Oxidized Phospholipids Negatively Regulate Dendritic Cell Maturation Induced by TLRs and CD40 1 Stefan Blu ¨ ml,* Stefanie Kirchberger,* Valery N. Bochkov, † Gerhard Kro ¨nke, 2† Karl Stuhlmeier, ‡ Otto Majdic,* Gerhard J. Zlabinger,* Walter Knapp, 3 * Bernd R. Binder, † Johannes Sto ¨ckl, 4 * and Norbert Leitinger 2† Maturation of dendritic cells (DCs) induced by pathogen-derived signals via TLRs is a crucial step in the initiation of an adaptive immune response and therefore has to be well controlled. In this study, we demonstrate that oxidized phospholipids (ox-PLs), which are generated during infections, apoptosis, and tissue damage, interfere with DC activation, preventing their maturation. ox-PLs blocked TLR-3- and TLR-4-mediated induction of the costimulatory molecules CD40, CD80, CD83, and CD86, the cy- tokines IL-12 and TNF, as well as lymphocyte stimulatory capacity. CD40 and TLR-2-mediated cytokine production was also inhibited, whereas up-regulation of costimulatory molecules via these receptors was not affected by ox-PLs. Thus, formation of ox-PLs during the course of an inflammatory response may represent a negative-feedback loop preventing excessive and sustained immune reactions through regulating DC maturation. The Journal of Immunology, 2005, 175: 501–508. D endritic cells (DCs) 5 are the key regulators of adaptive immunity. In the steady state, DCs reside as sentinels in peripheral tissues as immature APCs and are considered to be tolerogenic (1, 2). However, DCs are capable of sensing changes in their local environment. During infections, DCs are activated by stimulatory signals from invading pathogens, called pathogen-associated molecular patterns (PAMPs), which are rec- ognized by pattern recognition receptors (PRRs) (3–7). Classical PAMPs are LPS, an integral part of the cell wall of Gram-negative bacteria, which is recognized by TLR-4 (8 –10), and lipopeptides from bacterial cell walls and dsRNA, signaling through TLR-2 and TLR-3, respectively (11–14). Peptidoglycan (PGN) is another well-known PAMP, which is sensed by a number of PRRs includ- ing Nod proteins (15–17). Upon stimulation, DCs mature by translocating MHC-peptide complexes to the cell surface, up-regulating costimulatory mole- cules such as CD40, CD80, and CD86, and are thus becoming fully competent to activate T cells (2, 18 –22). In addition, proinflam- matory cytokines including IL-12, TNF, and IFN- are released by DCs during acute infections (3, 23). Host-derived factors associ- ated with tissue damage have been shown to activate or suppress DC function (24, 25). As a part of the body’s defense strategy against invading mi- croorganisms, cells of the innate immune system, mainly neutro- phil granulocytes, produce reactive oxygen species capable of kill- ing bacteria (26). Thereby, tissue damage including host lipid peroxidation occurs, resulting in the formation of bioactive oxi- dized lipids, which are recognized by the immune system (27, 28). Oxidized phospholipids (ox-PLs), which are present in oxidized low-density lipoprotein (29), in apoptotic cells (30, 31), and in inflamed tissue (32), profoundly modulate the course of an inflam- matory response by acting on the endothelium, inducing expres- sion of pro- and anti-inflammatory genes (33–36). Moreover, we and others demonstrated that ox-PLs are able to inhibit binding of LPS to CD14 and LPS-binding protein, thereby preventing recog- nition of LPS by its signaling receptor, TLR-4 (37, 38). However, the impact of ox-PLs on DC function and modulation of the adap- tive immune response is not known. In this paper, we demonstrate that ox-PLs, more precisely lipid oxidation products derived from 1-palmitoyl-2-arachidoyl-sn- glycero-3-phosphorylcholine (OxPAPC), represent microenviron- mental factors regulating DC activation. We show that OxPAPC efficiently prevents LPS- and polyinosinic:polycytidylic acid (poly(I:C))-induced activation of DC via TLR-4 and -3, respec- tively, thereby limiting their capacity to stimulate T cells. More- over, OxPAPC inhibited cytokine production induced by both TLR-2 and CD40L. Thus, oxidative modification of phospholipids leads to the formation of negative regulators of adaptive immune responses through limiting pathogen-induced activation and sub- sequent maturation of DC. Materials and Methods Media and reagents The cell culture medium RPMI 1640 (Invitrogen Life Technologies) was supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% FCS (Sigma-Aldrich). Recombinant human GM- CSF and IL-4 were kindly provided by Novartis Research Institute (Vi- enna, Austria). LPS from Escherichia coli (serotype 0127-B8), poly(I:C), and PGN from Staphylococcus aureus were obtained from Sigma Chemie. Ultrapure LPS (serotype 0111:B4) and Pam3CSK4 were from InvivoGen. *Institute of Immunology, Medical University of Vienna, † Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, and ‡ Ludwig Bolt- zmann Institute for Rheumatology, Vienna, Austria Received for publication June 2, 2004. Accepted for publication April 18, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Wiener Wissenschafts-, Forschungs-, und Technolo- giefond WWTF. 2 Current address: Cardiovascular Research Center, University of Virginia, 409 Lane Road, Charlottesville, VA 22908. 3 Deceased August 30, 2004. 4 Address correspondence and reprint requests to Dr. Johannes Sto ¨ckl, Institute of Immunology, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Aus- tria. E-mail address: Johannes.Stoeckl@meduniwien.ac.at 5 Abbreviations used in this paper: DC, dendritic cell; PAMP, pathogen-associated molecular pattern; PRR, pattern recognition receptor; PGN, peptidoglycan; ox-PL, oxidized phospholipid; (Ox)PAPC, (oxidized) 1-palmitoyl-2-arachidoyl-sn-glycero- 3-phosphorylcholine; TT, tetanus toxoid; Lyso-PC, lysophosphatidylcholine; COX-2, cyclooxygenase-2. The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00