Soluble TLR2 Reduces without Compromising Bacterial Clearance by Disrupting TLR2 Triggering

This information is current as Anne-Catherine Raby, Emmanuel Le Bouder, Chantal of September 29, 2021. Colmont, James Davies, Peter Richards, Barbara Coles, Christopher H. George, Simon A. Jones, Paul Brennan, Nicholas Topley and Mario O. Labéta J Immunol 2009; 183:506-517; ;

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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Soluble TLR2 Reduces Inflammation without Compromising Bacterial 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. Labe´ta5*

TLR overactivation may lead to end organ damage and serious acute and chronic inflammatory conditions. TLR responses must therefore 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 , are hyporesponsive to TLR2 ligands. Regulation was TLR2 specific, and affected NF-␬B activation, , and superoxide production. Natural sTLR2-depleted serum rendered leu- kocytes hypersensitive to TLR2-mediated stimulation. Mice administered sTLR2 together with Gram-positive -derived Downloaded from components showed lower peritoneal levels of the neutrophil (PMN) chemoattractant, -derived chemokine; lower PMN numbers; and a reduction in late apoptotic PMN. Mononuclear cell recruitment remained unaffected, and endogenous peritoneal 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 TLR2 mobilization to rafts for signaling, acted as a decoy microbial , and disrupted the interaction of TLR2 with its http://www.jimmunol.org/ coreceptor, CD14, by associating with CD14. These findings establish sTLR2 as a regulator of TLR2-mediated inflammatory responses, capable of blunting immune responses without abrogating microbial recognition and may inform the design of novel therapeutics against acute and chronic inflammatory conditions. The Journal of Immunology, 2009, 183: 506–517.

veractivation or dysregulation of the innate immune re- TLR engagement leads to the production of a variety of proin- sponse may lead to end organ damage and serious acute flammatory, cytotoxic, and immunoregulatory molecules. This O and chronic inflammatory conditions, such as myocar- process results in an immediate response to microbial chal- dial dysfunction, respiratory, renal and multiorgan failure, septic lenge. However, the excessive TLR-mediated release of some

shock, arthritis, asthma, and autoimmunity (1, 2). proinflammatory molecules, either by overactivation of the re- by guest on September 29, 2021 The TLR family plays a pivotal role in the prompt and effi- ceptor or by dysregulation of endogenous TLR-signaling inhib- cient innate immune recognition of and response to an array of itory mechanisms, may lead to the aforementioned disease con- microorganisms and their components, and also in controlling ditions. TLR responses therefore have to be tightly regulated to the activation of the adaptive immune response (3–5). Ten hu- limit these potentially deleterious consequences. man TLRs have to date been identified (TLR1-TLR10), and the A number of negative regulatory mechanisms controlling TLR ligand specificity described for most of them (3, 4). The effi- responses have been reported (1, 12, 13). These include general cient recognition of most microbial components activating via mechanisms aimed at the overall reduction in TLR expression and TLR2, TLR3, and TLR4 requires the activity of a coreceptor, function (e.g., receptor compartmentalization, down-modulation, CD14, which enhances cellular responses substantially (6–8). ubiquitinylation, and degradation; activity of anti-inflammatory CD14 is expressed as a cell surface molecule, and also as a such as TGF-␤ and IL-10), the destruction of the acti- soluble coreceptor in plasma and other biological fluids (9–11). vated cells by apoptosis, the activity of intracellular TLR signaling pathway inhibitors (e.g., MyD88s, IL-1R-associated kinase (IRAK)-M,6 IRAK2c and IRAK2d, suppressor of signal- *Department of Medical Biochemistry and Immunology and †Department of Medi- ing 1, SARM, PI3K, TOLLIP, and A20), cell membrane-bound cine, School of Medicine, Cardiff University, Cardiff, United Kingdom TLR suppressors (e.g., ST2, SIGIRR, TRAILR, and RP105), and Received for publication September 4, 2008. Accepted for publication May 4, 2009. extracellular soluble decoy microbial receptors (soluble TLRs The costs of publication of this article were defrayed in part by the payment of page (sTLRs)) (5). charges. This article must therefore be hereby marked advertisement in accordance Natural sTLRs are believed to play a crucial role in preventing with 18 U.S.C. Section 1734 solely to indicate this fact. the excessive initial triggering of the membrane-bound TLR and 1 This work was supported by grants from the Wellcome Trust of Great Britain and subsequent TLR overactivation (1, 12, 14–17); however, this Medical 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, 6 Abbreviations used in this paper: IRAK, IL-1R-associated kinase; BS3, bis(sulfos- F-75248 France. uccinimidyl)suberate; CHO, Chinese hamster ovary; EV, expression vector; FRET, 4 fluorescence resonance energy transfer; HEK, human embryonic kidney; KC, kera- Current address: Kuros Biosurgery AG, 8005 Zu¨rich, Switzerland. tinocyte-derived chemokine; LBP, LPS-binding protein; m, membrane bound; MNC, 5 Address correspondence and reprint requests to Dr. Mario O. Labe´ta, Infection and mononuclear cell; PMN, polymorphoneutrophil; poly(I:C), polyinosinic-polycytidylic Immunity, Department of Medical Biochemistry and Immunology, School of Medi- acid; SES, S. epidermidis cell-free supernatant; sTLR, soluble TLR. cine, Cardiff University, Henry Wellcome Research Building, Heath Park, Cardiff CF14 4XX, United Kingdom. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802909 The Journal of Immunology 507

supposition has yet to be proven. In the mouse, a splice variant of TNF-␣ (R&D Systems), or PMA plus ionomycin (Sigma-Aldrich), as de- the TLR4 mRNA coding for a putative partially secreted soluble scribed in Results. Following a 16-h incubation, the supernatants were ␬ TLR4 fragment has been described (16). The corresponding cDNA tested for IL-8 by ELISA (Duoset; R&D Systems). For NF- B reporter assays, cells were transiently transfected (Lipofectamine; Invitrogen) with was found to reduce cell sensitivity to LPS (a TLR4 agonist) when constructs directing expression of the firefly luciferase reporter and the introduced in a mouse cell line. However, it remains Renilla luciferase. Forty-eight hours posttransfection, the cells were stim- to be established whether this putative soluble TLR4 protein frag- ulated (16 h, 37°C) with Pam3Cys in the absence or presence of purified ment is naturally expressed and released by normal mouse cells, sTLR2, and luciferase activity was measured (Promega). sTLR2-depleted (Ն90%) AB serum (Fig. 2F) was prepared by sequential immunoprecipi- and has the capacity to modulate cellular responses in vivo. In tation (see below) with the anti-TLR2 Ab, sc8689, or an irrelevant, anti- humans, we have described the existence of a naturally occurring plexin-C1 Ab, for mock depletion (both from Santa Cruz Biotechnology), sTLR2 (17), the receptor involved in responses to Gram-positive as previously described (17). PBMC (2 ϫ 105 cells) were stimulated over- bacteria and their cell-wall components, as well as a wide range of night with Pam3Cys in the absence or presence of 2% sTLR2- or mock- other microbial components (3, 6). It was found that sTLR2, which depleted serum. consists of most of the TLR2 extracellular domain, is released by Overexpression of sTLR2 normal and present in plasma, breast milk (17–19), and A sTLR2 fragment consisting of the putative extracellular domain of hu- saliva (20, 21). The full extent of sTLR2’s negative regulatory man TLR2 (Met1-Arg587) with an N-terminal c-Myc tag was constructed. capacity, the mechanism underlying it, and its biological signifi- The plasmid pCRII-TOPO-c-myc-TLR2, previously engineered (17), was cance in vivo have not yet been determined. Other than sTLR2, no used as a template to generate a PCR fragment corresponding to aa 1–587 naturally occurring soluble form of a mammalian TLR has been of the TLR2 molecule. The sTLR2 cDNA was subcloned into the pDR2⌬EF1␣ expression vector. The recombinant plasmid was transfected identified to date. into HEK-TLR2 cells. Expression and release of sTLR2-Myc by the HEK- Downloaded from In view of the potentially severe pathological conditions that TLR2 plus sTLR2 cells were confirmed by Western blotting of culture may be caused by Gram-positive bacterial infections via TLR2 supernatants using a rabbit polyclonal anti-TLR2 Ab, TLR2p, generated in triggering, and the reported modulation of sTLR2 release in dis- our laboratory by immunization with the N terminus 20-mer human TLR2 peptide SKEESSNGASLSGDRNGIGK (17) and anti-c-Myc epitope mAb ease states (17–21), it is hypothesized that sTLR2 may serve as a clone 9E10 (Sigma-Aldrich). critical first-line regulator of TLR2-mediated responses (1, 12, 17, Production of human rsTLR2 18, 20). In this study, we have therefore sought to fully assess the http://www.jimmunol.org/ modulatory capacity of sTLR2 by evaluating its physiological ac- A TLR2 construct consisting of the putative human TLR2 extracellular 21 587 tivity and anti-inflammatory potential in vitro and in vivo, and shed domain (Glu -Arg ) with a C-terminal His6 tag tail was generated. The on the mechanism underlying sTLR2 regulatory activity. Our TLR2 cDNA was obtained by RT-PCR using RNA from Mono Mac-6 monocytes, and cloned into the pCR II-TOPO cloning vector, as previously results show that cells overexpressing sTLR2 are markedly hypo- described (17). The plasmid pCRII-TOPO-TLR2 cDNA was used as a sensitive to TLR2-mediated stimulation. The regulatory effect was template to generate a PCR fragment corresponding to aa 21–587 with a

reproduced by the purified rsTLR2 protein, was TLR2 specific, C-terminal His6 tag. The resulting sTLR2 cDNA was subcloned into the and affected NF-␬B activation, phagocytosis, and superoxide pro- baculovirus transfer vector pMELBacB (Invitrogen) in frame to the Hon- duction. Plasma sTLR2 depletion experiments indicated that eybee Melittin secretion signal. Sf-9 cells were cotransfected with the re-

combinant pMELBacB-sTLR2 cDNA transfer vector and Bac-N-Blue by guest on September 29, 2021 sTLR2 acts as a physiological regulator of TLR2 signaling in DNA by using the Bac-N-Blue Transfection and Expression system (In- PBMC, whereas administration of sTLR2 to mice resulted in mod- vitrogen). High Five cell cultures (Express Five serum-free medium; Life ulation of the inflammatory response to Gram-positive bacterial Technologies) were infected with the recombinant . After 72 h postin- components as well as to live Gram-positive bacteria, but impor- fection, supernatants were cleared by centrifugation, filtered (0.22-␮m fil- ters), and concentrated 25 times (CentriconPlus-70; Millipore) before tantly sTLR2 achieved this effect without compromising bacterial buffer exchange to 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole clearance. Mechanistically, sTLR2 was found to interfere with the (pH 8.0; binding buffer). The sTLR2-His protein in the concentrated sam- ligand-induced mobilization of TLR2 to lipid rafts for signaling, ple was purified by metal-affinity chromatography using Ni-NTA Super- act as a decoy receptor by binding to bacterial lipopeptide and flow resin (Qiagen; 2 h, 4°C, orbital rotation). The protein was eluted by whole Gram-positive bacteria, and disrupt the close proximity of increasing the concentration of imidazole in the binding buffer to 250 mM. sTLR2-containing fractions were pooled and concentrated, and the buffer membrane-bound TLR2 and CD14 by interacting with CD14. was exchanged to PBS. The protein concentration in the final sample was determined (Dc Protein Assay; Bio-Rad), and the purity of the sTLR2 Materials and Methods preparation was assessed by 10% SDS-PAGE under reducing condition, ␬ followed by Coomassie blue G250 staining (Bio-Rad), as described in Re- Cells, cell activation, and NF- B reporter assays sults. The sTLR2 preparation was also analyzed by Western blotting, as Human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO), previously described (17), using either a rabbit polyclonal anti-TLR2 Ab ␮ mouse RAW264 (American Type Culture Collection), HEK-TLR2, and (TLR2p) or an anti-His5 mAb (Qiagen). Typically, 200 g of purified CHO-CD14 (previously generated in our laboratory (17)) cells were cul- sTLR2 was obtained from 200 ml of High Five cell culture supernatant. Ϫ tured in DMEM (HEK293) or RPMI 1640 medium (Invitrogen) supple- The sTLR2 preparations were aliquoted and kept at 85°C until use. mented with 10% FCS (HyClone; Ͻ0.06 U/ml endotoxin), 2 mM glu- Phagocytosis and superoxide production assays tamine, 400 ␮g/ml hygromycin B (HEK-TLR2), 1 mM pyruvate, 0.5% ␮ ϫ 5 (v/v) NaHCO3, and 50 g/ml L-proline (CHO-CD14). The human mono- For phagocytosis experiments, RAW264 (4 10 ) were re- cytic cell line, Mono Mac-6 (provided by H. Ziegler-Heitbrock, Depart- suspended in 400 ␮l of binding buffer (phenol red-free RPMI 1640, 1% ment of Immunology, University of Leicester, Leicester, U.K.), was cul- sodium azide, 2.5% HEPES) and incubated with FITC-labeled Staphylo- tured, as previously described (17). Human monocytes were also prepared, coccus aureus (Molecular Probes) at a bacteria:cell ratio of 10:1 for 30 min as described (17). Human neutrophil (PMN) preparations were obtained at either 0°C or 37°C. To test the effect of sTLR2, the bacterial suspension through dextran sedimentation and Ficoll density-gradient centrifugation of was preincubated with 5 ␮g/ml sTLR2 or BSA for 30 min at 37°C. Fol- blood from healthy donors. Human peritoneal mesothelial cells were pre- lowing binding, cells were washed with cold washing buffer (PBS/1% so- pared and cultured, as described (22). For cell activation experiments (Fig. dium azide), resuspended in washing buffer, fixed (2% paraformaldehyde), 2), triplicate cell aliquots (1 ϫ 105 cells/well) were cultured in serum-free and analyzed by flow cytometry, as previously described (23). Cell surface medium supplemented or not (Fig. 2, A and B; and monocytes) with fluorescence was quenched with 125 ␮g/ml trypan blue before flow 500 ng/ml sCD14 (purified from human milk (11)) and stimulated with cytometry. ultra-pure LPS (Escherichia coli O111:B4 strain), heat-killed Listeria For superoxide production assays, 150 ␮l of assay buffer (13 mM

monocytogenes, peptidoglycan, polyinosinic-polycytidylic acid (poly(I: Na2HPO4,3mMNaH2PO4, 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, ␮ C)), flagellin (all from InvivoGen), the synthetic bacterial lipopeptide 11 mM dextrose, and 0.71 mM CaCl2 (pH 7.4)) containing 5 g/ml ␤ Pam3-Cys-Ser-(Lys)4 HCl (Pam3Cys; EMC Microcollections), IL-1 , Pam3Cys or heat-killed Staphylococcus epidermidis strain PCI 1200 508 NEGATIVE REGULATION BY sTLR2

(American Type Culture Collection; 5 ϫ 106/well) and 5 ␮g/ml sTLR2 human Fc (R&D Systems) or control CD46-Fc fusion protein (provided by was added in triplicate to microtiter well plates (microlite 2; Thermo Lab- C. Harris). Following washing (2ϫ PBS/0.01% sodium azide), samples Systems) kept at 37°C. To this mixture, 2 ␮M Luminol and 2 ϫ 105 PMN were incubated (0°C, 30 min) with a biotin-conjugated anti-human IgG Ab resuspended in PBS were added, and the chemiluminescence generated (0.5 ␮g; Southern Biotechnology Associates). After washing and fixing was measured at 2-min intervals for 1 h using a fluorometer (Fluorstar (2% paraformaldehyde), streptavidin-allophycocyanin (0.1 ␮g; Southern Optima plate reader; BMG Labtech). Biotechnology Associates) was added, and the samples were incubated for 20 min on ice before washing and analysis by flow cytometry. In vivo models of peritoneal inflammation Coimmunoprecipitation and chemical cross-linking experiments Inbred 8- to 12-wk-old C57BL/6 mice (Harlan) were maintained under barrier conditions and free. All experimental procedures were The immunoprecipitation technique was as previously described (17). In conducted under a Home Office project license. Lyophilized S. epidermidis this study, for membrane-bound (m)CD14-mTLR2 coimmunoprecipita- cell-free supernatant (SES) was prepared from suspension cultures of S. tions, 5 ϫ 106 freshly isolated human monocytes were resuspended in epidermidis, isolated from an end-stage renal failure patient under contin- phenol red-free RPMI 1640 medium and incubated (30 min at 37°C) in the uous ambulatory peritoneal dialysis, as previously described (24). SES presence of 5 ␮g/ml sTLR2 or 10 ␮g/ml BSA. After washing and lysis (1% preparations were reconstituted in PBS before in vivo use. Peritoneal in- (v/v) Nonidet P-40, 50 mM Tris-HCl, 150 mM NaCl, 1 ␮g/ml leupeptin flammation was induced in mice by i.p. injection of a defined 500 ␮l dose and pepstatin, 1 mM PMSF (pH 7.4) buffer), the cell lysate was precleared of SES, SES plus 100 ng of sTLR2, or control PBS. At the indicated time by successive incubations with the following: 80 ␮l of protein G-Sepharose points, the animals were sacrificed, and their peritoneal cavities were la- (50% suspension; Sigma-Aldrich), 4 ␮g of the isotype-matched control, vaged with 2 ml of ice-cold PBS. Leukocyte numbers in the lavages were mouse IgG2b, and protein G-Sepharose. The precleared samples were assessed by differential cell count on cytospin preparations and Coulter incubated (1 h, 4°C) with 5 ␮g of the anti-CD14 mAb, MY4, and the counting (Coulter Z2; Beckman Coulter), or by double staining (anti-F4/80 immunocomplexes were precipitated with 50 ␮l of protein G-Sepharose. FITC, Serotec; anti-CD11b allophycocyanin, BD Pharmingen), followed Following washing, samples were analyzed by Western blotting with the Downloaded from by flow cytometric analysis. Chemokine levels in the cell-free peritoneal anti-TLR2 mAb, IMG319. For chemical cross-linking experiments, 5 ϫ lavage samples were quantified by ELISA (R&D Systems). To test for 106 cells were resuspended in 500 ␮l of cold phenol red-free RPMI 1640 mouse sTLR2 in the peritoneal lavages, equal aliquots were diluted with medium and incubated with 5 ␮g of sTLR2 (sTLR2-His) or the irrelevant 3ϫ concentrated Laemmli reducing sample buffer and analyzed by West- His-tagged protein sCD55 for 30 min at room temperature. Following ern blotting using the TLR2-specific polyclonal Ab, TLR2p. Peritoneal washing (cold RPMI 1640), 3 mg/ml membrane-impermeable and non- inflammation was also induced by i.p. injection (500 ␮l) of 5 ϫ 107 or 5 ϫ cleavable cross-linker, bis(sulfosuccinimidyl)suberate (BS3; Pierce), was 108 CFU S. epidermidis PCI 1200 strain (American Type Culture Collec- added to the samples, and the mixture was incubated for an additional 30

tion) in the absence or presence of sTLR2. At the indicated time points, min at room temperature. Cross-linking was stopped by the addition of 10 http://www.jimmunol.org/ peritoneal cavity lavages were obtained. Blood was obtained by cardiac mM Tris-HCl (pH 7.4) buffer and incubation on ice for 15 min. The cells puncture. Bacterial CFU were determined by culturing blood and perito- were then lysed, and cell lysates were incubated (2 h, 4°C, orbital rotation) neal lavage samples on Mueller-Hinton agar plates (Oxoid) overnight with Ni-NTA beads (10 ␮l of beads/100 ␮l of lysate). The beads were at 37°C. washed, and the protein was eluted with Laemmli reducing sample buffer Preparation of lipid rafts containing 250 mM imidazole. The eluate was analyzed by 7.5% SDS- PAGE and Western blotting using anti-CD14 (69.4, rabbit polyclonal Ab Freshly isolated human monocytes (1 ϫ 108 cells) were resuspended in (11)) or anti-TLR2 (sc8689) Abs. warm phenol red-free RPMI 1640 medium and stimulated (1 h at 37°C) or not with 5 ␮g/ml Pam3Cys in the absence or presence of 5 ␮g/ml sTLR2. Fluorescence resonance energy transfer (FRET) measurements

Subsequently, protein solubilization was conducted (1% (v/v) Triton For FRET measurements, freshly isolated monocytes were allowed to ad- by guest on September 29, 2021 ␮ X-100, 150 mM NaCl, 50 mM Tris-HCl, 1 mM PMSF, 1 g/ml leupeptin, here (1 h; 37°C) to multispot slides (Shandon Multispot; Thermo Electron) and pepstatin (pH 7.4)) for 1 h at 0°C. Cell lysates (1.5 ml) were mixed in the absence or presence of 5 ␮g/ml sTLR2 or BSA in phenol red-free with an equal volume of cold 90% sucrose solution (90% sucrose/50 mM RPMI 1640 medium. The slides were then incubated with 20% normal Tris-HCl, 150 mM NaCl (pH 7.4)). Samples were overlaid with 7 ml of rabbit serum for 15 min at room temperature before washing and labeling 30%, followed by 2 ml of 5% cold sucrose solutions, and centrifuged at (1 h, 0°C) with the anti-CD14 mAb My4-Cy3 or its isotype-matched ϫ 200,000 g for 16 h at 4°C. One-milliliter fractions were removed from IgG2b-Cy3 control (acceptor fluorophore; 0.25 ␮g/spot). Both Abs (Beck- ␮ the gradient, and 60 M n-octylglucoside was added to each fraction. man Coulter) were Cy3 conjugated using the FluoroLink mAb Cy3 label- Equal fraction aliquots were analyzed by Western blotting using the anti- ing (GE Healthcare). Cell labeling was performed in the absence or CD14 mAb, MY4 (Beckman Coulter), or the anti-TLR2 mAb, IMG319 presence of 5 ␮g/ml sTLR2 or BSA. The slides were then washed (2ϫ (Imgenex). To define the lipid raft-containing fractions in the gradient, dot PBS/0.01% sodium azide), fixed (2% paraformaldehyde), and, following blots of fraction aliquots were tested with HRP-conjugated cholera toxin B washing, stained with an Alexa 488-conjugated anti-TLR2 (T2.5; eBio- (List Biological Laboratories), followed by ECL (Amersham Biosciences) science) or anti-C3aR (hC3aRZ1; Serotec) mAb (donor fluorophore), as to reveal the presence of the raft-associated ganglioside, GM1. described above for MY4-Cy3. After washing and fixing, the slides were Binding assays of sTLR2 to Pam3Cys, LPS, and bacteria mounted (Vectashield; Vector Laboratories). FRET was measured by the release of quenched donor fluorescence after acceptor photobleaching us- Triplicate wells of microtiter well plates (high binding; Costar) were coated ing a previously described technique (25). In this technique, the donor (50 ␮l) with the amounts of Pam3Cys or LPS, indicated in Results, dis- fluorescence intensity before and after acceptor photobleaching in the same solved in ethanol. Following solvent evaporation at room temperature, non- cell sample is compared. FRET efficiency was quantified by the following: ϭ Ϫ ϫ specific binding was blocked by incubation (2 h, room temperature) with E (%) ((IDA IDB)/IDA) 100, where E represents percentage of FRET PBS/1% BSA/5% sucrose/0.05% sodium azide. The plates were then efficiency; IDA and IDB, the donor’s intensity after and before photobleach- washed three times (PBS/0.05% Tween 20) and incubated (2 h, room tem- ing of the acceptor, respectively. In each cell to be analyzed, FRET effi- perature) with 5 ␮g/ml sTLR2-His, sCD55-His (donated by C. Harris, ciency was determined typically in four to five regions of the plasma mem- Cardiff University, Cardiff, U.K.), or LPS-binding protein (LBP; Alexis brane with different donor intensity. For each experiment, a minimum of Biochemicals) diluted in 0.05% Tween 20, 20 mM Trizma base, and 150 200 cells per condition was analyzed. E values from all regions of interest mM NaCl (pH 7.3) buffer (buffer A) supplemented with 0.1% BSA. Sub- were averaged. The Cy3 acceptor fluorophore was bleached by repeated sequently, the wells were washed and incubated (1 h on ice) with an anti- excitation (50 times for a total of 2 min), and the bleaching was Ն20% and ␮ ␮ His5 (5 g/ml; Qiagen) or anti-LBP (1 g/ml; Hycult Biotechnology) mAb up to 100% (depending on the experiment and the region of interest). The diluted in buffer A/2% BSA. Following washing, the wells were incubated bleaching conditions were set to avoid bleaching the donor fluorophore. (1 h on ice) with a biotin-conjugated anti-mouse IgG Ab (DakoCytomation) Cells were imaged using the Leica TCS SP2 resonant scanning confocal diluted in buffer A/2% BSA, before washing and incubation (20 min on system (Leica Microsystems). Signal-to-noise ratio was improved by re- ice) with streptavidin-HRP (Jackson ImmunoResearch Laboratories) di- cording images using the frame averaging method (average of 2 frames). luted in buffer A/0.5% skim milk. The wells were then washed, and color The donor fluorophore was excited at 488 nm, and emission was detected developed by addition of tetramethylbenzidine substrate (SureBlue; between 498 and 540 nm. The acceptor fluorophore was excited at 543 nm Kirkegaard & Perry Laboratories) was measured at 450 nm. and detected between 551 and 669 nm. Under these conditions, negligible To test the binding of sTLR2 to bacteria, 5 ϫ 104 heat-killed S. epi- fluorescence was observed from an Alexa 488-labeled specimen within the dermidis PCI 1200 strain was resuspended in 100 ␮l of PBS/0.05% BSA Cy3 emission spectrum, and from a Cy3-labeled specimen within the Alexa and incubated (30 min at room temperature) with 1 ␮g of mouse rsTLR2- 488 emission spectrum. The validity of each FRET dataset was confirmed The Journal of Immunology 509 by the lack of correlation between E% and acceptor or donor fluorescence intensity (data not shown). This suggested that the FRET values observed between mTLR2 and mCD14 were not dependent on acceptor or donor density, and thus resulted from genuine protein-protein interactions and not from randomly associated molecules (26, 27). Statistics Statistical analysis of the data was performed by using paired Student’s t test (Minitab 15 statistical software). Values of p of less than 0.05 were considered significant. Results sTLR2 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 occurring sTLR2 form found in plasma and milk (17). HEK293 cells stably expressing either the mTLR2 receptor (HEK-TLR2) or both mTLR2 and sTLR2, the latter tagged at the N terminus with a Myc epitope (HEK-TLR2 ϩ sTLR2), were generated. Initial analysis Downloaded from by 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 and HEK-TLR2 cells expressed similar levels of mTLR2 (Fig. 1, A and B). A C terminus His-tagged human sTLR2 protein was also en-

gineered and purified from insect cell culture supernatants. The http://www.jimmunol.org/ purity 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 either anti-TLR2 or anti-His5 Abs (Fig. 1, C and D). Coomassie staining and Western blots showed a major 72- to 75-kDa sTLR2 band. Minor sTLR2 bands of ϳ83 and 90 kDa, whose intensity depended on the preparation, were also detected, mainly with the anti-TLR2 Ab. sTLR2 preparations were estimated to be 85–95% pure (de- pending on the preparation) and mostly (ϳ95%) monomeric. Some by guest on September 29, 2021 of the low-intensity Ͻ75-kDa bands detected by Coomassie stain- ing may correspond to sTLR2 degradation products, because they can also be detected by either the anti-TLR2 or anti-His Abs. The HEK-TLR2 plus sTLR2 cells were found to be markedly insensitive to stimulation with different doses of the TLR2 agonist synthetic bacterial lipopeptide Pam3CysSer(Lys)4 (Pam3Cys), as FIGURE 1. Expression and purification of human rsTLR2. A, Detection judged by the release of the proinflammatory chemokine IL-8 of sTLR2 in HEK-TLR2 plus sTLR2 culture supernatants (2 ϫ 106 cells) (CXCL8) (Fig. 2A, left). The negative effect was sTLR2 concen- by Western blotting with the anti-TLR2 rabbit Ab, TLR2p, or the anti- tration dependent, because HEK-TLR2 cells transiently transfected cMyc epitope mAb, 9E10 (HEK-TLR2 ϩ sTLR2 cells express an N ter- with increasing amounts of the cDNA encoding sTLR2 showed a minus c-Myc-tagged sTLR2 protein). For control experiments, culture su- concomitant progressive reduction in sensitivity (Fig. 2A, right). pernatants from HEK-TLR2 plus empty expression vector (EV) cell transfectants were tested. B, Fluorescence profiles of mTLR2 expression in TLR2 signaling inhibition by sTLR2 was not limited to Pam3Cys HEK-TLR2 and HEK-TLR2 plus sTLR2 cell transfectants stained with the stimulation, because cell activation induced by another TLR2 ag- PE-conjugated anti-TLR2 mAb, TL2.1, or the isotype-matched control onist, peptidoglycan, and by the whole Gram-positive bacterium IgG. C and D, Coomassie blue staining (C) and Western blot (D) pattern of heat-killed L. monocytogenes, was also affected (Fig. 2B). purified His-tagged rsTLR2 following production by insect cells, purifica- The purified rsTLR2 protein also showed negative regulatory tion by Ni-NTA chromatography, and 10% SDS-PAGE (reducing condi- capacity. The inhibitory effect of rsTLR2 was observed in HEK- tions). For Western blots, an anti-His5 mAb and the anti-TLR2 Ab, TLR2p, TLR2 cell transfectants, human monocytes, PBMC (data not were used. shown), and (mTLR2ϩ) peritoneal mesothelial cells (Fig. 2C). The latter cells play a pivotal role during the course of a peritoneal infection, like the one studied in this work (see below), by secret- trans activation of the transcription factor NF-␬B was markedly ing chemokines that regulate leukocyte infiltration into the perito- inhibited, indicating that sTLR2 has a wide spectrum of effects neal cavity and by expressing adhesion molecules (22, 28). Fig. 2C (Fig. 2D). shows that release of IL-8, the archetypal human PMN chemoat- The specificity of the sTLR2 inhibitory effect was evaluated tractant, by mesothelial cells stimulated with Pam3Cys or a cell- next. We tested whether sTLR2 influences activation free supernatant prepared from the Gram-positive bacterium, S. induced by suboptimal doses of the TLR agonists, viral dsRNA epidermidis (termed SES), was reduced in the presence of sTLR2, mimic poly(I:C) (TLR3), LPS (TLR4), and flagellin (TLR5). In suggesting that during peritoneal infections sTLR2 may also target addition, the effect of sTLR2 on signaling via the IL-1R (which mesothelial cells for negative regulation. The effect of sTLR2 was shares with TLRs the MyD88-dependent signaling pathway), the not limited to modulation of IL-8 release: the Pam3Cys-driven TLR-nonrelated receptor TNFR, and nonreceptor-mediated cell 510 NEGATIVE REGULATION BY sTLR2

FIGURE 2. sTLR2 renders cells hyposensitive to TLR2-mediated stimulation. A and B, IL-8 levels in culture supernatants of HEK293 cells stably expressing mTLR2 (HEK- TLR2), mTLR2 and sTLR2 (HEK- TLR2 ϩ sTLR2), the empty vector (HEK-EV) (A, left panel), or HEK- TLR2 cells transiently transfected with EV or sTLR2 cDNA (A, right panel) or 250 ng of sTLR2 cDNA (B), and stimulated, as indicated. Re- sults are means Ϯ SD of one experi- ment representative of four (A)or three (B). The differences in IL-8 re- lease between sTLR2-expressing cells and HEK-TLR2 or HEK-TLR2 p Ͻ ,ءءء :plus EV were significant

0.0001. C–E, Cells were stimulated Downloaded from with the indicated concentrations of Pam3Cys or 200 ng/ml Pam3Cys (E), dilutions of SES, 80 ␮g/ml poly(I:C), 10 ng/ml LPS, 5 ␮g/ml flagellin, 5 ng/ml IL-1␤, 10 ng/ml TNF-␣,or50 ng/ml PMA plus 500 ng/ml inonomy- cin in the absence or presence of 5 http://www.jimmunol.org/ ␮g/ml sTLR2. For NF-␬B reporter assays, cells transiently transfected with firefly and Renilla luciferase re- porter plasmids were stimulated with Pam3Cys, followed by luciferase ac- tivity measurements. Results are of one experiment (ϮSD) representative p Ͻ ,ءءء ;p Ͻ 0.05 ,ء) of at least three

0.0001 sTLR2 vs control). F, IL-8 by guest on September 29, 2021 levels released by Pam3Cys-stimu- lated PBMC in the absence or pres- ence of sTLR2- or mocked-depleted 2% AB serum. Results are from one experiment performed in triplicates p Ͻ ,ء) ϮSD) representative of four) -p Ͻ 0.01; sTLR2 vs mock ,ءء ;0.05 depleted serum).

stimulation (PMA ϩ ionomycin) was also tested. Fig. 2E shows ciated with bacterial killing, namely phagocytosis and superoxide that only TLR2-mediated monocyte activation was inhibited by production. RAW264 macrophages were used to test macrophage sTLR2, indicating that sTLR2 targets monocyte TLR2 signaling phagocytic capacity in the absence and presence of sTLR2. The specifically. binding and phagocytic uptake of fluorescent bacteria were tested To evaluate the physiological relevance of the negative regula- at 0°C and 37°C, respectively, in the presence and absence of tory capacity of sTLR2, we compared the sensitivity of PBMC to trypan blue, to quench cell surface fluorescence. In this way, the stimulation via TLR2 in the presence of serum that had been de- amount of bacteria bound (0°C, trypan blue-sensitive fluorescence) pleted of naturally occurring sTLR2 (Ն90%) with that of PBMC and phagocytosed (37°C, trypan blue-resistant fluorescence) by stimulated in the presence of mock-depleted serum (Fig. 2F). Re- macrophages was evaluated separately. To assess the full potential ducing the amount of serum sTLR2 resulted in a significant in- of sTLR2 as a regulator of the phagocytic process, the experiments crease in cell sensitivity to TLR2-mediated stimulation. This result were performed in serum-free medium, thereby excluding the con- confirmed previous findings (17, 20), and suggested that naturally tribution of Fc and/or complement receptors. Fig. 3A shows that occurring sTLR2 may play an important immunomodulatory role sTLR2 interfered strongly with the macrophage binding (0°C) of in controlling TLR2-mediated activation in vivo. Gram-positive bacteria, S. aureus, while having a comparatively modest effect on phagocytosis (37°C). This effect on phagocytosis Phagocytosis and superoxide production can be affected by was, most likely, a consequence of the marked effect on bacterial sTLR2 binding. At 37°C, the activity of phagocytic receptors (e.g., scav- To extend the assessment of the negative regulatory potential of enger receptors, C-type lectins) most likely compensates for the sTLR2, we tested the capacity of sTLR2 to affect pathways asso- interfering effect of sTLR2. The Journal of Immunology 511

of a clinical bacterial peritonitis episode typically seen in end- stage renal failure patients on continuous ambulatory peritoneal dialysis is mimicked by the peritoneal injection of a previously defined dose of the cell-free supernatant SES, derived from cul- tures of S. epidermidis, the main causative pathogen of this type of peritonitis (30). Intraperitoneal administration of SES to mice resulted in a rapid and transient increase in the peritoneal levels of the PMN chemoattractants, keratinocyte-derived che- mokine (KC), and MIP-2, murine functional counterparts of human IL-8 and growth-related oncogene-␣ (CXCL1), with peak 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 the experiment) after SES administration (Fig. 4B). The simulta- neous administration of SES and sTLR2 (100 ng) resulted in reduced 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, Downloaded from sTLR2 administration resulted in a marked reduction in PMN numbers recruited to the peritoneum either over the whole time course or at the peak of their influx (Fig. 4B). The effect of sTLR2 on the SES-induced peritoneal levels of the mononu- clear cell (MNC) chemoattractant, MCP-1 (MCP-1/CCL2), and

on the relatively late recruitment of MNC, responsible for the http://www.jimmunol.org/ removal of the apoptotic PMN, was also tested (Fig. 4C). Under these conditions, sTLR2 was found to exert a positive and sig- nificant effect on MCP-1 levels over the time period post-SES injection. Total MNC recruitment, however, was not found to be affected. The suppressive effect of sTLR2 on early (PMN), but not late (MNC), leukocyte recruitment posed the question of whether such a disproportionate leukocyte influx influences PMN sur- vival and thus inflammatory resolution. We compared the apo- by guest on September 29, 2021 ptotic status of PMN at the peak of their peritoneal influx in FIGURE 3. Phagocytosis and superoxide production can be affected by SES-challenged mice with that in mice challenged with SES sTLR2. A, Extent of FITC-labeled bacteria bound (0°C) or phagocytosed (37°C) by RAW264 macrophages preincubated or not with 5 ␮g/ml sTLR2 plus sTLR2 (Fig. 4D). Profile comparison of the annexin V/pro- or an irrelevant protein (BSA, 2ϫ sTLR2 molarity), as determined by flow pidium iodide scatter plots showed no difference in the propor- cytometry. To distinguish between cell surface-bound and phagocytosed tion of early apoptotic PMN (lower right quadrant) between bacteria, the cell surface fluorescence was quenched with trypan blue be- SES- and SES plus sTLR2-treated mice. Examination of the fore flow cytometric analysis. Results are of one representative of three proportion of late apoptotic/early necrotic PMN (upper right independent experiments. B, Luminol-dependent chemiluminescence gen- quadrant), however, showed a marked and significant reduction erated by superoxide produced over the time by triplicate cultures of hu- (ϳ50%) of their numbers in the SES plus sTLR2-treated mice, man PMN stimulated with 5 ␮g/ml Pam3Cys or 5 ϫ 106 heat-killed S. suggesting a more efficient clearance of the dying PMN by the epidermidis in the absence or presence of 5 ␮g/ml sTLR2. Results are from one representative experiment of four. MNC in these animals. The effect of administering sTLR2 together with SES to mice on the levels of endogenous (mouse) sTLR2 in the peritoneal lavage Freshly isolated human PMN were used to test the effect of fluid was also tested, because we and others have demonstrated sTLR2 on microbial-induced superoxide production. In the pres- that sTLR2 release is affected by cell activation and infection (17, ence of sTLR2, the capacity of PMN to generate superoxide over 18, 21). The detection of endogenous sTLR2 was facilitated by the time in response to either the Pam3Cys lipopeptide or whole heat- absence of exogenous sTLR2 (sTLR2-His) in the peritoneal la- killed S. epidermidis was substantially reduced (Fig. 3B). vages (data not shown). At 1 h postinjection, no differences in the Collectively, the results shown in Figs. 2 and 3 demonstrated the levels of sTLR2 between SES- and SES plus sTLR2-challenged potential of sTLR2 to negatively regulate TLR2-mediated cell sig- mice were observed (Fig. 4E). At 3 h, i.e., when PMN influx was naling and effector functions that are critical during microbial high, mouse sTLR2 levels in the peritoneal lavages of the sTLR2- infection. treated mice were found increased. By 6 h postinjection, sTLR2 levels between sTLR2-treated and nontreated mice were compa- sTLR2 affects early leukocyte recruitment and endogenous rable and similar to those at the 1-h time point. This finding sug- sTLR2 release in a mouse model of peritoneal inflammation gested that the administration of sTLR2 together with SES induced To evaluate the biological activity of sTLR2 and assess its po- a positive feedback for the release of sTLR2, resulting in tran- tential as a modulator of inflammation in vivo, we first tested siently higher local concentrations of endogenous sTLR2, which the effect of sTLR2 on a well-established mouse model of acute may well contribute to maintaining its regulatory effect on peritoneal inflammation (24, 29). In this model, the progression inflammation. 512 NEGATIVE REGULATION BY sTLR2

FIGURE 4. sTLR2 affects early leukocyte recruitment and endoge- nous sTLR2 release in a mouse peri- toneal inflammation model. A–E, Mice were injected i.p. with a defined dose of SES, SES plus 100 ng of sTLR2, or PBS. At each time point, chemokine expression, PMN, and MNC numbers in the peritoneal la- vages were determined (A–C). Cell numbers were determined by differ- ential cell counts on cytospin prepa- rations (B, right; results from four in- dependent experiments) or leukocytes were double stained with anti-F4/80 and anti-CD11b mAbs and analyzed Downloaded from by flow cytometry (B and C, time courses). Values in A–C are ex- pressed as the mean Ϯ SEM (n ϭ p Ͻ ,ءء ;p Ͻ 0.05 ,ء ;condition/5 p Ͻ 0.0001; SES ϩ sTLR2 ,ءءء ;0.01 vs SES). D, Annexin V/propidium io- dide staining of leukocytes present in http://www.jimmunol.org/ the lavages at the peak time of PMN influx (shown, 3 h). The representa- tive scatter plots are from analyses of gated PMN. Apoptotic cells were identified according to the annexin Vϩ/PIϪ (lower right quadrant, early apoptosis) and annexin Vϩ/PIϩ (up- per right quadrant, late apoptosis/ne- crosis) staining. Percentage of cells in by guest on September 29, 2021 the apoptotic quadrants is shown ,ءءء ;mean Ϯ SEM, n ϭ 5/condition) p Ͻ 0.0001; significant reduction vs SES). E, Western blot of peritoneal lavages taken at the indicated times and tested for mouse sTLR2 (msTLR2) release. Densitometric scanning of msTLR2 levels at the peak of PMN influx is shown (right; p Ͻ 0.05; SES ϩ ,ء ;n ϭ 5/condition sTLR2 vs SES).

sTLR2 reduces peritoneal PMN infiltration without ever, affect the capacity of the mice to clear the infection, because compromising bacterial clearance no difference in bacterial load either in the peritoneal cavity (Fig. The in vitro and in vivo anti-inflammatory effects of sTLR2 de- 5B) or blood (Fig. 5C) between sTLR2-treated and -nontreated scribed previously raised the question of whether such effects mice was observed. To study the inflammation-modulating effect would be detrimental to bacterial clearance during infection. To of sTLR2 further, mice were injected with a higher dose of S. 8 address this issue, an experimental model of acute peritoneal in- epidermidis (5 ϫ 10 CFU), and the effect of increasing doses of flammation consisting of an i.p. challenge with 5 ϫ 107 CFU of S. sTLR2 (10–1000 ng) was tested (Fig. 5, D–F). All doses of sTLR2 epidermidis in the absence or presence of sTLR2 (100 ng) was tested showed a similar suppressive effect on the PMN numbers used first. At this bacterial inoculum, the infection almost com- recruited to the peritoneal cavity over the 18-h period postinjection pletely cleared by 12 h. PMN numbers in the peritoneum of mice (Fig. 5D). This effect was most significant at the peak time (12 h) injected with S. epidermidis showed peak levels at 12 h postinjec- of PMN influx. Despite this significant reduction in local PMN tion (Fig. 5A, inset). In the presence of sTLR2, peritoneal PMN numbers, bacterial clearance from the peritoneal cavity was not accumulation at the peak time of their influx was significantly negatively affected by sTLR2 treatment (Fig. 5E). All mice reduced (Fig. 5A). Such reduced early PMN influx did not, how- (sTLR2 treated and nontreated) showed a marked reduction in The Journal of Immunology 513

FIGURE 5. sTLR2 reduces perito- neal PMN infiltration without com- promising bacterial clearance. A–F, Mice (n ϭ 5/condition) were i.p. in- oculated with 5 ϫ 107 (A–C)or5ϫ 108 (D–F) CFU S. epidermidis alone or together with 100 ng (A–C)orthe indicated amounts of sTLR2. At the indicated times, mice were sacrificed, the peritoneal cavity was lavaged, and PMN numbers in the lavages (A and Downloaded from D) were determined by differential cell counts on cytospin preparations. Bacterial titers in the peritoneal fluid and blood (B, C, E, and F) were de- termined, as described in Materials and Methods. Values in A and D–F are expressed as the mean Ϯ SEM http://www.jimmunol.org/ ,ءء ;p Ͻ 0.05 ,ء ;n ϭ 5/condition) p Ͻ 0.0001; S. epi. ϩ ,ءءء ;p Ͻ 0.01 sTLR2 vs S. epi.). by guest on September 29, 2021

bacterial load between 3 and 6 h postinjection. In sTLR2-treated the rafts, are believed to be critical to signaling (27, 31–33). mice, there was an apparent increase in bacterial clearance; how- Analysis of lipid raft preparations from freshly isolated non- ever, this is most probably not physiologically significant, because stimulated (control) monocytes (Fig. 6A) confirmed the prefer- it was very modest in magnitude. In the peripheral circulation, ential association of CD14 with lipid rafts and TLR2 with although the mice treated with the highest dose of sTLR2 (1 ␮g) detergent-soluble (nonraft) fractions (31, 32). Pam3Cys stimu- showed increased bacterial load at the earliest time (3 h) postin- lation resulted in an enrichment of TLR2 in lipid rafts and re- jection (Fig. 5F), this effect does not appear to be physiologically duced levels of CD14, most likely as a consequence of the significant, because none of the animals showed peripheral bacte- activation-induced shedding of soluble CD14 (9). However, rial abscesses, symptoms of shock, or died from the infection, and when cells were stimulated in the presence of sTLR2, the pat- all animals had cleared the blood infection almost completely by tern of CD14 and TLR2 partition into membrane domains re- 6 h postinjection. Moreover, plasma levels of the acute-phase re- sembled that in nonstimulated cells (Fig. 6A), indicating that actant, serum amyloid-A, were similar in control (S. epidermidis sTLR2 interferes with the ligand-induced TLR2 mobilization to only), and sTLR2-treated mice at all time points examined (data lipid rafts for signaling, and consequently, with the approxima- not shown), indicating that the relatively small increase in bacterial tion of TLR2 to CD14 in the rafts. load in the bloodstream of 1 ␮g of sTLR2-treated mice was not A decoy receptor activity would explain, at least in part, such significant enough to impact on the level of the systemic acute- interference by sTLR2. We tested this possibility and found that phase response. sTLR2 specifically binds Pam3Cys lipopeptide (Fig. 6B, left panel) in a ligand concentration-dependent manner, confirming sTLR2 disrupts the interaction of mCD14 with mTLR2, acts as a previous reports (34–36). We also tested for a possible inter- decoy receptor, and associates with mCD14 action of sTLR2 with whole bacteria. A sTLR2-Fc fusion pro- We next examined the mechanism underlying the regulatory tein, but not an irrelevant control, specifically bound heat-killed capacity of sTLR2. We first tested whether sTLR2 affected the S. epidermidis (Fig. 6B, right) as well as S. aureus (data not ligand-induced clustering of mCD14 and mTLR2 in lipid rafts. shown). These findings indicated the potential of sTLR2 to act The ligand-induced mobilization of TLR2 and TLR4 to lipid as a decoy receptor; this activity may contribute to sTLR2’s rafts and their close proximity to CD14, which resides mainly in negative regulatory capacity. 514 NEGATIVE REGULATION BY sTLR2 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. sTLR2 disrupts mTLR2 triggering. A, mCD14 and mTLR2 partitioning into lipid raft and nonraft fractions following monocyte stimulation with Pam3Cys Ϯ 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-Fc binding 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 gave identical 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-Alexa 488 (donor). Monocytes were preincubated and labeled in the absence or presence of sTLR2 or BSA. Threshold for significant FRET was determined with an anti-C3aR-Alexa488 mAb used as FRET donor-negative control. Results are from four experiments. E, Chemical cross-linking (BS3) in cells (5 ϫ 106) incubated with 5 ␮g of sTLR2-His or sCD55-His. Cross-linked His-tagged in cell lysates were Ni-NTA pulled down and analyzed by Western blotting. Head arrows point at Ni-NTA-precipitated, CD14-cross-linked polypeptides. Left panel, Mobility of sTLR2 (5 ␮g) and mCD14 monomers pulled down (sTLR2) or immunoprecipitated (mCD14) from High Five cell culture supernatants or 5 ϫ 106 CHO-CD14 transfectants, respectively. Right panel, right lane, Control immunoprecipitation of mTLR2. Results are from four experiments.

We speculated that sTLR2 might also disrupt the close prox- cell lysates. The typical ϳ110-kDa mTLR2 polypeptide band imity of mCD14 to mTLR2 directly, i.e., in the absence of (17) was consistently detected by Western blotting in mCD14 ligand, by interacting with mTLR2 and/or mCD14. We tested immunoprecipitates from monocyte cell lysates (Fig. 6C, left this possibility by first examining the effect of sTLR2 on the track). In addition, TLR2 polypeptide bands most likely ligand-independent natural association of mTLR2 with mCD14 corresponding to an intracellular (ϳ95-kDa) glycoform of the in the detergent-soluble fractions of normal human monocyte mature protein (37) and to fully glycosylated (ϳ83-kDa) and The Journal of Immunology 515 intracellularly located sTLR2 (17) were detected. The ϳ110-kDa ulated highlight the importance of such regulation to the mainte- mTLR2 polypeptide band was not detected when the coimmuno- nance of immune homeostasis. sTLR2 is the only soluble form of precipitation experiments were performed following preincubation a mammalian TLR to date identified that occurs naturally, because of monocytes with sTLR2 (Fig. 6C, center track), indicating that it is constitutively released by normal monocytes, and present in sTLR2 interfered with the natural interaction of mCD14 with normal human plasma, breast milk (17–19), saliva (20, 21), mouse mTLR2, and that such interference takes place at the cell surface. peritoneal lavage fluids (this study), and plasma, as well as bovine We obtained confirmatory evidence of this interference by per- and porcine plasma (J. Rey-Nores, unpublished data). It has been forming FRET studies. FRET was used because it allows for the proposed that sTLR2 may protect the host from excessive initial evaluation of interactions between neighboring (colocalized) mol- triggering of TLR2, which may result in deleterious TLR2-medi- ecules by determining their proximity within Յ10-nm range (26). ated innate immune responses (1, 12, 17, 18, 20). The full extent In this study, FRET efficiency for the transfer of energy from the of sTLR2’s regulatory capacity, the mechanism(s) underlying it, anti-TLR2 Alexa488 (donor) mAb to the anti-CD14 Cy3 (accep- and its biological relevance in vivo have not, however, been ad- tor) mAb, used to label mTLR2 and mCD14, was measured. An dressed to date. In this study, we demonstrated that sTLR2 regu- increase in TLR2 (green, donor) fluorescence after CD14 (red, lates TLR2-mediated cellular responses induced by microbial acceptor) photobleaching was detected in monocytes (Fig. 6D, components and whole Gram-positive bacteria in vitro and in vivo, center), indicating energy transfer and, thus, close proximity be- and that sTLR2 also has the potential to modulate critical effector tween the two molecules. This was in agreement with the results of functions, namely phagocytosis and superoxide production. Two the coimmunoprecipitation experiments. In the presence of sTLR2, mechanisms contributing to such regulatory activity were identi- however, no increase in TLR2 fluorescence after CD14 photo- fied: first, the capacity of sTLR2 to act as a decoy microbial re- Downloaded from bleaching was observed, and FRET efficiency between TLR2 and ceptor, and second, its capacity to disrupt the interaction of TLR2 CD14 was reduced to almost background levels (i.e., E% ϭ 2.9 Ϯ with its coreceptor by binding to CD14. The physiological rele- 0.5, threshold for significant energy transfer defined with an anti- vance of such elaborate negative regulation is highlighted in ex- C3aR Alexa488 mAb used as FRET donor-negative control; Fig. periments that demonstrate the hypersensitivity of PBMC to li- 6D, right). These findings thus confirmed that sTLR2 perturbs the popeptide stimulation in the presence of sTLR2-depleted serum.

mCD14-mTLR2 interaction. These findings indicate that regulation by naturally occurring http://www.jimmunol.org/ The interfering effect exerted by sTLR2 in the absence of ligand sTLR2 is a physiological feature that contributes to a controlled, raised the question of whether this effect results from an interaction yet efficient, host innate immune response against microbial of sTLR2 with mCD14 and/or mTLR2. To address this issue, we . performed chemical cross-linking experiments by using a non- To assess sTLR2’s regulatory capacity in vivo, we used two cleavable, membrane-impermeable, cross-linking reagent, BS3 well-characterized mouse models of acute inflammation based on (Fig. 6E). The purified (Ni-NTA pulled-down) rHis-tagged sTLR2 the injection of a S. epidermidis-derived cell-free supernatant or protein (sTLR2-His) and mCD14, immunoprecipitated from CHO- live S. epidermidis into the peritoneal cavity. These models were CD14 cell transfectants, showed expected sizes of ϳ72–75 kDa chosen because they allowed us to evaluate the effect of sTLR2 on and ϳ54–56 kDa, respectively, when analyzed by SDS-PAGE, the temporal changes in leukocyte infiltration, inflammatory and by guest on September 29, 2021 followed by immunoblotting with specific Abs (Fig. 6E, left chemotactic mediator expression, and bacterial clearance kinetics panel). Incubation of CHO-CD14 transfectants with sTLR2-His, that have been extensively characterized in human peritonitis (24, followed by chemical cross-linking, Ni-NTA bead pull-down from 29). By using these models, we established that administration of the CHO-CD14 cell lysates, and anti-CD14 or anti-TLR2 Western sTLR2 reduced the level of PMN recruitment into the peritoneal blotting revealed bands of ϳ125–130 kDa and ϳ250–260 kDa, cavity in animals challenged either with Gram-positive bacteria- i.e., of lower mobility than that of mCD14 (Fig. 6E, center panel). derived microbial components or live bacteria. Notably, despite its The size of these bands was consistent with that estimated for ability to control the inflammatory response, and in vitro capacity mCD14/sTLR2 heterodimers (ϳ126–131 kDa) and mCD14/ to interfere with the phagocytic uptake of bacteria and microbial- sTLR2 dimer of dimers (ϳ252–262 kDa). To test for an interaction induced superoxide production, sTLR2 administration, irrespec- of sTLR2 with mTLR2, a similar cross-linking strategy was ap- tive of its amount or the dose of bacteria tested, did not have a plied to CHO-mTLR2 cell transfectants preincubated with sTLR2- detrimental impact on the clearance of bacteria. The maintenance His. In this study, however, Ni-NTA bead pull-down, followed by of efficient peritoneal removal of bacteria in the face of sTLR2 anti-TLR2 immunoblotting, did not show any cross-linked TLR2 modulation is likely to be due in part to the following: 1) the fact polypeptide band (Fig. 6E, right panel). that modulation by sTLR2 of PMN recruitment was most signif- Together, these findings indicated that sTLR2 may exert nega- icant at the peak (12 h) of their influx, when the animals had tive regulatory effects by acting as a decoy receptor, and also by cleared the infection almost completely, and 2) the activity of a disrupting the close proximity between the coreceptor (CD14) and number of other humoral mediator pathways that contribute to ef- the receptor (TLR2) that is crucial to highly efficient signaling. ficient bacterial clearance and killing, including complement com- Such disruption most likely results from the capacity of sTLR2 to ponents, mannose-binding lectin, and Igs, as well as cell surface Fc interact with the coreceptor. and scavenger receptors. The latter is consistent with our obser- vation that the negative effect of sTLR2 on phagocytosis in vitro Discussion was significantly reduced in the presence of serum (our unpub- Following the initial description of the crucial involvement of lished data). Clearly, in vivo other immune components make a TLRs in acute inflammation and and the more recent, substantial contribution to bacterial clearance mechanisms. The well-documented observations implicating TLRs in a number of possibility that sTLR2 affects bacterial clearance in certain pathol- autoimmune and chronic inflammatory diseases, such as lupus, ogies (e.g., complement deficiency) or when administered at arthritis, inflammatory bowel disease, and artherosclerosis, it has higher doses, however, remains to be investigated. become clear that TLR overactivation plays a prominent role in the By contrast to its inhibitory effect on PMN mobilization to the pathogenesis of a variety of acute and chronic inflammatory con- site of injury, sTLR2 did not influence MNC recruitment, despite ditions (2). The different levels at which TLR activity can be reg- causing increased production of MCP-1. We can only speculate on 516 NEGATIVE REGULATION BY sTLR2 the mechanism underlying this differential effect, because we have associating with CD14, together with sTLR2’s decoy receptor ac- not yet investigated the additional effects that sTLR2 might exert tivity, may affect the mobilization of mTLR2 to lipid rafts for on the complex chemokine network that controls MNC recruit- signaling upon cell stimulation, and lead to reduced proinflamma- ment. This would require the assessment of not only other MNC- tory responses, which in turn result in the observed reduction in specific chemokines such as MIP-1␣ (CCL3) and RANTES PMN recruitment to the site of infection. Similar to our findings, a (CCL5), but also the regulatory mechanisms controlling that net- peritoneal infection model using Salmonella spp. in CD14-defi- work, which include the activity of matrix metalloproteinases, cient mice has also shown impaired influx of PMN, but not MNC CD26, and the decoy chemokine receptors D6 and Duffy Ag re- (45). This observation raises the possibility that the interfering ceptor for chemokines (38–41). With regard to the increased lev- effect of sTLR2 on CD14 coreceptor activity demonstrated in this els of MCP-1 in the presence of sTLR2, it could be speculated, for study might constitute the predominant mechanism underlying the example, that it results from a negative effect of sTLR2 on the modulatory effect of sTLR2 on the inflammatory response ob- levels of D6 and Duffy Ag receptor for chemokines. These decoy served in the in vivo models we have studied. receptors are critically involved in the deactivation and elimination The ability of sTLR2 to affect the activity of CD14 raises the from the circulation of a number of chemokines, including MCP-1, question of why TLR4- and TLR3-mediated monocyte responses, but not KC or MIP-2. Notably, D6 production appears to be reg- which also require CD14 for efficient signaling, are not affected, as ulated, at least in part, by NF-␬B (42), which we demonstrate in indicated by the absence of a negative effect of sTLR2 on LPS or the present study to be negatively affected by sTLR2. poly(I:C)-stimulated Mono Mac-6 cells (Fig. 2E). It is possible Nevertheless, this differential effect of sTLR2 on early (PMN) that, when the effect of sTLR2 depends solely on its capacity to and late (MNC) leukocyte recruitment and the consequent skewing interact with CD14 (no decoy activity, i.e., TLR3 and TLR4/MD2 Downloaded from of the leukocyte influx in favor of MNC appear to promote the signaling), the extent of sTLR2 inhibition may critically depend more efficient removal of senescent PMN, as indicated by the sub- not only on the local concentration of sTLR2, but also on the stantially reduced proportion of late apoptotic/early necrotic PMN expression levels of CD14 (mCD14 or sCD14) and the mTLR found in the peritoneal cavity of sTLR2-treated mice (Fig. 4D). involved, as well as on the affinity and stoichiometry of the inter- This effect might ultimately favor more rapid resolution of inflam- actions of mTLR, CD14, and sTLR2, and those of the ligand with mation. A similar pattern of effects on the modulation of leukocyte mTLR and CD14. In support of this possibility, we observed that http://www.jimmunol.org/ trafficking and PMN apoptosis in resolving acute inflammation has sTLR2 exerts a significant negative effect on the LPS stimulation been previously observed for IL-6/soluble IL-6R signaling (29). of a number of cell lines of epithelial origin, which express very Such anti-inflammatory capacity of sTLR2 may prove to be a cru- low levels of TLR4, do not express mCD14, and require sCD14 for cial factor during septic shock. sensitive signaling (A.-C. Raby and M. Labe´ta, manuscript in This study has also shed light on the mechanisms underlying preparation). Clearly, a better knowledge of the parameters govern- sTLR2’s regulatory capacity. The fact that sTLR2 does not affect ing the interactions of TLRs, CD14, the ligands, and sTLR2 will im- signaling via other TLRs, the IL-1R and non-TLR-related recep- prove our understanding of the activity of sTLR2. With regard to tors, or nonreceptor-mediated signaling (Fig. 2E) suggested that TLR3, its mostly intracellular location and function (8) may limit the the primary effect of sTLR2 is exerted upstream of signaling prox- activity of sTLR2. Nevertheless, the modulatory activity of sTLR2 by guest on September 29, 2021 imal to TLR2 ligand recognition. We therefore first tested whether may not be limited to Gram-positive bacteria-induced responses. In- sTLR2 affected the ligand-induced clustering of mCD14 and deed, a recent report demonstrated the involvement of TLR2 in mTLR2 in lipid rafts. It has been demonstrated that the TLR co- antibiotic-treated Gram-negative bacterial sepsis (46). This finding receptor, CD14, resides mainly in cholesterol and sphingolipid- raises the possibility that sTLR2 also contributes to controlling rich detergent-resistant membrane microdomains, termed lipid Gram-negative bacteria-induced inflammation. rafts (31, 32). It has also been shown that, in resting conditions, In conclusion, the findings reported in this study define sTLR2 TLR4 and TLR2 are localized mainly outside lipid rafts in the as an efficient regulator of TLR2-mediated inflammatory re- detergent-soluble membrane fractions. Upon TLR ligand-induced sponses, because it is capable of reducing inflammation by con- cell stimulation, the specific TLR is recruited to lipid rafts, where trolling PMN influx while preserving MNC recruitment and with- it is found in close proximity to CD14 and other cell surface mol- out compromising bacterial clearance. The capacity of sTLR2 to ecules, thus forming a receptor cluster that is believed to be critical exert its regulatory effect not only by acting as a decoy microbial to signaling (32, 33). We found that sTLR2 interferes with the receptor, but also by targeting the coreceptor, may inform the de- ligand-induced mobilization of TLR2 to lipid rafts for signaling. sign of novel therapeutics against acute and chronic inflammatory Such interference would be explained, at least in part, by the ca- conditions that will aim at disrupting the coreceptor’s activity, thus pacity of sTLR2 to act as a decoy microbial receptor, demonstrated blunting, but not abrogating, microbial recognition and host innate in this study (Fig. 6B). This decoy activity may involve competi- immune responses. tion between sTLR2 and mTLR2 for binding not only the micro- bial ligand, but also TLR1, the heterodimerization partner for mTLR2 that is required for recognition of and responses to triacy- Acknowledgments lated lipopeptides, like the Pam3Cys lipopeptide used in this study We are indebted to J. E. Rey-Nores (School of Applied Sciences, Univer- (43, 44). Such a heterodimeric receptor complex involving sTLR2 sity of Wales Institute, Cardiff, U.K.) for critical insight, expert help, dis- may be unable to signal, because only one TIR domain (TLR1’s) cussions, and review of this manuscript. We also thank R. J. Matthews, would be involved. This possibility, however, remains to be tested. B. P. Morgan (Department of Medical Biochemistry and Immunology, We also found, however, that sTLR2 disrupts the close prox- School of Medicine, Cardiff University, Cardiff, U.K.), and N. Gay (De- imity of mCD14 to mTLR2 in the absence of ligand by associating partment of Biochemistry, University of Cambridge, Cambridge, U.K.) for with mCD14, as indicated by the coimmunoprecipitation, FRET, helpful discussions and critical reading of the manuscript. and chemical cross-linking experiments (Fig. 6, C–E). Such close proximity is crucial to CD14’s coreceptor function and highly ef- ficient TLR signaling. Thus, sTLR2’s capacity to interfere with the Disclosures mCD14-mTLR2 interaction and disrupt the coreceptor function by The authors have no financial conflict of interest. The Journal of Immunology 517

References IL-6 and its soluble receptor orchestrate a temporal switch in the pattern of leu- kocyte recruitment seen during acute inflammation. Immunity 14: 705–714. 1. Liew, F. Y., D. Xu, E. K. Brint, and L. A. O’Neill. 2005. Negative regulation of 25. Wouters, F. S., and P. I. Bastiaens. 2001. Imaging protein-protein interactions by Toll-like receptor-mediated immune responses. Nat. Rev. Immunol. 5: 446–458. fluorescence resonance energy transfer (FRET) microscopy. Curr. Protoc. Pro- 2. Kanzler, H., F. J. Barrat, E. M. Hessel, and R. L. Coffman. 2007. Therapeutic tein Sci. 3: 19.5.1–19.5.15. targeting of innate immunity with Toll-like receptor agonists and antagonists. 26. Kenworthy, A. K. 2001. Imaging protein-protein interactions using fluorescence Nat. Med. 13: 552–559. resonance energy transfer microscopy. Methods 24: 289–296. 3. Kaisho, T., and S. Akira. 2006. Toll-like receptor function and signaling. 27. Triantafilou, M., M. Manukyan, A. Mackie, S. Morath, T. Hartung, H. Heine, and J. Allergy Clin. Immunol. 117: 979–987. K. Triantafilou. 2004. and Toll-like receptor 2 internalization 4. Gay, N. J., and M. Gangloff. 2007. Structure and function of Toll receptors and and targeting to the Golgi are lipid raft-dependent. J. Biol. Chem. 279: their ligands. Annu. Rev. Biochem. 76: 23.1–23.25. 40882–40889. 5. Schnare, M., G. M. Barton, A. C. Holt, K. Takeda, S. Akira, and R. Medzhitov. 28. Park, J. H., Y. G. Kim, M. Shaw, T. D. Kanneganti, Y. Fujimoto, K. Fukase, 2001. Toll-like receptors control activation of adaptive immune responses. Nat. N. Inohara, and G. Nunez. 2007. Nod1/RICK and TLR signaling regulate che- Immunol. 2: 947–950. mokine and antimicrobial innate immune responses in mesothelial cells. J. Im- 6. Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, and munol. 179: 514–521. D. Golenbock. 1999. Cutting edge: recognition of Gram-positive bacterial cell 29. McLoughlin, R. M., J. Witowski, R. L. Robson, T. S. Wilkinson, S. M. Hurst, wall components by the innate occurs via Toll-like receptor 2. A. S. Williams, J. D. Williams, S. Rose-John, S. A. Jones, and N. Topley. 2003. J. Immunol. 163: 1–5. Interplay between IFN-␥ and IL-6 signaling governs neutrophil trafficking and 7. Haziot, A., E. Ferrero, F. Kontgen, N. Hijiya, S. Yamamoto, J. Silver, apoptosis during acute inflammation. J. Clin. Invest. 112: 598–607. C. L. Stewart, and S. M. Goyert. 1996. Resistance to endotoxin shock and re- 30. Topley, N., T. Liberek, A. Davenport, F. K. Li, H. Fear, and J. D. Williams. 1996. duced dissemination of Gram-negative bacteria in CD14-deficient mice. Immu- Activation of inflammation and leukocyte recruitment into the peritoneal cavity. nity 4: 407–414. Kidney Int. Suppl. 56: S17–S21. 8. Lee, H. K., S. Dunzendorfer, K. Soldau, and P. S. Tobias. 2006. Double-stranded 31. Triantafilou, M., K. Miyake, D. T. Golenbock, and K. Triantafilou. 2002. Medi- RNA-mediated TLR3 activation is enhanced by CD14. Immunity 24: 153–163. ators of innate immune recognition of bacteria concentrate in lipid rafts and 9. Durieux, J. J., N. Vita, O. Popescu, F. Guette, J. Calzada-Wack, R. Munker, facilitate -induced cell activation. J. Cell Sci. 115: 2603–2611. R. E. Schmidt, J. Lupker, P. Ferrara, H. W. Ziegler-Heitbrock, and M. O. Labe´ta. 32. Pfeiffer, A., A. Bottcher, E. Orso, M. Kapinsky, P. Nagy, A. Bodnar, I. Spreitzer, Downloaded from 1994. The two soluble forms of the lipopolysaccharide receptor: CD14: charac- G. Liebisch, W. Drobnik, K. Gempel, et al. 2001. Lipopolysaccharide and cer- terization and release by normal human monocytes. Eur. J. Immunol. 24: amide docking to CD14 provokes ligand-specific receptor clustering in rafts. Eur. 2006–2012. J. Immunol. 31: 3153–3164. 10. Frey, E. A., D. S. Miller, T. G. Jahr, A. Sundan, V. Bazil, T. Espevik, 33. Triantafilou, M., F. G. Gamper, R. M. Haston, M. A. Mouratis, S. Morath, B. B. Finlay, and S. D. Wright. 1992. Soluble CD14 participates in the response T. Hartung, and K. Triantafilou. 2006. Membrane sorting of Toll-like receptor of cells to lipopolysaccharide. J. Exp. Med. 176: 1665–1671. (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic 11. Labe´ta, M. O., K. Vidal, J. E. Nores, M. Arias, N. Vita, B. P. Morgan, associations with CD36 and intracellular targeting. J. Biol. Chem. 281:

J. C. Guillemot, D. Loyaux, P. Ferrara, D. Schmid, et al. 2000. Innate recognition 31002–31011. http://www.jimmunol.org/ of bacteria in human milk is mediated by a milk-derived highly expressed pattern 34. Iwaki, D., H. Mitsuzawa, S. Murakami, H. Sano, M. Konishi, T. Akino, and recognition receptor, soluble CD14. J. Exp. Med. 191: 1807–1812. Y. Kuroki. 2002. The extracellular Toll-like receptor 2 domain directly binds 12. Divanovic, S., A. Trompette, L. K. Petiniot, J. L. Allen, L. M. Flick, Y. Belkaid, peptidoglycan derived from Staphylococcus aureus. J. Biol. Chem. 277: R. Madan, J. J. Haky, and C. L. Karp. 2007. Regulation of TLR4 signaling and 24315–24320. the host interface with pathogens and danger: the role of RP105. J. Leukocyte 35. Sato, M., H. Sano, D. Iwaki, K. Kudo, M. Konishi, M. Takahashi, T. Takahashi, Biol. 82: 265–271. H. Imaizumi, Y. Asai, and Y. Kuroki. 2003. Direct binding of Toll-like receptor ␬ ␣ 13. O’Neill, L. A. 2008. When signaling pathways collide: positive and negative 2 to zymosan, and zymosan-induced NF- B activation and TNF- secretion are regulation of Toll-like receptor . Immunity 29: 12–20. down-regulated by lung surfactant protein A. J. Immunol. 171: 14. O’Neill, L. A. 2003. Therapeutic targeting of Toll-like receptors for inflammatory 417–425. and infectious diseases. Curr. Opin. Pharmacol. 3: 396–403. 36. Vasselon, T., P. A. Detmers, D. Charron, and A. Haziot. 2004. TLR2 recognizes a bacterial lipopeptide through direct binding. J. Immunol. 173: 7401–7405. 15. Zuany-Amorim, C., J. Hastewell, and C. Walker. 2002. Toll-like receptors as 37. Weber, A. N., M. A. Morse, and N. J. Gay. 2004. Four N-linked glycosylation by guest on September 29, 2021 potential therapeutic targets for multiple diseases. Nat. Rev. Drug Discov. 1: sites in human Toll-like receptor 2 cooperate to direct efficient biosynthesis and 797–807. secretion. J. Biol. Chem. 279: 34589–34594. 16. Iwami, K. I., T. Matsuguchi, A. Masuda, T. Kikuchi, T. Musikacharoen, and 38. Fillion, I., N. Ouellet, M. Simard, Y. Bergeron, S. Sato, and M. G. Bergeron. Y. Yoshikai. 2000. Cutting edge: naturally occurring soluble form of mouse 2001. Role of chemokines and formyl peptides in pneumococcal pneumonia- Toll-like receptor 4 inhibits lipopolysaccharide signaling. J. Immunol. 165: induced monocyte/macrophage recruitment. J. Immunol. 166: 7353–7361. 6682–6686. 39. Keepers, T. R., L. K. Gross, and T. G. Obrig. 2007. Monocyte chemoattractant 17. LeBouder, E., J. E. Rey-Nores, N. K. Rushmere, M. Grigorov, S. D. Lawn, protein 1, macrophage inflammatory protein 1␣, and RANTES recruit macro- M. Affolter, G. E. Griffin, P. Ferrara, E. J. Schiffrin, B. P. Morgan, and phages to the kidney in a mouse model of hemolytic-uremic syndrome. Infect. M. O. Labe´ta. 2003. Soluble forms of Toll-like receptor (TLR) 2 capable of Immun. 75: 1229–1236. modulating TLR2 signaling are present in human plasma and breast milk. J. Im- 40. Comerford, I., and R. J. Nibbs. 2005. Post-translational control of chemokines: a munol. 171: 6680–6689. role for decoy receptors? Immunol. Lett. 96: 163–174. 18. Heggelund, L., T. Flo, K. Berg, E. Lien, T. E. Mollnes, T. Ueland, P. Aukrust, 41. Locati, M., Y. Martinez de la Torre, E. Galliera, R. Bonecchi, H. Bodduluri, T. Espevik, and S. S. Froland. 2004. Soluble Toll-like receptor 2 in HIV infec- G. Vago, A. Vecchi, and A. Mantovani. 2005. Silent chemoattractant receptor: tion: association with disease progression. AIDS 18: 2437–2439. D6 as a decoy and scavenger receptor for inflammatory CC chemokines. Cytokine 19. Ueland, T., T. Espevik, J. Kjekshus, L. Gullestad, T. Omland, I. B. Squire, Growth Factor Rev. 16: 679–686. S. S. Froland, T. E. Mollnes, K. Dickstein, and P. Aukrust. 2006. Mannose bind- 42. McKimmie, C. S., A. R. Fraser, C. Hansell, L. Gutie´rrez, S. Philipsen, L. Connell, ing lectin and soluble Toll-like receptor 2 in heart failure following acute myo- A. Rot, M. Kurowska-Stolarska, P. Carreno, M. Pruenster, et al. 2008. Hemo- cardial infarction. J. Card. Fail. 12: 659–663. poietic cell expression of the chemokine decoy receptor D6 is dynamic and reg- 20. Kuroishi, T., Y. Tanaka, A. Sakai, Y. Sugawara, K. Komine, and S. Sugawara. ulated by GATA1. J. Immunol. 181: 3353–3363. 2007. Human parotid saliva contains soluble Toll-like receptor (TLR) 2 and mod- 43. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong, ulates TLR2-mediated -8 production by monocytic cells. Mol. Immu- R. L. Modlin, and S. Akira. 2002. Cutting edge: role of Toll-like receptor 1 in nol. 44: 1969–1976. mediating immune response to microbial . J. Immunol. 169: 10–14. 21. Srinivasan, M., K. N. Kodumudi, and S. L. Zunt. 2008. Soluble CD14 and Toll- 44. Jin, M. S., S. E. Kim, J. Y. Heo, M. E. Lee, H. M. Kim, S.-G. Paik, H. Lee, and like receptor-2 are potential salivary biomarkers for oral lichen planus and burn- J.-O. Lee. 2007. Crystal structure of the TLR1-TLR2 heterodimer induced by ing mouth syndrome. Clin. Immunol. 126: 31–37. binding of a tri-acylated lipopeptide. Cell 130: 1071–1082. 22. Topley, N., R. K. Mackenzie, and J. D. Williams. 1996. Macrophages and me- 45. Yang, K. K., B. G. Dorner, U. Merkel, B. Ryffel, C. Schutt, D. Golenbock, sothelial cells in bacterial peritonitis. Immunobiology 195: 563–573. M. W. Freeman, and R. S. Jack. 2002. Neutrophil influx in response to a peri- 23. Rey Nores, J. E., A. Bensussan, N. Vita, F. Stelter, M. A. Arias, M. Jones, toneal infection with Salmonella is delayed in lipopolysaccharide-binding protein S. Lefort, L. K. Borysiewicz, P. Ferrara, and M. O. Labe´ta. 1999. Soluble CD14 or CD14-deficient mice. J. Immunol. 169: 4475–4480. acts as a negative regulator of human activation and function. Eur. J. Im- 46. Spiller, S., G. Elson, R. Ferstl, S. Dreher, T. Mueller, M. Freudenberg, munol. 29: 265–276. B. Daubeuf, H. Wagner, and C. J. Kirschning. 2008. TLR4-induced IFN-␥ pro- 24. Hurst, S. M., T. S. Wilkinson, R. M. McLoughlin, S. Jones, S. Horiuchi, duction increases TLR2 sensitivity and drives Gram-negative sepsis in mice. N. Yamamoto, S. Rose-John, G. M. Fuller, N. Topley, and S. A. Jones. 2001. J. Exp. Med. 205: 1747–1754.