TLR and HIF in Human DCs Figure 11. Modulation of the secretion of proinflammatory cytokines as well as IL-10 by inhibition of HIF-1a by digoxin. (A), (B), (C) and (D), MoDC were stimulated with LPS (1 mg/ml), HA (20 mg/ml) and LTA (5 mg/ml). After 24 hours of incubation, cells were harvested and the supernatants examined for the amounts of IL-6 (A), TNF-a (B), IL-1b (C), IL-10 (D) and IL-8 (E) with a Luminex multiplex array system. w/o: without stimulus. Mean values 6 SD of 3 experiments with cells from different donors are shown. *p,0.05 vs. mature MoDC (unpaired Student’s t-test). doi:10.1371/journal.pone.0010983.g011 This begs the question of whether HIF-1a stabilization under normoxic conditions has other physiological consequences and whether the role of downstream events that may be activated are the same as in hypoxia. Current evidence suggests that different target genes may be affected by HIF-1a under normoxic vs hypoxic conditions with physiological roles that differ from those induced by ischemia. Further investigation of these aspects of HIF-1a action, however, lie outside the original scope and aims of the present study. Figure 12. Influence of LPS stimulation on mitochondrial respiration and the generation of ROS by MoDC. (A) MoDC were stimulated under normoxic conditions for 4 hours with LPS (1 mg/ml). Mitochondrial Respiration was evaluated using a high resolution respirometry system. Mean values 6 SD from 10 donors are shown as column graphs. (B) Prior to TLR stimulation, cells were incubated with 0.4 mM DCFH-DA for 15 minutes. Afterwards cells were washed and treated with LPS (1 mg/ml) or H2O2 (2 mM) for 15 and 30 minutes and fluorescence was evaluated by using flow cytometry. Y-axis shows the mean fluorescence intensity (MFI). Mean values 6 SD from 3 donors are shown as column graphs. doi:10.1371/journal.pone.0010983.g012 PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e10983