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  • TLR2 Ligands Induce NF-B Activation from EndosomalCompartments of Human MonocytesKarim J. Brandt, Cline Fickentscher, Egbert. K. O. Kruithof, Philippe de Moerloose*

    Division of Angiology and Hemostasis, University Hospital of Geneva and Faculty of Medicine, Geneva, Switzerland

    Abstract

    Localization of Toll-like receptors (TLR) in subcellular organelles is a major strategy to regulate innate immuneresponses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomalcompartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we showthat the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-B-dependent signaling from endosomalcompartments in human monocytes and in a NF-B sensitive reporter cell line, despite the expression of TLR2 at thecell surface. Further analyses indicate that TLR2-induced NF-B activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findingsestablish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-B activation.These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependentsignaling pathways.

    Citation: Brandt KJ, Fickentscher C, Kruithof EKO, de Moerloose P (2013) TLR2 Ligands Induce NF-B Activation from Endosomal Compartments ofHuman Monocytes. PLoS ONE 8(12): e80743. doi:10.1371/journal.pone.0080743

    Editor: Jrg Hermann Fritz, McGill University, CanadaReceived July 18, 2013; Accepted October 6, 2013; Published December 12, 2013Copyright: 2013 Brandt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

    Funding: This work was supported by two grants: the Swiss National Fonds (n310030127639) and the ISTH2007 Presidential Fund. The funders had norole 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: philippe.demoerloose@hcuge.ch

    Introduction

    The innate immune system provides a first line of defenseagainst various pathogens without the requirement of priorexposure to foreign antigens. Among the members of thepattern-recognition receptors, Toll-like receptors (TLR) play acentral role in innate immunity. A fundamental principle thatgoverns all aspects of TLR signal transduction is that themechanisms that ensure the fidelity of signaling are determinedby their cellular localization and selective regulators of TLRsignal transduction [1]. Endocytosis of plasma membranelocalized TLRs was initially thought to attenuate ligand-inducedresponses, but it is now widely accepted that receptorinternalization permits both the propagation of the signalingcascade from endosomal compartments and the generation ofdistinct signaling events [2-4]. Although less is known about theregulators that control TLR endocytosis after microbialdetection, new accumulating evidence indicates that accessoryproteins, such as CD14, are not only critical in ligandrecognition but may also fulfill additional functions [5]. TLR2recognizes various ligands, and makes use of differentmechanisms to provide specificity to each of them. In thiscontext, recognition by receptor heterodimerization and/orendocytosis gives rise to broader ligand specificity [3,6,7]. Inparticular, by association with TLR1 and/ or TLR6, TLR2 is able

    to recognize bacterial triacylated or diacylated lipopeptides [8].Indeed, the role of TLR1 and TLR6, as well as that of CD14, incell activation in response to lipoteichoic acid (LTA) and thesynthetic bacterial lipopeptide Pam3-Cys-Ser-Lys4(Pam3CSK4) is well established [7,9]. Like TLR4, TLR2 hasbeen demonstrated to associate with CD14 to increase cellularresponses to ligands [7,10,11]. CD14 regulates TLR4endocytosis and hence the subsequent endosomal-dependentsignaling pathway [5]. In addition to the TLR4 signaling thattakes place at the plasma membrane through MyD88/TIRAPadaptor proteins leading to early NF-B activation, a secondsignaling event was demonstrated to be initiated from theendosomal compartments through TRAM/TRIF adaptorproteins mediating late NF-B signaling and phosphorylation ofthe transcription factor Interferon Regulatory Factor-3 (IRF3),which in turn regulates type I interferon (IFN) [12]. While anintracellular localization of TLR2 has been observed [3,13,14],its relevance remains to be clearly established in TLR2signaling. Indeed, Nilsen et al. have shown that a dominantnegative form of Dynamin had no effect on TLR2 signalingwhereas several other studies have shown that production ofTNF and IL-6 is altered by inhibition of TLR2 internalization[11,15].

    In this study, we investigated the mechanism mediating theactivation of NF-B in TLR2-activated human monocytes. We

    PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e80743

    http://creativecommons.org/licenses/by/4.0/

  • demonstrate that NF-B activation in primary monocytes inresponse to LTA and Pam3CSK4 requires internalization ofTLR2 through clathrin-coated pits, and that this mechanism isregulated by CD14.

    Materials and Methods

    Ethics statementBuffy coats of blood of healthy donors were provided by the

    Geneva Hospital Blood Transfusion Center. In accordance withthe ethical committee of the Geneva Hospital and with theDeclaration of Helsinki, the blood bank obtained informedconsent from the donors, who are thus informed that part oftheir blood will be used for research purposes.

    ReagentsUltra-pure LPS K12, LPS conjugated-biotin; lipoteichoic acid

    (LTA) from Staphylococcus aureus, Pam3CSK4 conjugated-biotin, blocking peptide against MyD88 and TRIF were fromInvivoGen (San Diego, CA). Pam3CSK4 was from AlexisCorporation (San Diego, CA). IFN was from LaboraoireRoussel et Cie, SNC, Paris. Chloroquine diphosphate,Chlorpromazine hydrochloride, Dynasore and Ammoniumchloride are from Sigma (Sigma, St. Louis, MO). Monoclonalanti-human CD14 blocking antibody, AF488-conjugated mouseanti-human TLR2 and TLR4, anti-human CD32 antibodies werefrom Biolegend. Secondary antibodies were from JacksonImmunoResearch Laboratories, Inc (West Grove, PA).

    Cell cultureMonocytes were isolated from blood buffy coats of healthy

    volunteers as previously described [16]. Monocyte purityroutinely consisted of >90% CD14+ cells,

  • Flow cytometryFor staining of extracellular proteins, cells were stained with

    mouse anti-biotin/ goat anti-mouse-phycoerythrin (Jackson,West Grove, PA). For intracellular staining, cells were activatedas described above, trypsinized, fixed and permeabilized.Staining was assessed with ACCURI C6 flow cytometer (BDBiosciences, San Jose, CA).

    Statistical analysisWhere indicated, significance of differences between groups

    was assessed using Students paired t test. *:p 0.05; **:p 0.005; ***:p 0.0005. All data are represented as mean +/- SDof at least 3 independent experiments.

    Results

    Clathrin-dependent endocytosis controls TNFexpression induced by LTA and Pam3CSK4

    Recent data indicate a key role for the internalization ofTLR4 and TLR2 molecules expressed at the plasmamembrane by a process dependent on clathrin and theGTPase dynamin in the triggering of specific signals from earlyendosomes [2,18]. The previously described localization ofTLR2 ligands within endosomal compartments [18,19]suggests a possible role of endocytic pathways in TLR2signaling. To assess the role of TLR2 internalization in theinduction of TNF in monocytes, cells were treated with differentinhibitors: Chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis; Chloroquine (CHQ), an inhibitor ofendosomal maturation; and Dynasore (Dyn), a specific inhibitorof dynamin which is crucial for pinching off of clathrin-coatedpits and other vesicular trafficking processes. In addition, weused a non-specific inhibitor of vesicular acidification: NH4Cl.This weak base inhibits endosomes maturation by increasingthe vesicular pH [20,21]. As shown in Figure 1 and Figure S1A,CPZ, CHQ and Dyn significantly decreased the expression ofTNF in LTA- and Pam3CSK4-activated monocytes (Figures 1A-B and S1A). The effects of CPZ, CHQ, Dyn and NH4Cl werenot due to direct cytotoxic effects of the drugs on monocytes(Figure S1B), nor to a non-specific inhibition of cellularresponses, because these drugs had no effect on the TNFexpression in IFN-activated monocytes (Figure 1C) meaningthat translation and release of TNF are not affected byendocytosis inhibitors. Our results suggest that internalizationof TLR2 is required to increase TNF expression in LTA- andPam3CSK4-activated monocytes. The mean SD of allexperiments are presented in Table 1. The variations were dueto difference in response of the different monocytespreparations used.

    Clathrin mediated endocytosis controls NF-Bactivation by LTA and Pam3CSK4

    Taking in consideration the primary role of NF-B inregulating monocytic TNF expression in response to LPS[22,23] and the data in Figure 1 suggesting a key function ofendocytosis in favoring TNF production by monocytes, wehypothesized that TLR2 internalization is required for NF-B

    activation. To this end, we took advantage of HEK-Blue2cells which express TLR2, CD14 and a secreted embryonicalkaline phosphatase (SEAP) reporter gene under the controlof a promoter containing five NF-B binding sites. As shown inFigures 2A-C and S1C, the treatment of HEK-Blue2 cellswith CPZ, CHQ, Dyn and NH4Cl decreased the activation ofNF-B in response to TLR2 ligands and subsequent secretionof SEAP.

    As an alternative approach to t