TLR and HIF in Human DCs Figure 5. Effect of Digoxin (50 nM or 100 nM) 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 Digoxin for one hour, 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 3 experiments with cells from different donors are shown. doi:10.1371/journal.pone.0010983.g005 requirement of HA and LPS for distinct accessory molecules leading to alternate downstream signaling pathway activation (Taylor 2007). Figure 6. MoDC viability in response to CoCl2, CTM and LPS. Cells were treated with CoCl2, CTM and LPS for 24 hours. Thereafter cells were harvested and stained with PI (5 mg/ml). Unstained viable cells (PIneg) were enumerated by flow cytometry. Mean values 6 SD of 3 experiments with cells from different donors are shown. doi:10.1371/journal.pone.0010983.g006 In contrast, neither hypoxia nor CoCl2 induced HIF-1a accumulation lead to phenotypic maturation of MoDC examined after 24 hours suggesting that changes in HIF-1a per se do not necessarily lead to maturation. At first sight this may suggest methodological differences as an underlying factor. However, at the dose used, CoCl2 has clearly been shown to be an inducer of HIF-1a activation [34–36]. Furthermore, different ranges of oxygen levels have been considered as hypoxic conditions, starting from almost anoxic conditions of 0.1% up to 2% O2, oxygen levels in the body vary from around 16% in the pulmonary alveoli down to approximately 6% in other tissues [37]. Although there is no current consensus regarding a critical threshold level we used the commonly quoted value of 1.5% [38,39]. While this level of hypoxia did not directly lead to MoDC maturation we did observe a synergistic effect on LPS induced phenotypic maturation of MoDC in the presence of hypoxia. Consequently, hypoxia may augment an inflammatory response elicited in the presence of a danger signal associated with infection or ischemia. In contrast, hypoxia without pro-inflammatory stimuli does not induce activation, which may serve as a protection mechanism of the host preventing development of an autoimmune response. These data are consistent with the finding of Jantsch et al. in murine DC for hypoxia [23]. In contrast, a recent study has shown that human MoDC differentiated from monocytes under permanent hypoxic (1% O2) conditions exhibit a reduced up-regulation of CD40, CD80, CD83 and CD86 in response to LPS, whereas the secretion of TNF-a, CCL22 and IL-1b was increased [40]. Another study demonstrated, that MoDC generated under hypoxic conditions (1% O2) change their chemokine releasing profile and exhibit a reduced Ag-uptake capacity [41]. These differences may relate to PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e10983