TRIF Can Be a Suppressor of TLR Signaling Figure 4. TRIF negatively regulates rEA-mediated MyD88 dependent activation of pro-inflammatory cytokines and chemokines in vivo. C57BL/6 WT (N = 9), MyD88-KO (N = 3), TRIF-KO (N = 3–4), and MyD88/TRIF-DKO (N = 4) mice were injected with 100 ng of rEA. Plasma samples were collected at 6 hpi and were analyzed using a multiplexed bead array based quantitative system. All genotype mock-injected mice (N = 2–3) were included in analysis. One of two representative experiments is shown. Statistical analysis was completed using a one-way ANOVA with a Student- Newman-Keuls post-hoc test. The bars represent Mean 6 SD. *, ** - Indicate plasma cytokine values that are statistically different from those in mock injected animals, p,0.05, p,0.001 respectively. No significant differences between mock-injected animals of different genotypes were detected. No significant activation of cytokines was observed in MyD88-KO and MyD88/TRIF-DKO animals. doi:10.1371/journal.pone.0022064.g004 CD86 on the surface of DCs. In this study, we have shown that administration of rEA results in robust maturation of DCs, as evidenced by not only increased expression of co-stimulatory molecules on these DCs, but also that rEA increased cytokines and chemokine production. While these responses were completely abrogated in rEA- treated MyD88-KO mice, most of them were dramatically enhanced in TRIF-KO mice when compared to rEA treated WT mice. From this information, we are proposing a model of rEA signaling in DCs (see Figure 8). The rEA protein likely interacts with a PRR, (likely TLR11 in mice, unknown in humans [9]), that senses rEA. The MyD88 adaptor protein gets recruited which allows MAP kinases downstream of MyD88 to become activated (pErk1/2, p38). pErk1/2 is capable of activating various transcription factors (e.g. AP1) which further activates pro- inflammatory genes, including those of pro-inflammatory cyto- kines/chemokines [17,26,29,35]. In contrast, functional TRIF protein acts as a negative regulator of the rEA-induced signaling. As a result of TRIF’s inhibitory effects, DCs have a reduction in surface expression of maturation markers, as well mitigated release of pro-inflammatory cytokines/chemokines [16]. PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22064