Figure 7. Effect of HIF-1a inhibition by CTM, YC-1 and Digoxin on VEGF production of MoDC. MoDC were incubated with CTM (200 nM) for 3 hours, YC-1 (100 mM) for 5 minutes and Digoxin (50 nM or 100 nM) for one hour, then cells were stimulated with LPS (1 mg/ml), HA (20 mg/ml) and LTA (5 mg/ml). After 24 hours cells were harvested and the supernatant assayed for VEGF with a Luminex multiplex array system. w/ o: without stimulus. Mean values 6 standard deviation of 6 experiments (4 for YC-1 and 3 for Digoxin) 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.g007 TLR and HIF in Human DCs Figure 8. Digoxin inhibits LPS-, HA- and LTA-induced upregu- lation of HIF-1a (A) and does not modify TLR-4 expression (B). Cell lysates from MoDC that were incubated with digoxin (50 nM [D50] or 100 nM [D100]) and then stimulated with LPS (1 mg/ml), HA (20 mg/ ml) or LTA (20 mg/ml) for 24 hours. Equal amounts of protein were loaded and separated by SDS page, transferred onto membranes and probed for HIF-1a and TLR-4. doi:10.1371/journal.pone.0010983.g008 the fact that hypoxia during the differentiation process of monocytes into DC has additional effects on cells that differ from hypoxic exposure of already fully differentiated DC. In view of the complex interplay between these factors we subsequently evaluated a potential role of HIF-1a stabilization in the processes of MoDC maturation and cytokine production using three inhibitors of HIF-1a and/or NFkB, namely CTM [25], YC- 1 [27] and digoxin [28] (see Fig. 13). Taken together the data showed that there is no consistent effect of the three inhibitors on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1a stabilization or activity through different mechanisms. Thus the data of YC-1 and digoxin did not support the original hypothesis that HIF-1a activation by TLR ligands under normoxic conditions may have functional consequences for human MoDC maturation, VEGF and cytokine production. These events appear to occur indepen- dently of the observed changes in HIF-1a stabilization. This would suggest that other, as yet unknown effects of these inhibitors are responsible for differential responses to the individual inhibitors in relation to cell maturation (cell surface marker expression, or VEGF and cytokine production). This is reinforced by the observation that digoxin was without effect on any of the parameters measured despite preventing TLR induced stabiliza- tion of HIF-1a in response to LPS, LTA and HA. Similarly variable responses of the antagonists were seen for VEGF production by TLR ligands where only CTM showed any inhibition and responses to HA and LTA in the presence of YC-1 were even enhanced. In this regard, non-HIF-1a dependent stimuli including insulin- like growth factor-1 (IGF-1), also stimulate VEGF secretion which is phosphatidyl-inositol 3-kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin)-dependent. Other regulators include angio- tensin, nitric oxide and guanyl cyclases [42,43]. Interestingly YC-1 was originally characterized as a cGMP inducer because it stimulated soluble guanylyl cyclase activation in response to nitric oxide or carbon monoxide [44] in some cells, only low concentrations of YC-1 (1–20 mmol/L) are required for anti- HIF-1a activity, whereas cGMP elevation requires higher concentrations (.50 mmol/L; [44] as used in the present study. Whether and to what extent this may play a role in the differential responses of MoDC to YC-1 in the presence of different TLR ligands, particularly in relation to the observed stimulatory effects on VEGF remain to be determined. PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e10983