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

Nature Immunology volume 9, pages 14071414 (2008) | Download Citation

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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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References

  1. 1.

    , & Toll-like receptors. Annu. Rev. Immunol. 21, 335–376 (2003).

  2. 2.

    & Innate immune recognition. Annu. Rev. Immunol. 20, 197–216 (2002).

  3. 3.

    & TLR signaling. Cell Death Differ. 13, 816–825 (2006).

  4. 4.

    & Toll-like receptors in innate immunity. Int. Immunol. 17, 1–14 (2005).

  5. 5.

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

  6. 6.

    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).

  7. 7.

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

  8. 8.

    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).

  9. 9.

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

  10. 10.

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

  11. 11.

    , , , & Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529–1531 (2004).

  12. 12.

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

  13. 13.

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

  14. 14.

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

  15. 15.

    , , & TLR3 and TLR7 are targeted to the same intracellular compartments by distinct regulatory elements. J. Biol. Chem. 280, 37107–37117 (2005).

  16. 16.

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

  17. 17.

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

  18. 18.

    , , & The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins. FEBS Lett. 287, 189–192 (1991).

  19. 19.

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

  20. 20.

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

  21. 21.

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

  22. 22.

    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).

  23. 23.

    , , & UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 452, 234–238 (2008).

  24. 24.

    , & Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309, 581–585 (2005).

  25. 25.

    & Structures of the Toll-like receptor family and Its ligand complexes. Immunity 29, 182–191 (2008).

  26. 26.

    & Proteolysis in MHC class II antigen presentation: who's in charge? Immunity 12, 233–239 (2000).

  27. 27.

    , , , & A closer look at proteolysis and MHC-class-II-restricted antigen presentation. Curr. Opin. Immunol. 14, 15–21 (2002).

  28. 28.

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

  29. 29.

    , , , & Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs. Chem. Biol. 7, 27–38 (2000).

  30. 30.

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

  31. 31.

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

  32. 32.

    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).

  33. 33.

    , , , & CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433 (1990).

  34. 34.

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

  35. 35.

    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).

  36. 36.

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

  37. 37.

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

  38. 38.

    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).

  39. 39.

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

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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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  1. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02115, USA.

    • Boyoun Park
    • , Melanie M Brinkmann
    • , Eric Spooner
    • , Clarissa C Lee
    •  & Hidde L Ploegh
  2. Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA.

    • You-Me Kim

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Correspondence to Hidde L Ploegh.

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DOI

https://doi.org/10.1038/ni.1669

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