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TLR9 signals after translocating from the ER to CpG DNA in the lysosome

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Abstract

Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA–containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.

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Acknowledgements

We thank K. Halmen, L. Ryan, R. Vik and K. Egeberg for technical support; S. Akira (Osaka University, Osaka, Japan) for the full-length TLR9 cDNA and the MyD88-deficient mice; S. Ishizaka (Eisai Research Institute, Andover, Massachusetts, USA) for the HEK–TLR9–NF-κB–luc reporter cell line; B. Seed (Harvard University and Massachusetts General Hospital, Boston, Massachusetts, USA) for the retroviral vector peak12mmp; N. Aliverdi (eBiosciences, San Diego, California, USA) for TLR9 mAbs; A. Sundan (Norwegian Institute of Science and Technology, Trondheim, Norway) for antibody labeling; and H. Stenmark (Norwegian Radium Hospital, Oslo, Norway) for the FYVE-GFP construct. This work was supported by grants from the National Institute of Health (D.T.G), the Commission of the European Communities (T.E. and D.T.G.), the Norwegian Research Council and the Norwegian Cancer Society (T.E.), and by the German Academic Exchange Program (E.L).

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Competing interests

The authors declare no competing financial interests.

Correspondence to Douglas T Golenbock.

Supplementary information

  1. Supplementary Fig. 1 (PDF 418 kb)

  2. Supplementary Fig. 2 (PDF 535 kb)

  3. Supplementary Fig. 3 (PDF 2368 kb)

  4. Supplementary Fig. 4 (PDF 2628 kb)

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DOI

https://doi.org/10.1038/ni1028

Further reading

Figure 1: Fluorescently labeled TLR9 is expressed in the ER.
Figure 2: Fluorescent CpG DNA enters pDCs in vesicular structures and moves into a tubular compartment.
Figure 3: CpG DNA enters cells via a clathrin-dependent, caveolin-independent pathway.
Figure 4: CpG DNA binds to TLR9.
Figure 5: TLR9 and MyD88 translocate to sites of CpG DNA in a MyD88-independent manner.
Figure 6: Endogenous TLR9 redistributes to CpG-containing compartments.