TRIF Can Be a Suppressor of TLR Signaling knockout genotype. Mean Fluorescent Intensity (MFI) is shown and is indicative of amount of analyte per cell. The bars represent Mean 6 SEM. Statistical analysis was completed using two-tailed homoscedastic Student’s t-tests. *, ** - Indicate values statistically different from those in mock- injected animals (of the same genotype), p,0.05, p,0.001 respectively. doi:10.1371/journal.pone.0022064.g002 TRIF-KO mice, but significantly reduced the levels of CD69 expression per cell (Figure S6). Within 6 hours of administration, rEA protein triggers signif- icant production of pro-inflammatory cytokines and chemokines in mice. Specifically, circulating levels of IL12p70, IL6, TNFa, IFNc, IL2 were markedly elevated in rEA-treated mice [8,9]. Purified murine DCs exposed to 0.2 ng/ml of rEA were shown to release significant amounts of IL12p70, IL6, and IL2 [8]. We confirmed and extended the observation that rEA triggers the release of a wide spectrum of pro-inflammatory cytokines and chemokines, including IL6, IL12p40, IL12p70, GCSF, IFNc, IL2, IL1a, IL1b, IL10, IL13, GMCSF, KC, MCP1, MIP1a, MIP1b, RANTES, and TNFa. This effect was however, completely dependent on MyD88 given that all of these analytes were induced in rEA- treated WT, but not in rEA-treated MyD88-KO or MyD88/ TRIF-DKO mice (Figure 4). Again, paralleling our previous results, the release of all of these cytokines and chemokines was significantly increased in rEA-treated TRIF-KO mice, and the majority of these analytes were elevated to levels that were significantly higher than levels measured in rEA-treated WT mice. In particular, IL6 was induced to ,3 fold (p,0.001) higher levels and IL12p40, GCSF, and IFNc were induced to over 2 fold (p,0.001) higher levels when comparing rEA-treated TRIF-KO mice to rEA-treated WT mice. Moreover, IL12p70, IL2, IL1a, IL1b, and MIP1a were also produced at significantly higher levels in rEA-treated TRIF-KO mice as compared to rEA treated WT mice (Figure 4). Dendritic cells are a major cell type mediating rEA responses It is known that many of the rEA induced cytokines and chemokines are released by DCs (Figure 4) [16]. For that reason, we purified CD11c+ DCs from WT, MyD88-KO, TRIF-KO, and MyD88/TRIF-DKO mice, and then stimulated the cells ex vivo with escalating amounts of rEA protein. Utilizing an IL12p70 specific ELISA, we confirmed that in response to rEA, DC production of this cytokine was completely dependent upon functional MyD88 (Figure 5A). The minimal rEA dose in which WT mouse-derived DCs produced significant amounts of IL12p70 was found to be 100 pg/mL, whereas TRIF-KO mouse-derived DCs only required a 10 pg/mL dose (10 fold less) for significant IL12p70 release. In an overall comparison to WT mouse derived DCs, TRIF-KO mouse derived DCs had a significantly higher production of IL12p70 in response to rEA. The most dramatic difference between these two groups was noted when DCs were stimulated with a 0.2 ng/ml dose of rEA, which caused ,1300 pg/ml of IL12p70 to be released from DCs derived from TRIF-KO mice as compared to ,600 pg/ml from DCs derived from WT mice (p,0.01) (Figure 5A.) We have verified our ELISA-based data for IL12p70 indepen- dently, by Bioplex bead array. Again, we confirmed that MyD88- KO and the MyD88/TRIF-DKO mouse derived DCs each respectively failed to produce significant levels of pro-inflamma- tory cytokines and chemokines in response to rEA stimulation. Interestingly, we also confirmed that these rEA mediated DC responses were also partially suppressed by TRIF, as, IL2, IL6, IL12p40, IL12p70, IL1a, IL1b, and MIP1a were released to significantly (p,0.05) higher levels in rEA-treated DCs derived from TRIF-KO mice, as compared to those derived from WT mice (Figure 5B). TRIF negatively regulates cytokine production by DCs, triggered by several common TLR agonists To more fully investigate if TRIF suppressive effects were rEA- specific or a more global phenomenon, we have specifically stimulated CD11c+ DCs, isolated from WT, TRIF-KO, MyD88- KO or MyD88/TRIF-DKO mice with various common TLR4, TLR7/8, an TLR9 agonists. Specifically, LPS, R848 and ODN2006 were utilized in these experiments. All of these TLR agonists were able to induce pro-inflammatory cytokine produc- tion after administration to DCs, as compared to unstimulated DCs, all derived from WT mice. Importantly, however, DCs derived from TRIF-KO mice had dramatically higher levels of secretion of pleiotropic pro-inflammatory cytokines, when stimu- lated with these same TLR agonists (Figure 6AB). Specifically, ODN2006 stimulation resulted in significantly higher IL6, IL12p40, IL12p70 and MIP1a levels; LPS stimulation significantly increased IL1a, IL3, IL6, IL12p70 levels; R848 treatment resulted in significantly higher production of IL1a, IL1b, IL6, and MIP1a; in DCs, derived from TRIF-KO mice as compared to DCs derived from WT mice, as tested both by ELISA (Figure 6A) and Bioplex analysis (Figure 6B and data not shown). DCs derived from MyD88-KO or MyD88/TRIF-DKO mice did not show any significant cytokine activations when stimulated with any of these TLR agonists (data not shown). Interestingly, lack of the TRIF adaptor protein resulted in up to 2 fold increases in cytokine production from DCs, when stimulated with LPS or ODN2006 (i.e. IL12p70), indicating that the suppressive role of TRIF in this cell type might be immunologically significant. rEA-triggered Erk1/2 phosphorylation is MyD88 dependent Extensive past research has demonstrated that the major signaling pathways activated downstream of TLR adaptor proteins (e.g. MyD88, TRIF) are the NFkB and MAPK pathways [17]. Along with many other biological roles, these pathways promote production of cytokines and chemokines as well as proliferation, maturation, and development of various immune cells. To identify which of the several signaling pathways may be activated in response to rEA in vivo, we IP injected WT or MyD88-KO mice with 100 ng of rEA, then collected spleen and liver tissues at various time points (0–120 minutes) post-injection. We found that rEA-triggered Erk1/2 phosphorylation was MyD88 dependent, as confirmed by increased Erk1/2 phosphorylation in rEA-treated WT mice, but not in the MyD88-KO mice (Figure 7). Discussion The ideal adjuvant (from Latin ‘‘adjuvare’’, meaning ‘‘to enhance’’) is an agent that is capable of dramatically enhancing both cellular and humoral adaptive immune responses to co- administered antigens, thereby providing more efficient and long- term protection against specific pathogens. Aluminum salts, discovered to be potent adjuvants in 1920s, remained the only FDA approved adjuvant for many decades and still represent one of the few in clinical use. A barrier in adjuvant research has been that the mechanism of action of many adjuvants remained poorly PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22064