Mycobacterium Tuberculosis Activation by Human Toll-Like Receptors
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Human Toll-Like Receptors Mediate Cellular Activation by Mycobacterium tuberculosis 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 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 macrophages 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 bacteria 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 k the transcription factor Dorsal, the fly homologue of NF- B. The of the NF-kB-dependent ELAM-1 promoter. Deletion mutants of k 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-1b 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 3 107 CHO or RAW264.7 cells were washed and 1 1 We have extended these earlier studies to test the hypothesis that harvested by scraping in Ca2 - and Mg2 -free PBS (BioWhittaker). Cells 3 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 ml 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- b-glycerol phosphate, 0.1 mM EGTA, 1 mM PMSF, and 5 mg/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 ml of 10% Nonidet P-40 (Sigma) were added to each sample before vortexing. The nuclei were centrifuged for 1 min at 5000 3 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 b 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.