TRAF6 poly-ubiquitinylation and proteasomal degradation is triggered by DC exposure to LXA₄ and L-kynurenine.

<p>WT spleen-derived DCs were cultured in the presence of medium alone (M) or with LXA₄ (Lx) (1000 ng/mL), L-kynurenine (Ky) (60 µM) or IL-10 (10) (100 ng/mL) for 6 hrs. Cells were then homogenized and immuno-precipitated with an anti-TRAF6-agarose-bead mAb. The precipitates were analyzed by western blot for the presence of TRAF6, poly-ubiquitin (Lys47) and poly-ubiquitin (Lys63). (B) Total TRAF6/beta-actin western blot analysis in WT DC cell homogenates after incubation with medium alone (M), LXA₄ (Lx) (1000 ng/mL) or L-kynurenine (Ky) (60 µM) for the indicated time points. (C) Time-course, western blot detection of total TRAF6 in spleen-derived cells incubated with LXA₄ (3 nM) with or without pre-exposure to the proteasomal inhibitor PR11 (3 nM for 90 mins). (D) WT spleen-derived cells were incubated with increasing concentrations of IL-10 (black squares) or LXA₄ (red triangles) for 6 hrs, followed by fixation/permeabilization and staining for CD11c and TRAF6 and analyzed by flow cytometry. Data shown represent plotted MFI’s for TRAF6 staining in CD11c+ cells. (E) WT (squares), SOCS2-KO (triangles) or AhR-deficient (circles) spleen-derived cells were purified and incubated with medium alone or with increasing concentrations of L-kynurenine (6–60 µM) for 6 hrs, cells were then fixed, permeabilized and stained for CD11c and TRAF6. The mean fluorescence intensity of TRAF6 staining within CD11c+ cells was then analyzed by flow cytometry. (F) WT spleen-derived cells were pre-incubated with PR11 (0.03 to 3 nM), followed by incubation with medium alone (filled squares) or with LXA₄ (upright triangles –3 nM), IL-10 (inverted triangles –100 ng/mL), L-kynurenine (diamonds –60 µM), 3-OH-kynurenine (circles –3 µM) and growth hormone (empty squares –100 ng/mL) for 6 hrs. Cells were then fixed, permeabilized and stained for CD11c, CD8α and TRAF6. Cells were then analyzed by flow cytometry for their TRAF6 mean fluorescence intensity within the CD11c+CD8α+ subset. Data shown is representative of three independent experiments performed.