TRIF Can Be a Suppressor of TLR Signaling a 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.g001 be involved in rEA signaling [9]. Additionally, it is not known what cell types are primarily responsible for mediating rEA-triggered responses in vivo. MyD88 and TRIF are two adaptor proteins which primarily mediate the signaling derived from activation of many pattern recognition receptors (PRRs), including TLRs [6]. We have investigated if rEA requires either of these two proteins to trigger immune responses in vivo, and have found that all rEA-triggered immune responses are dependent on MyD88 functionality (including the rapid activation of DCs, macrophages, NK, NKT, T and B cells, the induction of pro-inflammatory cytokine/ chemokine releases, as well as Erk1/2 phosphorylation). Surpris- ingly, we discovered that functional TRIF protein acts to suppress rEA induction of these same responses; thereby unveiling a novel inhibitory role for TRIF during rEA mediated signaling. We also present evidence that TRIF may similarly suppress TLR activations by other known TLR ligands. Together the findings highlight the complexities underlying adjuvant activation of the innate immune system, as well suggests that simple notions of augmenting or modifying adjuvant activity by use of TLR system agonists or antagonists may be complicated by these complex molecular mechanisms. Results TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation of dendritic cells and macrophages in vivo The purpose of this study was to identify the impact of rEA stimulation on host immune systems, to define important cell types that respond to or modulate rEA-driven activation, and to identify signaling pathways responsible for activation of the immune system in response to rEA. To investigate this, C57BL/6 mice were each IP injected with 100 ng of purified, rEA protein. Splenocytes were harvested at 6 hpi and flow cytometry was performed as detailed in Materials and Methods. We identified that activation of murine DCs (CD11c+, CD192, CD32) in response to rEA was completely dependent on the presence of full MyD88 functionality. Specifically, we found significant increases in the percent of CD40+, CD80+ and CD86+ DCs (as well as induction of the expression of these molecules per cell as measured by Mean Fluorescent Intensity [MFI]) in rEA treated wild type (WT) mice, but no such increases were observed in rEA treated MyD88 knockout (KO) or MyD88/TRIF double knockout (DKO) mice, each as compared to mock-injected animals (Figures 1, 2 and Figures S1, S2). Surprisingly, we detected significant (p,0.001) increases in DC activation after identical rEA treatments of TRIF-KO mice (as compared to WT mice), as measured by CD40, CD80 and CD86 surface staining. Further- more, the amount of CD86 expression per cell was significantly (p,0.001) increased in rEA treated TRIF-KO mice, as compared to rEA treated WT mice (Figures 1, 2 and Figures S1, S2). rEA stimulation also increased the percent of MHC-II presenting DCs in WT and TRIF-KO mice, increases that were not seen in the rEA-treated MyD88-KO or the MyD88/TRIF-DKO mice. The DCs from rEA-treated TRIF-KO mice also had significantly (p,0.05) higher MHC-II surface expression levels as compared to rEA treated WT mice (Figure 1). rEA-mediated activation of splenic macrophages (CD11b+, CD192, CD3 2 ) was also completely dependent on MyD88, as confirmed by lack of macrophage activation in response to rEA stimulation in the MyD88-KO or the MyD88/TRIF-DKO mice. In contrast, WT mice injected with rEA experienced a dramatic increase in the levels of CD80, CD86, and MHC-II, on the surface of splenic macrophages (MFI), as well as in the percent of macrophages, expressing the CD40, CD80, and CD86 activation markers. Similar to observations in DCs, macrophages derived from rEA-treated TRIF-KO mice were activated to levels that were significantly higher than levels measured in WT mice treated with rEA. Not only were the amounts of CD40-expressing macrophages increased, but also both the percentages of CD80 and CD86 expressing macrophages (p,0.01) and amount of these markers per cell (MFI, p,0.05) were significantly increased in rEA treated TRIF-KO mice as compared to rEA-treated wild type mice, (p,0.05) (Figures S3, S4, S5). These experiments unveiled an important, not previously described, role of TRIF as a suppressor of rEA-induced TLR/MyD88 signaling in DCs and macrophages in vivo. Interestingly, we found that baseline levels of MHC-II expression on splenic DCs were significantly increased in untreated, MyD88-KO and MyD88/TRIF-DKO mice (amount of MHCII per cell, p,0.01, Figure 2) as compared to untreated WT mice or untreated TRIF-KO mice (Figures 1, 2 and Figures S3, S4). Additionally, the MyD88/TRIF-DKO mice also had higher baseline levels of CD40 expression in DCs (Figure 2). This phenomenon was not unexpected, as we had previously noted increased baseline levels of the MHCII b-chain (three-fold higher) in MyD88-KO mice, as confirmed by microarray transcriptome analysis and flow cytometry based analyses ([15] and data not shown). Potentially, these baseline changes may be due to lack of presence of these adaptors during normal mouse development. TRIF negatively regulates rEA-mediated activation of NK, NKT, T and B cells in vivo The unexpected result of TRIF being a negative regulator of rEA-triggered activation of DCs and macrophages, prompted us to evaluate if TRIF reduces rEA-induced activation of other important immune cells, including effector NK cells, as well as NKT, T and B cells. We have previously shown that rEA protein activates splenic and hepatic NK and NKT cells within 6 hours post-injection in WT mice [11]. In this study, rEA injections into MyD88-KO and MyD88/TRIF-DKO mice yielded only baseline activation levels of CD69+ NK, NKT, T and B cells. This contrasted with a significant activation of these same cell types in WT mice treated with rEA protein (Figure 3 and Figures S6, S7). Similarly, the amount of IFNc production from NK and NKT cells was not increased in the MyD88-KO or the MyD88/TRIF- DKO mice in response to rEA, whereas WT mice had dramatically increased numbers of IFNc secreting NK (from 1% to 20–30%) and NKT (from 1% to 2–5%) cells. In contrast, rEA treatment of TRIF-KO mice revealed significant increases in the number of IFNc producing NK cells (p,0.05), the amount of CD69 expression per NK cell (MFI, p,0.01), the number of cells (p,0.05) and amount (p,0.05) of IFNc-production from NKT cells, the number of cells (p,0.01) and amount (p,0.01) of CD69 expression from T cells, and the number of B cells expressing CD69 (p,0.01); all in comparison to rEA-treated WT mice (Figure 3 and Figures S6, S7). Note, rEA stimulation significantly increased the number of CD69-expressing B cells in WT and PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22064