HMGN1 activates DCs in a TRIF-dependent manner. (A and B) Human MoDCs were incubated in the absence or presence of 1 µg/ml LPS or recombinant HMGN1 prepared in insect (riN1) cells at 10 µg/ml for 48 h before measurement of surface marker expression (A) or cytokine production (n = 3; B). In A, the results of one experiment representative of three are shown. Gray histograms indicate sham-treated DCs. (C) Flow cytometric analysis of the expression of DC surface molecules after treatment without or with 1 µg/ml LPS or 10 µg/ml riN1 for 48 h. Shown are the overlay histograms of C3H/HeN or C3H/HeJ DCs of one experiment representative of two (gray histograms indicate sham-treated DCs). (D) C3HeN and C3HeJ mouse DCs were cultured at 5 × 10⁵/ml in the absence or presence of 5 µg/ml riN1 for 48 h, and cytokines were measured in the supernatants. (E) Cytokine production by MyD88^+/+ or MyD88^−/− DCs (5 × 10⁵/ml) after 48-h treatment with or without riN1. (F) DCs from TRIF^+/+ or TRIF^−/− mice were incubated at 10⁶/ml without or with 5 µg/ml riN1 for 48 h, and cytokines in the supernatants were measured. (D–F) Shown is the mean (±SD) of two (E and F) or three (D) independent experiments. *, P < 0.05 by ANOVA.