Glycolipids Treponema Initiation of Innate Immune Responses by the Protein, CD14, and Toll-Like Receptors in Involvement of Lipo

Involvement of Lipopolysaccharide Binding Protein, CD14, and Toll-Like Receptors in the Initiation of Innate Immune Responses by Treponema Glycolipids This information is current as of September 29, 2021. Nicolas W. J. Schröder, Bastian Opitz, Norbert Lamping, Kathrin S. Michelsen, Ulrich Zähringer, Ulf B. Göbel and Ralf R. Schumann J Immunol 2000; 165:2683-2693; ; doi: 10.4049/jimmunol.165.5.2683 Downloaded from http://www.jimmunol.org/content/165/5/2683

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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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Involvement of Lipopolysaccharide Binding Protein, CD14, and Toll-Like Receptors in the Initiation of Innate Immune Responses by Treponema Glycolipids1

Nicolas W. J. Schro¨der,* Bastian Opitz,* Norbert Lamping,2* Kathrin S. Michelsen,* Ulrich Za¨hringer,† Ulf B. Go¨bel,* and Ralf R. Schumann3*

Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-␣ synthesis in human monocytes increasing with culture time. This activity could be reduced signiﬁcantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-␣ secretion

in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, Downloaded from while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-␬B translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent http://www.jimmunol.org/ fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs. The Journal of Immunology, 2000, 165: 2683–2693.

arly recognition of pathogenic microorganisms and sub- ing evidence that these compounds use similar host receptor path- sequent initiation of an innate immune response is essen- ways as LPS, i.e., it has been reported that PG is also recognized E tial for the host to survive acute and chronic infections by cellular CD14 (5, 6), and LTA from Bacillus subtilis interacts (1). Crucial elements of the reaction cascade initiated by Gram- with LPS binding protein (LBP) (7). The structural basis for the by guest on September 29, 2021 negative bacteria have been elucidated by chemical and biological immunostimulating properties of spirochetes has not yet been analysis of the major toxic cell wall component LPS. So far, the completely elucidated. The presence of LPS in spirochetes remains molecular mechanisms of host defense stimulation by Gram-pos- controversial; however, the analysis of the entire Treponema pal- itive bacteria and spirochetes have not been completely elucidated, lidum genome revealed the lack of LPS synthesis genes (8–11). and some of the results are yet controversial. Two compounds Lipoproteins of spirochetes clearly have been shown to induce found in the cell wall of Gram-positive microorganisms, lipotei- proinflammatory responses in host cells involving CD14 (12, 13). 4 choic acid (LTA) and peptidoglycan (PG), have been shown to Treponemes are strictly anaerobic bacteria not easily maintained induce inflammatory responses in host cells (2–4). There is grow- in culture. By molecular genetic analysis, several formerly “non- cultivable” treponemes have been identified recently (14–16),

*Institut fu¨r Mikrobiologie und Hygiene, Universita¨tsklinikum Charite´Medizinische some of which were associated with human periodontitis, a chronic Fakulta¨t der Humboldt-Universita¨t zu Berlin, Berlin, Germany; and †Laborgruppe inﬂammation of the periodontium causing severe costs to health Immunchemie, Forschungszentrum Borstel, Borstel, Germany care systems (17), or digital dermatitis, a disease commonly found Received for publication June 2, 1999. Accepted for publication June 20, 2000. in cattle (15). Two Treponema species were used in this study, The costs of publication of this article were defrayed in part by the payment of page Treponema brennaborense and Treponema maltophilum (15, 16, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 18). Previous analyses of Treponema denticola, another putative 1 This work was supported in part by grants given by the Deutsche Forschungsge- periodontal pathogen, have shown that its cell wall contained a meinschaft (Schu 828/1-5, to R.R.S.) and the Bundesministerium fu¨r Bildung und glycolipid chemically different from LPS (19). In this study, we Forschung (01 KI 94750, to R.R.S.; 01 KI 9471/9 and 01 KI 9851/0, to U.Z.; and 01 focus on the interaction of treponeme glycolipids and host cells. KI 9318, to U.B.G.). K.S.M. was supported by the Boehringer-Ingelheim Stiftung. The host response to bacterial compounds is regulated and mod- 2 Current address: Department of Lipid Biochemistry, Merck Research Laboratories, Rahway, NJ 07065. ulated by certain serum proteins and cellular receptor molecules 3 Address correspondence and reprint requests to Dr. Ralf R. Schumann, Institut fu¨r (20–22). LBP and soluble CD14, both able to bind LPS, are Mikrobiologie und Hygiene, Universita¨tsklinikum Charite´, Humboldt-Universita¨t zu present in serum in high quantities (23, 24). LBP is an acute-phase Berlin, Dorotheenstrasse 96, D-10117 Berlin, Germany. E-mail address: protein synthesized in the liver, the concentration of which rises [email protected] dramatically during systemic infection and the acute-phase re- 4 Abbreviations used in this paper: LTA, lipoteichoic acid; CHO, Chinese hamster ovary; ENP, endotoxin neutralizing protein; Kdo, 3-deoxy-D-manno-octulosonic acid; sponse (25, 26). It monomerizes LPS vesicles and transports LPS LAL, Limulus amoebocyte lysate; LBP, LPS binding protein; OMIZ-Pat, Treponema to the CD14 part of the cellular LPS receptor enabling inﬂamma- culture medium; PCP, phenol/chlorophorm/petroleum ether; PEM, peritoneal elicited ␣ macrophages; PG, peptidoglycan; TLR, Toll-like receptor; h, human; m, murine; tory responses, such as TNF- synthesis. LPS effects can be GLC, gas-liquid chromatography; MS, mass spectrometry. blocked by a range of inhibitors including polymyxin B, a

polypeptide known to bind lipid A, the active moiety of LPS (27). phenol/water method, higher yields were obtained (T. brennaborense, LPS effects can also be blocked by the mAb My 4 directed against 0.42%; T. maltophilum, 0.47%), therefore, the latter material was used for CD14 (21). In the last years, it has been shown convincingly that additional experiments. members of the Toll-like receptor (TLR) family are involved in the Stimulation of human monocytes and the murine macrophage recognition of pathogens by a wide variety of host organisms (28). cell line RAW 264.7 In Drosophila, Toll has been shown to be involved in antifungal responses (29), while a homologous protein, 18-wheeler, induces Peripheral blood samples obtained from healthy volunteers were mixed antibacterial responses (30). In vertebrates, strong evidence has with 50 U/ml of heparin as anticoagulant and diluted 1:2 with RPMI 1640. Then 30 ml of the diluted blood was carefully layered on top of 15 ml been presented that TLR-4 recognizes LPS of Gram-negative bac- Lymphoprep (Nycomed, Oslo, Norway) and centrifuged at 600 ϫ g with- teria (31–33), while TLR-2 recognizes PG of Gram-positive bac- out brake at 21°C for 15 min. The intermediate phase was recovered, teria, as well as lipoproteins of mycobacteria or Borrelia (34–39). washed twice with RPMI 1640, and recentrifuged at 600 ϫ g at 21°C for ϫ Regarding LTA, results for an involvement of TLR-2 or -4 have 5 min. To separate platelets, cells were further spun at 100 g at 21°C for 15 min. Remaining cells were diluted in RPMI 1640 containing 5% human been controversial (34, 35, 40). AB serum (Sigma) to a final concentration of 1 ϫ 106 cells/ml. Cells were Here we analyze the ability of two Treponema species isolated transferred to 96-well cell-culture plates (100 ␮l/well) and incubated at from a patient suffering from periodontitis and from a digital der- 37°C for 1.5 h, followed by two washing steps with RPMI 1640 to remove matitis lesion, respectively, to activate human monocytes, a murine nonadherent cells. Remaining monocytes were stimulated with culture su- macrophage cell line, macrophages obtained from C3H/HeJ mice, pernatants, butanol extracts of Treponema isolates, extracts of whole treponeme cells, Escherichia coli 0111:B4 LPS (Sigma), or PMA (Sigma) in the and Chinese hamster ovary (CHO) cells. First, we analyzed the presence or absence of 5% human AB serum in a total volume of 100 ␮l. involvement of the host LPS binding and receptor molecules LBP, For certain experiments, polymyxin B (Sigma) at a concentration of 5 Downloaded from CD14, as well as TLR-2 and -4. Second, a partial chemical puri- ␮g/ml was added directly before stimulation, as indicated. For selected fication was performed for structural analysis of the active com- experiments, monoclonal anti-CD14 Ab My 4 (Coulter, Hamburg, Ger- many) was incubated with the cells at a concentration of 5 ␮g/ml at 37°C pound. The results presented here should help to elucidate the role for 20 min before addition of stimuli to block CD14. After 4 h, superna- of spirochetes in chronic inflammatory reactions and to identify the tants were harvested and viability of cells was assessed via trypan blue mechanisms involved. staining. Additionally, 5 ϫ 104 RAW 264.7 cells per well (kindly provided by T. Blankenstein, Max-Delbru¨ck-Centrum, Berlin, Germany) were cul- http://www.jimmunol.org/ tured overnight in 96-well tissue culture plates using RPMI 1640 supple- Materials and Methods mented with 10% FCS. After repeated washing with RPMI 1640, stimu- Treponeme culture and processing of culture supernatants lation was performed in the presence or absence of 1 ␮g/ml recombinant ␮ Frozen stocks of T. brennaborense and T. maltophilum cells (300 ␮l, each murine LBP (rmLBP) in a total volume of 100 l. RAW 264.7 superna- stored at Ϫ80°C) were inoculated in 3 ml of culture medium (OMIZ-Pat) tants were harvested after4hofincubation, and cells were stained with as described previously (16). Bacteria were cultured under anaerobic con- trypan blue, ensuring integrity of cells. ditions (Anaerogen, Oxoid, Germany) at 37°C for 3–4 days. The cultures were then transferred to a larger volume of OMIZ-Pat (20–100 ml) and Preparation and stimulation of peritoneal elicited macrophages further incubated for 1–2 days. Viability of treponemes and possible pres- (PEM) ence of contaminating bacteria were assessed by dark field microscopy (400-fold magnification, BH2-RFCA microscope, Olympus, Hamburg, Peritoneal macrophages were isolated from C3H/HeJ or C3H/HeN mice by guest on September 29, 2021 Germany). Sterility controls of the medium were performed by incubating (Charles River, Sulzbach, Germany), by thioglycollate elicidation. Female OMIZ-Pat medium under aerobic and anaerobic conditions at 37°C for 1 7-wk-old mice were injected i.p. with 1.5 ml of 3% thioglycollate broth wk. The pH value of the culture medium was measured repeatedly. Cul- (Sifin, Berlin, Germany). After 3 days, mice were sacrificed and peritoneal tures were stopped at pH 6.0 and centrifuged at 12,000 ϫ g at 4°C for 20 macrophages were harvested by injection of 10 ml of ice-cold HBSS (Life min. The supernatant was passed through 0.2-␮m sterile filters (Schleicher Technologies) i.p. followed by aspiration. Cells were washed twice with ϫ 5 & Schuell, Dassel, Germany). For some studies, culture supernatants were RPMI 1640, and 2 10 cells were plated in 96-well tissue culture plates heat-inactivated at 100°C for 20 min and passed again through 0.2-␮m in RPMI 1640 containing 5% FCS. After 2 h, plates were washed twice sterile filters. OMIZ-Pat medium (16), treated similarly, was used as with RPMI 1640 to remove nonadherent cells, and remaining cells were control. stimulated with treponeme phenol/water extracts or LPS for 24 h in RPMI 1640 containing 5% non-heat-inactivated FCS followed by NO detection Extraction of culture supernatants and whole Treponema cells as described below. We used a modification of a published protocol for the extraction of LPS Quantitative detection of human and murine TNF-␣ and NO from Gram-negative cell walls using n-butanol (41). Briefly, filtered and heat-inactivated culture supernatants were mixed with an equal volume of Nunc MaxiSorp ELISA plates (Nunc, Roskilde, Denmark) were coated n-butanol and incubated at 4°C for1hona180° shaker. Subsequently, the with 0.5 mg/ml of anti-human TNF (anti-hTNF) Ab (PharMingen, Heidel- ϫ mixture was centrifuged at 26,000 g at 4°C for 1 h, and the upper butanol berg, Germany) in 100 mM NaHCO3, pH 8.3, and blocked with PBS con- phase was recovered. These steps were repeated once. Combined butanol taining 0.05% Tween 20 and 10% FCS. Cell supernatants and rhTNF stan- phases were recentrifuged and lyophilized. For stimulation experiments, dard (R&D Systems, Wiesbaden, Germany) in PBS containing 10% FCS lyophilized extracts were dissolved in RPMI 1640 medium (Life Technol- were incubated at 4°C overnight. Bound hTNF was detected using a bio- ogies, Eggenstein, Germany) to the original volume of supernatants before tinylated mouse anti-hTNF Ab (PharMingen) at a concentration of 0.5 extraction. For silver stain analysis, butanol extracts corresponding to 1 ml mg/ml. Subsequently, 1 ␮g/ml streptavidin peroxidase conjugate (Sigma) of culture supernatant were dissolved in 50 ␮l of distilled water. OMIZ-Pat was added with ortho-phenylene-diphosphate (Sigma) as substrate. The medium was processed similarly and used as control. For an extraction of detection limit of this assay was ϳ10 pg/ml. For quantitation of murine whole treponeme cells, aqueous suspensions of treponeme cells were di- TNF-␣, MaxiSorp ELISA plates were coated with 3 ␮g/ml anti-murine

gested with RNase (Sigma, Deisenhofen, Germany), DNase (Merck, TNF (anti-mTNF) Ab (PharMingen) in 100 mM Na3PO4, pH 6.0. Samples Darmstadt, Germany), and proteinase K (Merck). The suspensions were and rmTNF standard (R&D Systems) were incubated at room temperature dialyzed and extracted using a hot phenol/water extraction method (42) or, for 3 h, followed by detection with a biotin-conjugated anti-mTNF-␣ Ab after drying, a phenol/chloroform/petroleum ether (PCP) method (43). In (PharMingen) and streptavidin-peroxidase with ortho-phenylene-diphos- brief, the phenol/water extraction was performed by mixing the cell sus- phate as substrate. The detection limit was ϳ15 pg/ml. All in vitro TNF-␣ pension with an equal volume of 90% phenol and stirring at 68°C for 10 results were assessed statistically by the Student’s t test, and the inhibitory min. After cooling on ice, the mixture was centrifuged at 3000 ϫ g for 10 effects of polymyxin B and My 4 as well as the enhancing effects of LBP Ͻ Ϫ min at 0°C, and the upper phase was collected. This procedure was re- were highly signiﬁcant ( p 0.001). NO2 accumulation in culture me- peated twice, and combined phases were dialyzed and lyophilized. PCP dium was assessed according to a published protocol (44). In brief, 100 ␮l extraction was conducted with a mixture of 90% PCP in a volume ratio of of Griess reagent (Sigma) was added to 100 ␮l of culture medium in 96- 2.5:2.5:4. PCP extraction resulted in yields of 0.075% for T. brennaborense well plates and measured in a microplate reader at 540 nm with a standard

and 0.036% for T. maltophilum according to wet weight. By using the of NaNO2 diluted in RPMI 1640. The Journal of Immunology 2685

Estimation of NF-␬B translocation 50% by volume). For GLC-MS analysis, fatty acids were released from the glycolipid by alkaline hydrolysis (50 ␮l 0.5 M NaOH,1hat65°C), and the CHO cells transfected with human CD14 (CHO/CD14, generously pro- product was permethylated (51). GLC-MS analysis of the permethylated vided by L. Hamann, Forschungszentrum Borstel, Germany) (45) were and deacylated glycolipids was performed using a gradient of 150°C (3 ϫ 5 cultured overnight in six-well tissue culture plates at 4 10 cells per well min) to 330°C at 10°/min. with Ham’s nutrient medium F12 (PAA Laboratories, Linz, Austria) sup- plemented with 10% FCS and 400 ␮g/ml hygromycin B (Calbiochem, San Binding of phenol/water extracts to rmLBP, endotoxin Diego, CA). Before stimulation, cells were starved in FCS-free Ham’s neutralizing protein (ENP), and polymyxin B medium for 3 h and incubated with LPS or treponeme extracts in the presence of 2% non-heat-inactivated FCS. After 1 h, cells were washed Binding of phenol/water extracts to mLBP was investigated with a slightly ␮ with ice-cold PBS containing 1 mM Na3VO4 and incubated in 150 l modiﬁed competition assay published elsewhere (52). Brieﬂy, MaxiSorp of buffer A (1 mM Na3VO4, 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, ELISA plates were coated with E. coli 0111:B4 LPS. Free protein binding 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1 mM NaF). After 20 sites were blocked by incubation with 10 mg/ml BSA in 150 mM NaCl, 50 min, cells were harvested mechanically, transferred to 1.5-ml tubes, mixed mM HEPES, pH 7.4, at 37°C for 30 min. Washing and dilution steps were with 25 ␮l Nonidet P-40, and centrifuged at 13,000 ϫ g at 4°C for 1 min. performed with blocking buffer containing 1 mg/ml BSA. E. coli 0111:B4 Pellets were resuspended in 50 ␮l of buffer B (400 mM NaCl, 1 mM LPS and S. minnesota Re 595 LPS, and phenol/water extracts of T. bren- Na3VO4, 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM naborense and T. maltophilum were assayed for their ability to bind to 100 PMSF, and 1 mM NaF), incubated for 30 min at 4°C, and spun at 13,000 ϫ ng/ml mLBP by inhibiting binding of LBP to LPS-coated plates. LPS- g at 4°C for 5 min. Supernatants containing nuclear proteins were col- bound LBP was detected by a polyclonal rabbit-anti-mLBP Ab and incu- lected, and nuclear extracts were analyzed by EMSA as described previ- bated with goat anti-rabbit IgG-Ab, conjugated with HRP (Biogenes, Ber- ously (46) using two synthetic oligonucleotides (Eurogentec, Seraing, Bel- lin, Germany). Ortho-phenylene-diphosphate was used as a substrate. gium) containing the NF-␬B binding sequence of the murine Ig ␬ light E. coli 0111:B4 LPS and treponeme phenol/water extracts were precipi-

chain gene enhancer. tated by polymyxin B-coupled Agarose beads (Sigma) or Sepharose beads Downloaded from conjugated with ENP (Associates of Cape Cod, Falmouth, MA), respectively. Limulus assay LPS and extracts in a volume of 500 ␮l at a ﬁnal concentration of 10 ␮g/ml ␮ Treponema culture supernatants, both native and butanol extracted, as well were mixed with 50 l of polymyxin B beads or with ENP beads, respectively, as extracts of whole cells were assayed for endotoxin contamination by as recommended by the manufacturer. Samples were incubated at 4°C for 24 h ϫ using a chromogenic Limulus amoebocyte lysate (LAL) assay (LPS, using a 180° shaker. After centrifugation at 3000 g at 4°C for 10 min, Sinntal-Oberzell, Germany). The endotoxin content of the OMIZ-Pat cul- supernatants were collected and loaded onto 15% SDS-PAGE gels, followed ture medium was below 2 ng/ml regardless of whether treponemes were by silver staining as described above. Control samples were treated accord- http://www.jimmunol.org/ cultured or the medium was incubated without bacteria for control. Butanol ingly, however, without addition of any beads. Murine LBP was expressed in extracts of the culture supernatants contained endotoxin of ϳ25 pg/ml. The a baculovirus system and puriﬁed as described (26). phenol/water extracts of T. maltophilum and T. brennaborense exhibited a LAL activity of 16.3 pg/␮g corresponding to 0.16 endotoxin units (EU) Results and 35.2 pg/␮g corresponding to 0.35 EU, respectively. Induction of TNF-␣ in myeloid cells by treponeme culture Electrophoresis and silver staining of treponeme extracts supernatants in the presence of serum, polymyxin B, mAb My 4, or rmLBP Stacking gels (5%) and separating gels (15, 16, and 20%, respectively) were prepared without SDS. Prestained and unstained low molecular mass Treponema culture supernatants induced TNF-␣ in freshly isolated markers ranging from 3 to 43 kDa (Life Technologies), 30 ␮l of culture

human monocytes. This activity increased with culture time reach- by guest on September 29, 2021 ␮ ␮ supernatants, 30 l of butanol extracts, and 30 l of LPS solutions derived ing a maximum at day 3 (Fig. 1). OMIZ-Pat culture medium alone, from E. coli 0111:B4 or Salmonella minnesota Re 595 LPS (Sigma) were boiled in sample buffer (2 ml 1 M Tris, 4 ml 1 M DTT, 800 mg SDS, 40 incubated with monocytes for the same period of time, failed to ␣ mg bromophenol blue, and 4 g glycerol ad 10 ml H2O) for 5 min, loaded induce any detectable amounts of TNF- (data not shown). For the onto the gels, and submitted to electrophoresis. Gels were stained with the silver stain plus kit (Bio-Rad, Munich, Germany) according to the manu- facturers instructions. In addition to the original protocol, gels were oxi- dized with 0.7% periodic acid after ﬁxation (47). For some experiments, glycolipids were hydrolyzed and/or dephosphorylated as explained below. Chemical analysis of the phenol/water-extracted cell wall fractions of T. maltophilum and T. brennaborense

Phosphate was determined according to Lowry (73), and 3-deoxy-D- manno-octulosonic acid (Kdo) was estimated by the thiobarbituric acid method (43). Amino acids were identified as their phenyl isothiocyanate derivatives by reversed-phase HPLC using a Waters PICO-TAG system (Waters, Eschborn, Germany) under conditions described previously (48). Amino sugars were analyzed using HPLC (49). Gas-liquid chromatography (GLC) and combined gas-liquid chromatography/mass spectrometry (GLC-MS) were applied for the analysis of neutral sugar alditol acetates (50) and fatty acid methyl esters liberated after strong methanolysis (2 M HCl/MeOH, 120°C, 24 h) and extraction with chloroform. GLC was performed on a model 3700 Varian gas chromatograph (Varian Associates, Palo Alto, CA), and GLC-MS was performed on a Hewlett Packard 5989A instrument equipped with a gas chromatograph (model 5890 Series II, Hewlett-Packard, Palo Alto, CA) operating under identical conditions as for GLC. For structural analysis of treponeme glycolipids, 3.8 mg of T. maltophilum and 4.9 mg of T. brennaborense phenol/water extracts were FIGURE 1. Induction of TNF-␣ in human monocytes by Treponema dephosphorylated in 100 ␮l HF (48% by volume) at 4°C for 24 h in a culture supernatants. Freshly isolated human monocytes were incubated in sealed Teflon tube. Samples were extensively dialyzed (cut-off at 10–14 a total volume of 80 ␮l. Then 20 ␮lofTreponema culture supernatants kDa) against water and lyophilized. The yields obtained were 1.3 mg for T. taken at the incubation time indicated from T. maltophilum and T. bren- maltophilum and 1.8 mg for T. brennaborense. Dephosphorylated glycolipids were peracetylated in 0.8 ml pyridine/acetic acid anhydride (5:3 by naborense cultures were added (20%). All experiments were performed in ␣ volume, 85°C, 30 min) and subjected to GLC-MS analysis. For analysis of the presence of 5% human AB serum. TNF- concentrations were mea- the glycosyl part, peracetalyted glycolipids were further purified on a silica sured by ELISA as described in Materials and Methods. Shown are mean gel column (3.5 ϫ 1.5 cm, Kieselgel 60, Merck, 230–400 mesh), eluted values and SD of duplicate measurements. Experiments were conducted in with a stepwise gradient of increasing amounts of ethanol in toluene (1– duplicates with similar results. 2686 MECHANISMS OF Treponema GLYCOLIPID-INDUCED IMMUNE RESPONSES following experiments, bacteria were cultured for 3 days. Cultures were monitored by pH measurement (6.0) to guarantee similar growth conditions. Both viability and motility of treponemes were assessed by dark field microscopy. To compare the activity of the treponeme supernatants with LPS, we performed experiments with monocytes in the presence and absence of serum. Cytokine induction caused by treponeme culture supernatants increased significantly in the presence of 5% human serum (Fig. 2A). As compared with T. brennaborense, serum-independent stimulation was significantly stronger for T. maltophilum culture supernatants (Fig. 2A). Both polymyxin B and the inhibitory monoclonal anti-CD14 Ab My 4 were able to significantly reduce cytokine levels induced by both treponeme cultures. However, the effect was more pronounced for LPS (Fig. 2B). The cytokine-inducing activity of T. brennaborense and T. maltophilum culture supernatants was inhibited in the presence of polymyxin B or My 4 mAb at least by 50%. In contrast, polymyxin B and My 4 did not influence cytokine induction caused by PMA, a phorbol ester causing cytokine induction by activating protein kinase C directly without receptor in- Downloaded from teraction (53) (data not shown). To investigate LBP effects on cytokine induction caused by treponemes, purified rmLBP and the murine macrophage cell line RAW 264.7 were used. In RAW 264.7 cells, TNF-␣-induction caused by both treponeme culture supernatants was significantly increased by addition of mLBP (Fig. 2C). http://www.jimmunol.org/ Induction of cytokines by butanol extracts of culture supernatants and phenol/water extracts of whole cells in the presence of serum, polymyxin B, My 4, and rmLBP Based upon the notion that the stimulatory activity found within the supernatants shared characteristics with LPS, we purified the compounds from culture supernatants as well as from whole cells using extraction methods commonly used for LPS. Supernatants were treated with butanol, while whole cells were subjected to phenol/water or the PCP extraction. Yields obtained from whole by guest on September 29, 2021 cells with the phenol/water method were clearly higher; therefore, this method was used in the following experiments. To identify possible LPS contamination during the preparation, a mock extraction including all media and chemicals used during the procedure, was performed. TNF-␣ induction in human monocytes caused by butanol extracted Treponema supernatants was clearly reduced by both polymyxin B and My 4 (Fig. 3A). In RAW 264.7, an LBP-dependent cytokine induction was observed (Fig. 3B). In contrast to the culture supernatants, no TNF-␣ was induced in the absence of LBP. Similarly, phenol/water extracts of whole treponeme cells revealed a serum-dependent cell-stimulating capacity in human monocytes (data not shown). However, to achieve a TNF-␣ release equivalent to that caused by LPS, the concentrations of the extracts had to be increased by 1000-fold. Addition of polymyxin B and mAb My 4 led to a significant decrease of cytokine levels (Fig. 3C). The cytokine induction in RAW 264.7 cells was greatly increased by the addition of rmLBP (Fig. 3D). In case of T. maltophilum, it was LBP dependent. In all experiments, the mock extracts did not cause cytokine induction.

Induction of NO synthesis by treponeme phenol/water extracts in FIGURE 2. Effect of serum, LBP, polymyxin B, and anti-CD14 mAb My 4 PEM derived from C3H/HeJ and C3H/HeN mice on TNF-␣ induction in human monocytes and RAW 264.7 by Treponema super- The role of TLR-4 in treponeme-mediated cell stimulation was natants. Freshly isolated human monocytes were stimulated with 10% of the Treponema cultures, and 1 ng/ml of E. coli 0111:B4 LPS both in the absence and analyzed using PEM derived from LPS hyporesponsive C3H/HeJ presence of 5% human serum (A), 5 ␮g/ml polymyxin B, or 5 ␮g/ml of the mice, a strain bearing a dominant negative mutation in the gene anti-CD14 mAb My 4 (B). The murine macrophage cell line RAW 264.7 was encoding TLR-4 (31). We isolated PEM from C3H/HeJ mice, as stimulated with 10% of culture supernatants or 1 ng/ml of E. coli 0111:B4 LPS in well as from the control C3H/HeN strain normally responsive to the presence or absence of 1 ␮g/ml mLBP (C). Human and murine TNF-␣ con- LPS. Cells were stimulated with increasing amounts of LPS and centrations were assessed by ELISA as described in Materials and Methods. treponeme phenol/water extracts, followed by measurement of Shown are mean values and SD of quadruplicate measurements. Experiments NO. LPS exhibited a signiﬁcantly stronger stimulatory activity to- were repeated in quadruplicate (A) and twice (B) with similar results. The Journal of Immunology 2687 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Effect of polymyxin B, anti-CD14 mAb My 4, and rmLBP on TNF-␣ induction in human monocytes and RAW 264.7 by butanol extracts of Treponema culture supernatants and phenol/water extracts of whole cells. Freshly isolated human monocytes were stimulated with 25% (v/v) of butanol extracts (A) or with phenol/water extracts of whole cells (C)oftheTreponema strains or LPS as indicated in the presence of 5% human serum with or without 5 ␮g/ml polymyxin B or 5 ␮g/ml of the anti-CD14 mAb My 4, respectively. RAW 264.7 cells were stimulated either with butanol extracts of treponeme culture supernatants (B) or with phenol/water extracts (D) in the presence or absence of rmLBP. LPS derived from E. coli 0111:B4 was added as control. TNF-␣ levels were measured by ELISA as described in Materials and Methods. Shown are mean values and SD of quadruplicate measurements. Experiments were repeated in quadruplicate (A), twice (B), and once (C and D) with similar results. ward C3H/HeN as compared with C3H/HeJ PEM (Fig. 4). While CD14) were stimulated with treponeme phenol/water extracts as the mock extract failed to stimulate cells, phenol/water extracts well as with LPS. LPS induced a strong translocation of NF-␬Bin derived from T. brennaborense revealed a stimulation pattern com- CHO/CD14 cells as shown in an EMSA (Fig. 5). T. brennaborense parable to LPS, leading to a signiﬁcantly weaker NO release by phenol/water extracts induced a translocation of NF-␬B at concen- C3H/HeJ macrophages as compared with C3H/HeN cells. In con- trations of 1 ␮g/ml comparable to the LPS effect. In contrast, trast, extracts derived from T. maltophilum led to a comparable NO T. maltophilum-derived extracts, at 1 ␮g/ml, failed to induce production in PEM of both strains, suggesting a less important role NF-␬B translocation, indicating an involvement of TLR-2. of TLR-4. SDS-PAGE silver stain analysis of native and butanol-extracted Translocation of NF-␬B in CHO cells by treponeme phenol/ culture supernatants and whole cell phenol/water extracts water extracts To identify and further characterize the active components in pure To elucidate the role of TLR-2 in treponeme-mediated signaling, or butanol-extracted treponeme culture supernatants, the material we investigated CHO cells. These cells carry a mutation for TLR-2 was analyzed on 15% polyacrylamide gels that were silver stained leading to a defective receptor expression (54). CHO cells trans- subsequently. Visible bands appeared in the range of about 4 and fected with human CD14 inducing responsiveness to LPS (CHO/ 6 kDa in butanol-extracted supernatants from T. maltophilum and 2688 MECHANISMS OF Treponema GLYCOLIPID-INDUCED IMMUNE RESPONSES

FIGURE 5. Translocation of NF-␬B in CHO/CD14 cells stimulated with treponeme phenol/water extracts. CHO cells transfected with human CD14 were stimulated with LPS from E. coli 0111:B4 (10 ng/ml) in comparison to phenol/water extracts derived from T. maltophilum (TM) and T. brennaborense (TB) (1 ␮g/ml) or a mock extract in the presence of 2% non-heat-inactivated FCS. After 1 h, incubation was terminated and nuclear extracts of the cells were prepared as described in Materials and Methods. Nuclear extracts were assayed by EMSA and exposed to x-ray ﬁlm. Shown is one representative of two experiments. Downloaded from

about twice as much of the low molecular compound as the T. maltophilum extract. Digestion of both butanol extracts with pro-

nase failed to change the proﬁle of bands observed in SDS-PAGE, http://www.jimmunol.org/ suggesting a nonproteinaceous nature of the immunostimulatory compound (data not shown). Phenol/water extracts of both strains were analyzed by silver-stained SDS-PAGE, revealing striking differences (Fig. 6B). The material obtained from T. brennaborense displayed a ladder-like pattern similar to that of smooth LPS pre- sumably containing numerous repeating carbohydrate units. In contrast, T. maltophilum extracts exhibited few repeating units of larger molecular size. For both butanol-extracted culture superna-

tants and phenol/water extracts of whole cells, the size of their by guest on September 29, 2021 smallest units was similar.

Compositional and structural analysis of the phenol/water- extracted cell wall fractions of T. maltophilum and T. brennaborense The results of a chemical analysis of phenol/water extracts from T. maltophilum and T. brennaborense are shown in Table I. Most of the components in both fractions could be analyzed and quantified (56% (w/w) for T. maltophilum and 71% for T. brennaborense). As expected from SDS-PAGE analysis, the amount of total fatty acids in the smaller glycolipid of T. maltophilum was significantly higher (8.3%, w/w) as compared with the high molecular T. brennaborense glycolipid (2.8%). LPS-characteristic ␤-hydroxy- lated fatty acids, as well as Kdo and heptose, were completely FIGURE 4. NO release by C3H/HeJ and C3H/HeN PEM after stimu- lacking in both strains. In T. maltophilum, galactosamine was iden- lation with treponeme phenol/water extracts. Freshly isolated PEM were tified to be the main sugar component (13%), whereas in T. bren- stimulated with increasing amounts of LPS derived from E. coli 0111:B4, naborense, glucose was identified as the dominating sugar mock extract, and treponeme phenol/water extracts in the presence of 5% (50.8%). In both preparations, a characteristically high amount of Ϫ non-heat-inactivated FCS for 24 h. NO2 levels were assessed using the phosphate could be identified (5–10%). Besides traces of contam- Griess reagent. Shown are the results obtained with C3H/HeN PEM (A) inating residual amino acids, alanine was the only amino acid iden- and those obtained with C3H/HeJ PEM (B). Given are mean values and SD tified in T. maltophilum, whereas T. brennaborense completely of quadruplicate measurements. Experiments were repeated twice with lacked amino acids. similar results. After alkaline hydrolysis of both glycolipids by treatment with KOH, the material could not be visualized by silver staining, indicating the loss of ester-bound fatty acids (Fig. 7, A and B). To T. brennaborense, respectively (Fig. 6A). The low molecular ma- cleave phosphate-interlinked sugar chains, glycolipids were de- terial reflected rough LPS derived from S. minnesota Re 595 con- phosphorylated by HF, resulting in material of ϳ1 kDa size, which sisting only of lipid A and the inner core region with a size of 2.5 also could not be stained after KOH treatment (Fig. 7, A and B). kDa (lane 1). The relative concentration present in the butanol This result suggests the presence of diacylglycerol as lipid anchor extract of T. brennaborense revealed that this material contained in both glycolipids and excludes sphingosine and steroids. For The Journal of Immunology 2689

Binding of treponeme phenol/water extracts to mLBP, ENP, and polymyxin B To analyze the potential interaction of Treponema glycolipids with LBP, we performed competition assays with rmLBP (Fig. 8A). These studies revealed that the glycolipids are able to compete with LPS-LBP binding. Both extracts displayed a comparable af- ﬁnity, while LPS derived from S. minnesota Re 595 and LPS from E. coli 0111:B4 exhibited a stronger activity. Next, we investigated possible interactions of Treponema extracts by precipitation with polymyxin B and beads coated with ENP, an endotoxin binding protein used in the Limulus assay. These experiments indicate that the glycolipids studied interact with both, polymyxin B, and ENP, as precipitation led to a marked reduction of bands shown in SDS- PAGE analysis (Fig. 8B). While for ENP no differences were observed comparing LPS and the treponeme extracts, the effect was less pronounced for polymyxin B, especially regarding T. maltophilum interaction. FIGURE 6. Analysis of butanol-extracted culture supernatants and PCP Downloaded from or phenol/water extracts of treponeme whole cells by SDS-PAGE and sil- Discussion ver staining. S. minnesota Re 595 LPS (“rough”-LPS, r-LPS, 25 ng) was The interaction of microorganisms with the host defense system is loaded in comparison to butanol extracts of T. brennaborense corresponding to 900, 450, and 225 ␮l of culture supernatants and extracts derived a hallmark during infection, deciding over survival of the microbe from T. maltophilum corresponding to 900 ␮l(A) on a 15% SDS poly- and/or development of an inﬂammation. Bacteria exhibit certain acrylamide gel and subjected to electrophoresis. On a separate gel, 5 ␮gof features enabling the host to discriminate them as “foreign” and to ␮

E. coli 0111:B4 LPS (“smooth”-LPS, s-LPS) and 2.5 gofS. minnesota mount responses leading to elimination of the pathogen. There is http://www.jimmunol.org/ Re 595 LPS (“rough”-LPS, r-LPS) were loaded in comparison to PCP and a number of bacteria causing chronic inflammations leading to phenol/water (PW) extracts of whole treponemes (B). Then 5 ␮gofT. tissue destruction and severe, although not acute life-threatening, brennaborense extracts (TB) and 2.5 ␮gofT. maltophilum extracts (TM) diseases. Treponema, generally known for their lack of antigenic- were run. Molecular mass standards ranging from 3.0 to 42.0 kDa in size ity, induce a moderate innate immune response leading to chronic were loaded as control. rather than acute, often systemic, infections. However, a distinct local inflammatory reaction pattern can be observed in many spirochete infections, i.e., the primary local stage of Lyme disease. identification of the lipid anchor in the glycolipid of T. maltophi- Local release of cytokines causing tissue inflammation is likely, lum, extracts were peracetylated after dephosphorylation with HF. by guest on September 29, 2021 GLC-MS analysis (CI mode) of the peracetylated glycolipids from ϳ T. maltophilum revealed one major set of peaks (tR 17 min) Table I. Chemical analysis of T. maltophilum and T. brennaborense ϩ ϩ ϭ expressing pseudomolecular ions ([M NH4] )ofm/z 572 and glycolipids ϭ 586, respectively. Their molecular masses (Mr 554 and 568 kDa, respectively) are consistent with monoacetylated diacyl-glycerol Concentration carrying two tetradecanoic acid residues (14:0) and one pentade- T. maltophilum T. brennaborense canoic acid (15:0), respectively (Fig. 7C). However, attempts to identify a similar lipid anchor in T. brennaborense by using the Componenta nmol/mg % (w/w) nmol/mg % (w/w) same approach were unsuccessful, most likely due to the small Carbohydrates proportion of the lipid in the whole molecule (see Table I). Glucose 350 6.3 2282 50.8 For identification of the glycosyl part within the glycolipid, the Galactose 443 8.0 144 2.6 peracetylated glycolipids were further purified by silica gel chro- Mannose – – 143 2.6 matography, and fatty acids were released by alkaline hydrolysis, Fucose 248 4.1 – – Rhamnose – – 275 4.5 followed by permethylation of the remaining material. GLC-MS Galactosamine 728 13.0 127 2.3 analysis of T. maltophilum, revealed two glycosyl derivatives with Glucosamine 224 4.0 – – retention times of 24.9 and 28.7 min expressing in the CI-mode Amino-dideoxy-hexoseb ϩ ϩ ϩ ϭ ϭ Amino acids pseudomolecular ions [M NH4] with m/z 764 (Mr 746 ϩ ϩ ϩ ϭ ϭ Alanine 209 1.8 – – kDa) and [M H ] m/z 788 (Mr 787 kDa). These could be Polar head groups assigned to a permethylated trisaccharide glyceride with three hex- Phosphate 1076 10.5 518 5.1 oses (Hex3-Gro), and a trisaccharide consisting of two hexoses and Ethanolamine 30 0.8 30 0.8 one hexosamine (Hex -HexNAc-Gro) (Fig. 7D). Both permethyl- Fatty Acids 2 Ϻ ated glycosylglycerides were present in ϳ1:9 proportion. The elec- 12 0 22 0.4 12 0.2 13Ϻ0 28 0.6 1 Ͻ0.1 tron impact-mass spectrometry of the major permethylated glyco- 14Ϻ0 241 5.5 79 1.8 sylglyceride showed diagnostic fragments derived from the 15Ϻ0 38 0.9 4 0.1 reducing part of the molecule (m/z ϭ 307, 557) as well as those 16Ϻ0 30 0.8 24 0.6 Ϻ Ͻ from the nonreducing part (m/z ϭ 464, 668), thus allowing the 18 0 3 0.1 1 0.1 Total 56 71 sequence of the trisaccharide to be assigned to Hex-HexNAc-Hex- Gro (Fig. 7D). Despite several attempts, the structure of the gly- a Chemical analysis was performed employing GLC and HPLC as described in Materials and Methods. cosyl part of the putative glycolipid isolated from T. brennabo- b The amino-dideoxy-hexose could not be quantified due to the lack of reference rense could not be revealed. compound. 2690 MECHANISMS OF Treponema GLYCOLIPID-INDUCED IMMUNE RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. Silver stain analysis of hydrolyzed and dephosphorylated treponeme glycolipids, GLC-MS analysis of T. maltophilum-derived glycolipid, and proposed schematic structure. Treponeme glycolipids were either hydrolyzed by treatment with KOH, dephosphorylated by HF, or both, as explained in Materials and Methods. Resulting material, in comparison to untreated glycolipid, was further analyzed by silver staining (A, T. brennaborense, 16% gel; B, T. maltophilum, 20% gel). Before GLC-MS analysis, glycolipids of T. maltophilum were dephosphorylated (HF), dialyzed, and peracetylated. C, EI-MS of the major lipid mono-acetylated (deglycosylated) glycerol carrying two tetradecanoic acid residues (14:0). Dephosphorylated glycolipid was defrayed from fatty acids by alkaline hydrolysis and permethylated. D, The major glycosyl part of the T. maltophilum glycolipid carrying the Hex- HexNAc-Hex-Gro unit. E, A schematic structure proposal of the treponeme glycolipids investigated. The Journal of Immunology 2691

and macrophages have been proposed to play a major role during this process (55). Spirochetes may also directly stimulate resident defense cells for mediator release, although clear molecular mechanisms for this process have to be defined. Immunostimulatory elements of bacteria interact with soluble and cell-bound receptor molecules of the host organism, a key element of the host’s repertoire to modulate an inflammatory reaction. As we have shown recently, the acute-phase response to a systemic infection leading to elevated levels of the hepatic acute-phase protein LBP can greatly modulate the hosts response to a systemic challenge with LPS (26). It is likely that LBP serves for toxic Treponema cell wall products as well as a modulator in vivo and may be able to reduce or enhance the inflammatory reaction. Recent results by others and us provide evidence that LBP interacts not only with LPS, but also with other bacterial products, i.e., LTA (7). Furthermore, our results showing an involvement of the CD14 part of the LPS receptor are in agreement with CD14 acting as a pattern recognition receptor (56, 57). Downloaded from The immunostimulatory treponeme cell wall compounds described here are apparently released by live bacteria or after cell death. We present evidence that the compounds retained from the supernatants correspond to the glycolipids extracted from whole cells regarding size and biological characteristics. After cell death, bacteria release immunostimulating particles such as LPS in http://www.jimmunol.org/ Gram-negative bacteria, causing a strong inflammatory response potentially leading to septic shock and subsequent death of the host (58, 59). In Gram-positive bacteria, elements like PG and LTA of the outer cell wall, released after cell disintegration often following antibiotic treatment, also stimulate cytokine release in host cells (3, 60). However, for spirochetes, the predominant inflammatory active element of their cell wall has not yet been clearly identified.

For some spirochetes like T. hyodysenteriae and T. innocens, the by guest on September 29, 2021 presence of LPS-like molecules has been described, while for others it has been clearly ruled out (10). Borrelia burgdorferi fails to contain LPS in the cell wall (8, 9); however, it contains PG (61) and a set of outer membrane proteins eliciting inflammatory responses in immune cells (62–64). It is likely that these proteins are identical with lipoproteins described to induce TNF-␣-synthesis in human monocytes (12, 65). T. pallidum possesses a number of TNF-␣-inducing membrane proteins, and for this spirochete the presence of LPS was definitively ruled out after the completion of the whole genome sequence (8, 11, 66). Our observations provide evidence that treponemes contain a glycolipid-like material within their membranes that is chemically different from LPS while exhibiting comparable biological characteristics including, for one of FIGURE 8. Phenol/water extracts of Treponema bind to mLBP, ENP, our isolates, involvement of TLR-4. and polymyxin B. A, LPS derived from E. coli 0111:B4 (“smooth”-LPS, Analysis of the role of TLRs is important in light of the recent s-LPS) and S. minnesota Re 595 (“rough”-LPS, r-LPS) and treponeme paradigm of Gram-negative bacteria using TLR-4 via LPS and phenol/water extracts competed with immobilized LPS for binding to rm- LBP. After addition of increasing concentrations of the competitors, LBP other bacteria stimulating cells via TLR-2. Recently, two immu- bound to immobilized LPS was detected by an Ab followed by colorimetric nostimulatory fractions isolated from the cell walls of T. denticola detection. The results shown as OD on the y-axis reflect the amount of have been compared, lipoproteins on one hand and lipooligosac- mLBP bound to the LPS-coated plates. Samples were incubated at con- charides, comparable to the glycolipids isolated in our study, on centrations ranging from 250 to 0.03 ␮g/ml, and mLBP was used at a the other hand (67). Both fractions were able to induce cytokines concentration of 100 ng/ml. Shown is one representative of three experi- and NO in host cells of normal and C3H/HeJ mice, suggesting a ments. B, Depletion of LPS and phenol/water extracts by ENP- or poly- TLR-4-independent activity, which is in line with our results for T. ␮ myxin B-conjugated beads. Solutions containing 100 g/ml of E. coli maltophilum. Lipoproteins isolated from Borrelia recently also 0111:B4 LPS or phenol/water extracts were incubated with ENP- or poly- have been found to stimulate host cells via TLR-2 similar to li- myxin B (PB)-conjugated beads. After centrifugation, 30 ␮l of supernatant was mixed with loading buffer and loaded onto a 15% SDS-PAGE gel poproteins isolated from mycobacteria (36, 37, 39). According to followed by silver staining. Molecular mass standards ranging from 2.35 to our results, active cell wall compounds from genetically closely 46 kDa in size were loaded as control. Shown is one representative of two related spirochetes stimulate cells via different TLRs. However, experiments. chemical analysis revealed differences in the composition of the glycolipids isolated. Thus structural differences may explain the 2692 MECHANISMS OF Treponema GLYCOLIPID-INDUCED IMMUNE RESPONSES differential use of pattern recognition receptors. While the T. bren- will be performed in our laboratories to further support this naborense glycolipid contained significantly more carbohydrates interpretation. and revealed the presence of a high number of small “repeating Our data complement the list of bacterial cell wall components units” in silver gel analysis, T. maltophilum glycolipids displayed recognized by TLRs and CD14, explaining results by others show- a small number of larger “repeats.” Recently, the TLR-2/TLR-4 ing a CD14 involvement for spirochete-mediated host cell stimu- paradigm was questioned by two other studies showing involve- lation (13, 64). The differential use of TLRs by the treponeme ment of TLR-4 in non-LPS-mediated cell stimulation. Viable my- glycolipids may help in understanding basic mechanisms of innate cobacteria, in contrast to isolated lipoarabinomannan, stimulated immunity caused by spirochetes as well as other microorganisms. CHO cells overexpressing both TLR-2 or TLR-4 (68). A recent study comparing the TLR-2 and the TLR-4 knockout mouse pro- Acknowledgments vided evidence that LTA from Gram-positive bacteria also stim- We acknowledge the excellent technical assistance of Nicole Siegemund, ulate macrophages via TLR-4 (40). Furthermore, this group com- Fra¨nzi Creutzburg, Marco Kachler, and Cyndi Hefenbrock (Charite´), as pared different types of LPS leading to a different degree of use of well as Hermann Moll, and Ursula Schombel (Forschungszentrum members of the TLR family (69). Borstel). We describe here the molecules involved in the reaction pattern of myelo-monocytic host cells to contact with cell wall compo- References nents of recently identified spirochetes. Certain features of this 1. Medzhitov, R., and C. A. Janeway, Jr. 1997. Innate immunity: the virtues of a interaction, i.e., involvement of LBP, CD14, and the use of TLR-4 nonclonal system of recognition. Cell 91:295. 2. Bhakdi, S., T. Klonisch, P. Nuber, and W. Fischer. 1991. Stimulation of by T. brennaborense, as well as the inhibitory effect of polymyxin monokine production by lipoteichoic acids. Infect. Immun. 59:4614. Downloaded from B, resemble the cell stimulation pattern induced by LPS of Gram- 3. Heumann, D., C. Barras, A. Severin, M. P. Glauser, and A. Tomasz. 1994. negative bacteria. However, for polymyxin B, it recently has been Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes. Infect. Immun. 62:2715. shown that it interacts with numerous structures including phos- 4. De Kimpe, S. J., M. Kengatharan, C. Thiemermann, and J. R. Vane. 1995. pholipids (70). Furthermore, the Treponema glycolipids described The cell wall components peptidoglycan and lipoteichoic acid from Staphy- lococcus aureus act in synergy to cause shock and multiple organ failure. here bound to ENP, a protein usually considered to bind specifi- Proc. Natl. Acad. Sci. USA 92:10359. cally to LPS. However, it is known that agents other than LPS 5. Dziarski, R. 1991. Peptidoglycan and lipopolysaccharide bind to the same http://www.jimmunol.org/ cross-react in the Limulus assay, potentially due to similar physical binding site on lymphocytes. J. Biol. Chem. 266:4719. 6. Weidemann, B., H. Brade, E. T. Rietschel, R. Dziarski, V. Bazil, properties (71, 72). Because the glycolipids described in this study S. Kusumoto, H.-D. Flad, and A. J. Ulmer. 1994. Soluble peptidoglycan- interact with a range of other LPS-binding structures, it is likely induced monokine production can be blocked by anti-CD14 monoclonal an- that the discrete LAL activity observed is caused by the extracted tibodies and by lipid A partial structures. Infect. Immun. 62:4709. 7. Fan, X., F. Stelter, R. Menzel, R. Jack, I. Spreitzer, T. Hartung, and C. Schu¨tt. compounds themselves and not by contaminating LPS. Further- 1999. Structures in Bacillus subtilis are recognized by CD14 in a lipopoly- more, precipitation studies using ENP revealed a specific affinity saccharide binding protein-dependent reaction. Infect. Immun. 67:2964. of the treponeme extracts to this protein. 8. Hardy, P. J., and J. Levin. 1983. Lack of endotoxin in Borrelia hispanica and Treponema pallidum. Proc. Soc. Exp. Biol. Med. 174:47. Our chemical analysis suggests a glycolipid structure in T. mal- 9. Takayama, K., R. J. Rothenberg, and A. G. Barbour. 1987. Absence of lipo- tophilum and T. brennaborense differing significantly from that of polysaccharide in the Lyme disease spirochete, Borrelia burgdorferi. Infect. by guest on September 29, 2021 LPS. This is based on the absence of structural components char- Immun. 55:2311. 10. Greer, J. M., and M. J. Wannemuehler. 1989. Comparison of the biological acteristic for LPS, such as heptose, Kdo, and ␤-hydroxy fatty ac- responses induced by lipopolysaccharide and endotoxin of Treponema hyo- ids. In contrast, Treponema glycolipids displayed LTA-like ele- dysenteriae and Treponema innocens. Infect. Immun. 57:717. 11. Fraser, C. M., S. J. Norris, G. M. Weinstock, O. White, G. G. Sutton, ments such as sugar, high phosphate, and alanine similar to that R. Dodson, M. Gwinn, E. K. Hickey, R. Clayton, K. A. Ketchum, et al. 1998. previously identified in T. denticola (19). This similarity was fur- Complete genome sequence of Treponema pallidum, the syphilis spirochete. ther supported by isolation and analysis of the dephosphorylated Science 281:375. 12. Radolf, J. D., M. V. Norgard, M. E. Brandt, R. D. Isaacs, P. A. Thompson, glycosyl part of the repeating units, being a hexasaccharide in and B. Beutler. 1991. Lipoproteins of Borrelia burgdorferi and Treponema T. maltophilum and a glucan in T. brennaborense (data not shown). pallidum activate cachectin/tumor necrosis factor synthesis: analysis using a Moreover, in T. maltophilum we identified two glycolipids com- CAT reporter construct. J. Immunol. 147:1968. 13. Sellati, T. J., D. A. Bouis, R. L. Kitchens, R. P. Darveau, J. Pugin, posed of Hex3Gro and Hex-HexN-Hex-Gro (Fig. 7D). GLC-MS R. J. Ulevitch, S. C. Gangloff, S. M. Goyert, M. V. Norgard, and J. D. Radolf. analysis of the lipid anchor revealed two monoacetylated diacyl- 1998. Treponema pallidum and Borrelia burgdorferi lipoproteins and syn- glycerols, the predominant one containing two tetradecanoic acids thetic lipopeptides activate monocytic cells via a CD14-dependent pathway distinct from that used by lipopolysaccharide. J. Immunol. 160:5455. (14:0) (Fig. 7C). Our interpretation that T. brennaborense contains 14. Choi, B. K., B. J. Paster, F. E. Dewhirst, and U. B. Go¨bel. 1994. Diversity of a glycolipid of similar structure is based on results obtained from cultivable and uncultivable oral spirochetes from a patient with severe de- structive periodontitis. Infect. Immun. 62:1889. SDS-PAGE (Fig. 7, A and B) and from TLC analysis (data not 15. Choi, B. K., H. Nattermann, S. Grund, W. Haider, and U. B. Go¨bel. 1997. shown). Spirochetes from digital dermatitis lesions in cattle are closely related to Taken together our chemical results indicate that T. maltophilum treponemes associated with human periodontitis. Int. J. Syst. Bacteriol. 47: 175. and T. brennaborense both exhibit a glycolipid consisting of a 16. Wyss, C., B. K. Choi, P. Schu¨pbach, B. Guggenheim, and U. B. Go¨bel. 1996. diacylglycerol-lipid anchor, a core region, in the case of T. mal- Treponema maltophilum sp. nov., a small oral spirochete isolated from human tophilum consisting of three sugars, and carbohydrate repeating periodontal lesions. Int. J. Syst. Bacteriol. 46:745. 17. Riviere, G. 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