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Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis

Nature volume 450, pages 1253–1257 (2007)Cite this article

Abstract

Phagocytosis and autophagy are two ancient, highly conserved processes involved, respectively, in the removal of extracellular organisms and the destruction of organisms in the cytosol1,2,3. Autophagy, for either metabolic regulation or defence, involves the formation of a double membrane called the autophagosome, which then fuses with lysosomes to degrade the contents4, a process that has similarities with phagosome maturation. Toll-like-receptor (TLR) engagement activates a variety of defence mechanisms within phagocytes5, including facilitation of phagosome maturation6, and also engages autophagy7. Therefore we speculated that TLR signalling might link these processes to enhance the function of conventional phagosomes. Here we show that a particle that engages TLRs on a murine macrophage while it is phagocytosed triggers the autophagosome marker LC3 to be rapidly recruited to the phagosome in a manner that depends on the autophagy pathway proteins ATG5 and ATG7; this process is preceded by recruitment of beclin 1 and phosphoinositide-3-OH kinase activity. Translocation of beclin 1 and LC3 to the phagosome was not associated with observable double-membrane structures characteristic of conventional autophagosomes, but was associated with phagosome fusion with lysosomes, leading to rapid acidification and enhanced killing of the ingested organism.

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Figure 1: TLR signalling activates components of the autophagy pathway.
Figure 2: TLR-induced LC3 localization to the phagosome appears to occur independently of autophagy.
Figure 3: Components of the autophagic pathway induce localization of LC3 to the phagosome on TLR stimulation.
Figure 4: TLR signalling induces phagosome maturation using components of the autophagic pathway.

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Acknowledgements

We thank P. Murray, M. del Mar Lozano-Cedeño, C.-S. Li, M. Smeltzer, M. Bix and D. Vignali for discussions, and the St Jude Flow Cytometry and Scientific Imaging shared resource facilities for technical assistance. M. Colonna and H. Haecker provided us with essential reagents. This work is supported by grants from the US National Institutes of Health.

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Authors and Affiliations

  1. Department of Immunology,,

    Miguel A. Sanjuan, Christopher P. Dillon, Stephen W. G. Tait, Samuel Connell, Sebo Withoff & Douglas R. Green

  2. Department of Tumor Cell Biology, and,

    Simon Moshiach

  3. Department of Biochemistry, St Jude Children’s Research Institute, Memphis, Tennessee 38105, USA,

    Frank Dorsey

  4. Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan,

    Masaaki Komatsu & Keiji Tanaka

  5. Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida 33458, USA,

    John L. Cleveland

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Corresponding author

Correspondence to Douglas R. Green.

Supplementary information

Supplementary Information

This file contains Supplementary Methods and Supplementary Figures S1-S8 with Legends. (PDF 5160 kb)

Supplementary Movie 1

This file contains Supplementary Movie 1 showing RAW cells expressing GFP-LC3 were fed with uncolored E. coli and confocal images were taken every minute. Representative frames of this movie are shown in Figure S3. (MOV 12912 kb)

Supplementary Movie 2

This file contains Supplementary Movie 2 showing GFP-LC3 in RAW cells translocates to phagosomes containing zymosan (red). Representative frames of this movie are shown in Figure 1c. (MOV 11335 kb)

Supplementary Movie 3

This file contains Supplementary Movie 3 showing RAW cells expressing GFP-LC3 were treated with chloroquine for 5 hours. Endoplasmatic reticulum was then stained with ER-Tracker (red). This movie depicts a full z-stack analysis of the macrophage shown in Figure S4b (MOV 7369 kb)

Supplementary Movie 4

This file contains Supplementary Movie 4 showing RAW cells expressing GFP-LC3 were stained with ER-Tracker (red) and then fed with uncolored zymosan. Note that a small area in the center of the yeast is already red before uptake by the macrophage. A representative frame of this video is shown in Figure S4c. (MOV 50299 kb)

Supplementary Movie 5

This file contains Supplementary Movie 5 showing RAW cells expressing GFP-LC3 and the PX-domain of p40 (phox) fused to mCherry (red) were fed with uncolored zymosan. The internalization of a yeast particle was followed at 1 min intervals. Representative frames of this movie are shown in Figure 3g. (MOV 566 kb)

Supplementary Movie 6

This file contains Supplementary Movie 6 showing GFP-LC3/PX-mCherry expressing macrophages were fed with uncolored zymosan and confocal images were taken every minute. Representative frames of this movie are shown in Figure S7. (MOV 15740 kb)

Supplementary Movie 7

This file contains Supplementary Movie 7 showing GFP-LC3/PX-mCherry expressing macrophages were fed with uncolored beads and confocal images were taken every minute. Representative frames of this movie are shown in Figure S7. (MOV 8104 kb)

Supplementary Movie 8

This file contains Supplementary Movie 8 showing Internalization of zymosan (red) was followed at 1.5 min. intervals in RAW cells transiently transfected with GFP-Beclin-1. Representative images of this video are shown in Figure 3h. (MOV 7792 kb)

Supplementary Movie 9

This file contains Supplementary Movie 9 showing Phagocytosis of beads (uncolored) was followed at 1.5 min. intervals in RAW cells transiently transfected with GFP-Beclin-1. (MOV 15584 kb)

Supplementary Movie 10

This file contains Supplementary Movie 10 showing GFP-LC3 expressing RAW cells were preloaded with lysotracker (red) and the phagocytosis of control beads was followed at 3 min. intervals. Representative frames of this video are shown in Figure 4a. (MOV 18892 kb)

Supplementary Movie 11

This file contains Supplementary Movie 11 showing GFP-LC3 expressing RAW cells were preloaded with lysotracker (red) and the internalization of PAM3CSK4-covered beads was followed. Representative frames from this video are shown in Figure 4a. (MOV 28052 kb)

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Sanjuan, M., Dillon, C., Tait, S. et al. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450, 1253–1257 (2007). https://doi.org/10.1038/nature06421

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