Cellular trafficking of lipoteichoic acid and Toll-like receptor 2 in relation to signaling; role of CD14 and CD36

Nadra J. Nilsen^*, Susanne Deininger, Unni Nonstad^*, Frode Skjeldal, Harald Husebye^*, Dmitrii Rodionov, Sonja von Aulock, Thomas Hartung^,, Egil Lien^¶, Oddmund Bakke and Terje Espevik^*^,1

^* Norwegian University of Science and Technology, Institute of Cancer Research and Molecular Medicine, Trondheim, Norway;
University of Konstanz, Biochemical Pharmacology, Konstanz, Germany;
University of Oslo, Department of Molecular Biosciences, Oslo, Norway;
European Commission of Joint Research Centre, Institute for Health and Consumer Protection, European Centre for the Validation of Alternative Methods, Ispra, Italy; and
^¶ University of Massachusetts Medical School, Department of Medicine, Division of Infectious Diseases and Immunology, Worcester, Massachusetts, USA

¹Correspondence: Institute of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Olav Kyrres gt. 9, N-7489 Trondheim, Norway. E-mail: terje.espevik{at}ntnu.no

Lipoteichoic acid (LTA) is a central inducer of inflammatoryresponses caused by Gram-positive bacteria, such as Staphylococcusaureus, via activation of TLR2. Localization of TLR2 in relationto its coreceptors may be important for function. This studyexplores the signaling, uptake, and trafficking pattern of LTAin relation to expression of TLR2 and its coreceptors CD36 andCD14 in human monocytes. We found TLR2 expressed in early endosomes,late endosomes/lysosomes, and in Rab-11-positive compartmentsbut not in the Golgi apparatus or endoplasmic reticulum (ER).Rapid internalization of fluorescently labeled LTA was observedin human monocytes, colocalizing with markers for early andlate endosomes, lysosomes, ER, and Golgi network. Blocking CD14and CD36 with antibodies inhibited LTA binding and LTA-inducedTNF release from monocytes, emphasizing an important role forboth molecules as coreceptors for TLR2. Importantly, blockingCD36 did not affect TNF release induced by N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysineor LPS. Expression of CD14 markedly enhanced LTA binding tothe plasma membrane and also enhanced NF- ${kappa}$ B activation. LTA internalization,but not NF- ${kappa}$ B activation, was inhibited in Dynamin-I K44A dominant-negativetransfectants, suggesting that LTA is internalized by receptor-mediatedendocytosis but that internalization is not required for signaling.In fact, immobilizing LTA and thereby inhibiting internalizationresulted in enhanced TNF release from monocytes. Our resultssuggest that LTA signaling preferentially occurs at the plasmamembrane, is independent of internalization, and is facilitatedby CD36 and CD14 as coreceptors for TLR2.

Key Words: TLR • LTA • endocytosis