Effects of TLR Agonists on the Hypoxia-Regulated Transcription Factor HIF-1a and Dendritic Cell Maturation under Normoxic Conditions Rolf Spirig1., Siamak Djafarzadeh2,5., Tomas Regueira2,5, Sidney G. Shaw3, Christophe von Garnier4, Jukka Takala2,5, Stephan M. Jakob2,5, Robert Rieben1, Philipp M. Lepper2,5* 1 Laboratory of Cardiovascular Research, Department of Clinical Research, University of Bern, Bern, Switzerland, 2 Laboratory of Intensive Care, Department of Clinical Research, University of Bern, Bern, Switzerland, 3 Laboratory of Vasoactive Peptides, Department of Clinical Research, University of Bern, Bern, Switzerland, 4 Department of Pneumology, Bern University Hospital, Bern, Switzerland, 5 Department of Intensive Care Medicine, Bern University Hospital, Bern, Switzerland Abstract Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1a in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1a using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1a stabilization or activity through different mechanisms. Stabilization of HIF-1a protein by hypoxia or CoCl2 did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1a controlled VEGF secretion. These 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. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1a under normoxic conditions with physiological roles that differ from those induced by hypoxia. Citation: Spirig R, Djafarzadeh S, Regueira T, Shaw SG, von Garnier C, et al. (2010) Effects of TLR Agonists on the Hypoxia-Regulated Transcription Factor HIF-1a and Dendritic Cell Maturation under Normoxic Conditions. PLoS ONE 5(6): e10983. doi:10.1371/journal.pone.0010983 Editor: Patricia T. Bozza, Fundac¸a˜o Oswaldo Cruz, Brazil Received August 17, 2009; Accepted May 9, 2010; Published June 7, 2010 Copyright: 2010 Spirig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Foundation for Research in Anaesthesia and Intensive Care Medicine (to PL, to SD), the EU 6th Framework Integrated Research Project ‘‘Reprogramming the Immune System for the Establishment of Tolerance’’ (RISET) and the Swiss National Science Foundation grant no. 3200B0- 116618 (to RR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: philipp.lepper@gmx.de . These authors contributed equally to this work. Introduction DC are a unique leukocyte population of professional antigen presenting cells (APC) that play an important role in bridging innate and adaptive immunity [1]. They are crucial for inducing T-cell mediated immune responses, as seen in infection, allograft rejection, as well as the induction of peripheral tolerance [2,3]. DC continuously scan their environment. For this purpose, they express pattern recognition receptors (PRR) including Toll-like receptors (TLR), nucleotide-binding oligomerization domain (NOD)-like receptors, C-type lectin receptors and others. Activat- ing signals such as pathogen-derived molecules, e.g. LPS or lipoteichoic acid (LTA), induce maturation of the cells. Recent studies have shown that some of these PRR can also sense the presence of endogenous danger molecules, which are released in the context of tissue injury [4]. Ischemia/reperfusion (I/R) results in shedding and degradation of the glycosaminoglycans heparan sulfate (HS) [5–8] and hyaluronic acid (HA) [9,10] from the endothelial cell surface. It has been shown that HS and HA induce maturation of DC via TLR4 in vitro [11,12]. Furthermore, it has been demonstrated that TLR2 and TLR4 are crucially involved in I/R injury in vivo [13–17]. Additionally, maturation of DC is influenced by their microenvironment. Recent evidence suggests, that Toll-like receptor ligation can lead to stabilization of the transcription factor hypoxia-inducible factor 1a (HIF-1a) under normoxic conditions, most likely via NFkB [18,19]. HIF-1a has been described as a key regulator of a broad range of cellular and systemic responses to hypoxic conditions. HIF-1a is also involved in myeloid cell-mediated inflammation [20]. Furthermore, HIF-1a is a master regulator of the bactericidal capacity of phagocytes [21]. Lipopolysaccharides (LPS) induce the expression of HIF-1a in murine macrophages [22] and dendritic cells (DC) [23]. Additionally, HIF-1a knock-out mice develop less clinical signs of sepsis [24]. These findings suggest the hypothesis that HIF-1a may also be an important mediator of inflammatory responses in the absence of hypoxia and that HIF-1a activation by TLR ligands under normoxic conditions might have functional consequences for human DC maturation and cytokine production. Thus, we investigated the effect of TLR2 and TLR4 activation by endogenous and exogenous ligands on the upregulation of the transcription factor HIF-1a, dendritic cell maturation and cytokine production in human monocyte-derived DC (MoDC) PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e10983