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Science 329 (5998): 1530-1534

Copyright © 2010 by the American Association for the Advancement of Science

Bifurcation of Toll-Like Receptor 9 Signaling by Adaptor Protein 3

Miwa Sasai, Melissa M. Linehan, and Akiko Iwasaki*

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.


Figure 1
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Fig. 1. Type-I IFN production is impaired in AP-3–deficient pDCs after endosomal TLR stimulation. Flt3L-cultured BM pDCs from WT and Ap3b1–/– mice were stimulated with (top) CpG-A DNA (3 µM), (middle) VSV (MOI 10), or (bottom) influenza A virus infection (MOI 5) for 24 hours. Amounts of (A) IFN-{alpha} and (B) IL-12p40 were measured by means of enzyme-linked immunosorbent assay (ELISA). WT and Ap3b1–/– mice were injected intravenously with CpG-A DNA (25 µg), and at the indicated time points serum concentrations of (C) IFN-{alpha} and (D) IL-12p40 were measured with ELISA. *P < 0.05. **P < 0.01. Results are mean ± SEM (n = 4 mice) and are representative of [(A) and (B)] four and [(C) and (D)] three independent experiments.

 

Figure 2
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Fig. 2. TLR9 fails to enter the acidic LRO compartment in AP-3–deficient cells after CpG-A stimulation. BMM from WT and Ap3b1–/– mice were transduced with TLR9-GFP retrovirus. Cells were stimulated with DOTAP–CpG-A and analyzed for TLR9-GFP (green) and VAMP-3 (an early endosomal marker) (red) at (A) 3 hours or (B) 6 hours by means of confocal microscopy. (C and D) TLR9-GFP–transduced BMM were stimulated with DOTAP–CpG-A for 6 hours and analyzed by means of confocal microscopy to detect (C) TLR9-GFP (green) and LAMP-2 (late endosomal marker) (red). The histograms depict cross-line scans of the fluorescence intensities of the merged panel. (D) To visualize the acidic compartment, the cells were incubated with LysoTracker (red) followed by staining for TLR9-GFP (green) and LAMP-2 (white/blue). The data are representative of three independent experiments. Scale bars, 5 µm.

 

Figure 3
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Fig. 3. AP-3 forms a complex with TLR9 and promotes association of TRAF3 and IRF7. (A) RAW264.7 cells expressing TLR9-GFP and AP-3µ3A were left unstimulated or stimulated with DOTAP–CpG-A (3 µM) for 1 hour. The cells were stained and analyzed for TLR9-GFP (green) and AP-3µ3A (red). Scale bars, 5 µm. (B) RAW264.7 cells transduced with a combination of retroviruses encoding TLR9-GFP or AP-3µ3A-Flag or both were stimulated with DOTAP–CpG-A for the indicated time points, and lysates were prepared and analyzed either (bottom) directly or (top) after immunoprecipitation with an antibody to Flag and immunoblotted with an antibody to GFP. (C) Human embryonic kidney (HEK) 293 T cells transfected with the indicated plasmids for 24 hours were lysed and immunoprecipitated with antibody to Flag, separated by means of SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted with the indicated antibodies. The data are representative of (A) four, (B) five, and (C) three independent experiments.

 

Figure 4
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Fig. 4. Targeting of TRAF3 to PI(3,5)P2+ compartment bypasses the requirement for AP-3 in TLR9 signaling. (A) BMM from Ap3b1–/– mice transduced with lentivirus encoding YFP-IRF7 were stimulated with DOTAP–CpG-A-Cy5 for 6 hours and analyzed by means of confocal microscopy to detect YFP-IRF7 (green), LysoTracker (red), and CpG-A (white/blue). Scale bars, 5 µm. (B) BMM from Ap3b1–/– mice transduced with retrovirus encoding TLR9-GFP and PH-TRAF3 were stimulated with DOTAP–CpG-A for 4 hours (VAMP-3 staining) or 6 hours (LAMP-2 staining) and analyzed by means of confocal microscopy in order to detect TLR9-GFP (green), VAMP3 (red), or LAMP-2 (red), and PH-TRAF3 (blue). Scale bars, 5 µm. (C) BMMs from Ap3b1–/– mice were transduced with lentivirus encoding TRAF3 or PH-TRAF3. IFN-{alpha} and IL-12p40 mRNA expression was assessed by means of quantitative RT-PCR at 12 hours after DOTAP–CpG-A stimulation. Results are representative of four independent experiments.

 


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