under normoxia. Results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1a in a time-dependent manner. Interestingly, hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Furthermore, we have investigated the effect of three previously described HIF-1a inhibitors (chetomin [CTM], digoxin and an inhibitor of both, NFkB and HIF-1a signaling, YC-1 on MoDC maturation and function. Results Upregulation of HIF-1a by exogenous and endogenous TLR4 agonists in human MoDC under normoxia To investigate if HIF-1a is upregulated by TLR stimulation under normoxic conditions, MoDC were incubated with selective TLR ligands for the indicated time periods in a normal humidified cell incubator. We used LPS (1 mg/ml, Fig. 1A) and HA (20 mg/ ml, Fig. 1B) as exogenous and endogenous TLR4 agonists, respectively. HIF-1a expression at the protein level was deter- mined by Western blot analyses. As shown in Fig. 1A and B, both TLR4 agonists induced the expression of HIF-1a in a time- dependent manner. In parallel, cells were stained for the expression of the co-stimulatory molecules CD80 and CD86 as markers of cell maturation to confirm activation of the cells (Fig. 2A). To rule out contamination of HA with LPS, we incubated the cells with polymyxin B, an inhibitor of the biological activities of endotoxin. As shown in Fig. 2B, polymyxin B did not affect maturation of MoDC induced by HA whereas the effect of LPS was abolished. HIF-1a expression is induced by TLR2 To investigate, if stabilization of HIF-1a protein is restricted to TLR4 signaling, we tested the effect of the TLR2 agonist LTA on the expression of HIF-1a. As shown in Fig. 1C, LTA also induced the stabilization of HIF-1a protein in a time dependent manner under normoxic conditions. Hypoxia and stabilization of HIF-1a by the agonist CoCl2 does not induce maturation of MoDC We further analyzed, if stabilization of HIF-1a protein by CoCl2 alone, without stimulation of TLR, leads to an activation of MoDC. As shown in Fig. 1D, treatment with CoCl2 led to the stabilization of HIF-1a but did not lead to a significant increase of the expression of the co-stimulatory molecules CD80 and CD86 (Fig. 2A). In addition we investigated the effect of hypoxic conditions (1.5% oxygen) on the maturation of MoDC. Hypoxia alone did not lead to phenotypic maturation of human MoDC (Fig. 2A), whereas the cells still matured if they were incubated together with LPS (Fig. 2A). MoDC stimulated with LPS under hypoxic conditions showed a synergistic response with a significant increase of double positive CD80+/CD86+ cells (Fig. 2A) com- pared to LPS stimulation under normoxia. Analysis of commonly used inhibitors of HIF-1a/NFkB (CTM, digoxin and YC-1) on phenotypic maturation of human MoDC To further characterize the potential involvement of the transcription factor HIF-1a and NFkB in the maturation process of human MoDC, we used CTM (Chetomin), YC-1 and digoxin to attenuate HIF-1a and/or NFkB responses. CTM has been shown to block the interaction of HIF1a with transcriptional co- activators p300 and cAMP response element binding protein TLR and HIF in Human DCs (CREB), thereby attenuating hypoxia-inducible transcription [25]. CTM, however, did not prevent the increase in NFkB p65 subunit nuclear levels or NFkB transactivation activity in response to LPS- IFNc challenge [19]. YC-1 in contrast has been reported to abolish constitutive nuclear translocation and activation of NFkB/p65 [26] an upstream regulator of HIF-1a. However, YC-1 might also act on the PI3K/Akt/mTOR pathway, which can also regulate HIF- 1a expression at the translational step [27]. Digoxin is thought to block accumulation of HIF-1a protein [28]. MoDC were pretreated with CTM for 3 hours and then stimulated with LPS, LTA or HA for 24 hours. Afterwards, cells were analyzed for expression of CD40, CD80, CD86 and ICAM-1 (CD54) by flow cytometry. CTM consistently inhibited the up- regulation of all markers compared to the positive controls in response to all TLR ligands tested (Fig. 3A-D). In contrast, pretreatment of MoDC with YC-1 for 5 minutes before exposure to LPS, LTA or HA for 24 hours significantly inhibited only the up-regulation of CD80, CD86 and ICAM-1 primarily in response to LPS. No significant inhibitory response of YC-1 was observed for HA or LTA except for ICAM-1 induction by LTA (Fig. 4A-D). In a second series of experiments digoxin was used as an additional inhibitor of HIF-1a in an attempt to confirm the above observations. MoDC were pretreated with Digoxin (50 nM, 100nM) for one hour then stimulated with LPS, LTA or HA for 24 hours. As before, cells were analyzed for expression of CD40, CD80, CD86 and ICAM-1 by flow cytometry. Under these conditions, there was no significant effect of digoxin on the expression of any of the maturation markers analysed. (Figure 5A-D). In all studies, exposure of MoDC to the used concentrations of CTM, CoCl2, LPS or YC-1 for 24hr did not affect viability of the cells as determined by flow cytometry (Figure 6). As a general rule any dead cells were always excluded from flow cytometric analysis by PI staining and there was no indication of synergistic loss of cell viability in co-incubation studies. Modification of VEGF secretion by MoDC by CTM, YC-1 and digoxin Under hypoxic conditions, the vegf-gene is known to be at least partly under control of the transcription factor HIF-1a. To analyze if a similar dependency could be observed in response to TLR agonists under normoxic conditions, VEGF stimulation by selective TLR ligands was measured in the supernatants of MoDC. As shown in Fig. 7A, treatment with CTM significantly inhibited the TLR-induced production of VEGF whereas no inhibition was observed in the presence of YC-1 or digoxin. In fact, responses to HA and LTA in the presence of YC-1 were enhanced (Fig. 7B and C). As expected hypoxia-induced secretion of VEGF by MoDC was increased by incubation for 24 hours under hypoxic conditions (data not shown). Digoxin inhibits LPS-, HA- and LTA-induced increases in HIF-1a but does not alter TLR4 expression Given the lack of effect of digoxin on expression of DC maturation markers or VEGF production in response to TLR ligands we determined the effect of digoxin on cellular HIF-1a protein levels. Cells were exposed to various concentrations of digoxin and thereafter stimulated with TLR ligands. As shown in Fig. 8A, pretreatment of cells with 50 and 100 nM digoxin dose dependently inhibited TLR agonist stimulated cellular HIF-1a protein increases whereas TLR4 levels were unaffected (Fig. 8B). PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e10983