Figure 5.

Rapamycin inhibits the spatial interaction of TLR9 with the MyD88-IRF7 complex and affects the phosphorylation and nuclear translocation of IRF7. (a) Immunoassay of RAW cells (4 × 10⁶) transfected with hemagglutinin-tagged MyD88 and YFP-tagged TLR9 and then, 36 h later, stimulated for 90 min with CpG-A–DOTAP; cell extracts were immunoprecipitated (IP) with anti-hemagglutinin and analyzed by immunoblot (IB) for coprecipitated TLR9. MyD88 serves as a loading control. (b) Flow cytometry of human pDCs pretreated with rapamycin (left) or transfected with control (con) siRNA or siRNA specific for p70S6K1 and p70S6K2 (S6K), then stimulated for 45 min with CpG-A; cells were fixed, made permeable and stained with phycoerythrin-conjugated mouse antibody to IRF7 phosphorylated at Ser477 and Ser479 (phosphor-IRF7) or control isotype antibody (Control Ab; phycoerythrin-conjugated mouse immunoglobulin G1). (c) Confocal microscopy of purified mouse pDCs stimulated for 12 h with CpG-A in the presence of rapamycin or vehicle, then fixed with formaldehyde, made permeable with saponin, blocked with 20% (vol/vol) goat serum, stained with anti-IRF7 (green) and mounted with ProLong Gold antifade reagent with DAPI (blue). Scale bar, 2 μm. (d) Luciferase assay of HEK293 cells transiently transfected with 0 ng (−), 40 ng or 200 ng of constitutively active IRF7 (IRF7-D477,479) together with luciferase-tagged IFN-β (plasmid p125-Luc; 50 ng), renilla luciferase (0.5 ng), wild-type IRF7 (3 ng), MyD88 (20 ng) and TLR9 (50 ng), cultured for 24 h, then pretreated for 3 h with 100 nM rapamycin and then stimulated overnight with CpG-A (10 μg/ml). Empty vector, vector without IRF7. Data are representative of three (a,d) or two (b,c) independent experiments (error bars (d), s.d.).