Figure 2. Evaluation of phenotypic maturation of MoDC. (A) Percentages of CD80+ CD86+ double-positive cells stimulated with LPS (1 mg/ml), HA (20 mg/ml), LTA (5 mg/ml), CoCl2 (200 mM) or cultured under hypoxia 6 LPS are shown as mean values 6 SD of 3–5 experiments with cells from different donors. N: Normoxia, H: Hypoxia. (B) Polymyxin B does not affect stimulatory activity of HA. MoDC were incubated with polymyxin B (1 or 10 mg/ml) and then stimulated with HA (20 mg/ml) or LPS (1 mg/ml) for 24 hours. Afterwards, cells were washed and analyzed for the expression of CD86 as a maturation marker. Y-axis shows the median fluorescence intensity (MFI). The dotted line indicates the MFI of non-stimulated MoDC. Data are representative of three independent experiments. doi:10.1371/journal.pone.0010983.g002 Treatment of MoDC with CTM, YC-1 or digoxin differentially modulates secretion of proinflammatory cytokines and IL-10 The effect of HIF-1a/NFkB inhibitors on the secretion of cytokines by MoDC was evaluated using a Luminex multiplex array system. Supernatants of LPS, HA and LTA stimulated cells were assayed for IL-1b, IL-6, IL-10 and TNF-a (for digoxin IL-8 was also analyzed). TLR and HIF in Human DCs Stimulation of MoDC with LPS resulted in a marked increase in the proinflammatory cytokine IL-6. which was inhibited approx. 75% by CTM treatment after 24 hours of stimulation. The trend towards a similar attenuated response to HA did not reach statistical significance (Fig. 9A). None of the TLR agonists significantly affected TNF-a or IL-1b production (Fig 9B and C) but treatment with CTM reduced IL-10 secretion in response to stimulation with the endogenous TLR4 ligands LPS and HA (Fig. 9D). In contrast no significant inhibitory effects of YC-1 or digoxin were observed for any of the cytokines analyzed (Fig. 10, 11). Effect of LPS stimulation on mitochondrial respiration of MoDC As maturation is an energy dependent process, we analyzed the functional effect of TLR receptor stimulation on energy metabolism, and measured the mitochondrial respiration of MoDC, which leads to generation of ATP. DC were incubated with 1 mg/ml of LPS under normoxic conditions. After 4 hours of incubation no significant changes in maximal oxygen consumption (state 3) was observed (Fig. 12A) Influence of LPS on the generation of ROS in human MoDC Since LPS can induce generation of ROS in human primary MoDC, which in turn might play a role in the maturation process we analyzed the generation of ROS using flow cytometry. We could not observe an increase in ROS after 15 and 30 minutes stimulation with LPS, whereas levels of ROS treated with the positive control hydrogen peroxide were increased (Fig. 12B). Discussion The present study addressed the question of whether HIF-1a could be induced by different TLR agonists under normoxia and if stabilization is responsible for the subsequent MoDC maturation and TLR ligand-induced cytokine production under these conditions. The results demonstrate that under normoxic conditions, endogenous and exogenous TLR2- and TLR4-ligands induce maturation of human MoDC, which is associated with cell surface expression of CD40, CD80, CD86 and ICAM-1 in a time dependent manner together with secretion of VEGF and proinflammatory cytokines, and stabilization of the transcription factor HIF-1a. Recent evidence suggests that endogenous TLR agonists play an important role in human disease states such as I/R injury which occurs in solid organ transplantation, myocardial infarction, autoimmunity or trauma. HA, a non-sulfated glycosaminoglycan, is a major constituent of the extracellular matrix (ECM). During tissue injury or inflammation, HA is degraded and shed from the ECM and cell surface and serves as an endogenous danger signal [9,29]. Subsequent DC maturation and activation, with induction of proinflammatory cytokines, then contributes to a prolongation and exacerbation of tissue damage. Inflammatory responses of mouse macrophages to endogenous and exogenous TLR2 and TLR4 ligands [30] have previously been reported [31]. A potential role, however, or involvement of HIF-1a in these processes has not been considered. Untill now, HIF-1a protein stabilization under normoxic conditions by TLR ligands has been shown only for stimulation with LPS in murine bone-marrow derived DC, [23] and several studies have highlighted distinct differences between mouse and human immunology [32]. Hence the relevance of these earlier observations for human disease has largely remained unknown. In the present experiments, LTA from the gram- PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e10983