TLR and HIF in Human DCs Figure 3. Effect of CTM on phenotypic maturation of MoDC. Monocytes isolated from buffy coat were cultured in the presence of GM-CSF and IL-4 for 6 days. Inhibition of HIF-1a expression by CTM inhibited maturation of MoDC in response to LPS. MoDC were incubated with CTM (200 nM) for 3 hours, afterwards cells were stimulated for 24 hours with LPS (1 mg/ml), HA (20 mg/ml) and LTA (5 mg/ml). Cells were washed and analyzed for the expression of CD40 (A), CD80 (B), CD86 (C) and ICAM-1 (D). Y-axis shows the median fluorescence intensity (MFI). Mean values 6 SD of 6 experiments with cells from different donors are shown. *p,0.05; **p,0.01; ***p,0.001 vs. mature MoDC (unpaired Student’s t-test). doi:10.1371/journal.pone.0010983.g003 positive cocci S. aureus, a TLR2 ligand [33], induced DC maturatuion and HIF-1a protein stabilization, comparable to the stimulation induced by LPS from the gram-negative rod E. coli. Similar effects were induced by the endogenous TLR4 ligand HA although some differences were observed in the time course. A possible explanation may relate to the known differences in Figure 4. Effect of YC-1 on phenotypic maturation of MoDC. Monocytes isolated from buffy coat were cultured in the presence of GM-CSF and IL-4 for 6 days. MoDC were incubated with YC-1 (100 mM) for 5 min, afterwards cells were stimulated for 24 hours with LPS (1 mg/ml), HA m20 (g/ ml) and LTA (5 mg/ml). Cells were washed and analyzed for the expression of CD40 (A), CD80 (B), CD86 (C) and ICAM-1 (D). Y-axis shows the median fluorescence intensity (MFI). Mean values 6 SD of 4 experiments with cells from different donors are shown. *p,0.05; **p,0.01 vs. mature MoDC (unpaired Student’s t-test). doi:10.1371/journal.pone.0010983.g004 PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e10983