-DR. Mature MoDC exhibit a very potent T cell proliferation capacity and reduced Ag-uptake as previously described [46,47,49]. Cells were kept at 37uC in a 5% CO2 humidified atmosphere. On day 3, the culture medium was replaced with fresh medium. For induction of maturation 1 mg/ml LPS (Sigma Aldrich, Buchs, Switzerland), 5 mg/ml LTA or 20 mg/ ml HA (Sigma Aldrich, Buchs, Switzerland) were added to the MoDC cultures for the indicated periods. FACS analysis and cell viability Cells were incubated with FITC- or PE-labeled monoclonal antibody (mAb) against CD80, CD86 (BD, Franklin Lakes, NJ, USA), isotype control IgG1 (BD), or unlabeled mAb against CD40, ICAM-1 (Diaclone, Besanc¸on, France) followed by a FITC-labeled polyclonal goat anti-mouse IgG (Sigma). TLR and HIF in Human DCs For determination of viability, the cells were stained with 5 mg/ ml of propidium iodide (PI; Invitrogen) and analyzed by flow cytometry. As positive control for PI staining, cells were treated with PBS containing 0.1% BSA and 0.1% saponin (Sigma Aldrich, Buchs, Switzerland). Measurements were performed with a BD FACScan flow cytometer and analyzed using the FlowJo software (Tree Star Inc., Ashland, OR, USA). Hypoxic conditions Cells were incubated in a hypoxia chamber (1.5% oxygen, 37uC) for the indicated time-periods. In some experiments (as indicated), LPS (1 mg/ml) was given in the hypoxia-chamber one hour after putting the cells under hypoxic conditions. SDS-PAGE and Western blotting DC were lysed in 60 mM Tris-HCl, 8.5% glycerol, and 2% SDS. Protein concentration was determined with the Quanti-IT assay kit and read with the Qubit fluorometer (Invitrogen). Equal amounts of protein (20 mg per lane) were loaded and separated by 4–12% SDS-PAGE. Gels were then transferred to nitrocellulose membranes with the iBlot dry blotting system (Invitrogen). Equal loading was verified by staining the extracted gel with SimplyBlue SafeStain (Invitrogen). Afterwards, the membranes were blocked for 30 min with incubation buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl and 0.1% w/v Tween 20) supplemented with 5% non- fat dry skim milk, and incubated overnight with the primary antibodies against HIF-1a (Novus Biologicals, Littleton, CO, USA; dilution 1:2000) or primary antibodies against TLR-4 (Invitrogen, Basel, Switzerland; dilution 1:1000) and actin (Sigma Aldrich, Buchs, Switzerland; dilution 1:3000). Membranes were washed with incubation buffer and incubated for 1 hour with horseradish peroxidase-coupled goat polyclonal anti-rabbit IgG (dilution 1:3000). Finally, the membranes were developed with a chemiluminescence detection kit (Pierce, Rockford, IL, USA). All Western blotting experiments were performed in triplicates. Protein Densiometry was quantified using Adobe Photoshop CS3. Cytokine assays MoDC (106 cells/ml) were treated with LPS, LTA or HA for 24 hours. Cell culture supernatants were analyzed using a Luminex multiplex suspension array system from Bio-Rad (Bio-Rad, Hercules, CA, USA) for IL-1b, IL-6, IL-8, IL-10, TNF-a and VEGF (all kits from BioSource, Invitrogen, Carlsbad, CA, USA) according the manufacturer’s instructions. Inhibitors and agonists of HIF-1a and/or NFkB Blocking of HIF-1a was achieved using the commercially available inhibitors CTM (Chetomin, NSC 289491, Invitrogen), and digoxin (Sigma-Aldrich, Buchs, Switzerland). Chetomin is a natural metabolite produced by several species of the genus Chaetomium. To inhibit NFkB/p65 nuclear translocation, YC-1 (3- (59-Hydroxymethyl-29-furyl)-1-benzyl indazole; Calbiochem-No- vabiochem, Zug, Switzerland) was used. Stabilization of HIF-1a protein in control experiments was achieved using CoCl2 (Sigma Aldrich, Buchs, Switzerland), which is a potent inhibitor of post- translational hydroxylation of proline residues in the oxygen- dependent degradation (ODD) domain of HIF-1a and thus inhibits HIF-1a degradation. High-resolution respirometry DC were centrifuged for 5 min (350 g) and resuspended in respiration buffer (110 mM sucrose, 0.5 mM EGTA, 3.0 mM MgCl2, 80 mM KCl, 60 mM K-lactobionate, 10 mM KH2PO4, PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e10983