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Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA


Toll-like receptors (TLRs) sense infection by detecting molecular structures of microbial origin. TLR3, TLR7 and TLR9 recognize nucleic acids and are localized to intracellular compartments where they normally respond to viral nucleic acids. The purpose for this intracellular localization, however, is not clear. Here we describe a chimeric TLR9 receptor that localized to the cell surface and responded normally to synthetic TLR9 ligands but not to viral nucleic acids. However, the 'relocated' chimeric TLR9 receptor was able to recognize self DNA, which does not stimulate wild-type TLR9. These data demonstrated that intracellular localization of TLR9 was not required for ligand recognition. Instead, localization of the nucleic acid-sensing TLRs is critical in discriminating between self and nonself nucleic acid.

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Figure 1: Defining the TLR domains responsible for localization.
Figure 2: The TLR9N4C chimeric receptor is expressed at cell surface.
Figure 3: Relocalized TLR9 responds to CpG DNA independently of endosomal acidification.
Figure 4: The intracellular localization of TLR9 is necessary for recognition of viral DNA.
Figure 5: TLR9N4C recognizes self DNA.

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  1. Akira, S., Takeda, K. & Kaisho, T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675–680 (2001).

    Article  CAS  Google Scholar 

  2. Janeway, C.A., Jr. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb. Symp. Quant. Biol. 54, 1–13 (1989).

    Article  CAS  Google Scholar 

  3. Funami, K. et al. The cytoplasmic 'linker region' in Toll-like receptor 3 controls receptor localization and signaling. Int. Immunol. 16, 1143–1154 (2004).

    Article  CAS  Google Scholar 

  4. Latz, E. et al. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5, 190–198 (2004).

    Article  CAS  Google Scholar 

  5. Leifer, C.A. et al. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J. Immunol. 173, 1179–1183 (2004).

    Article  CAS  Google Scholar 

  6. Matsumoto, M. et al. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J. Immunol. 171, 3154–3162 (2003).

    Article  CAS  Google Scholar 

  7. Nagai, Y. et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat. Immunol. 3, 667–672 (2002).

    Article  CAS  Google Scholar 

  8. Underhill, D.M. et al. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401, 811–815 (1999).

    Article  CAS  Google Scholar 

  9. Ahmad-Nejad, P. et al. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32, 1958–1968 (2002).

    Article  CAS  Google Scholar 

  10. Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

    CAS  Google Scholar 

  11. Hacker, H. et al. CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. EMBO J. 17, 6230–6240 (1998).

    Article  CAS  Google Scholar 

  12. Leadbetter, E.A. et al. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603–607 (2002).

    Article  CAS  Google Scholar 

  13. Viglianti, G.A. et al. Activation of autoreactive B cells by CpG dsDNA. Immunity 19, 837–847 (2003).

    Article  CAS  Google Scholar 

  14. Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088 (1998).

    Article  CAS  Google Scholar 

  15. Qureshi, S.T. et al. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J. Exp. Med. 189, 615–625 (1999).

    Article  CAS  Google Scholar 

  16. Krieg, A.M. et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–549 (1995).

    Article  CAS  Google Scholar 

  17. Nishiya, T. & DeFranco, A.L. Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling properties of the Toll-like receptors. J. Biol. Chem. 279, 19008–19017 (2004).

    Article  CAS  Google Scholar 

  18. Lund, J., Sato, A., Akira, S., Medzhitov, R. & Iwasaki, A. Toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells. J. Exp. Med. 198, 513–520 (2003).

    Article  CAS  Google Scholar 

  19. Krug, A. et al. Herpes simplex virus type 1 activates murine natural interferon-producing cells through Toll-like receptor 9. Blood 103, 1433–1437 (2004).

    Article  CAS  Google Scholar 

  20. Krug, A. et al. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21, 107–119 (2004).

    Article  CAS  Google Scholar 

  21. Tabeta, K. et al. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc. Natl. Acad. Sci. USA 101, 3516–3521 (2004).

    Article  CAS  Google Scholar 

  22. Gilliet, M. et al. The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 195, 953–958 (2002).

    Article  CAS  Google Scholar 

  23. Honda, K. et al. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 434, 1035–1040 (2005).

    Article  CAS  Google Scholar 

  24. Krieg, A.M. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20, 709–760 (2002).

    Article  CAS  Google Scholar 

  25. Shlomchik, M.J., Craft, J.E. & Mamula, M.J. From T to B and back again: positive feedback in systemic autoimmune disease. Nat. Rev. Immunol. 1, 147–153 (2001).

    Article  CAS  Google Scholar 

  26. Boule, M.W. et al. Toll-like receptor 9-dependent and -independent dendritic cell activation by chromatin-immunoglobulin G complexes. J. Exp. Med. 199, 1631–1640 (2004).

    Article  CAS  Google Scholar 

  27. Spitzer, S. & Eckstein, F. Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides. Nucleic Acids Res. 16, 11691–11704 (1988).

    Article  CAS  Google Scholar 

  28. Napirei, M. et al. Features of systemic lupus erythematosus in DNase1-deficient mice. Nat. Genet. 25, 177–181 (2000).

    Article  CAS  Google Scholar 

  29. Yasutomo, K. et al. Mutation of DNASE1 in people with systemic lupus erythematosus. Nat. Genet. 28, 313–314 (2001).

    Article  CAS  Google Scholar 

  30. Reggiori, F. & Pelham, H.R. A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies. Nat. Cell Biol. 4, 117–123 (2002).

    Article  CAS  Google Scholar 

  31. Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S. & Reis e Sousa, C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529–1531 (2004).

    Article  CAS  Google Scholar 

  32. Lund, J.M. et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 101, 5598–5603 (2004).

    Article  CAS  Google Scholar 

  33. Horng, T., Barton, G.M. & Medzhitov, R. TIRAP: an adapter molecule in the Toll signaling pathway. Nat. Immunol. 2, 835–841 (2001).

    Article  CAS  Google Scholar 

  34. Barton, G.M. & Medzhitov, R. Retroviral delivery of small interfering RNA into primary cells. Proc. Natl. Acad. Sci. USA 99, 14943–14945 (2002).

    Article  CAS  Google Scholar 

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We thank S. Akira for TLR9-deficient mice; A. Iwasaki for HSV-2; A. Unni, C. Pasare and A. Rudensky for discussions and advice; and W. Yuan for technical advice. R.M. is an investigator of the Howard Hughes Medical Institute. Supported by the National Institutes of Health (AI46688 and AI055502 to R.M. and AI07019 to J.K.).

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Correspondence to Ruslan Medzhitov.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Immunofluorescence images of MEFs expressing CD4-TLR4 (left), ARF1T31N (center), and the merged image (right). (PDF 156 kb)

Supplementary Fig. 2

The TLR4-T4TM chimeric receptor does not signal. (PDF 226 kb)

Supplementary Fig. 3

TLR9N4C does not signal in pDC. (PDF 298 kb)

Supplementary Fig. 4

Equivalent expression of TLR9 and TLR9N4C from bicistronic retroviral vectors. (PDF 228 kb)

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Barton, G., Kagan, J. & Medzhitov, R. Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA. Nat Immunol 7, 49–56 (2006).

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