Human Toll-Like Receptors Mediate Cellular Activation by Terry K. Means, Shuyan Wang, Egil Lien, Atsutoshi Yoshimura, Douglas T. Golenbock and Matthew J. Fenton This information is current as of September 27, 2021. J Immunol 1999; 163:3920-3927; ; http://www.jimmunol.org/content/163/7/3920 Downloaded from References This article cites 32 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/163/7/3920.full#ref-list-1

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Human Toll-Like Receptors Mediate Cellular Activation by Mycobacterium tuberculosis1

Terry K. Means,* Shuyan Wang,* Egil Lien,† Atsutoshi Yoshimura,† Douglas T. Golenbock,† and Matthew J. Fenton2*

Recent studies have implicated a family of mammalian Toll-like receptors (TLR) in the activation of by Gram- negative and Gram-positive bacterial products. We have previously shown that different TLR proteins mediate cellular activation by the distinct CD14 ligands Gram-negative bacterial LPS and mycobacterial glycolipid lipoarabinomannan (LAM). Here we show that viable Mycobacterium tuberculosis bacilli activated both Chinese hamster ovary cells and murine macrophages that overexpressed either TLR2 or TLR4. This contrasted with Gram-positive and Mycobacterium avium, which activated cells via TLR2 but not TLR4. Both virulent and attenuated strains of M. tuberculosis could activate the cells in a TLR-dependent

manner. Neither membrane-bound nor soluble CD14 was required for bacilli to activate cells in a TLR-dependent manner. We Downloaded from also assessed whether LAM was the mycobacterial cell wall component responsible for TLR-dependent cellular activation by M. tuberculosis. We found that TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobac- teria. In contrast, LAM isolated from M. tuberculosis or Mycobacterium bovis bacillus Calmette-Gue´rin failed to induce TLR- dependent activation. Lastly, both soluble and cell wall-associated mycobacterial factors were capable of mediating activation via distinct TLR proteins. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation,

whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4-dependent activation. Together, our data demon- http://www.jimmunol.org/ strate that Toll-like receptors can mediate cellular activation by M. tuberculosis via CD14-independent ligands that are distinct from the mycobacterial cell wall glycolipid LAM. The Journal of Immunology, 1999, 163: 3920–3927.

he discovery of mammalian homologues of the Drosoph- mains of Toll proteins share significant sequence similarity with ila Toll receptor protein has elicited interest in the role of the type I IL-1 receptor, the known mammalian Toll-like receptors, T these proteins in innate immunity (reviewed in Ref. 1). and the cytosolic adapter protein MyD88 (4–6). Several published reports have illustrated the potential importance Recent data have demonstrated that mammalian TLR proteins of Toll-like receptors (TLR)3 in intracellular signaling. Janeway participate in intracellular signaling initiated by Gram-negative by guest on September 27, 2021 and colleagues (2) reported a human homologue of the Drosophila bacterial LPS. CD14 has been recognized for many years as the Toll protein, a protein later to be designated Toll-like receptor 4 major receptor responsible for the effects of LPS on macrophages, (TLR4). There were three features of Toll that linked this protein monocytes, and neutrophils (reviewed in Ref. 7). Two groups in- with innate immunity and intracellular signaling. First, Drosophila dependently reported that TLR2 could function as a signaling re- Toll participates in an antifungal response in the adult fly (3), and ceptor for LPS in the presence of CD14 (8, 9). These investigators it is likely that mammalian homologues would participate in sim- reported that human HEK293 cells stably transfected with TLR2 ilar innate immune responses. Second, the Drosophila Toll partic- could respond to LPS in the presence of CD14 and LPS-binding ipates in a signal transduction pathway leading to the activation of protein, as judged by activation of a reporter gene under the control ␬ the transcription factor Dorsal, the fly homologue of NF- B. The of the NF-␬B-dependent ELAM-1 promoter. Deletion mutants of ␬ central role played by NF- B in signal transduction pathways ac- TLR2 that lack a region of the intracellular domain that shares tivated by cytokines, and in the regulation of cytokine genes them- sequence homology with the IL-1 receptor failed to mediate LPS selves, implicates mammalian Toll proteins in cellular responses responsiveness in this assay. similar to those evoked by cytokines. Third, the intracellular do- Subsequent to these findings, two other groups identified the gene responsible for the LPS-hyporesponsive phenotype of the C3H/HeJ mouse (10, 11). Macrophages from this mouse are hy- *Pulmonary Center, Boston University School of Medicine, and †Infectious Disease Section, Boston Medical Center, Boston, MA 02118 poresponsive to LPS, even though they express normal amounts of Received for publication April 23, 1999. Accepted for publication July 19, 1999. CD14 on their surface. The gene locus responsible for this defect (Lpsd) mapped to the Tlr4 gene. In the C3H/HeJ mice, a single The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance missense mutation within the Tlr4 coding sequence was identified with 18 U.S.C. Section 1734 solely to indicate this fact. (P712H). Supporting evidence for the hypothesis that this mutation 1 This work was supported by National Institutes of Health Grants GM54060 (to is responsible for the LPS-hyporesponsive phenotype of the C3H/ D.T.G. and A.Y.) and HL55681 (to M.J.F. and T.K.M.). E.L. was supported by the Norwegian Cancer Society and the Research Council of Norway. HeJ mouse comes from the finding that the C57BL/10ScCr LPS- nonresponsive mouse does not express TLR4. These results raised 2 Address correspondence and reprint requests to Dr. Matthew J. Fenton, Pulmonary Center, Room R-220, Boston University School of Medicine, 80 E. Concord Street, the possibility that, at least in mice, TLR2 is not sufficient to confer Boston, MA 02118. E-mail address: [email protected] LPS responsiveness. Data from our own laboratories have shown 3 Abbreviations used in this paper: TLR, Toll-like receptor; LAM, lipoarabinoman- that TLR4 is predominantly responsible for LPS signaling in mu- nan; Mtb, Mycobacterium tuberculosis; CHO, Chinese hamster ovary; LBP, LPS- binding protein; BCG, bacillus Calmette-Gue´rin; EMSA, electrophoretic mobility rine and hamster cells, whereas TLR2 mediates cellular activation shift assays. by a distinct CD14 ligand, the mycobacterial cell wall glycolipid

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 3921 lipoarabinomannan (LAM).4 This conclusion was further sup- ferent mycobacteria (CS-35) was also provided by Dr. Belisle and was ported by our demonstration that LPS hyporesponsive macro- previously described (16). Recombinant human IL-1␤ was purchased from phages from C3H/HeJ mice were not hyporesponsive to LAM.4 Genzyme (Cambridge, MA). FITC- and PE-conjugated anti-human CD25 mAbs were purchased from Becton Dickinson (Bedford, MA). Most recently, Chow et al. (13) reported that human TLR4 could mediate LPS responsiveness in HEK293 cells. Together, these re- Electrophoretic mobility shift assays (EMSA) sults suggest that TLR2 and TLR4 mediate CD14-dependent sig- Nuclear extracts were prepared essentially as described by Schreiber et al. nals in both a ligand- and species-specific manner. (17). Approximately 1.0 ϫ 107 CHO or RAW264.7 cells were washed and ϩ ϩ We have extended these earlier studies to test the hypothesis that harvested by scraping in Ca2 - and Mg2 -free PBS (BioWhittaker). Cells ϫ Mycobacterium tuberculosis, bacteria that do not synthesize LPS, were pelleted by centrifugation at 800 g for 10 min at 4°C. Cell pellets were resuspended in 400 ␮l of a buffer containing 10 mM KCl, 10 mM might also be recognized by TLR proteins. It was recently found Tris-HCl (pH 7.8), 5 mM MgCl2, 0.5 mM DTT, 0.3 M sucrose, 10 mM that cellular activation by Gram-positive bacteria and Mycobacte- ␤-glycerol phosphate, 0.1 mM EGTA, 1 mM PMSF, and 5 ␮g/ml each of rium avium was mediated by TLR2, but not TLR4 (Ref. 14 and aprotinin, leupeptin, chymostatin, and antipain and incubated on ice for 10 data not shown).5 Here we report that M. tuberculosis activates min. Subsequently, 25 ␮l of 10% Nonidet P-40 (Sigma) were added to each sample before vortexing. The nuclei were centrifuged for 1 min at 5000 ϫ cells in a TLR-dependent manner, but unlike Gram-positive bac- g to pellet the nuclei. Nuclear pellets were resuspended in a nuclear ex- teria and M. avium, these organisms utilize both TLR2 and TLR4 traction buffer containing 320 mM KCl, 10 mM Tris-HCl (pH 7.8), 5 mM ␤ proteins. Unlike Gram-positive bacteria, TLR-dependent cellular MgCl2, 0.5 mM DTT, 10 mM -glycerol phosphate, 0.1 mM EGTA, 25% activation by M. tuberculosis does not appear to depend on the glycerol, 1 mM PMSF, and 5 ␮g/ml each of aprotinin, leupeptin, antipain, presence of CD14. Furthermore, the mycobacterial ligands respon- and chymostatin. Samples were extracted on ice for 15 min followed by centrifugation at 16,000 ϫ g for 10 min at 4°C. Protein concentration was sible for this activation appear to be distinct from LAM. determined using the Bio-Rad assay kit (Bio-Rad, Hercules, CA). All nu- Downloaded from clear extracts were stored at Ϫ70°C, and multiple freeze-thawing cycles Materials and Methods were avoided. Cells and reagents A double-stranded oligonucleotide containing a single copy of the IL-2 receptor ␣-chain NF-␬B (GGGGAATTCC) was used as an EMSA probe. M. tuberculosis strains H37Rv (ATCC 25618), H37Ra (ATCC 25177), and DNA probes were labeled with [␣-32P]dNTPs (DuPont-NEN, Boston, MA) Mycobacterium bovis BCG (ATCC 35734) were purchased from the using E. coli DNA polymerase Klenow fragment (U.S. Biochemicals,

American Type Culture Collection (ATCC, Manassas, VA). Bacterial cul- Cleveland, OH) as recommended by the manufacturer. Unincorporated nu- http://www.jimmunol.org/ tures were grown in Middlebrook 7H9 medium supplemented with Tween cleotides were removed with Sephadex G-25 columns (5 Prime33 Prime, 80 and ADC (Difco, Detroit, MI) at 37°C under biosafety level 3 condi- Boulder, CO). Nuclear extracts (typically 3 ␮g) were incubated with ra- tions. Bacterial culture medium was prepared in LPS-free flasks using LPS- diolabeled probe DNA (0.1 ng, typically 10,000 cpm) in the presence of 2 free water (Baxter-Travenol, Deerfield, IL). Bacterial cultures were grown ␮g poly(dI-dC) (Pharmacia, Piscataway, NJ), 1.0 mM EDTA, 10 mM Tris- ϭ to midlogarithmic phase (OD620 nm 0.4), and CFU per ml were deter- HCl (pH 7.9), 25 mM glycerol, and 0.5 mM DTT in a final volume of 20 mined by growth on Middlebrook agar plates. The CHO-K1 fibroblast ␮l, as previously described (18). Binding reactions were then incubated at (CCL-61) and RAW264.7 murine cell lines (TIB-71) were room temperature for 30 min. After incubation, a portion of each binding purchased from the ATCC. RAW264.7 cells were maintained in DMEM reaction (typically 6 ␮l) was loaded onto 7% nondenaturing low ionic (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inacti- strength polyacrylamide gel. The gels were then dried and visualized by vated fetal bovine serum (HyClone Laboratories, Logan, UT), 10 mM autoradiography. by guest on September 27, 2021 HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml strepto- mycin (BioWhittaker). Chinese hamster ovary (CHO) cells were main- Plasmids tained in Ham’s F-12 culture medium (BioWhittaker) supplemented with The ELAM-luc reporter plasmid was generated by subcloning the promoter 10% heat-inactivated FBS (HyClone, Logan, UT), 10 mM HEPES, 2 mM of pUMS(ELAM)-Tac (15) into the promoterless pGL3 luciferase reporter L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (BioWhit- plasmid (Promega, Madison, WI). The human TLR2 and TLR4 cDNAs taker). LPS levels in all medium components were Ͻ10 pg/ml final con- cloned into the pFlag-CMV-1 mammalian expression plasmid were gifts of centration as indicated by BioWhittaker or measured by the Limulus ame- Drs. Carsten Kirschning and Mike Rothe (Tularik, South San Francisco, bocyte lysate kit (BioWhittaker). Cells were cultured at 37°C in the CA) and were previously described (9). These gene products are expressed presence of 5% CO2 in a humidified incubator. CHO/CD14, 3E10 (CHO/ as fusion proteins containing an N-terminal FLAG epitope tag. A second CD14/ELAM-CD25), 3E10/TLR2, 3E10/TLR4, and CHO/TLR2 were pre- TLR4 expression plasmid that expresses native TLR4 (termed hToll) was 4 viously described (14, 15) and cultured as described above. All of these a gift of Dr. Charles Janeway (Yale University, New Haven, CT) and was stable cell lines used expressed a similar level of surface CD14, TLR2, and previously described (2). Plasmids were prepared using Qiagen (Valencia, TLR4. As described below, cells that were stably transfected with the CA) plasmid DNA purification columns, DNA was eluted from the col- TLR2 and TLR4 expression plasmids express FLAG-tagged TLR proteins. umns using LPS-free buffers, and contaminating LPS levels were found to After transfection and selection of stable lines, clones were selected that be Ͻ10 pg/ml. Furthermore, all plasmid preparations were unable to acti- expressed similar levels of TLR proteins, based on mean fluorescence in- vate the LPS-sensitive CHO/CD14 cells, demonstrating that the plasmids tensity determined with the same anti-FLAG Ab. Furthermore, the TLR2- were not contaminated with LPS. and TLR4-expressing cell lines were generated from the same CHO/CD14 parental clonal cell line, and thus each line also expresses the same amount Transfection and reporter assays of CD14. LPS (purified from Escherichia coli 055:B5) was purchased from Sigma Transient transfections were performed using SuperFect reagent (Qiagen, (St. Louis, MO). Mycobacterial LAM, purified from rapidly growing avir- Valencia, CA) as per the manufacturer’s instructions. Briefly, cells were ulent mycobacteria (AraLAM), M. bovis BCG, and M. tuberculosis strains plated on six-well dishes 1–2 days before transfection, and transfections were performed when cells plated reached 80% confluence. Plasmid DNA H37Rv and H37Ra were all provided by Dr. John Belisle (Colorado State ␮ University, Fort Collins, CO) under the provisions of National Institutes of was added to 100 l of Opti-Mem reduced serum media (Life Technolo- gies, Gaithersburg, MD). All transfections utilized a total of 4 ␮g of plas- Health Contract NO1 AI25147. Levels of contaminating LPS in the LAM ␮ ␮ preparations were determined using a quantitative Limulus lysate assay mid DNA consisting of 2 g of reporter plasmid, 1 g of each expression Ͻ vector, and the balance made up with empty vector described above. Su- (BioWhittaker) and were 1 pg/ml final concentration in all experiments. ␮ A neutralizing anti-LAM IgG3 mAb that recognizes LAM from these dif- perFect, 10 l, was added to the DNA-medium mixture and incubated for 10 min at ambient temperature. Subsequently, 600 ␮l of serum-containing medium were added to the reaction mixture and added to the individual 4 T. K. Means, E. Lien, A. Yoshimura, S. Wang, D. T. Golenbock, and M. J. Fenton. wells. Each reaction was prepared individually, and each condition was 1999. The CD14 ligands lipoarabinomannan and differ in their performed in triplicate. Reactions were incubated with the cells for 2–3 h, requirement for Toll-like receptors. Submitted for publication. whereupon the reaction was removed from the cells and fresh medium 5 E. Lien, T. J. Sellati, A. Yoshimura, J. D. Carroll, R. R. Ingalls, J. D. Radolf, and containing serum was added. On the following day, individual wells were D. T. Golenbock. 1999. Toll-like receptor 2 mediates pattern recognition by Borrelia left untreated or were stimulated with either LPS or LAM as indicated in burgdorferi, Treponema pallidum, and Mycobacterium avium. Submitted for publi- the figures. Cells were then incubated for an additional 5 h before harvest- cation.Author: any further publication information? ing. Luciferase assays were performed as described below. All transfection 3922 M. tuberculosis ACTIVATES CELLS VIA TLR PROTEINS

FIGURE 1. Expression of TLR2 by CHO/CD14 cells confers responsiveness to Mtb. CHO/CD14 and CHO/CD14/TLR2 reporter cell lines were Downloaded from stimulated with the Mtb strains H37Rv (Rv) or H37Ra (Ra, 10 bacilli/CHO cell) for 16 h. Both reporter lines contain a stably transfected ELAM-CD25 reporter gene and express human CD25 on their surface as a consequence of NF-␬B activation (14). Stimulated cells were stained with a PE-labeled anti-CD25 mAb and subjected to flow cytometry analysis to measure the expression of CD25. Data are expressed as the ratio (fold activation) of the percent of CD25ϩ cells in unstimulated and stimulated cell populations that were gated to exclude the lowest 5% of cells based on mean FL2 fluorescence. Representative overlaying histograms that correspond to the graphic data are also shown (insets). Four independent experiments were performed. http://www.jimmunol.org/ experiments were repeated at least three times with different plasmid prep- TLR-independent stimuli that activate NF-␬B (e.g., IL-1␤ protein) arations, and a single representative experiment is shown. Each single ex- also activate CD25 expression. Here we incubated viable Mtb with periment represents triplicate independent transfections and data are ex- CHO/CD14 and CHO/CD14/TLR2 cells (10 Mtb/CHO cell) for pressed as average values Ϯ S.D. Luciferase activity was measured using the Luciferase Assay System 16 h and subsequently measured CD25 expression by flow cytom- (Promega), according to manufacturer’s instructions. Briefly, cells were etry. As shown in Fig. 1, both the virulent H37Rv and attenuated washed and scraped on ice in cold PBS, pelleted by centrifugation, and H37Ra strains of Mtb did not activate the CHO/CD14 cells, and resuspended in 100 ␮l of reporter lysis buffer. Samples were freeze-thawed

overexpression of TLR2 conferred Mtb responsiveness on these by guest on September 27, 2021 once and centrifuged at 14,000 ϫ g for 10 min at 4°C to remove cellular debris. Supernatants were recovered and assayed for total protein using the cells. This TLR-dependent activation did not appear to vary with Bio-Rad protein assay according to manufacturer’s instructions. An equal the virulence of the Mtb strain used. Similar data were obtained amount of total protein from each lysate was assayed for luciferase activity with avirulent M. bovis BCG bacilli (data not shown). CD25 ex- as measured by light emissions in a scintillation counter. pression was also measured at 1, 4, 8, 16, and 24 h after stimula- In experiments with 3E10 cells, which contain a stably transfected CD25 reporter gene under the control of the ELAM-1 promoter, reporter tion by H37Ra and H37Rv. At each time point analyzed, we found gene expression was measured by flow cytometry as previously described4 that both Mtb strains activated the cells to a similar extent (data not (14, 15). Data were collected using FACScan software (Becton Dickinson, shown). Lastly, surface expression of either CD14 or TLR proteins Mountain View, CA) and expressed as the ratio (fold activation) of the did not alter Mtb binding or uptake by the CHO cells compared percent of CD25ϩ cells in unstimulated and stimulated cell populations. The 95% confidence limit for nonspecific fluorescence was established with untransfected CHO cells (data not shown). with the use of isotype control Abs. Each experiment was repeated at least three times in all cases. TLR-dependent activation by M. tuberculosis does not Results require LBP M. tuberculosis bacilli activate cells in a TLR-dependent Our earlier data showed that TLR-dependent activation of cells by manner the mycobacterial cell wall glycolipid LAM was substantially en- 4 Previous studies demonstrated that both bacterial cell wall com- hanced in the presence of LPS-binding protein (LBP (20)). To ponents (LPS, LAM, and peptidoglycan) and heat-killed Gram- determine whether LBP present in serum was required for TLR- positive bacteria (Staphylococcus aureus and Streptococcus pneu- dependent activation of cells by Mtb, we repeated the preceding moniae) could activate cells in a TLR-dependent manner4 (14, 19). experiment in the presence and absence of serum. As shown in Fig. Here we tested whether live M. tuberculosis bacilli (Mtb) could 2, the presence of serum did not significantly affect responsiveness also activate cells in a TLR-dependent manner. To measure TLR- of cells to Mtb, suggesting that LBP is not required for TLR- dependent activation, we used CHO fibroblast cell lines that were dependent activation by Mtb. Because the FBS used in these stud- stably transfected with expression plasmids encoding human ies was heat inactivated, our data also demonstrate that neither CD14 (CHO/CD14) and human TLR2 proteins (CHO/CD14/ opsonization nor activated complement was required for this TLR- TLR2). These lines were also stably transfected with a reporter dependent activation. This is not surprising because CHO cells do plasmid consisting of a human CD25 cDNA under the control of not express either complement receptors or other receptors known an NF-␬B-dependent ELAM-1 promoter. As we previously pub- to mediate the binding of Mtb to macrophages (e.g., macrophage lished, activation of these cells in a CD14-dependent manner leads ). Because LBP, at least in part, transfers bacte- to the rapid activation of NF-␬B and the subsequent expression of rial glycolipids to CD14, our data also suggested that CD14 was CD25 on the cell surface4 (14, 15). Furthermore, CD14- and not required for this TLR-mediated activation of cells by Mtb. The Journal of Immunology 3923

FIGURE 2. TLR-dependent activation by M. tuberculosis does not re- quire LBP. CHO/CD14 and CHO/CD14/TLR2 cell lines were stimulated with H37Ra Mtb (10 bacilli/CHO cell) in medium containing 10% FBS or in serum-free medium for 16 h. The stimulated cells were then stained with a FITC-labeled anti-CD25 mAb and subjected to flow cytometry analysis to measure the expression of CD25. Data are expressed as the ratio (fold activation) of the percent of CD25ϩ cells in unstimulated and stimulated Downloaded from cell populations that were gated to exclude the lowest 5% of cells based on FIGURE 3. TLR-dependent activation by M. tuberculosis does not re- mean FL1 fluorescence. quire CD14. A, CHO/CD14 and CHO/TLR2 cells were stimulated with Mtb (10 H37Ra bacilli per CHO cell) in medium containing 10% FBS or TLR-dependent activation by M. tuberculosis does not require in serum-free medium for 1 h. B, CHO/CD14/TLR2 and CHO/TLR2 cells were stimulated with Mtb (1 and 10 H37Ra bacilli per CHO cell) for 1 h. CD14

Nuclear extracts from stimulated and unstimulated cells were prepared as http://www.jimmunol.org/ We recently showed that TLR-dependent activation of cells by described in the text and then assessed for the presence of NF-␬B using an either LAM or Gram-positive bacteria required the presence of EMSA. The migration of the DNA-protein complex containing NF-␬Bis CD14 as either a membrane-bound or soluble protein4 (14). To test indicated. This complex was found to be specific as judged using super- ␬ the hypothesis that Mtb activate cells in a TLR-dependent, but shifting Abs and unlabeled NF- B competitor oligonucleotides (data not CD14-independent manner, we compared the capacity of Mtb to shown). activate CHO/TLR2 cells (i.e., CHO fibroblasts that express TLR2, but not CD14) in the absence and presence of serum, which expression plasmids. After transfection, the cells were stimulated serves as a source of soluble CD14. Nuclear extracts were prepared with Mtb (10 Mtb/RAW264.7 cell) and harvested 5 h later. Lysates

from the CHO/TLR2 cells 1 h after Mtb stimulation, and NF-␬B were prepared from the harvested cells, and luciferase activity was by guest on September 27, 2021 levels were subsequently measured by EMSA. As shown in Fig. measured as described in Materials and Methods. As shown in Fig. 3A, similar levels of NF-␬B were induced in a TLR-dependent 4B, Mtb activated the macrophages in a TLR-dependent manner. manner in the presence and absence of soluble CD14. NF-␬B lev- Like the CHO cells, this activation could be mediated by both els in CHO/CD14 cells were not altered by exposure to Mtb. Fur- TLR2 and TLR4. thermore, we compared the capacity of Mtb to activate NF-␬Bin CHO/CD14/TLR2 vs CHO/TLR2 cells and found that the pres- AraLAM, but not ManLAM, activate cells in a TLR-dependent ence of membrane-bound CD14 did not augment Mtb respon- manner siveness (Fig. 3B). These data demonstrate that TLR-dependent Our previous studies revealed that LAM purified from rapidly activation by Mtb is not mediated by CD14. This contrasts with growing Mycobacteria could activate cells in a TLR-dependent Gram-positive bacteria where activation is dependent on both manner.4 This form of LAM contains highly branched arabino- TLR2 and CD14 (14, 19). furanosyl side chains (termed AraLAM), whereas Mtb and M. bo- vis BCG contain LAM that is terminally capped with mannose Both TLR2 and TLR4 mediate cell activation by M. tuberculosis residues (ManLAM). These chemical differences are believed to Recent studies revealed that both Gram-positive bacteria and M. be responsible for the different biological activities of these gly- avium can activate cells via TLR2, but not TLR4 (Ref. 14 and data colipids (reviewed in Ref. 21). We sought to determine whether not shown).5 We sought to determine whether Mtb also activated LAM on the surface of the mycobacteria was the ligand respon- cells via the same selective use of TLR proteins. Mtb were added sible for TLR-dependent activation of the cells. First, we compared to CHO/CD14/TLR cells. As shown in Fig. 4A, we found that Mtb the capacity of LAM isolated from rapidly growing Mycobacteria could activate the cells via both TLR2 and TLR4. Thus, Mtb dif- (AraLAM), Mtb (ManLAM), and M. bovis BCG (BCG LAM) to fered from Gram-positive bacteria and M. avium in their utilization activate cells in a TLR-dependent manner. As shown in Fig. 5, of TLR proteins during cellular activation. Virtually identical re- AraLAM was capable of activating CHO/CD14 cells that ex- sults were obtained with either H37Ra or H37Rv Mtb, or when the pressed TLR2, but not TLR4. In contrast to AraLAM, neither Mtb experiment was performed in the presence or absence of serum LAM nor BCG LAM were capable of activating any of the cell (data not shown). Furthermore, activation via either TLR2 or lines tested. This finding is consistent with the known relative in- TLR4 was not dependent on the presence of membrane-bound ability of these ManLAMs to activate macrophages (22–24). CD14 (data not shown). We subsequently examined whether Mtb could also activate macrophages via both TLR proteins. To test LAM is not responsible for TLR-dependent cellular activation by this possibility, we transiently cotransfected RAW264.7 murine M. tuberculosis macrophages with a luciferase reporter plasmid under the control To confirm that TLR-dependent cellular activation by Mtb was not of the NF-␬B-dependent ELAM-1 promoter and the various TLR mediated by LAM, we determined whether an anti-LAM Ab could 3924 M. tuberculosis ACTIVATES CELLS VIA TLR PROTEINS

FIGURE 5. AraLAM, but not ManLAM, activates cells in a TLR-de- pendent manner. CHO/CD14, CHO/CD14/TLR2, and CHO/CD14TLR4 reporter cell lines were treated with AraLAM (1 ␮g/ml), BCG LAM (1 ␮g/ml), or H37Rv Mtb LAM (1 ␮g/ml) for 16 h. Stimulated cells were stained with a FITC-labeled anti-CD25 mAb and subjected to flow cytom- etry analysis to measure the expression of CD25. Data are expressed as the Downloaded from ratio (fold activation) of the percent of CD25ϩ cells in unstimulated and stimulated cell populations that were gated to exclude the lowest 5% of cells based on mean FL1 fluorescence.

Both soluble and cell-associated mycobacterial factors mediate http://www.jimmunol.org/ TLR-dependent activation We sought to determine whether soluble factors released by cul- tured Mtb could activate the cells in a TLR-dependent manner. FIGURE 4. Both TLR2 and TLR4 mediate cell activation by M. tuber- culosis. A, CHO/CD14, CHO/CD14/TLR2, and CHO/CD14/TLR4 reporter H37Ra Mtb were grown to mid-log phase as described in Mate- cell lines were stimulated with Mtb (10 H37Ra bacilli per CHO cell) for rials and Methods, bacilli were removed by centrifugation and 16 h. These reporter lines contain a stably transfected ELAM-CD25 re- filtration, and the Mtb-free culture medium was used to stimulate porter gene and express human CD25 on their surface as a consequence of the CHO/CD14/TLR cells. As shown in Fig. 7A, we found that NF-␬B activation. Stimulated cells were stained with a FITC-labeled anti- Mtb-conditioned culture medium activated the cells via TLR2, but CD25 mAb and subjected to flow cytometry analysis to measure the ex- not TLR4. Fresh culture medium did not activate the cells under by guest on September 27, 2021 pression of CD25. Data are expressed as the ratio (fold activation) of the ϩ any conditions tested (data not shown). We subsequently examined percent of CD25 cells in unstimulated and stimulated cell populations that whether the soluble factors responsible for TLR-dependent acti- were gated to exclude the lowest 5% of cells based on mean FL1 fluores- vation were proteins. The Mtb-conditioned culture medium was cence. B, RAW264.7 cells were transiently cotransfected with the ELAM- digested with proteinase K (50 ␮g/ml, 55°C, 1 h) and boiled for 5 Luc reporter plasmid and expression plasmids encoding either human TLR2 or TLR4. A portion of the cells were then stimulated with Mtb (10 min to inactivate the protease. This protein-free Mtb-conditioned H37Ra bacilli per RAW264.7 cell) for 5 h. Cells were then harvested and culture medium was then evaluated for its capacity to activate cells luciferase activity was measured as described in the text. All transfection in a TLR2-dependent manner. As shown in Fig. 7B, the protein- experiments were performed in triplicate, repeated at least three times with free and heat-stable preparation was still capable of inducing the use of different plasmid preparations, and a single representative ex- TLR2-dependent cellular activation. Thus, it appears that a non- periment is shown. Data are expressed as average luciferase values from a proteinaceous heat-stable factor is the TLR2 ligand. Based on the single experiment (subtracted for background) Ϯ SD. chemical nature of other known TLR ligands (e.g LPS, LAM, pep- tidoglycan), it is likely that this factor is a polysaccharide or a glycolipid. It is also possible that this preparation contains several block activation of cells by Mtb bacilli. Mtb were pretreated with distinct ligands for TLR2. the neutralizing CS-35 anti-LAM mAb (107 H37Ra bacilli treated To test whether LAM present in the Mtb-conditioned culture with 500 ␮g/ml CS-35 for 15 min), and then added to the CHO/ medium was responsible for the TLR2-dependent cellular activa- CD14/TLR cells (10 Mtb/CHO cell, 12.5 ␮g/ml CS-35 final con- tion, we assessed the capacity of the CS-35 anti-LAM neutralizing centration). As shown in Fig. 6A, the anti-LAM mAb was incapa- Ab to block TLR2-dependent cellular activation by the Mtb-con- ble of blocking Mtb activation of cells via TLR2 or TLR4. The ditioned culture medium. CHO/CD14/TLR2 cells were stimulated ability of CS-35 to block LAM-induced cellular activation was with Mtb-conditioned culture medium in the presence or absence confirmed by the finding that this mAb could block TLR2-depen- of either CS-35, or a matching isotype control Ab. The CS-35 Ab dent activation by AraLAM (Fig. 6B). Normal rabbit serum had no had no effect on the capacity of Mtb-conditioned culture medium effect on either Mtb- or AraLAM-induced cellular activation (data to activate the CHO/CD14/TLR2 cells (data not shown). This find- not shown). Thus, using two distinct experimental approaches ing demonstrates that LAM is not responsible for the observed (Figs. 5 and 6), we have demonstrated that TLR-dependent acti- activation, and is consistent with our earlier finding that purified vation of cells by Mtb was not mediated by the cell wall glycolipid Mtb LAM could not activate cells in a TLR-dependent manner LAM. Although we have shown that CD14 does not appear to play (Fig. 5). a role in TLR-dependent cellular activation by Mtb, it remains to Because viable Mtb bacilli could induce both TLR2- and TLR4- be determined whether the mycobacterial ligands activate TLR dependent signaling, but the soluble mycobacterial factor only ac- proteins directly or in association with other cell surface receptors. tivated cells via TLR2, we sought to determine whether it was a The Journal of Immunology 3925

distinct bacterial products (LPS and LAM) bind to a common re- ceptor (CD14) but utilize different TLR proteins (TLR4 and TLR2, respectively). We have now shown that Mtb bacilli can activate both CHO cells and murine macrophages via both TLR2 and TLR4. This contrasts with Gram-positive bacteria and M. avium which activate cells via TLR2, but not TLR4. Both virulent and attenuated Mtb strains can activate the cells in a TLR-dependent manner. Neither membrane-bound nor soluble CD14 appears to be required for Mtb to activate cells in a TLR-dependent manner. We also found that TLR2, but not TLR4, can confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, nei- ther LAM isolated from Mtb nor M. bovis BCG can activate cells in a TLR-dependent manner. Lastly, both soluble and cell wall- associated mycobacterial factors can mediate activation via dis- tinct TLR proteins. Together, our data demonstrate that mamma- lian Toll-like receptors can mediate cellular activation by Mtb via CD14-independent ligands that are distinct from LAM. M. tuberculosis, the etiological agent of tuberculosis, is a major

worldwide public health threat. Mtb bacilli are uniquely adapted to Downloaded from survive and grow within macrophages (reviewed in Ref. 25). Par- adoxically, macrophages are the primary effector cells of the innate immune response. On binding microbial pathogens, these cells are activated to release a variety of cytokines, nitric oxide, and reac- tive oxygen intermediates. Cytokines released by activated mac-

rophages can augment both innate and cell-mediated immune re- http://www.jimmunol.org/ sponses. The precise mechanisms by which Mtb activate macrophages remain unclear. Our data revealed two unexpected features of these Mtb-induced responses. The first feature is that CD14 does not appear to be required for cellular activation by Mtb. This contrasts with both Gram-positive and Gram-negative bacte- FIGURE 6. TLR-dependent activation by M. tuberculosis is not medi- ria that predominantly activate cells in a CD14-dependent manner ated by LAM. A, Mtb were pretreated with the neutralizing CS-35 anti- (14, 24, 26). Although CD14 has been reported to function as an LAM mAb (107 H37Ra bacilli treated with 500 ␮g/ml CS-35 for 15 min) adhesion receptor for Mtb bacilli in some cell types (27), blocking and then added to the CHO/CD14, CHO/CD14/TLR2, or CHO/CD14/ Abs against the complement receptors CR3, CR4, the macrophage by guest on September 27, 2021 ␮ TLR4 cells (10 H37Ra bacilli per CHO cell, 12.5 g/ml CS-35 final con- mannose receptor, and the class A scavenger receptor can together centration) for 16 h. B, AraLAM was pretreated with the CS35 anti-LAM prevent almost all binding of Mtb to macrophages (28–30). It is mAb (2 ␮g AraLAM treated with 500 ␮g/ml CS-35 for 15 min) and then possible that complement receptors, like CD14, can serve as co- added to cells (AraLAM 1 ␮g/ml, 12.5 ␮g/ml CS-35 final concentration) for 16 h. Normal rabbit serum had no effect on either Mtb- or AraLAM- receptors for TLR proteins. Support for this possibility comes from induced cellular activation (data not shown). Stimulated cells were stained the finding that the CR3 ligand taxol, a plant-derived antitumor with a FITC-labeled anti-CD25 mAb and subjected to flow cytometry anal- agent, cannot activate macrophages from the TLR4-deficient C3H/ ysis to measure the expression of CD25. Data are expressed as the ratio HeJ mouse whereas taxol can activate macrophages from the nor- (fold activation) of the percent of CD25ϩ cells in unstimulated and stim- mal C3H/FeJ mouse (31). The second novel feature of our findings ulated cell populations that were gated to exclude the lowest 5% of cells is that Mtb differs in the utilization of TLR proteins to initiate based on mean FL1 fluorescence. intracellular signaling compared with Gram-positive bacteria (14).5 Whether these differences in TLR signaling induced by Mtb cell-associated factor that induced TLR4-dependent signaling. As and other bacteria result in different qualitative or quantitative an- shown in Fig. 7C, we found that heat-killed Mtb that were washed timicrobial responses remains to be determined. free of culture medium activated cells via TLR2. In contrast to the Like Gram-positive bacteria, the cell wall of Mtb does not con- viable Mtb, the heat-killed bacilli could not activate cells via tain LPS. Nevertheless, both types of bacteria can activate cells in TLR4. These differences imply that the ligand for TLR4-depen- a TLR-dependent manner via chemically distinct bacterial prod- dent activation is a heat-labile cell-associated mycobacterial factor. ucts that serve as ligands to mediate this activation. Previous stud- Cellular activation via TLR4 was not caused by LPS contamina- ies have shown that LAM, LPS, and peptidoglycan all activate tion because all materials used to perform this experiment were TLR-dependent signaling in association with CD14 (14, 19).4 In essentially LPS free and cellular activation by the bacilli was not contrast, we have shown that TLR proteins can be activated by CD14 dependent. Thus, soluble mycobacterial factors selectively CD14-independent ligands. We found both soluble and cell-asso- activate cells in a TLR2-dependent manner, whereas heat-labile ciated mycobacterial factors can activate TLR-dependent signaling cell-associated mycobacterial factors selectively activate cells in a in a CD14-independent manner. A heat-labile cell-associated my- TLR4-dependent manner. These findings demonstrate that whole cobacterial factor activated cells in a TLR4-dependent manner, Mtb and soluble mycobacterial factors differ in their utilization of whereas a soluble heat-stable mycobacterial factor activated cells TLR proteins leading to cellular activation. in a TLR2-dependent manner. This soluble factor was also resis- tant to protease treatment, suggesting that it is a polysaccharide or Discussion a glycolipid. In addition, we found that a heat-stable cell-associ- Mammalian TLR proteins are novel mediators of cellular activa- ated factor could activate cells in a TLR2-dependent manner (data tion by bacterial products. Our previous studies have shown that not shown), although it remains to be determined whether this 3926 M. tuberculosis ACTIVATES CELLS VIA TLR PROTEINS

FIGURE 7. Both soluble and cell-associated mycobacterial fac- tors mediate TLR-dependent acti- vation. A, CHO/CD14, CHO/CD14/ TLR2, and CHO/CD14TLR4 reporter cell lines were stimulated with 100 ␮l of soluble Mtb-condi- tioned culture supernatant (soluble tuberculosis factor (STF)) for 16 h. This final volume of STF corre- sponds to 10 Mtb equivalents per CHO cell. The stimulated cells were then stained with a FITC-la- beled anti-CD25 mAb and sub- jected to flow cytometry analysis to measure the expression of CD25. Representative overlaying histo- grams that were gated to exclude

the lowest 5% of cells based on Downloaded from mean FL1 fluorescence are shown. B, The soluble Mtb-conditioned culture supernatant (STF) was pre- treated with proteinase K (50 ␮g/ ml, 55°C, 1 h) and boiled for 5 min to inactivate the protease. The re- porter cell lines were stimulated http://www.jimmunol.org/ with 100 ␮l of protease-treated STF for 16 h. The stimulated cells were then stained with a PE-labeled anti- CD25 mAb, subjected to flow cy- tometry analysis to measure the ex- pression of CD25, and the data were analyzed as described above. C, CHO/CD14/TLR2 and CHO/ CD14/TLR4 cell lines were stimu- by guest on September 27, 2021 lated with live and heat-killed Mtb (10 H37Ra bacilli per CHO cell) for 16 h. The stimulated cells were then stained with a FITC-labeled anti- CD25 mAb and subjected to flow cytometry analysis to measure the expression of CD25. Data are ex- pressed as the ratio (fold activation) of the percent of CD25ϩ cells in un- stimulated and stimulated cell pop- ulations that were gated to exclude the lowest 5% of cells based on mean FL1 fluorescence.

factor is identical with the soluble heat-stable factor. Studies are the host macrophage without evoking a strong antimicrobial re- currently under way to identify these factors. Interestingly, myco- sponse. Our data demonstrate that Mtb can induce TLR-dependent bacterial envelopes contain peptidoglycan, a known CD14 ligand cellular activation, but the extent of this activation may differ qual- that can activate cells in a TLR2-dependent manner (14, 19). Our itatively from TLR signaling induced by other types of bacteria. finding that Mtb activates cells in a CD14-independent manner CD14 ligands present in the cell walls of Gram-positive and Gram- suggests that peptidoglycan is not the heat-stable factor we found negative bacilli that activate TLR signaling may induce potent an- that activated cells via TLR2. The extensive waxy cell wall of Mtb timicrobial responses that are not induced by Mtb. Our studies may mask the underlying peptidoglycan, although this possibility have only assessed signaling via TLR2 and TLR4, but how Mtb remains to be tested. and other types of bacteria might differentially utilize other TLR Pathogenic mycobacteria, such as Mtb, are part of a family of proteins remain to be determined. slow-growing mycobacteria that contain mannose-capped LAM The Drosophila Toll and 18-wheeler proteins are receptors that (i.e., ManLAM) in their cell walls. In contrast, rapidly growing mediate antifungal and antibacterial immune responses, respec- mycobacteria are nonpathogenic in immunocompetent hosts and tively (3, 32). These findings support the possibility that mamma- possess arabinofuranosyl-capped LAM (i.e., AraLAM). Part of the lian TLR proteins also participate in innate immunity. Mammalian survival strategy of Mtb may depend on bacilli gaining entry into cells possess at least 10 distinct TLR genes, and it is possible that The Journal of Immunology 3927 these proteins can bind a wide variety of bacterial products (Patent 15. Delude, R. L., A. Yoshimura, R. R. Ingalls, and D. T. Golenbock. 1998. Con- Cooperation Treaty Publication number WO9850547A3). Given struction of a lipopolysaccharide reporter cell line and its use in identifying mu- tants defective in endotoxin, but not TNF-␣, signal transduction. J. Immunol. the dimeric nature of the Drosophila Toll receptor, it is also pos- 161:3001. sible that mammalian TLR proteins might heterodimerize, thereby 16. Prinzis, S., D. Chatterjee, and P. J. Brennan. 1993. Structure and antigenicity of lipoarabinomannan from Mycobacterium bovis BCG. J. Gen. Microbiol. 139: further extending the variety of ligands that might be capable of 2649. inducing TLR signaling. Furthermore, mammalian ligands for 17. Schreiber, E., P. Matthias, M. M. Muller, and W. Schaffner. 1989. Rapid detec- TLR proteins may also exist. In Drosophila, the natural ligand for tion of octamer binding proteins with “mini-extracts,” prepared from a small number of cells. Nucleic Acids Res. 17:6419. Toll is the Spatzle protein (33). Spatzle, a primitive member of the 18. Hunninghake, G. W., B. G. Monks, L. J. Geist, M. M. Monick, M. A. Monroy, NGF family of cysteine-knot proteins (34), is secreted as an inac- M. F. Stinski, A. C. Webb, J. M. Dayer, P. E. Auron, and M. J. Fenton. 1992. The tive precursor protein that is cleaved into a biologically active form functional importance of a cap site-proximal region of the human prointerleukin 1␤ gene is defined by viral trans-activation. Mol. Cell. Biol. 12:3439. by the serine protease Easter (12). Similar paradigms are well 19. Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning. 1999. known in mammalian innate immune responses, and natural mam- Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll- malian ligands for TLR proteins may yet be discovered. Our data like receptor 2. J. Biol. Chem. 274:17406. 20. Savedra, R., R. J. Delude, R. R. Ingalls, M. J. Fenton, and D. T. Golenbock. 1996. do not exclude the possibility that Mtb, and mycobacterial factors Mycobacterial lipoarabinomannan recognition requires a receptor that shares do not bind directly to TLR proteins, but instead stimulate the components of the endotoxin signaling system. J. Immunol. 157:2549. 21. Strohmeiher, G., and M. J. Fenton. 1999. Role of lipoarabinomannan in myco- production of a mammalian Spatzle-like factor that binds directly bacterial pathogenesis and immunity. Microbes Inf. 2:1.Author: please confirm to the TLR proteins. journal title; not in our sources. 22. Chatterjee, D., A. D. Roberts, K. Lowell, P. J. Brennan, and I. M. Orme. 1992. 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