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Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9

Abstract

Toll-like receptors (TLRs) activate the innate immune system in response to pathogens. Here we show that TLR9 proteolytic cleavage is a prerequisite for TLR9 signaling. Inhibition of lysosomal proteolysis rendered TLR9 inactive. The carboxy-terminal fragment of TLR9 thus generated included a portion of the TLR9 ectodomain, as well as the transmembrane and cytoplasmic domains. This cleavage fragment bound to the TLR9 ligand CpG DNA and, when expressed in Tlr9−/− dendritic cells, restored CpG DNA–induced cytokine production. Although cathepsin L generated the requisite TLR9 cleavage products in a cell-free in vitro system, several proteases influenced TLR9 cleavage in intact cells. Lysosomal proteolysis thus contributes to innate immunity by facilitating specific cleavage of TLR9.

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Figure 1: TLR9 is cleaved into two distinct polypeptides by cathepsins.
Figure 2: Trafficking of TLR9 to the endolysosomal compartment is required for its fragmentation.
Figure 3: The C-terminal TLR9 fragment directly interacts with CpG DNA.
Figure 4: The C-terminal TLR9 fragment is the active form responsible for binding CpG DNA and for subsequent TLR9 signal transduction.
Figure 5: TLR9 cleavage requires many lysosomal proteases.
Figure 6: Cathepsin L cleaves TLR9 in vitro but fails to cleave the TLR9 deletion mutant lacking the region encompassing the putative cathepsin cleavage site(s).

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References

  1. Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. 21, 335–376 (2003).

    Article  CAS  Google Scholar 

  2. Janeway, C.A. Jr & Medzhitov, R. Innate immune recognition. Annu. Rev. Immunol. 20, 197–216 (2002).

    Article  CAS  Google Scholar 

  3. Kawai, T. & Akira, S. TLR signaling. Cell Death Differ. 13, 816–825 (2006).

    Article  CAS  Google Scholar 

  4. Takeda, K. & Akira, S. Toll-like receptors in innate immunity. Int. Immunol. 17, 1–14 (2005).

    Article  CAS  Google Scholar 

  5. 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 

  6. Takeuchi, O. et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11, 443–451 (1999).

    Article  CAS  Google Scholar 

  7. Takeuchi, O. et al. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13, 933–940 (2001).

    Article  CAS  Google Scholar 

  8. Ozinsky, A. et al. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc. Natl. Acad. Sci. USA 97, 13766–13771 (2000).

    Article  CAS  Google Scholar 

  9. Hayashi, F. et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103 (2001).

    Article  CAS  Google Scholar 

  10. Hemmi, H. et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3, 196–200 (2002).

    Article  CAS  Google Scholar 

  11. 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 

  12. Heil, F. et al. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 303, 1526–1529 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. 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 

  15. Nishiya, T., Kajita, E., Miwa, S. & Defranco, A.L. TLR3 and TLR7 are targeted to the same intracellular compartments by distinct regulatory elements. J. Biol. Chem. 280, 37107–37117 (2005).

    Article  CAS  Google Scholar 

  16. Barton, G.M., Kagan, J.C. & 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).

    Article  CAS  Google Scholar 

  17. 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 

  18. Kominami, E., Ueno, T., Muno, D. & Katunuma, N. The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins. FEBS Lett. 287, 189–192 (1991).

    Article  CAS  Google Scholar 

  19. 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 

  20. Matsumoto, F. et al. Cathepsins are required for Toll-like receptor 9 responses. Biochem. Biophys. Res. Commun. 367, 693–699 (2008).

    Article  CAS  Google Scholar 

  21. Asagiri, M. et al. Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science 319, 624–627 (2008).

    Article  CAS  Google Scholar 

  22. Brinkmann, M.M. et al. The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLR signaling. J. Cell Biol. 177, 265–275 (2007).

    Article  CAS  Google Scholar 

  23. Kim, Y.M., Brinkmann, M.M., Paquet, M.E. & Ploegh, H.L. UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 452, 234–238 (2008).

    Article  CAS  Google Scholar 

  24. Choe, J., Kelker, M.S. & Wilson, I.A. Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309, 581–585 (2005).

    Article  CAS  Google Scholar 

  25. Jin, M.S. & Lee, J.O. Structures of the Toll-like receptor family and Its ligand complexes. Immunity 29, 182–191 (2008).

    Article  CAS  Google Scholar 

  26. Villadangos, J.A. & Ploegh, H.L. Proteolysis in MHC class II antigen presentation: who's in charge? Immunity 12, 233–239 (2000).

    Article  CAS  Google Scholar 

  27. Lennon-Dumenil, A.M., Bakker, A.H., Wolf-Bryant, P., Ploegh, H.L. & Lagaudriere-Gesbert, C. A closer look at proteolysis and MHC-class-II-restricted antigen presentation. Curr. Opin. Immunol. 14, 15–21 (2002).

    Article  CAS  Google Scholar 

  28. Greenbaum, D.C. et al. Small molecule affinity fingerprinting. A tool for enzyme family subclassification, target identification, and inhibitor design. Chem. Biol. 9, 1085–1094 (2002).

    Article  CAS  Google Scholar 

  29. Bogyo, M., Verhelst, S., Bellingard-Dubouchaud, V., Toba, S. & Greenbaum, D. Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs. Chem. Biol. 7, 27–38 (2000).

    Article  CAS  Google Scholar 

  30. Felbor, U. et al. Neuronal loss and brain atrophy in mice lacking cathepsins B and L. Proc. Natl. Acad. Sci. USA 99, 7883–7888 (2002).

    Article  CAS  Google Scholar 

  31. Huppa, J.B. & Ploegh, H.L. In vitro translation and assembly of a complete T cell receptor-CD3 complex. J. Exp. Med. 186, 393–403 (1997).

    Article  CAS  Google Scholar 

  32. Kawai, T. et al. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167, 5887–5894 (2001).

    Article  CAS  Google Scholar 

  33. Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J. & Mathison, J.C. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433 (1990).

    Article  CAS  Google Scholar 

  34. Shimazu, R. et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189, 1777–1782 (1999).

    Article  CAS  Google Scholar 

  35. Triantafilou, M. et al. Membrane sorting of toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting. J. Biol. Chem. 281, 31002–31011 (2006).

    Article  CAS  Google Scholar 

  36. Rogers, N.C. et al. Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity 22, 507–517 (2005).

    Article  CAS  Google Scholar 

  37. Yang, M. et al. Cathepsin L activity controls adipogenesis and glucose tolerance. Nat. Cell Biol. 9, 970–977 (2007).

    Article  CAS  Google Scholar 

  38. Maehr, R. et al. Asparagine endopeptidase is not essential for class II MHC antigen presentation but is required for processing of cathepsin L in mice. J. Immunol. 174, 7066–7074 (2005).

    Article  CAS  Google Scholar 

  39. Liu, L. et al. Structural basis of Toll-like receptor 3 signaling with double-stranded RNA. Science 320, 379–381 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank G.-P. Shi (Brigham and Women's Hospital and Harvard Medical School) for the selective inhibitors of cathepsins S, L and K and for mice deficient in cathepsins K, S and L; S. Akira (Osaka University), A. Marshak-Rothstein (Boston University) and K. Kiefer (Boston University) for Tlr9−/− mice; S.K. Dougan and C. Schlieker for critical reading of the manuscript; and T. DiCesare for graphic design. Supported by the National Institutes of Health (H.L.P.), Novartis (H.L.P.), the Charles A. King Trust, Bank of America (M.M.B.) and the Whitehead Institute for Biomedical Research, Landon T. Clay (B.R.).

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Correspondence to Hidde L Ploegh.

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Park, B., Brinkmann, M., Spooner, E. et al. Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9. Nat Immunol 9, 1407–1414 (2008). https://doi.org/10.1038/ni.1669

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