TRIF Can Be a Suppressor of TLR Signaling Figure 6. TRIF negatively regulates cytokine production by DCs, triggered by several common TLR agonists. (A) CD11c+ dendritic cells were isolated from C57BL/6 WT (N = 3), MyD88-KO (N = 3), TRIF-KO (N = 3), and MyD88/TRIF-DKO (N = 3) mice, in vitro stimulated with various TLR agonists, and used to perform a IL12p70 ELISA as described in Materials and Methods. The bars represent Mean 6 SEM. Statistical analysis was completed using a one-way ANOVA with Student-Newman-Keuls post-hoc test. ** - Indicate values that are statistically different from those in unstimulated DCs (for the same genotype), p,0.001. No significant differences between mock-injected animals of different genotypes were detected. No significant activation of IL12p70 was observed in MyD88-KO and MyD88/TRIF-DKO DCs. (B) DC culture media was collected at 15 hours post stimulation with various TLR agonists (rEA, LPS, ODN2006) and was analyzed for cytokines/chemokines levels using a multiplexed bead array based quantitative system. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. The bars represent Mean 6 SD. *, ** - Indicate cytokine values that are statistically different from those in mock injected animals, p,0.05, p,0.001 respectively. doi:10.1371/journal.pone.0022064.g006 PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e22064