of July 10, 2018.This information is current as

Disrupting TLR2 TriggeringCompromising Bacterial Clearance by Soluble TLR2 Reduces Inflammation without

Nicholas Topley and Mario O. LabétaChristopher H. George, Simon A. Jones, Paul Brennan,Colmont, James Davies, Peter Richards, Barbara Coles, Anne-Catherine Raby, Emmanuel Le Bouder, Chantal

http://www.jimmunol.org/content/183/1/506doi: 10.4049/jimmunol.0802909

2009; 183:506-517; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/183/1/506.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Soluble TLR2 Reduces Inflammation without CompromisingBacterial Clearance by Disrupting TLR2 Triggering1

Anne-Catherine Raby,2* Emmanuel Le Bouder,2,3* Chantal Colmont,* James Davies,*Peter Richards,4* Barbara Coles,* Christopher H. George,† Simon A. Jones,* Paul Brennan,*Nicholas Topley,* and Mario O. Labeta5*

TLR overactivation may lead to end organ damage and serious acute and chronic inflammatory conditions. TLR responses musttherefore be tightly regulated to control disease outcomes. We show in this study the ability of the soluble form of TLR2 (sTLR2)to regulate proinflammatory responses, and demonstrate the mechanisms underlying sTLR2 regulatory capacity. Cells overex-pressing sTLR2, or stimulated in the presence of the sTLR2 protein, are hyporesponsive to TLR2 ligands. Regulation was TLR2specific, and affected NF-�B activation, phagocytosis, and superoxide production. Natural sTLR2-depleted serum rendered leu-kocytes hypersensitive to TLR2-mediated stimulation. Mice administered sTLR2 together with Gram-positive bacteria-derivedcomponents showed lower peritoneal levels of the neutrophil (PMN) chemoattractant, keratinocyte-derived chemokine; lowerPMN numbers; and a reduction in late apoptotic PMN. Mononuclear cell recruitment remained unaffected, and endogenousperitoneal sTLR2 levels increased. Notably, the capacity of sTLR2 to modulate acute inflammatory parameters did not compro-mise the ability of mice to clear live Gram-positive bacteria-induced infection. Mechanistically, sTLR2 interfered with TLR2mobilization to lipid rafts for signaling, acted as a decoy microbial receptor, and disrupted the interaction of TLR2 with itscoreceptor, CD14, by associating with CD14. These findings establish sTLR2 as a regulator of TLR2-mediated inflammatoryresponses, capable of blunting immune responses without abrogating microbial recognition and may inform the design of noveltherapeutics against acute and chronic inflammatory conditions. The Journal of Immunology, 2009, 183: 506–517.

O veractivation or dysregulation of the innate immune re-sponse may lead to end organ damage and serious acuteand chronic inflammatory conditions, such as myocar-

dial dysfunction, respiratory, renal and multiorgan failure, septicshock, arthritis, asthma, and autoimmunity (1, 2).

The TLR family plays a pivotal role in the prompt and effi-cient innate immune recognition of and response to an array ofmicroorganisms and their components, and also in controllingthe activation of the adaptive immune response (3–5). Ten hu-man TLRs have to date been identified (TLR1-TLR10), and theligand specificity described for most of them (3, 4). The effi-cient recognition of most microbial components activating viaTLR2, TLR3, and TLR4 requires the activity of a coreceptor,CD14, which enhances cellular responses substantially (6 – 8).CD14 is expressed as a cell surface molecule, and also as asoluble coreceptor in plasma and other biological fluids (9 –11).

TLR engagement leads to the production of a variety of proin-flammatory, cytotoxic, and immunoregulatory molecules. Thisprocess results in an immediate response to microbial chal-lenge. However, the excessive TLR-mediated release of someproinflammatory molecules, either by overactivation of the re-ceptor or by dysregulation of endogenous TLR-signaling inhib-itory mechanisms, may lead to the aforementioned disease con-ditions. TLR responses therefore have to be tightly regulated tolimit these potentially deleterious consequences.

A number of negative regulatory mechanisms controlling TLRresponses have been reported (1, 12, 13). These include generalmechanisms aimed at the overall reduction in TLR expression andfunction (e.g., receptor compartmentalization, down-modulation,ubiquitinylation, and degradation; activity of anti-inflammatorycytokines such as TGF-� and IL-10), the destruction of the acti-vated cells by apoptosis, the activity of intracellular TLR signalingpathway inhibitors (e.g., MyD88s, IL-1R-associated kinase(IRAK)-M,6 IRAK2c and IRAK2d, suppressor of cytokine signal-ing 1, SARM, PI3K, TOLLIP, and A20), cell membrane-boundTLR suppressors (e.g., ST2, SIGIRR, TRAILR, and RP105), andextracellular soluble decoy microbial receptors (soluble TLRs(sTLRs)) (5).

Natural sTLRs are believed to play a crucial role in preventingthe excessive initial triggering of the membrane-bound TLR andsubsequent TLR overactivation (1, 12, 14–17); however, this

*Department of Medical Biochemistry and Immunology and †Department of Medi-cine, School of Medicine, Cardiff University, Cardiff, United Kingdom

Received for publication September 4, 2008. Accepted for publication May 4, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Wellcome Trust of Great Britain andMedical Research Council U.K.–I3 Interdisciplinary Research Group, Cardiff Uni-versity, Cardiff, U.K.2 A.-C.R. and E.L.B. contributed equally to this study.3 Current address: Institut Curie, Centre de Recherche and INSERM, U653, Paris,F-75248 France.4 Current address: Kuros Biosurgery AG, 8005 Zurich, Switzerland.5 Address correspondence and reprint requests to Dr. Mario O. Labeta, Infection andImmunity, Department of Medical Biochemistry and Immunology, School of Medi-cine, Cardiff University, Henry Wellcome Research Building, Heath Park, CardiffCF14 4XX, United Kingdom. E-mail address: wmdmol@cardiff.ac.uk

6 Abbreviations used in this paper: IRAK, IL-1R-associated kinase; BS3, bis(sulfos-uccinimidyl)suberate; CHO, Chinese hamster ovary; EV, expression vector; FRET,fluorescence resonance energy transfer; HEK, human embryonic kidney; KC, kera-tinocyte-derived chemokine; LBP, LPS-binding protein; m, membrane bound; MNC,mononuclear cell; PMN, polymorphoneutrophil; poly(I:C), polyinosinic-polycytidylicacid; SES, S. epidermidis cell-free supernatant; sTLR, soluble TLR.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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supposition has yet to be proven. In the mouse, a splice variant ofthe TLR4 mRNA coding for a putative partially secreted solubleTLR4 fragment has been described (16). The corresponding cDNAwas found to reduce cell sensitivity to LPS (a TLR4 agonist) whenintroduced in a mouse macrophage cell line. However, it remainsto be established whether this putative soluble TLR4 protein frag-ment is naturally expressed and released by normal mouse cells,and has the capacity to modulate cellular responses in vivo. Inhumans, we have described the existence of a naturally occurringsTLR2 (17), the receptor involved in responses to Gram-positivebacteria and their cell-wall components, as well as a wide range ofother microbial components (3, 6). It was found that sTLR2, whichconsists of most of the TLR2 extracellular domain, is released bynormal monocytes and present in plasma, breast milk (17–19), andsaliva (20, 21). The full extent of sTLR2’s negative regulatorycapacity, the mechanism underlying it, and its biological signifi-cance in vivo have not yet been determined. Other than sTLR2, nonaturally occurring soluble form of a mammalian TLR has beenidentified to date.

In view of the potentially severe pathological conditions thatmay be caused by Gram-positive bacterial infections via TLR2triggering, and the reported modulation of sTLR2 release in dis-ease states (17–21), it is hypothesized that sTLR2 may serve as acritical first-line regulator of TLR2-mediated responses (1, 12, 17,18, 20). In this study, we have therefore sought to fully assess themodulatory capacity of sTLR2 by evaluating its physiological ac-tivity and anti-inflammatory potential in vitro and in vivo, and shedlight on the mechanism underlying sTLR2 regulatory activity. Ourresults show that cells overexpressing sTLR2 are markedly hypo-sensitive to TLR2-mediated stimulation. The regulatory effect wasreproduced by the purified rsTLR2 protein, was TLR2 specific,and affected NF-�B activation, phagocytosis, and superoxide pro-duction. Plasma sTLR2 depletion experiments indicated thatsTLR2 acts as a physiological regulator of TLR2 signaling inPBMC, whereas administration of sTLR2 to mice resulted in mod-ulation of the inflammatory response to Gram-positive bacterialcomponents as well as to live Gram-positive bacteria, but impor-tantly sTLR2 achieved this effect without compromising bacterialclearance. Mechanistically, sTLR2 was found to interfere with theligand-induced mobilization of TLR2 to lipid rafts for signaling,act as a decoy receptor by binding to bacterial lipopeptide andwhole Gram-positive bacteria, and disrupt the close proximity ofmembrane-bound TLR2 and CD14 by interacting with CD14.

Materials and MethodsCells, cell activation, and NF-�B reporter assays

Human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO),mouse RAW264 (American Type Culture Collection), HEK-TLR2, andCHO-CD14 (previously generated in our laboratory (17)) cells were cul-tured in DMEM (HEK293) or RPMI 1640 medium (Invitrogen) supple-mented with 10% FCS (HyClone; �0.06 U/ml endotoxin), 2 mM glu-tamine, 400 �g/ml hygromycin B (HEK-TLR2), 1 mM pyruvate, 0.5%(v/v) NaHCO3, and 50 �g/ml L-proline (CHO-CD14). The human mono-cytic cell line, Mono Mac-6 (provided by H. Ziegler-Heitbrock, Depart-ment of Immunology, University of Leicester, Leicester, U.K.), was cul-tured, as previously described (17). Human monocytes were also prepared,as described (17). Human neutrophil (PMN) preparations were obtainedthrough dextran sedimentation and Ficoll density-gradient centrifugation ofblood from healthy donors. Human peritoneal mesothelial cells were pre-pared and cultured, as described (22). For cell activation experiments (Fig.2), triplicate cell aliquots (1 � 105 cells/well) were cultured in serum-freemedium supplemented or not (Fig. 2, A and B; and monocytes) with500 ng/ml sCD14 (purified from human milk (11)) and stimulated withultra-pure LPS (Escherichia coli O111:B4 strain), heat-killed Listeriamonocytogenes, peptidoglycan, polyinosinic-polycytidylic acid (poly(I:C)), flagellin (all from InvivoGen), the synthetic bacterial lipopeptidePam3-Cys-Ser-(Lys)4 HCl (Pam3Cys; EMC Microcollections), IL-1�,

TNF-� (R&D Systems), or PMA plus ionomycin (Sigma-Aldrich), as de-scribed in Results. Following a 16-h incubation, the supernatants weretested for IL-8 by ELISA (Duoset; R&D Systems). For NF-�B reporterassays, cells were transiently transfected (Lipofectamine; Invitrogen) withconstructs directing expression of the firefly luciferase reporter and theRenilla luciferase. Forty-eight hours posttransfection, the cells were stim-ulated (16 h, 37°C) with Pam3Cys in the absence or presence of purifiedsTLR2, and luciferase activity was measured (Promega). sTLR2-depleted(�90%) AB serum (Fig. 2F) was prepared by sequential immunoprecipi-tation (see below) with the anti-TLR2 Ab, sc8689, or an irrelevant, anti-plexin-C1 Ab, for mock depletion (both from Santa Cruz Biotechnology),as previously described (17). PBMC (2 � 105 cells) were stimulated over-night with Pam3Cys in the absence or presence of 2% sTLR2- or mock-depleted serum.

Overexpression of sTLR2

A sTLR2 fragment consisting of the putative extracellular domain of hu-man TLR2 (Met1-Arg587) with an N-terminal c-Myc tag was constructed.The plasmid pCRII-TOPO-c-myc-TLR2, previously engineered (17), wasused as a template to generate a PCR fragment corresponding to aa 1–587of the TLR2 molecule. The sTLR2 cDNA was subcloned into thepDR2�EF1� expression vector. The recombinant plasmid was transfectedinto HEK-TLR2 cells. Expression and release of sTLR2-Myc by the HEK-TLR2 plus sTLR2 cells were confirmed by Western blotting of culturesupernatants using a rabbit polyclonal anti-TLR2 Ab, TLR2p, generated inour laboratory by immunization with the N terminus 20-mer human TLR2peptide SKEESSNGASLSGDRNGIGK (17) and anti-c-Myc epitope mAbclone 9E10 (Sigma-Aldrich).

Production of human rsTLR2

A TLR2 construct consisting of the putative human TLR2 extracellulardomain (Glu21-Arg587) with a C-terminal His6 tag tail was generated. TheTLR2 cDNA was obtained by RT-PCR using RNA from Mono Mac-6monocytes, and cloned into the pCR II-TOPO cloning vector, as previouslydescribed (17). The plasmid pCRII-TOPO-TLR2 cDNA was used as atemplate to generate a PCR fragment corresponding to aa 21–587 with aC-terminal His6 tag. The resulting sTLR2 cDNA was subcloned into thebaculovirus transfer vector pMELBacB (Invitrogen) in frame to the Hon-eybee Melittin secretion signal. Sf-9 cells were cotransfected with the re-combinant pMELBacB-sTLR2 cDNA transfer vector and Bac-N-BlueDNA by using the Bac-N-Blue Transfection and Expression system (In-vitrogen). High Five cell cultures (Express Five serum-free medium; LifeTechnologies) were infected with the recombinant virus. After 72 h postin-fection, supernatants were cleared by centrifugation, filtered (0.22-�m fil-ters), and concentrated 25 times (CentriconPlus-70; Millipore) beforebuffer exchange to 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole(pH 8.0; binding buffer). The sTLR2-His protein in the concentrated sam-ple was purified by metal-affinity chromatography using Ni-NTA Super-flow resin (Qiagen; 2 h, 4°C, orbital rotation). The protein was eluted byincreasing the concentration of imidazole in the binding buffer to 250 mM.sTLR2-containing fractions were pooled and concentrated, and the bufferwas exchanged to PBS. The protein concentration in the final sample wasdetermined (Dc Protein Assay; Bio-Rad), and the purity of the sTLR2preparation was assessed by 10% SDS-PAGE under reducing condition,followed by Coomassie blue G250 staining (Bio-Rad), as described in Re-sults. The sTLR2 preparation was also analyzed by Western blotting, aspreviously described (17), using either a rabbit polyclonal anti-TLR2 Ab(TLR2p) or an anti-His5 mAb (Qiagen). Typically, 200 �g of purifiedsTLR2 was obtained from 200 ml of High Five cell culture supernatant.The sTLR2 preparations were aliquoted and kept at �85°C until use.

Phagocytosis and superoxide production assays

For phagocytosis experiments, RAW264 macrophages (4 � 105) were re-suspended in 400 �l of binding buffer (phenol red-free RPMI 1640, 1%sodium azide, 2.5% HEPES) and incubated with FITC-labeled Staphylo-coccus aureus (Molecular Probes) at a bacteria:cell ratio of 10:1 for 30 minat either 0°C or 37°C. To test the effect of sTLR2, the bacterial suspensionwas preincubated with 5 �g/ml sTLR2 or BSA for 30 min at 37°C. Fol-lowing binding, cells were washed with cold washing buffer (PBS/1% so-dium azide), resuspended in washing buffer, fixed (2% paraformaldehyde),and analyzed by flow cytometry, as previously described (23). Cell surfacefluorescence was quenched with 125 �g/ml trypan blue before flowcytometry.

For superoxide production assays, 150 �l of assay buffer (13 mMNa2HPO4, 3 mM NaH2PO4, 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4,11 mM dextrose, and 0.71 mM CaCl2 (pH 7.4)) containing 5 �g/mlPam3Cys or heat-killed Staphylococcus epidermidis strain PCI 1200

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(American Type Culture Collection; 5 � 106/well) and 5 �g/ml sTLR2was added in triplicate to microtiter well plates (microlite 2; Thermo Lab-Systems) kept at 37°C. To this mixture, 2 �M Luminol and 2 � 105 PMNresuspended in PBS were added, and the chemiluminescence generatedwas measured at 2-min intervals for 1 h using a fluorometer (FluorstarOptima plate reader; BMG Labtech).

In vivo models of peritoneal inflammation

Inbred 8- to 12-wk-old C57BL/6 mice (Harlan) were maintained underbarrier conditions and pathogen free. All experimental procedures wereconducted under a Home Office project license. Lyophilized S. epidermidiscell-free supernatant (SES) was prepared from suspension cultures of S.epidermidis, isolated from an end-stage renal failure patient under contin-uous ambulatory peritoneal dialysis, as previously described (24). SESpreparations were reconstituted in PBS before in vivo use. Peritoneal in-flammation was induced in mice by i.p. injection of a defined 500 �l doseof SES, SES plus 100 ng of sTLR2, or control PBS. At the indicated timepoints, the animals were sacrificed, and their peritoneal cavities were la-vaged with 2 ml of ice-cold PBS. Leukocyte numbers in the lavages wereassessed by differential cell count on cytospin preparations and Coultercounting (Coulter Z2; Beckman Coulter), or by double staining (anti-F4/80FITC, Serotec; anti-CD11b allophycocyanin, BD Pharmingen), followedby flow cytometric analysis. Chemokine levels in the cell-free peritoneallavage samples were quantified by ELISA (R&D Systems). To test formouse sTLR2 in the peritoneal lavages, equal aliquots were diluted with3� concentrated Laemmli reducing sample buffer and analyzed by West-ern blotting using the TLR2-specific polyclonal Ab, TLR2p. Peritonealinflammation was also induced by i.p. injection (500 �l) of 5 � 107 or 5 �108 CFU S. epidermidis PCI 1200 strain (American Type Culture Collec-tion) in the absence or presence of sTLR2. At the indicated time points,peritoneal cavity lavages were obtained. Blood was obtained by cardiacpuncture. Bacterial CFU were determined by culturing blood and perito-neal lavage samples on Mueller-Hinton agar plates (Oxoid) overnightat 37°C.

Preparation of lipid rafts

Freshly isolated human monocytes (1 � 108 cells) were resuspended inwarm phenol red-free RPMI 1640 medium and stimulated (1 h at 37°C) ornot with 5 �g/ml Pam3Cys in the absence or presence of 5 �g/ml sTLR2.Subsequently, protein solubilization was conducted (1% (v/v) TritonX-100, 150 mM NaCl, 50 mM Tris-HCl, 1 mM PMSF, 1 �g/ml leupeptin,and pepstatin (pH 7.4)) for 1 h at 0°C. Cell lysates (1.5 ml) were mixedwith an equal volume of cold 90% sucrose solution (90% sucrose/50 mMTris-HCl, 150 mM NaCl (pH 7.4)). Samples were overlaid with 7 ml of30%, followed by 2 ml of 5% cold sucrose solutions, and centrifuged at200,000 � g for 16 h at 4°C. One-milliliter fractions were removed fromthe gradient, and 60 �M n-octylglucoside was added to each fraction.Equal fraction aliquots were analyzed by Western blotting using the anti-CD14 mAb, MY4 (Beckman Coulter), or the anti-TLR2 mAb, IMG319(Imgenex). To define the lipid raft-containing fractions in the gradient, dotblots of fraction aliquots were tested with HRP-conjugated cholera toxin B(List Biological Laboratories), followed by ECL (Amersham Biosciences)to reveal the presence of the raft-associated ganglioside, GM1.

Binding assays of sTLR2 to Pam3Cys, LPS, and bacteria

Triplicate wells of microtiter well plates (high binding; Costar) were coated(50 �l) with the amounts of Pam3Cys or LPS, indicated in Results, dis-solved in ethanol. Following solvent evaporation at room temperature, non-specific binding was blocked by incubation (2 h, room temperature) withPBS/1% BSA/5% sucrose/0.05% sodium azide. The plates were thenwashed three times (PBS/0.05% Tween 20) and incubated (2 h, room tem-perature) with 5 �g/ml sTLR2-His, sCD55-His (donated by C. Harris,Cardiff University, Cardiff, U.K.), or LPS-binding protein (LBP; AlexisBiochemicals) diluted in 0.05% Tween 20, 20 mM Trizma base, and 150mM NaCl (pH 7.3) buffer (buffer A) supplemented with 0.1% BSA. Sub-sequently, the wells were washed and incubated (1 h on ice) with an anti-His5 (5 �g/ml; Qiagen) or anti-LBP (1 �g/ml; Hycult Biotechnology) mAbdiluted in buffer A/2% BSA. Following washing, the wells were incubated(1 h on ice) with a biotin-conjugated anti-mouse IgG Ab (DakoCytomation)diluted in buffer A/2% BSA, before washing and incubation (20 min onice) with streptavidin-HRP (Jackson ImmunoResearch Laboratories) di-luted in buffer A/0.5% skim milk. The wells were then washed, and colordeveloped by addition of tetramethylbenzidine substrate (SureBlue;Kirkegaard & Perry Laboratories) was measured at 450 nm.

To test the binding of sTLR2 to bacteria, 5 � 104 heat-killed S. epi-dermidis PCI 1200 strain was resuspended in 100 �l of PBS/0.05% BSAand incubated (30 min at room temperature) with 1 �g of mouse rsTLR2-

human Fc (R&D Systems) or control CD46-Fc fusion protein (provided byC. Harris). Following washing (2� PBS/0.01% sodium azide), sampleswere incubated (0°C, 30 min) with a biotin-conjugated anti-human IgG Ab(0.5 �g; Southern Biotechnology Associates). After washing and fixing(2% paraformaldehyde), streptavidin-allophycocyanin (0.1 �g; SouthernBiotechnology Associates) was added, and the samples were incubated for20 min on ice before washing and analysis by flow cytometry.

Coimmunoprecipitation and chemical cross-linking experiments

The immunoprecipitation technique was as previously described (17). Inthis study, for membrane-bound (m)CD14-mTLR2 coimmunoprecipita-tions, 5 � 106 freshly isolated human monocytes were resuspended inphenol red-free RPMI 1640 medium and incubated (30 min at 37°C) in thepresence of 5 �g/ml sTLR2 or 10 �g/ml BSA. After washing and lysis (1%(v/v) Nonidet P-40, 50 mM Tris-HCl, 150 mM NaCl, 1 �g/ml leupeptinand pepstatin, 1 mM PMSF (pH 7.4) buffer), the cell lysate was preclearedby successive incubations with the following: 80 �l of protein G-Sepharose(50% suspension; Sigma-Aldrich), 4 �g of the isotype-matched control,mouse IgG2b, and protein G-Sepharose. The precleared samples wereincubated (1 h, 4°C) with 5 �g of the anti-CD14 mAb, MY4, and theimmunocomplexes were precipitated with 50 �l of protein G-Sepharose.Following washing, samples were analyzed by Western blotting with theanti-TLR2 mAb, IMG319. For chemical cross-linking experiments, 5 �106 cells were resuspended in 500 �l of cold phenol red-free RPMI 1640medium and incubated with 5 �g of sTLR2 (sTLR2-His) or the irrelevantHis-tagged protein sCD55 for 30 min at room temperature. Followingwashing (cold RPMI 1640), 3 mg/ml membrane-impermeable and non-cleavable cross-linker, bis(sulfosuccinimidyl)suberate (BS3; Pierce), wasadded to the samples, and the mixture was incubated for an additional 30min at room temperature. Cross-linking was stopped by the addition of 10mM Tris-HCl (pH 7.4) buffer and incubation on ice for 15 min. The cellswere then lysed, and cell lysates were incubated (2 h, 4°C, orbital rotation)with Ni-NTA beads (10 �l of beads/100 �l of lysate). The beads werewashed, and the protein was eluted with Laemmli reducing sample buffercontaining 250 mM imidazole. The eluate was analyzed by 7.5% SDS-PAGE and Western blotting using anti-CD14 (69.4, rabbit polyclonal Ab(11)) or anti-TLR2 (sc8689) Abs.

Fluorescence resonance energy transfer (FRET) measurements

For FRET measurements, freshly isolated monocytes were allowed to ad-here (1 h; 37°C) to multispot slides (Shandon Multispot; Thermo Electron)in the absence or presence of 5 �g/ml sTLR2 or BSA in phenol red-freeRPMI 1640 medium. The slides were then incubated with 20% normalrabbit serum for 15 min at room temperature before washing and labeling(1 h, 0°C) with the anti-CD14 mAb My4-Cy3 or its isotype-matchedIgG2b-Cy3 control (acceptor fluorophore; 0.25 �g/spot). Both Abs (Beck-man Coulter) were Cy3 conjugated using the FluoroLink mAb Cy3 label-ing kit (GE Healthcare). Cell labeling was performed in the absence orpresence of 5 �g/ml sTLR2 or BSA. The slides were then washed (2�PBS/0.01% sodium azide), fixed (2% paraformaldehyde), and, followingwashing, stained with an Alexa 488-conjugated anti-TLR2 (T2.5; eBio-science) or anti-C3aR (hC3aRZ1; Serotec) mAb (donor fluorophore), asdescribed above for MY4-Cy3. After washing and fixing, the slides weremounted (Vectashield; Vector Laboratories). FRET was measured by therelease of quenched donor fluorescence after acceptor photobleaching us-ing a previously described technique (25). In this technique, the donorfluorescence intensity before and after acceptor photobleaching in the samecell sample is compared. FRET efficiency was quantified by the following:E (%) � ((IDA � IDB)/IDA) � 100, where E represents percentage of FRETefficiency; IDA and IDB, the donor’s intensity after and before photobleach-ing of the acceptor, respectively. In each cell to be analyzed, FRET effi-ciency was determined typically in four to five regions of the plasma mem-brane with different donor intensity. For each experiment, a minimum of200 cells per condition was analyzed. E values from all regions of interestwere averaged. The Cy3 acceptor fluorophore was bleached by repeatedexcitation (50 times for a total of 2 min), and the bleaching was �20% andup to 100% (depending on the experiment and the region of interest). Thebleaching conditions were set to avoid bleaching the donor fluorophore.Cells were imaged using the Leica TCS SP2 resonant scanning confocalsystem (Leica Microsystems). Signal-to-noise ratio was improved by re-cording images using the frame averaging method (average of 2 frames).The donor fluorophore was excited at 488 nm, and emission was detectedbetween 498 and 540 nm. The acceptor fluorophore was excited at 543 nmand detected between 551 and 669 nm. Under these conditions, negligiblefluorescence was observed from an Alexa 488-labeled specimen within theCy3 emission spectrum, and from a Cy3-labeled specimen within the Alexa488 emission spectrum. The validity of each FRET dataset was confirmed

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by the lack of correlation between E% and acceptor or donor fluorescenceintensity (data not shown). This suggested that the FRET values observedbetween mTLR2 and mCD14 were not dependent on acceptor or donordensity, and thus resulted from genuine protein-protein interactions and notfrom randomly associated molecules (26, 27).

Statistics

Statistical analysis of the data was performed by using paired Student’s ttest (Minitab 15 statistical software). Values of p of less than 0.05 wereconsidered significant.

ResultssTLR2 renders cells hyposensitive to TLR2-mediated stimulation

To assess the negative regulatory capacity of sTLR2, we engi-neered a soluble form of human TLR2 consisting of its full extra-cellular domain, thus resembling the main naturally occurringsTLR2 form found in plasma and milk (17). HEK293 cells stablyexpressing either the mTLR2 receptor (HEK-TLR2) or bothmTLR2 and sTLR2, the latter tagged at the N terminus with a Mycepitope (HEK-TLR2 � sTLR2), were generated. Initial analysisby Western blotting and flow cytometry confirmed that the engi-neered sTLR2 protein was secreted into the medium by HEK-TLR2 plus sTLR2 cells, and that the HEK-TLR2 plus sTLR2 andHEK-TLR2 cells expressed similar levels of mTLR2 (Fig. 1, A andB). A C terminus His-tagged human sTLR2 protein was also en-gineered and purified from insect cell culture supernatants. Thepurity of the sTLR2 preparation was assessed by 10% SDS-PAGE(reducing conditions), followed by Coomassie blue staining. Par-allel samples were also analyzed by Western blotting using eitheranti-TLR2 or anti-His5 Abs (Fig. 1, C and D). Coomassie stainingand Western blots showed a major 72- to 75-kDa sTLR2 band.Minor sTLR2 bands of �83 and 90 kDa, whose intensity dependedon the preparation, were also detected, mainly with the anti-TLR2Ab. sTLR2 preparations were estimated to be 85–95% pure (de-pending on the preparation) and mostly (�95%) monomeric. Someof the low-intensity �75-kDa bands detected by Coomassie stain-ing may correspond to sTLR2 degradation products, because theycan also be detected by either the anti-TLR2 or anti-His Abs.

The HEK-TLR2 plus sTLR2 cells were found to be markedlyinsensitive to stimulation with different doses of the TLR2 agonistsynthetic bacterial lipopeptide Pam3CysSer(Lys)4 (Pam3Cys), asjudged by the release of the proinflammatory chemokine IL-8(CXCL8) (Fig. 2A, left). The negative effect was sTLR2 concen-tration dependent, because HEK-TLR2 cells transiently transfectedwith increasing amounts of the cDNA encoding sTLR2 showed aconcomitant progressive reduction in sensitivity (Fig. 2A, right).TLR2 signaling inhibition by sTLR2 was not limited to Pam3Cysstimulation, because cell activation induced by another TLR2 ag-onist, peptidoglycan, and by the whole Gram-positive bacteriumheat-killed L. monocytogenes, was also affected (Fig. 2B).

The purified rsTLR2 protein also showed negative regulatorycapacity. The inhibitory effect of rsTLR2 was observed in HEK-TLR2 cell transfectants, human monocytes, PBMC (data notshown), and (mTLR2�) peritoneal mesothelial cells (Fig. 2C). Thelatter cells play a pivotal role during the course of a peritonealinfection, like the one studied in this work (see below), by secret-ing chemokines that regulate leukocyte infiltration into the perito-neal cavity and by expressing adhesion molecules (22, 28). Fig. 2Cshows that release of IL-8, the archetypal human PMN chemoat-tractant, by mesothelial cells stimulated with Pam3Cys or a cell-free supernatant prepared from the Gram-positive bacterium, S.epidermidis (termed SES), was reduced in the presence of sTLR2,suggesting that during peritoneal infections sTLR2 may also targetmesothelial cells for negative regulation. The effect of sTLR2 wasnot limited to modulation of IL-8 release: the Pam3Cys-driven

trans activation of the transcription factor NF-�B was markedlyinhibited, indicating that sTLR2 has a wide spectrum of effects(Fig. 2D).

The specificity of the sTLR2 inhibitory effect was evaluatednext. We tested whether sTLR2 influences monocyte activationinduced by suboptimal doses of the TLR agonists, viral dsRNAmimic poly(I:C) (TLR3), LPS (TLR4), and flagellin (TLR5). Inaddition, the effect of sTLR2 on signaling via the IL-1R (whichshares with TLRs the MyD88-dependent signaling pathway), theTLR-nonrelated receptor TNFR, and nonreceptor-mediated cell

FIGURE 1. Expression and purification of human rsTLR2. A, Detectionof sTLR2 in HEK-TLR2 plus sTLR2 culture supernatants (2 � 106 cells)by Western blotting with the anti-TLR2 rabbit Ab, TLR2p, or the anti-cMyc epitope mAb, 9E10 (HEK-TLR2 � sTLR2 cells express an N ter-minus c-Myc-tagged sTLR2 protein). For control experiments, culture su-pernatants from HEK-TLR2 plus empty expression vector (EV) celltransfectants were tested. B, Fluorescence profiles of mTLR2 expression inHEK-TLR2 and HEK-TLR2 plus sTLR2 cell transfectants stained with thePE-conjugated anti-TLR2 mAb, TL2.1, or the isotype-matched controlIgG. C and D, Coomassie blue staining (C) and Western blot (D) pattern ofpurified His-tagged rsTLR2 following production by insect cells, purifica-tion by Ni-NTA chromatography, and 10% SDS-PAGE (reducing condi-tions). For Western blots, an anti-His5 mAb and the anti-TLR2 Ab, TLR2p,were used.

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stimulation (PMA � ionomycin) was also tested. Fig. 2E showsthat only TLR2-mediated monocyte activation was inhibited bysTLR2, indicating that sTLR2 targets monocyte TLR2 signalingspecifically.

To evaluate the physiological relevance of the negative regula-tory capacity of sTLR2, we compared the sensitivity of PBMC tostimulation via TLR2 in the presence of serum that had been de-pleted of naturally occurring sTLR2 (�90%) with that of PBMCstimulated in the presence of mock-depleted serum (Fig. 2F). Re-ducing the amount of serum sTLR2 resulted in a significant in-crease in cell sensitivity to TLR2-mediated stimulation. This resultconfirmed previous findings (17, 20), and suggested that naturallyoccurring sTLR2 may play an important immunomodulatory rolein controlling TLR2-mediated activation in vivo.

Phagocytosis and superoxide production can be affected bysTLR2

To extend the assessment of the negative regulatory potential ofsTLR2, we tested the capacity of sTLR2 to affect pathways asso-

ciated with bacterial killing, namely phagocytosis and superoxideproduction. RAW264 macrophages were used to test macrophagephagocytic capacity in the absence and presence of sTLR2. Thebinding and phagocytic uptake of fluorescent bacteria were testedat 0°C and 37°C, respectively, in the presence and absence oftrypan blue, to quench cell surface fluorescence. In this way, theamount of bacteria bound (0°C, trypan blue-sensitive fluorescence)and phagocytosed (37°C, trypan blue-resistant fluorescence) bymacrophages was evaluated separately. To assess the full potentialof sTLR2 as a regulator of the phagocytic process, the experimentswere performed in serum-free medium, thereby excluding the con-tribution of Fc and/or complement receptors. Fig. 3A shows thatsTLR2 interfered strongly with the macrophage binding (0°C) ofGram-positive bacteria, S. aureus, while having a comparativelymodest effect on phagocytosis (37°C). This effect on phagocytosiswas, most likely, a consequence of the marked effect on bacterialbinding. At 37°C, the activity of phagocytic receptors (e.g., scav-enger receptors, C-type lectins) most likely compensates for theinterfering effect of sTLR2.

FIGURE 2. sTLR2 renders cellshyposensitive to TLR2-mediatedstimulation. A and B, IL-8 levels inculture supernatants of HEK293 cellsstably expressing mTLR2 (HEK-TLR2), mTLR2 and sTLR2 (HEK-TLR2 � sTLR2), the empty vector(HEK-EV) (A, left panel), or HEK-TLR2 cells transiently transfectedwith EV or sTLR2 cDNA (A, rightpanel) or 250 ng of sTLR2 cDNA(B), and stimulated, as indicated. Re-sults are means � SD of one experi-ment representative of four (A) orthree (B). The differences in IL-8 re-lease between sTLR2-expressingcells and HEK-TLR2 or HEK-TLR2plus EV were significant: ���, p �0.0001. C–E, Cells were stimulatedwith the indicated concentrations ofPam3Cys or 200 ng/ml Pam3Cys (E),dilutions of SES, 80 �g/ml poly(I:C),10 ng/ml LPS, 5 �g/ml flagellin, 5ng/ml IL-1�, 10 ng/ml TNF-�, or 50ng/ml PMA plus 500 ng/ml inonomy-cin in the absence or presence of 5�g/ml sTLR2. For NF-�B reporterassays, cells transiently transfectedwith firefly and Renilla luciferase re-porter plasmids were stimulated withPam3Cys, followed by luciferase ac-tivity measurements. Results are ofone experiment (�SD) representativeof at least three (�, p � 0.05; ���, p �0.0001 sTLR2 vs control). F, IL-8levels released by Pam3Cys-stimu-lated PBMC in the absence or pres-ence of sTLR2- or mocked-depleted2% AB serum. Results are from oneexperiment performed in triplicates(�SD) representative of four (�, p �0.05; ��, p � 0.01; sTLR2 vs mock-depleted serum).

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Freshly isolated human PMN were used to test the effect ofsTLR2 on microbial-induced superoxide production. In the pres-ence of sTLR2, the capacity of PMN to generate superoxide overtime in response to either the Pam3Cys lipopeptide or whole heat-killed S. epidermidis was substantially reduced (Fig. 3B).

Collectively, the results shown in Figs. 2 and 3 demonstrated thepotential of sTLR2 to negatively regulate TLR2-mediated cell sig-naling and effector functions that are critical during microbialinfection.

sTLR2 affects early leukocyte recruitment and endogenoussTLR2 release in a mouse model of peritoneal inflammation

To evaluate the biological activity of sTLR2 and assess its po-tential as a modulator of inflammation in vivo, we first testedthe effect of sTLR2 on a well-established mouse model of acuteperitoneal inflammation (24, 29). In this model, the progression

of a clinical bacterial peritonitis episode typically seen in end-stage renal failure patients on continuous ambulatory peritonealdialysis is mimicked by the peritoneal injection of a previouslydefined dose of the cell-free supernatant SES, derived from cul-tures of S. epidermidis, the main causative pathogen of this typeof peritonitis (30). Intraperitoneal administration of SES tomice resulted in a rapid and transient increase in the peritoneallevels of the PMN chemoattractants, keratinocyte-derived che-mokine (KC), and MIP-2, murine functional counterparts ofhuman IL-8 and growth-related oncogene-� (CXCL1), withpeak levels occurring at 1 h postinjection (Fig. 4A). Corre-sponding determinations of PMN numbers recruited to the peri-toneal cavity showed peak levels at 2–3 h (depending on theexperiment) after SES administration (Fig. 4B). The simulta-neous administration of SES and sTLR2 (100 ng) resulted inreduced levels of PMN chemoattractants. These levels were sig-nificantly reduced in the case of KC, but not MIP-2 (Fig. 4A).Consistent with the inhibitory effect on PMN chemoattractants,sTLR2 administration resulted in a marked reduction in PMNnumbers recruited to the peritoneum either over the whole timecourse or at the peak of their influx (Fig. 4B). The effect ofsTLR2 on the SES-induced peritoneal levels of the mononu-clear cell (MNC) chemoattractant, MCP-1 (MCP-1/CCL2), andon the relatively late recruitment of MNC, responsible for theremoval of the apoptotic PMN, was also tested (Fig. 4C). Underthese conditions, sTLR2 was found to exert a positive and sig-nificant effect on MCP-1 levels over the time period post-SESinjection. Total MNC recruitment, however, was not found tobe affected.

The suppressive effect of sTLR2 on early (PMN), but not late(MNC), leukocyte recruitment posed the question of whethersuch a disproportionate leukocyte influx influences PMN sur-vival and thus inflammatory resolution. We compared the apo-ptotic status of PMN at the peak of their peritoneal influx inSES-challenged mice with that in mice challenged with SESplus sTLR2 (Fig. 4D). Profile comparison of the annexin V/pro-pidium iodide scatter plots showed no difference in the propor-tion of early apoptotic PMN (lower right quadrant) betweenSES- and SES plus sTLR2-treated mice. Examination of theproportion of late apoptotic/early necrotic PMN (upper rightquadrant), however, showed a marked and significant reduction(�50%) of their numbers in the SES plus sTLR2-treated mice,suggesting a more efficient clearance of the dying PMN by theMNC in these animals.

The effect of administering sTLR2 together with SES to mice onthe levels of endogenous (mouse) sTLR2 in the peritoneal lavagefluid was also tested, because we and others have demonstratedthat sTLR2 release is affected by cell activation and infection (17,18, 21). The detection of endogenous sTLR2 was facilitated by theabsence of exogenous sTLR2 (sTLR2-His) in the peritoneal la-vages (data not shown). At 1 h postinjection, no differences in thelevels of sTLR2 between SES- and SES plus sTLR2-challengedmice were observed (Fig. 4E). At 3 h, i.e., when PMN influx washigh, mouse sTLR2 levels in the peritoneal lavages of the sTLR2-treated mice were found increased. By 6 h postinjection, sTLR2levels between sTLR2-treated and nontreated mice were compa-rable and similar to those at the 1-h time point. This finding sug-gested that the administration of sTLR2 together with SES induceda positive feedback for the release of sTLR2, resulting in tran-siently higher local concentrations of endogenous sTLR2, whichmay well contribute to maintaining its regulatory effect oninflammation.

FIGURE 3. Phagocytosis and superoxide production can be affected bysTLR2. A, Extent of FITC-labeled bacteria bound (0°C) or phagocytosed(37°C) by RAW264 macrophages preincubated or not with 5 �g/ml sTLR2or an irrelevant protein (BSA, 2� sTLR2 molarity), as determined by flowcytometry. To distinguish between cell surface-bound and phagocytosedbacteria, the cell surface fluorescence was quenched with trypan blue be-fore flow cytometric analysis. Results are of one representative of threeindependent experiments. B, Luminol-dependent chemiluminescence gen-erated by superoxide produced over the time by triplicate cultures of hu-man PMN stimulated with 5 �g/ml Pam3Cys or 5 � 106 heat-killed S.epidermidis in the absence or presence of 5 �g/ml sTLR2. Results are fromone representative experiment of four.

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sTLR2 reduces peritoneal PMN infiltration withoutcompromising bacterial clearance

The in vitro and in vivo anti-inflammatory effects of sTLR2 de-scribed previously raised the question of whether such effectswould be detrimental to bacterial clearance during infection. Toaddress this issue, an experimental model of acute peritoneal in-flammation consisting of an i.p. challenge with 5 � 107 CFU of S.epidermidis in the absence or presence of sTLR2 (100 ng) wasused first. At this bacterial inoculum, the infection almost com-pletely cleared by 12 h. PMN numbers in the peritoneum of miceinjected with S. epidermidis showed peak levels at 12 h postinjec-tion (Fig. 5A, inset). In the presence of sTLR2, peritoneal PMNaccumulation at the peak time of their influx was significantlyreduced (Fig. 5A). Such reduced early PMN influx did not, how-

ever, affect the capacity of the mice to clear the infection, becauseno difference in bacterial load either in the peritoneal cavity (Fig.5B) or blood (Fig. 5C) between sTLR2-treated and -nontreatedmice was observed. To study the inflammation-modulating effectof sTLR2 further, mice were injected with a higher dose of S.epidermidis (5 � 108 CFU), and the effect of increasing doses ofsTLR2 (10–1000 ng) was tested (Fig. 5, D–F). All doses of sTLR2tested showed a similar suppressive effect on the PMN numbersrecruited to the peritoneal cavity over the 18-h period postinjection(Fig. 5D). This effect was most significant at the peak time (12 h)of PMN influx. Despite this significant reduction in local PMNnumbers, bacterial clearance from the peritoneal cavity was notnegatively affected by sTLR2 treatment (Fig. 5E). All mice(sTLR2 treated and nontreated) showed a marked reduction in

FIGURE 4. sTLR2 affects earlyleukocyte recruitment and endoge-nous sTLR2 release in a mouse peri-toneal inflammation model. A–E,Mice were injected i.p. with a defineddose of SES, SES plus 100 ng ofsTLR2, or PBS. At each time point,chemokine expression, PMN, andMNC numbers in the peritoneal la-vages were determined (A–C). Cellnumbers were determined by differ-ential cell counts on cytospin prepa-rations (B, right; results from four in-dependent experiments) or leukocyteswere double stained with anti-F4/80and anti-CD11b mAbs and analyzedby flow cytometry (B and C, timecourses). Values in A–C are ex-pressed as the mean � SEM (n �5/condition; �, p � 0.05; ��, p �0.01; ���, p � 0.0001; SES � sTLR2vs SES). D, Annexin V/propidium io-dide staining of leukocytes present inthe lavages at the peak time of PMNinflux (shown, 3 h). The representa-tive scatter plots are from analyses ofgated PMN. Apoptotic cells wereidentified according to the annexinV�/PI� (lower right quadrant, earlyapoptosis) and annexin V�/PI� (up-per right quadrant, late apoptosis/ne-crosis) staining. Percentage of cells inthe apoptotic quadrants is shown(mean � SEM, n � 5/condition; ���,p � 0.0001; significant reduction vsSES). E, Western blot of peritoneallavages taken at the indicated timesand tested for mouse sTLR2(msTLR2) release. Densitometricscanning of msTLR2 levels at thepeak of PMN influx is shown (right;n � 5/condition; �, p � 0.05; SES �sTLR2 vs SES).

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bacterial load between 3 and 6 h postinjection. In sTLR2-treatedmice, there was an apparent increase in bacterial clearance; how-ever, this is most probably not physiologically significant, becauseit was very modest in magnitude. In the peripheral circulation,although the mice treated with the highest dose of sTLR2 (1 �g)showed increased bacterial load at the earliest time (3 h) postin-jection (Fig. 5F), this effect does not appear to be physiologicallysignificant, because none of the animals showed peripheral bacte-rial abscesses, symptoms of shock, or died from the infection, andall animals had cleared the blood infection almost completely by6 h postinjection. Moreover, plasma levels of the acute-phase re-actant, serum amyloid-A, were similar in control (S. epidermidisonly), and sTLR2-treated mice at all time points examined (datanot shown), indicating that the relatively small increase in bacterialload in the bloodstream of 1 �g of sTLR2-treated mice was notsignificant enough to impact on the level of the systemic acute-phase response.

sTLR2 disrupts the interaction of mCD14 with mTLR2, acts as adecoy receptor, and associates with mCD14

We next examined the mechanism underlying the regulatorycapacity of sTLR2. We first tested whether sTLR2 affected theligand-induced clustering of mCD14 and mTLR2 in lipid rafts.The ligand-induced mobilization of TLR2 and TLR4 to lipidrafts and their close proximity to CD14, which resides mainly in

the rafts, are believed to be critical to signaling (27, 31–33).Analysis of lipid raft preparations from freshly isolated non-stimulated (control) monocytes (Fig. 6A) confirmed the prefer-ential association of CD14 with lipid rafts and TLR2 withdetergent-soluble (nonraft) fractions (31, 32). Pam3Cys stimu-lation resulted in an enrichment of TLR2 in lipid rafts and re-duced levels of CD14, most likely as a consequence of theactivation-induced shedding of soluble CD14 (9). However,when cells were stimulated in the presence of sTLR2, the pat-tern of CD14 and TLR2 partition into membrane domains re-sembled that in nonstimulated cells (Fig. 6A), indicating thatsTLR2 interferes with the ligand-induced TLR2 mobilization tolipid rafts for signaling, and consequently, with the approxima-tion of TLR2 to CD14 in the rafts.

A decoy receptor activity would explain, at least in part, suchinterference by sTLR2. We tested this possibility and found thatsTLR2 specifically binds Pam3Cys lipopeptide (Fig. 6B, leftpanel) in a ligand concentration-dependent manner, confirmingprevious reports (34 –36). We also tested for a possible inter-action of sTLR2 with whole bacteria. A sTLR2-Fc fusion pro-tein, but not an irrelevant control, specifically bound heat-killedS. epidermidis (Fig. 6B, right) as well as S. aureus (data notshown). These findings indicated the potential of sTLR2 to actas a decoy receptor; this activity may contribute to sTLR2’snegative regulatory capacity.

FIGURE 5. sTLR2 reduces perito-neal PMN infiltration without com-promising bacterial clearance. A–F,Mice (n � 5/condition) were i.p. in-oculated with 5 � 107 (A–C) or 5 �108 (D–F) CFU S. epidermidis aloneor together with 100 ng (A–C) or theindicated amounts of sTLR2. At theindicated times, mice were sacrificed,the peritoneal cavity was lavaged, andPMN numbers in the lavages (A andD) were determined by differentialcell counts on cytospin preparations.Bacterial titers in the peritoneal fluidand blood (B, C, E, and F) were de-termined, as described in Materialsand Methods. Values in A and D–Fare expressed as the mean � SEM(n � 5/condition; �, p � 0.05; ��,p � 0.01; ���, p � 0.0001; S. epi. �sTLR2 vs S. epi.).

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We speculated that sTLR2 might also disrupt the close prox-imity of mCD14 to mTLR2 directly, i.e., in the absence ofligand, by interacting with mTLR2 and/or mCD14. We testedthis possibility by first examining the effect of sTLR2 on theligand-independent natural association of mTLR2 with mCD14in the detergent-soluble fractions of normal human monocyte

cell lysates. The typical �110-kDa mTLR2 polypeptide band(17) was consistently detected by Western blotting in mCD14immunoprecipitates from monocyte cell lysates (Fig. 6C, lefttrack). In addition, TLR2 polypeptide bands most likelycorresponding to an intracellular (�95-kDa) glycoform of themature protein (37) and to fully glycosylated (�83-kDa) and

FIGURE 6. sTLR2 disrupts mTLR2 triggering. A, mCD14 and mTLR2 partitioning into lipid raft and nonraft fractions following monocyte stimulationwith Pam3Cys � sTLR2. Dot blots (top) show the position of the raft marker, GM1 ganglioside. Results are from five independent experiments. B, Left,Binding of sTLR2-His to Pam3Cys-, but not to LPS-coated wells. Control assays show no binding of sCD55-His to Pam3Cys, and binding of LBP to LPS.For detection, anti-His5 or anti-LBP mAbs, anti-mouse IgG biotin, streptavidin-HRP, and substrate were used. Right, Analysis of sTLR2-Fc or CD46-Fcbinding to S. epidermidis following detection with anti-IgG biotin and streptavidin-allophycocyanin. Results are from six (Pam3Cys) or five (S. epidermidis)experiments. C, Western blots of CD14 immunoprecipitates following monocyte incubation with sTLR2 or BSA. Monocytes from four donors gaveidentical results. H, Ig H chain. D, FRET analysis on monocytes labeled with the anti-CD14 mAb, MY4-Cy3 (acceptor), and anti-TLR2 mAb, TL2.5-Alexa488 (donor). Monocytes were preincubated and labeled in the absence or presence of sTLR2 or BSA. Threshold for significant FRET was determined withan anti-C3aR-Alexa488 mAb used as FRET donor-negative control. Results are from four experiments. E, Chemical cross-linking (BS3) in cells (5 � 106)incubated with 5 �g of sTLR2-His or sCD55-His. Cross-linked His-tagged proteins in cell lysates were Ni-NTA pulled down and analyzed by Westernblotting. Head arrows point at Ni-NTA-precipitated, CD14-cross-linked polypeptides. Left panel, Mobility of sTLR2 (5 �g) and mCD14 monomers pulleddown (sTLR2) or immunoprecipitated (mCD14) from High Five cell culture supernatants or 5 � 106 CHO-CD14 transfectants, respectively. Right panel,right lane, Control immunoprecipitation of mTLR2. Results are from four experiments.

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intracellularly located sTLR2 (17) were detected. The �110-kDamTLR2 polypeptide band was not detected when the coimmuno-precipitation experiments were performed following preincubationof monocytes with sTLR2 (Fig. 6C, center track), indicating thatsTLR2 interfered with the natural interaction of mCD14 withmTLR2, and that such interference takes place at the cell surface.We obtained confirmatory evidence of this interference by per-forming FRET studies. FRET was used because it allows for theevaluation of interactions between neighboring (colocalized) mol-ecules by determining their proximity within �10-nm range (26).In this study, FRET efficiency for the transfer of energy from theanti-TLR2 Alexa488 (donor) mAb to the anti-CD14 Cy3 (accep-tor) mAb, used to label mTLR2 and mCD14, was measured. Anincrease in TLR2 (green, donor) fluorescence after CD14 (red,acceptor) photobleaching was detected in monocytes (Fig. 6D,center), indicating energy transfer and, thus, close proximity be-tween the two molecules. This was in agreement with the results ofthe coimmunoprecipitation experiments. In the presence of sTLR2,however, no increase in TLR2 fluorescence after CD14 photo-bleaching was observed, and FRET efficiency between TLR2 andCD14 was reduced to almost background levels (i.e., E% � 2.9 �0.5, threshold for significant energy transfer defined with an anti-C3aR Alexa488 mAb used as FRET donor-negative control; Fig.6D, right). These findings thus confirmed that sTLR2 perturbs themCD14-mTLR2 interaction.

The interfering effect exerted by sTLR2 in the absence of ligandraised the question of whether this effect results from an interactionof sTLR2 with mCD14 and/or mTLR2. To address this issue, weperformed chemical cross-linking experiments by using a non-cleavable, membrane-impermeable, cross-linking reagent, BS3

(Fig. 6E). The purified (Ni-NTA pulled-down) rHis-tagged sTLR2protein (sTLR2-His) and mCD14, immunoprecipitated from CHO-CD14 cell transfectants, showed expected sizes of �72–75 kDaand �54–56 kDa, respectively, when analyzed by SDS-PAGE,followed by immunoblotting with specific Abs (Fig. 6E, leftpanel). Incubation of CHO-CD14 transfectants with sTLR2-His,followed by chemical cross-linking, Ni-NTA bead pull-down fromthe CHO-CD14 cell lysates, and anti-CD14 or anti-TLR2 Westernblotting revealed bands of �125–130 kDa and �250–260 kDa,i.e., of lower mobility than that of mCD14 (Fig. 6E, center panel).The size of these bands was consistent with that estimated formCD14/sTLR2 heterodimers (�126–131 kDa) and mCD14/sTLR2 dimer of dimers (�252–262 kDa). To test for an interactionof sTLR2 with mTLR2, a similar cross-linking strategy was ap-plied to CHO-mTLR2 cell transfectants preincubated with sTLR2-His. In this study, however, Ni-NTA bead pull-down, followed byanti-TLR2 immunoblotting, did not show any cross-linked TLR2polypeptide band (Fig. 6E, right panel).

Together, these findings indicated that sTLR2 may exert nega-tive regulatory effects by acting as a decoy receptor, and also bydisrupting the close proximity between the coreceptor (CD14) andthe receptor (TLR2) that is crucial to highly efficient signaling.Such disruption most likely results from the capacity of sTLR2 tointeract with the coreceptor.

DiscussionFollowing the initial description of the crucial involvement ofTLRs in acute inflammation and septic shock and the more recent,well-documented observations implicating TLRs in a number ofautoimmune and chronic inflammatory diseases, such as lupus,arthritis, inflammatory bowel disease, and artherosclerosis, it hasbecome clear that TLR overactivation plays a prominent role in thepathogenesis of a variety of acute and chronic inflammatory con-ditions (2). The different levels at which TLR activity can be reg-

ulated highlight the importance of such regulation to the mainte-nance of immune homeostasis. sTLR2 is the only soluble form ofa mammalian TLR to date identified that occurs naturally, becauseit is constitutively released by normal monocytes, and present innormal human plasma, breast milk (17–19), saliva (20, 21), mouseperitoneal lavage fluids (this study), and plasma, as well as bovineand porcine plasma (J. Rey-Nores, unpublished data). It has beenproposed that sTLR2 may protect the host from excessive initialtriggering of TLR2, which may result in deleterious TLR2-medi-ated innate immune responses (1, 12, 17, 18, 20). The full extentof sTLR2’s regulatory capacity, the mechanism(s) underlying it,and its biological relevance in vivo have not, however, been ad-dressed to date. In this study, we demonstrated that sTLR2 regu-lates TLR2-mediated cellular responses induced by microbialcomponents and whole Gram-positive bacteria in vitro and in vivo,and that sTLR2 also has the potential to modulate critical effectorfunctions, namely phagocytosis and superoxide production. Twomechanisms contributing to such regulatory activity were identi-fied: first, the capacity of sTLR2 to act as a decoy microbial re-ceptor, and second, its capacity to disrupt the interaction of TLR2with its coreceptor by binding to CD14. The physiological rele-vance of such elaborate negative regulation is highlighted in ex-periments that demonstrate the hypersensitivity of PBMC to li-popeptide stimulation in the presence of sTLR2-depleted serum.These findings indicate that regulation by naturally occurringsTLR2 is a physiological feature that contributes to a controlled,yet efficient, host innate immune response against microbialpathogens.

To assess sTLR2’s regulatory capacity in vivo, we used twowell-characterized mouse models of acute inflammation based onthe injection of a S. epidermidis-derived cell-free supernatant orlive S. epidermidis into the peritoneal cavity. These models werechosen because they allowed us to evaluate the effect of sTLR2 onthe temporal changes in leukocyte infiltration, inflammatory andchemotactic mediator expression, and bacterial clearance kineticsthat have been extensively characterized in human peritonitis (24,29). By using these models, we established that administration ofsTLR2 reduced the level of PMN recruitment into the peritonealcavity in animals challenged either with Gram-positive bacteria-derived microbial components or live bacteria. Notably, despite itsability to control the inflammatory response, and in vitro capacityto interfere with the phagocytic uptake of bacteria and microbial-induced superoxide production, sTLR2 administration, irrespec-tive of its amount or the dose of bacteria tested, did not have adetrimental impact on the clearance of bacteria. The maintenanceof efficient peritoneal removal of bacteria in the face of sTLR2modulation is likely to be due in part to the following: 1) the factthat modulation by sTLR2 of PMN recruitment was most signif-icant at the peak (12 h) of their influx, when the animals hadcleared the infection almost completely, and 2) the activity of anumber of other humoral mediator pathways that contribute to ef-ficient bacterial clearance and killing, including complement com-ponents, mannose-binding lectin, and Igs, as well as cell surface Fcand scavenger receptors. The latter is consistent with our obser-vation that the negative effect of sTLR2 on phagocytosis in vitrowas significantly reduced in the presence of serum (our unpub-lished data). Clearly, in vivo other immune components make asubstantial contribution to bacterial clearance mechanisms. Thepossibility that sTLR2 affects bacterial clearance in certain pathol-ogies (e.g., complement deficiency) or when administered athigher doses, however, remains to be investigated.

By contrast to its inhibitory effect on PMN mobilization to thesite of injury, sTLR2 did not influence MNC recruitment, despitecausing increased production of MCP-1. We can only speculate on

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the mechanism underlying this differential effect, because we havenot yet investigated the additional effects that sTLR2 might exerton the complex chemokine network that controls MNC recruit-ment. This would require the assessment of not only other MNC-specific chemokines such as MIP-1� (CCL3) and RANTES(CCL5), but also the regulatory mechanisms controlling that net-work, which include the activity of matrix metalloproteinases,CD26, and the decoy chemokine receptors D6 and Duffy Ag re-ceptor for chemokines (38–41). With regard to the increased lev-els of MCP-1 in the presence of sTLR2, it could be speculated, forexample, that it results from a negative effect of sTLR2 on thelevels of D6 and Duffy Ag receptor for chemokines. These decoyreceptors are critically involved in the deactivation and eliminationfrom the circulation of a number of chemokines, including MCP-1,but not KC or MIP-2. Notably, D6 production appears to be reg-ulated, at least in part, by NF-�B (42), which we demonstrate inthe present study to be negatively affected by sTLR2.

Nevertheless, this differential effect of sTLR2 on early (PMN)and late (MNC) leukocyte recruitment and the consequent skewingof the leukocyte influx in favor of MNC appear to promote themore efficient removal of senescent PMN, as indicated by the sub-stantially reduced proportion of late apoptotic/early necrotic PMNfound in the peritoneal cavity of sTLR2-treated mice (Fig. 4D).This effect might ultimately favor more rapid resolution of inflam-mation. A similar pattern of effects on the modulation of leukocytetrafficking and PMN apoptosis in resolving acute inflammation hasbeen previously observed for IL-6/soluble IL-6R signaling (29).Such anti-inflammatory capacity of sTLR2 may prove to be a cru-cial factor during septic shock.

This study has also shed light on the mechanisms underlyingsTLR2’s regulatory capacity. The fact that sTLR2 does not affectsignaling via other TLRs, the IL-1R and non-TLR-related recep-tors, or nonreceptor-mediated signaling (Fig. 2E) suggested thatthe primary effect of sTLR2 is exerted upstream of signaling prox-imal to TLR2 ligand recognition. We therefore first tested whethersTLR2 affected the ligand-induced clustering of mCD14 andmTLR2 in lipid rafts. It has been demonstrated that the TLR co-receptor, CD14, resides mainly in cholesterol and sphingolipid-rich detergent-resistant membrane microdomains, termed lipidrafts (31, 32). It has also been shown that, in resting conditions,TLR4 and TLR2 are localized mainly outside lipid rafts in thedetergent-soluble membrane fractions. Upon TLR ligand-inducedcell stimulation, the specific TLR is recruited to lipid rafts, whereit is found in close proximity to CD14 and other cell surface mol-ecules, thus forming a receptor cluster that is believed to be criticalto signaling (32, 33). We found that sTLR2 interferes with theligand-induced mobilization of TLR2 to lipid rafts for signaling.Such interference would be explained, at least in part, by the ca-pacity of sTLR2 to act as a decoy microbial receptor, demonstratedin this study (Fig. 6B). This decoy activity may involve competi-tion between sTLR2 and mTLR2 for binding not only the micro-bial ligand, but also TLR1, the heterodimerization partner formTLR2 that is required for recognition of and responses to triacy-lated lipopeptides, like the Pam3Cys lipopeptide used in this study(43, 44). Such a heterodimeric receptor complex involving sTLR2may be unable to signal, because only one TIR domain (TLR1’s)would be involved. This possibility, however, remains to be tested.

We also found, however, that sTLR2 disrupts the close prox-imity of mCD14 to mTLR2 in the absence of ligand by associatingwith mCD14, as indicated by the coimmunoprecipitation, FRET,and chemical cross-linking experiments (Fig. 6, C–E). Such closeproximity is crucial to CD14’s coreceptor function and highly ef-ficient TLR signaling. Thus, sTLR2’s capacity to interfere with themCD14-mTLR2 interaction and disrupt the coreceptor function by

associating with CD14, together with sTLR2’s decoy receptor ac-tivity, may affect the mobilization of mTLR2 to lipid rafts forsignaling upon cell stimulation, and lead to reduced proinflamma-tory responses, which in turn result in the observed reduction inPMN recruitment to the site of infection. Similar to our findings, aperitoneal infection model using Salmonella spp. in CD14-defi-cient mice has also shown impaired influx of PMN, but not MNC(45). This observation raises the possibility that the interferingeffect of sTLR2 on CD14 coreceptor activity demonstrated in thisstudy might constitute the predominant mechanism underlying themodulatory effect of sTLR2 on the inflammatory response ob-served in the in vivo models we have studied.

The ability of sTLR2 to affect the activity of CD14 raises thequestion of why TLR4- and TLR3-mediated monocyte responses,which also require CD14 for efficient signaling, are not affected, asindicated by the absence of a negative effect of sTLR2 on LPS orpoly(I:C)-stimulated Mono Mac-6 cells (Fig. 2E). It is possiblethat, when the effect of sTLR2 depends solely on its capacity tointeract with CD14 (no decoy activity, i.e., TLR3 and TLR4/MD2signaling), the extent of sTLR2 inhibition may critically dependnot only on the local concentration of sTLR2, but also on theexpression levels of CD14 (mCD14 or sCD14) and the mTLRinvolved, as well as on the affinity and stoichiometry of the inter-actions of mTLR, CD14, and sTLR2, and those of the ligand withmTLR and CD14. In support of this possibility, we observed thatsTLR2 exerts a significant negative effect on the LPS stimulationof a number of cell lines of epithelial origin, which express verylow levels of TLR4, do not express mCD14, and require sCD14 forsensitive signaling (A.-C. Raby and M. Labeta, manuscript inpreparation). Clearly, a better knowledge of the parameters govern-ing the interactions of TLRs, CD14, the ligands, and sTLR2 will im-prove our understanding of the activity of sTLR2. With regard toTLR3, its mostly intracellular location and function (8) may limit theactivity of sTLR2. Nevertheless, the modulatory activity of sTLR2may not be limited to Gram-positive bacteria-induced responses. In-deed, a recent report demonstrated the involvement of TLR2 inantibiotic-treated Gram-negative bacterial sepsis (46). This findingraises the possibility that sTLR2 also contributes to controllingGram-negative bacteria-induced inflammation.

In conclusion, the findings reported in this study define sTLR2as an efficient regulator of TLR2-mediated inflammatory re-sponses, because it is capable of reducing inflammation by con-trolling PMN influx while preserving MNC recruitment and with-out compromising bacterial clearance. The capacity of sTLR2 toexert its regulatory effect not only by acting as a decoy microbialreceptor, but also by targeting the coreceptor, may inform the de-sign of novel therapeutics against acute and chronic inflammatoryconditions that will aim at disrupting the coreceptor’s activity, thusblunting, but not abrogating, microbial recognition and host innateimmune responses.

AcknowledgmentsWe are indebted to J. E. Rey-Nores (School of Applied Sciences, Univer-sity of Wales Institute, Cardiff, U.K.) for critical insight, expert help, dis-cussions, and review of this manuscript. We also thank R. J. Matthews,B. P. Morgan (Department of Medical Biochemistry and Immunology,School of Medicine, Cardiff University, Cardiff, U.K.), and N. Gay (De-partment of Biochemistry, University of Cambridge, Cambridge, U.K.) forhelpful discussions and critical reading of the manuscript.

DisclosuresThe authors have no financial conflict of interest.

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